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1.  HAI-2 suppresses the invasive growth and metastasis of prostate cancer through regulation of matriptase 
Oncogene  2013;33(38):4643-4652.
Dysregulation of cell surface proteolysis has been strongly implicated in tumorigenicity and metastasis. In this study, we delineated the role of hepatocyte growth factor activator inhibitor-2 (HAI-2) in prostate cancer (PCa) cell migration, invasion, tumorigenicity and metastasis using a human PCa progression model (103E, N1, and N2 cells) and xenograft models. N1 and N2 cells were established through serial intraprostatic propagation of 103E human PCa cells and isolation of the metastatic cells from nearby lymph nodes. The invasion capability of these cells was revealed to gradually increase throughout the serial isolations (103E
doi:10.1038/onc.2013.412
PMCID: PMC4314694  PMID: 24121274
prostate cancer; hepatocyte growth factor activator inhibitor-2; cancer cell invasion; tumorigenicity and metastasis
The Journal of Neuroscience  2013;33(48):18806-18813.
Proper movement execution relies on precise input processing by spinal motoneurons (MNs). Spinal MNs are activated by limb joint rotations. Typically, their movement-related receptive fields (MRRFs) are sharply focused and joint-specific. After acute spinal transection MRRFs become wide, but their manifestation is not apparent, as intrinsic excitability, primarily resulting from the loss of persistent inward currents (PICs), dramatically decreases. PICs undergo a remarkable recovery with time after injury. Here we investigate whether MRRFs undergo a recovery that parallels that of the PIC. Using the chronic spinal cat in acute terminal decerebrate preparations, we found that MRRFs remain expanded 1 month after spinal transaction, whereas PICs recovered to >80% of their preinjury amplitudes. These recovered PICs substantially amplified the expanded inputs underlying the MRRFs. As a result, we show that single joint rotations lead to the activation of muscles across the entire limb. These results provide a potential mechanism for the propagation of spasms throughout the limb.
doi:10.1523/JNEUROSCI.2609-13.2013
PMCID: PMC3841450  PMID: 24285887
Genetic defects in matriptase are linked to two congenital ichthyosis, autosomal recessive ichthyosis with hypotrichosis (ARIH, OMIM 610765) and, ichthyosis, follicular atrophoderma, hypotrichosis, and hypohidrosis (IFAH, OMIM602400). Mouse models with matriptase deficiency indicate an involvement of matriptase in suprabasal keratinocytes in the maintenance of the epidermal barrier. In contrast to what has been reported for mouse skin, we show that in human skin, matriptase is primarily expressed in the basal and spinous keratinocytes, but not in the more differentiated keratinocytes of the granular layer. In addition, matriptase zymogen activation was predominantly detected in the basal cells. Furthermore, using skin organotypic cultures as a model system to monitor the course of human epidermal differentiation, we found elevated matriptase zymogen activation during early stages of epidermal differentiation, coupled with a loss of matriptase expression in the late stages of this process. We also show here that matriptase deficiency in HaCaT cells modestly reduces cell proliferation and temporally affects calcium-induced expression of differentiation markers. These collective data suggests that, unlike mouse matriptase, human matriptase may be involved in regulation of keratinocyte growth and early differentiation, rather than terminal differentiation, providing mechanistic insights for the pathology of the two congenital ichthyoses, ARIH and IFAH.
doi:10.1038/jid.2013.320
PMCID: PMC3925676  PMID: 23900022
Extra forces or torques are defined as forces or torques that are larger than would be expected from the input or stimuli, which can be mediated by properties intrinsic to motoneurons and/or to the muscle. The purpose of this study was to determine whether extra forces/torques evoked during electrical stimulation of the muscle or its nerve with variable frequency stimulation are modulated by muscle length/joint angle. A secondary aim was to determine whether extra forces/torques are generated by an intrinsic neuronal or muscle property. Experiments were conducted in 14 able-bodied human subjects and in eight adult decerebrate cats. Torque and force were measured in human and cat experiments, respectively. Extra forces/torques were evoked by stimulating muscles with surface electrodes (human experiments) or by stimulating the nerve with cuff electrodes (cat experiments). In humans and cats, extra forces/torques were larger at short muscle lengths, indicating that a similar regulatory mechanism is involved. In decerebrate cats, extra forces and length-dependent modulation were unaffected by intrathecal methoxamine injections, despite evidence of increased spinal excitability, and by transecting the sciatic nerve proximal to the nerve stimulations. Anesthetic nerve block experiments in two human subjects also failed to abolish extra torques and the length-dependent modulation. Therefore, these data indicate that extra forces/torques evoked during electrical stimulation of the muscle or nerve are muscle length-dependent and primarily mediated by an intrinsic muscle property.
doi:10.1523/JNEUROSCI.6641-10.2011
PMCID: PMC4115248  PMID: 21490198
Motoneurons provide the only conduit for motor commands to reach muscles. For many years, motoneurons were in fact considered to be little more than passive “wires”. Systematic studies in the past 25 years however have clearly demonstrated that the intrinsic electrical properties of motoneurons are under strong neuromodulatory control via multiple sources. The discovery of potent neuromodulation from the brainstem and its ability to change the gain of motoneurons shows that the “passive” view of the motor output stage is no longer tenable. A mechanism for gain control at the motor output stage makes good functional sense considering our capability of generating an enormous range of forces, from very delicate (e.g., putting in a contact lens) to highly forceful (emergency reactions). Just as sensory systems need gain control to deal with a wide dynamic range of inputs, so to might motor output need gain control to deal with the wide dynamic range of the normal movement repertoire. Two problems emerge from the potential use of the brainstem monoaminergic projection to motoneurons for gain control. First, the projection is highly diffuse anatomically, so that independent control of the gains of different motor pools is not feasible. In fact, the system is so diffuse that gain for all the motor pools in a limb likely increases in concert. Second, if there is a system that increases gain, probably a system to reduce gain is also needed. In this review, we summarize recent studies that show local inhibitory circuits within the spinal cord, especially reciprocal and recurrent inhibition, have the potential to solve both of these problems as well as constitute another source of gain modulation.
doi:10.3389/fncir.2014.00081
PMCID: PMC4114207  PMID: 25120435
gain; serotonin; motoneuron; spinal cord; spinal cord injury
Infection and Immunity  2013;81(11):4280-4289.
Neisseria gonorrhoeae PilC1 is a member of the PilC family of type IV pilus-associated adhesins found in Neisseria species and other type IV pilus-producing genera. Previously, a calcium-binding domain was described in the C-terminal domains of PilY1 of Pseudomonas aeruginosa and in PilC1 and PilC2 of Kingella kingae. Genetic analysis of N. gonorrhoeae revealed a similar calcium-binding motif in PilC1. To evaluate the potential significance of this calcium-binding region in N. gonorrhoeae, we produced recombinant full-length PilC1 and a PilC1 C-terminal domain fragment. We show that, while alterations of the calcium-binding motif disrupted the ability of PilC1 to bind calcium, they did not grossly affect the secondary structure of the protein. Furthermore, we demonstrate that both full-length wild-type PilC1 and full-length calcium-binding-deficient PilC1 inhibited gonococcal adherence to cultured human cervical epithelial cells, unlike the truncated PilC1 C-terminal domain. Similar to PilC1 in K. kingae, but in contrast to the calcium-binding mutant of P. aeruginosa PilY1, an equivalent mutation in N. gonorrhoeae PilC1 produced normal amounts of pili. However, the N. gonorrhoeae PilC1 calcium-binding mutant still had partial defects in gonococcal adhesion to ME180 cells and genetic transformation, which are both essential virulence factors in this human pathogen. Thus, we conclude that calcium binding to PilC1 plays a critical role in pilus function in N. gonorrhoeae.
doi:10.1128/IAI.00493-13
PMCID: PMC3811810  PMID: 24002068
PLoS ONE  2014;9(5):e97666.
Mammary epithelial (ME) cells cultured under conventional conditions senesce after several passages. Here, we demonstrate that mouse ME cells isolated from normal mammary glands or from mouse mammary tumor virus (MMTV)-Neu–induced mammary tumors, can be cultured indefinitely as conditionally reprogrammed cells (CRCs) on irradiated fibroblasts in the presence of the Rho kinase inhibitor Y-27632. Cell surface progenitor-associated markers are rapidly induced in normal mouse ME-CRCs relative to ME cells. However, the expression of certain mammary progenitor subpopulations, such as CD49f+ ESA+ CD44+, drops significantly in later passages. Nevertheless, mouse ME-CRCs grown in a three-dimensional extracellular matrix gave rise to mammary acinar structures. ME-CRCs isolated from MMTV-Neu transgenic mouse mammary tumors express high levels of HER2/neu, as well as tumor-initiating cell markers, such as CD44+, CD49f+, and ESA+ (EpCam). These patterns of expression are sustained in later CRC passages. Early and late passage ME-CRCs from MMTV-Neu tumors that were implanted in the mammary fat pads of syngeneic or nude mice developed vascular tumors that metastasized within 6 weeks of transplantation. Importantly, the histopathology of these tumors was indistinguishable from that of the parental tumors that develop in the MMTV-Neu mice. Application of the CRC system to mouse mammary epithelial cells provides an attractive model system to study the genetics and phenotype of normal and transformed mouse epithelium in a defined culture environment and in vivo transplant studies.
doi:10.1371/journal.pone.0097666
PMCID: PMC4022745  PMID: 24831228
Journal of Bacteriology  2013;195(4):886-895.
Kingella kingae is an emerging bacterial pathogen that is being recognized increasingly as an important etiology of septic arthritis, osteomyelitis, and bacteremia, especially in young children. The pathogenesis of K. kingae disease begins with bacterial adherence to respiratory epithelium, which is dependent on type IV pili and is influenced by two PilC-like proteins called PilC1 and PilC2. Production of either PilC1 or PilC2 is necessary for K. kingae piliation and bacterial adherence. In this study, we set out to further investigate the role of PilC1 and PilC2 in type IV pilus-associated phenotypes. We found that PilC1 contains a functional 9-amino-acid calcium-binding (Ca-binding) site with homology to the Pseudomonas aeruginosa PilY1 Ca-binding site and that PilC2 contains a functional 12-amino-acid Ca-binding site with homology to the human calmodulin Ca-binding site. Using targeted mutagenesis to disrupt the Ca-binding sites, we demonstrated that the PilC1 and PilC2 Ca-binding sites are dispensable for piliation. Interestingly, we showed that the PilC1 site is necessary for twitching motility and adherence to Chang epithelial cells, while the PilC2 site has only a minor influence on twitching motility and no influence on adherence. These findings establish key differences in PilC1 and PilC2 function in K. kingae and provide insights into the biology of the PilC-like family of proteins.
doi:10.1128/JB.02186-12
PMCID: PMC3562114  PMID: 23243304
PLoS ONE  2014;9(4):e93899.
The ability of cells to rapidly detect and react to alterations in their chemical environment, such as pH, ionic strength and redox potential, is essential for cell function and survival. We present here evidence that cells can respond to such environmental alterations by rapid induction of matriptase autoactivation. Specifically, we show that matriptase autoactivation can occur spontaneously at physiological pH, and is significantly enhanced by acidic pH, both in a cell-free system and in living cells. The acid-accelerated autoactivation can be attenuated by chloride, a property that may be part of a safety mechanism to prevent unregulated matriptase autoactivation. Additionally, the thio-redox balance of the environment also modulates matriptase autoactivation. Using the cell-free system, we show that matriptase autoactivation is suppressed by cytosolic reductive factors, with this cytosolic suppression being reverted by the addition of oxidizing agents. In living cells, we observed rapid induction of matriptase autoactivation upon exposure to toxic metal ions known to induce oxidative stress, including CoCl2 and CdCl2. The metal-induced matriptase autoactivation is suppressed by N-acetylcysteine, supporting the putative role of altered cellular redox state in metal induced matriptase autoactivation. Furthermore, matriptase knockdown rendered cells more susceptible to CdCl2-induced cell death compared to control cells. This observation implies that the metal-induced matriptase autoactivation confers cells with the ability to survive exposure to toxic metals and/or oxidative stress. Our results suggest that matriptase can act as a cellular sensor of the chemical environment of the cell that allows the cell to respond to and protect itself from changes in the chemical milieu.
doi:10.1371/journal.pone.0093899
PMCID: PMC3976350  PMID: 24705933
PLoS ONE  2014;9(3):e92244.
The type 2 transmembrane serine protease matriptase is broadly expressed in human carcinomas and hematological cancers. The proteolytic activity of matriptase is a potential target of drugs and imaging probes. We assessed the fate of active matriptase following the induction of matriptase zymogen activation. Exposing eight human carcinoma cells to pH 6.0 buffer induced robust matriptase zymogen activation followed by rapid inhibition of the nascent active matriptase by hepatocyte growth factor activator inhibitor (HAI)-1. Consequently, no enzymatically active matriptase was detected in these cells. Some active matriptase is, however, rapidly shed to the extracellular milieu by these carcinoma cells. The lack of cell-associated active matriptase and the shedding of active matriptase were also observed in two hematological cancer lines. Matriptase shedding is correlated closely with the induction of matriptase activation, suggesting that matriptase activation and shedding are kinetically coupled. The coupling allows a proportion of active matriptase to survive HAI-1 inhibition by rapid shedding from cell surface. Our study suggests that cellular free, active matriptase is scarce and might not be an effective target for in vivo imaging and drug development.
doi:10.1371/journal.pone.0092244
PMCID: PMC3963879  PMID: 24663123
Breast cancer research and treatment  2012;134(3):1027-1039.
Despite the success of the aromatase inhibitors (AIs) in treating estrogen receptor positive breast cancer, 15–20 % of patients receiving adjuvant AIs will relapse within 5–10 years of treatment initiation. Long-term estrogen deprivation (LTED) of breast cancer cells in culture mimics AI-induced estrogen depletion to dissect mechanisms of AI resistance. However, we hypothesized that a subset of patients receiving AI therapy may maintain low circulating concentrations of estrogens that influence the development of endocrine resistance. We expanded established LTED models to account for incomplete suppression of estrogen synthesis during AI therapy. MCF-7 cells were grown in medium with charcoal-stripped serum supplemented with defined concentrations of 17β-estradiol (E2) or the estrogenic androgen metabolite 5α-androstane-3β,17β-diol (3βAdiol), an endogenous selective estrogen receptor modulator. Cells were selected in concentrations of E2 or 3βAdiol that induce 10 or 90 percent of maximal proliferation (EC10 and EC90, respectively), or estrogen deprived. Estrogen independence was evaluated during selection by assessing cell growth in the absence or presence of E2 or 3βAdiol. Following >7 months of selection, estrogen independence developed in estrogen-deprived cells and EC10-selected cells. Functional analyses demonstrated that estrogen-deprived and EC10-selected cells developed estrogen independence via unique mechanisms, ERα-independent and dependent, respectively. Estrogen-independent proliferation in EC10-selected cells could be blocked by kinase inhibitors. However, these cells were resistant to kinase inhibition in the presence of low steroid concentrations. These data demonstrate that further understanding of the total estrogen environment in patients on AI therapy who experience recurrence is necessary to effectively treat endocrine-resistant disease.
doi:10.1007/s10549-012-2032-6
PMCID: PMC3951731  PMID: 22456984
Breast cancer; Estrogen; Androgen; Aromatase inhibitor; Endocrine resistance
Oncotarget  2013;4(12):2487-2501.
Ewing sarcoma (ES) is an aggressive malignancy driven by an oncogenic fusion protein, EWS-FLI1. Neuropeptide Y (NPY), and two of its receptors, Y1R and Y5R are up-regulated by EWS-FLI1 and abundantly expressed in ES cells. Paradoxically, NPY acting via Y1R and Y5R stimulates ES cell death. Here, we demonstrate that these growth-inhibitory actions of NPY are counteracted by hypoxia, which converts the peptide to a growth-promoting factor. In ES cells, hypoxia induces another NPY receptor, Y2R, and increases expression of dipeptidyl peptidase IV (DPPIV), an enzyme that cleaves NPY to a shorter form, NPY3-36. This truncated peptide no longer binds to Y1R and, therefore, does not stimulate ES cell death. Instead, NPY3-36 acts as a selective Y2R/Y5R agonist. The hypoxia-induced increase in DPPIV activity is most evident in a population of ES cells with high aldehyde dehydrogenase (ALDH) activity, rich in cancer stem cells (CSCs). Consequently, NPY, acting via Y2R/Y5Rs, preferentially stimulates proliferation and migration of hypoxic ALDHhigh cells. Hypoxia also enhances the angiogenic potential of ES by inducing Y2Rs in endothelial cells and increasing the release of its ligand, NPY3-36, from ES cells. In summary, hypoxia acts as a molecular switch shifting NPY activity away from Y1R/Y5R-mediated cell death and activating the Y2R/Y5R/DPPIV/NPY3-36 axis, which stimulates ES CSCs and promotes angiogenesis. Hypoxia-driven actions of the peptide such as these may contribute to ES progression. Due to the receptor-specific and multifaceted nature of NPY actions, these findings may inform novel therapeutic approaches to ES.
PMCID: PMC3926843  PMID: 24318733
Neuropeptide Y; Ewing sarcoma; hypoxia; cancer stem cells; angiogenesis
The Journal of Neuroscience  2012;32(13):4592-4599.
Inhibition usually decreases input-output excitability of neurons. If, however, inhibition is coupled to excitation in a push-pull fashion, where inhibition decreases as excitation increases, neuron excitability can be increased. Although the presence of push-pull organization has been demonstrated in single cells, its functional impact on neural processing depends on its effect on the system level. We studied push-pull in the motor output stage of the feline spinal cord, a system which hallows in dependent control of inhibitory and excitatory components. Push-pull organization was clearly present in ankle extensor motoneurons, producing increased peak to peak modulation of synaptic currents. The effect at the system level was equally strong. Independent control of the inhibitory component showed that the stronger the background of inhibition, the greater the peak force production. This illustrates the paradox at the heart of push-pull organization: increased force output can be achieved by increasing background inhibition to provide greater disinhibition.
doi:10.1523/JNEUROSCI.4709-11.2012
PMCID: PMC3335194  PMID: 22457505
PLoS ONE  2013;8(11):e82298.
The attenuation of sedimentation and convection in microgravity can sometimes decrease irregularities formed during macromolecular crystal growth. Current terrestrial protein crystal growth (PCG) capabilities are very different than those used during the Shuttle era and that are currently on the International Space Station (ISS). The focus of this experiment was to demonstrate the use of a commercial off-the-shelf, high throughput, PCG method in microgravity. Using Protein BioSolutions’ microfluidic Plug Maker™/CrystalCard™ system, we tested the ability to grow crystals of the regulator of glucose metabolism and adipogenesis: peroxisome proliferator-activated receptor gamma (apo-hPPAR-γ LBD), as well as several PCG standards. Overall, we sent 25 CrystalCards™ to the ISS, containing ~10,000 individual microgravity PCG experiments in a 3U NanoRacks NanoLab (1U = 103 cm.). After 70 days on the ISS, our samples were returned with 16 of 25 (64%) microgravity cards having crystals, compared to 12 of 25 (48%) of the ground controls. Encouragingly, there were more apo-hPPAR-γ LBD crystals in the microgravity PCG cards than the 1g controls. These positive results hope to introduce the use of the PCG standard of low sample volume and large experimental density to the microgravity environment and provide new opportunities for macromolecular samples that may crystallize poorly in standard laboratories.
doi:10.1371/journal.pone.0082298
PMCID: PMC3836816  PMID: 24278480
Journal of neurophysiology  2008;99(4):1643-1652.
We investigated summation of steady excitatory and inhibitory inputs in spinal motoneurons using an in vivo preparation, the decerebrate cat, in which neuromodulatory input from the brain stem facilitated a strong persistent inward current (PIC) in dendritic regions. This dendritic PIC amplified both excitatory and inhibitory synaptic currents two-to threefold, but within different voltage ranges. Amplification of excitatory synaptic current peaked at voltage-clamp holding potentials near spike threshold (about −55 to −50 mV), whereas amplification of inhibitory current peaked at significantly more depolarized levels (about −45 to −40 mV). Thus the linear sum of excitatory and inhibitory currents tended to vary from net excitatory to net inhibitory as holding potential was depolarized. The actual summed currents, however, diverged from the predicted linear currents. At the peak of excitation, summation averaged about 15% sublinear (actual sum was less positive than the linear sum). In contrast, at the peak of inhibition, summation averaged about 18% supralinear (actual more positive than linear). Moreover, these nonlinear effects were substantially larger in cells where the variation from peak excitation to peak inhibition for linear summation was larger. When descending neuromodulatory input was eliminated by acute spinalization, PIC amplification was not observed and summation tended to be either sublinear or approximately linear, depending on input source. Overall, in cells with strong PICs, nonlinear summation of excitation and inhibition does occur, but this nonlinearity results in a more consistent relationship between membrane potential and the summed excitatory and inhibitory current.
doi:10.1152/jn.01253.2007
PMCID: PMC3803147  PMID: 18234978
Purpose
Exposure to ionizing radiation is an established risk factor for breast cancer. Radiation exposure during infancy, childhood, and adolescence confers the highest risk. Although radiation is a proven mammary carcinogen, it remains unclear where it acts in the complex multistage process of breast cancer development. In this study, we investigated the long-term pathophysiologic effects of ionizing radiation at a dose (2 Gy) relevant to fractionated radiotherapy.
Methods and Materials
Adolescent (6–8 weeks old; n = 10) female C57BL/6J mice were exposed to 2 Gy total body γ-radiation, the mammary glands were surgically removed, and serum and urine samples were collected 2 and 12 months after exposure. Molecular pathways involving estrogen receptor-α (ERα) and phosphatidylinositol-3-OH kinase (PI3K)-Akt signaling were investigated by immunohistochemistry and Western blot.
Results
Serum estrogen and urinary levels of the oncogenic estrogen metabolite (16αOHE1) were significantly increased in irradiated animals. Immunostaining for the cellular proliferative marker Ki-67 and cyclin-D1 showed increased nuclear accumulation in sections of mammary glands from irradiated vs. control mice. Marked increase in p85α, a regulatory sub-unit of the PI3K was associated with increase in Akt, phospho-Akt, phospho-BAD, phospho-mTOR, and c-Myc in irradiated samples. Persistent increase in nuclear ERα in mammary tissues 2 and 12 months after radiation exposure was also observed.
Conclusions
Taken together, our data not only support epidemiologic observations associating radiation and breast cancer but also, specify molecular events that could be involved in radiation-induced breast cancer.
doi:10.1016/j.ijrobp.2011.12.033
PMCID: PMC3580184  PMID: 22381906
Radiation; Estradiol; Estrogen; Estrogen receptor α; Estrogen metabolite
Growth factor-induced activation of Akt occursin the majority of human breast cancer cell lines resulting in a variety of cellular outcomes, including suppression of apoptosis and enhanced survival. We demonstrate that epidermal growth factor (EGF)-initiated activation of Akt is mediated by the ubiquitous calcium sensing molecule, calmodulin, in the majority of human breast cancer cell lines. Specifically, in estrogen receptor (ER)-negative, but not ER-positive, breast cancer cells, Akt activation is abolished by treatment with the calmodulin antagonist, W-7. Suppression of calmodulin expression by siRNAs against all three calmodulin genes in c-Myc-overexpressing mouse mammary carcinoma cells results in significant inhibition of EGF-induced Akt activation. Additionally, transient expression of constitutively active Akt (Myr-Akt) can overcome W-7-mediated suppression of Akt activation. These results confirm the involvement of calmodulin in the Akt pathway. The calmodulin independence of EGF-initiated Akt signaling in some cells was not explained by calmodulin expression level. Additionally, it was not explained by ER status or activation, since removal of estrogen and ablation of the ER did not convert the ERpositive, W-7 insensitive, MCF-7 cell line to calmodulin dependent signaling. However, forced overexpression of either epidermal growth factor receptor (EGFR) or ErbB2 did partially restore calmodulin dependent EGF-stimulated Akt activation. This is consistent with observation that W-7 sensitive cells tend to be estrogen independent and express high levels of EGFR family members. In an attempt to address how calmodulin is regulating Akt activity, we looked at localization of fluorescently tagged Akt and calmodulin in MCF-7 and SK-BR-3 cells. We found that both Akt and calmodulin translocate to the membrane after EGFstimulation, and this translocation to the same sub-cellular compartment is inhibited by the calmodulin inhibitor W-7. Thus, calmodulin may be regulating Akt activity by modulating its sub-cellular location and is a novel target in the poor prognosis, ER-negative subset of breast cancers.
doi:10.1007/s10549-008-0097-z
PMCID: PMC3736740  PMID: 18587642
Akt; Calmodulin; Estrogen receptor; EGFR; ErbB2
BMC Cancer  2013;13:373.
Background
The applications of multiphoton microscopy for deep tissue imaging in basic and clinical research are ever increasing, supplementing confocal imaging of the surface layers of cells in tissue. However, imaging living tissue is made difficult by the light scattering properties of the tissue, and this is extraordinarily apparent in the mouse mammary gland which contains a stroma filled with fat cells surrounding the ductal epithelium. Whole mount mammary glands stained with Carmine Alum are easily archived for later reference and readily viewed using bright field microscopy to observe branching architecture of the ductal network. Here, we report on the advantages of multiphoton imaging of whole mount mammary glands. Chief among them is that optical sectioning of the terminal end bud (TEB) and ductal epithelium allows the appreciation of abnormalities in structure that are very difficult to ascertain using either bright field imaging of the stained gland or the conventional approach of hematoxylin and eosin staining of fixed and paraffin-embedded sections. A second advantage is the detail afforded by second harmonic generation (SHG) in which collagen fiber orientation and abundance can be observed.
Methods
GFP-mouse mammary glands were imaged live or after whole mount preparation using a Zeiss LSM510/META/NLO multiphoton microscope with the purpose of obtaining high resolution images with 3D content, and evaluating any structural alterations induced by whole mount preparation. We describe a simple means for using a commercial confocal/ multiphoton microscope equipped with a Ti-Sapphire laser to simultaneously image Carmine Alum fluorescence and collagen fiber networks by SHG with laser excitation set to 860 nm. Identical terminal end buds (TEBs) were compared before and after fixation, staining, and whole mount preparation and structure of collagen networks and TEB morphologies were determined. Flexibility in excitation and emission filters was explored using the META detector for spectral emission scanning. Backward scattered or reflected SHG (SHG-B) was detected using a conventional confocal detector with maximum aperture and forward scattered or transmitted SHG (SHG-F) detected using a non-descanned detector.
Results
We show here that the developing mammary gland is encased in a thin but dense layer of collagen fibers. Sparse collagen layers are also interspersed between stromal layers of fat cells surrounding TEBs. At the margins, TEBs approach the outer collagen layer but do not penetrate it. Abnormal mammary glands from an HAI-1 transgenic FVB mouse model were found to contain TEBs with abnormal pockets of cells forming extra lumens and zones of continuous lateral bud formation interspersed with sparse collagen fibers.
Parameters influencing live imaging and imaging of fixed unstained and Carmine Alum stained whole mounts were evaluated. Artifacts induced by light scattering of GFP and Carmine Alum signals from epithelial cells were identified in live tissue as primarily due to fat cells and in whole mount tissue as due to dense Carmine Alum staining of epithelium. Carmine Alum autofluorescence was detected at excitation wavelengths from 750 to 950 nm with a peak of emission at 623 nm (~602-656 nm). Images of Carmine Alum fluorescence differed dramatically at emission wavelengths of 565–615 nm versus 650–710 nm. In the latter, a mostly epithelial (nuclear) visualization of Carmine Alum predominates. Autofluorescence with a peak emission of 495 nm was derived from the fixed and processed tissue itself as it was present in the unstained whole mount. Contribution of autofluorescence to the image decreases with increasing laser excitation wavelengths. SHG-B versus SHG-F signals revealed collagen fibers and could be found within single fibers, or in different fibers within the same layer. These differences presumably reflected different states of collagen fiber maturation. Loss of SHG signals from layer to layer could be ascribed to artifacts rendered by light scattering from the dense TEB structures, and unless bandpass emissions were selected, contained unfiltered non-SHG fluorescence and autofluorescent emissions. Flexibility in imaging can be increased using spectral emission imaging to optimize emission bandwidths and to separate SHG-B, GFP, and Carmine Alum signals, although conventional filters were also useful.
Conclusions
Collagen fibril arrangement and TEB structure is well preserved during the whole mount procedure and light scattering is reduced dramatically by extracting fat resulting in improved 3D structure, particularly for SHG signals originating from collagen. In addition to providing a bright signal, Carmine Alum stained whole mount slides can be imaged retrospectively such as performed for the HAI-1 mouse gland revealing new aspects of abnormal TEB morphology. These studies demonstrated the intimate contact, but relatively sparse abundance of collagen fibrils adjacent to normal and abnormal TEBS in the developing mammary gland and the ability to obtain these high resolution details subject to the discussed limitations. Our studies demonstrated that the TEB architecture is essentially unchanged after processing.
doi:10.1186/1471-2407-13-373
PMCID: PMC3750743  PMID: 23919456
Experimental Neurology  2012;235(2):588-598.
Spasticity is a condition that can include increased muscle tone, clonus, spasms, and hyperreflexia. In this study, we report the effect of manually stimulating the dorsal lumbosacral skin on spontaneous locomotor-like activity and on a variety of reflex responses in 5 decerebrate chronic spinal cats treated with clonidine. Cats were spinalized 1 month before the terminal experiment. Stretch reflexes were evoked by stretching the left triceps surae muscles. Crossed reflexes were elicited by electrically stimulating the right tibial or superficial peroneal nerves. Windup of reflex responses was evoked by electrically stimulating the left tibial or superficial peroneal nerves. We found that pinching the skin of the back abolished spontaneous locomotor-like activity. We also found that back pinch abolished the rhythmic activity observed during reflex testing without eliminating the reflex responses. Some of the rhythmic episodes of activity observed during reflex testing were consistent with clonus with an oscillation frequency greater than 3 Hz. Pinching the skin of the back effectively abolished rhythmic activity occurring spontaneously or evoked during reflex testing, irrespective of oscillation frequency. The results are consistent with the hypothesis that locomotion and clonus are produced by common central pattern-generators. Stimulating the skin of the back could prove helpful in managing undesired rhythmic activity in spinal cord-injured humans.
doi:10.1016/j.expneurol.2012.03.013
PMCID: PMC3345060  PMID: 22487200
central pattern generator; locomotion; spinal cord injury; clonus; spinal reflexes; cutaneous; spasticity
PLoS ONE  2013;8(5):e62826.
Matriptase, a membrane-associated serine protease, plays an essential role in epidermal barrier function through activation of the glycosylphosphatidylinositol (GPI)-anchored serine protease prostasin. The matriptase-prostasin proteolytic cascade is tightly regulated by hepatocyte growth factor activator inhibitor (HAI)-1 such that matriptase autoactivation and prostasin activation occur simultaneously and are followed immediately by the inhibition of both enzymes by HAI-1. However, the mechanisms whereby matriptase acts on extracellular substrates remain elusive. Here we report that some active matriptase can escape HAI-1 inhibition by being rapidly shed from the cell surface. In the pericellular environment, shed active matriptase is able to activate hepatocyte growth factor (HGF), accelerate plasminogen activation, and shed syndecan 1. The amount of active matriptase shed is inversely correlated with the amount of antithrombin (AT) bound to the surface of the keratinocytes. Binding of AT to the surface of keratinocytes is dependent on a functional heparin binding site, Lys-125, and that the N-glycosylation site Asn-135 be unglycosylated. This suggests that β-AT, and not α-AT, is responsible for regulation of pericellular matriptase activity in keratinocytes. Keratinocytes appear to rely on AT to regulate the level of pericellular active matriptase much more than breast and prostate epithelial cells in which AT regulation of matriptase activity occurs at much lower levels than keratinocytes. These results suggest that keratinocytes employ two distinct serine protease inhibitors to control the activation and processing of two different sets of matriptase substrates leading to different biological events: 1) HAI-1 for prostasin activation/inhibition, and 2) AT for the pericellular proteolysis involved in HGF activation, accelerating plasminogen activation, and shedding of syndecans.
doi:10.1371/journal.pone.0062826
PMCID: PMC3652837  PMID: 23675430
For bacterial pathogens whose sole environmental reservoir is the human host, the acquisition of essential nutrients, particularly transition metals, is a critical aspect of survival due to tight sequestration and limitation strategies deployed to curtail pathogen outgrowth. As such, these bacteria have developed diverse, specialized acquisition mechanisms to obtain these metals from the niches of the body in which they reside. To oppose the spread of infection, the human host has evolved multiple mechanisms to counter bacterial invasion, including sequestering essential metals away from bacteria and exposing bacteria to lethal concentrations of metals. Hence, to maintain homeostasis within the host, pathogens must be able to acquire necessary metals from host proteins and to export such metals when concentrations become detrimental. Furthermore, this acquisition and efflux equilibrium must occur in a tissue-specific manner because the concentration of metals varies greatly within the various microenvironments of the human body. In this review, we examine the functional roles of the metal import and export systems of the Gram-positive pathogen Streptococcus pneumoniae in both signaling and pathogenesis.
doi:10.3389/fcimb.2013.00092
PMCID: PMC3849628  PMID: 24364001
Streptococcus pneumoniae; pathogenesis; metal transport; virulence factors; infection
Background
Breast tissue is among the most sensitive tissues to the carcinogenic actions of ionizing radiation and epidemiological studies have linked radiation exposure to breast cancer. Currently, molecular understanding of radiation carcinogenesis in mammary gland is hindered due to the scarcity of in vivo long-term follow up data. We undertook this study to delineate radiation-induced persistent alterations in gene expression in mouse mammary glands 2-month after radiation exposure.
Methods
Six to eight week old female C57BL/6J mice were exposed to 2 Gy of whole body γ radiation and mammary glands were surgically removed 2-month after radiation. RNA was isolated and microarray hybridization performed for gene expression analysis. Ingenuity Pathway Analysis (IPA) was used for biological interpretation of microarray data. Real time quantitative PCR was performed on selected genes to confirm the microarray data.
Results
Compared to untreated controls, the mRNA levels of a total of 737 genes were significantly (p<0.05) perturbed above 2-fold of control. More genes (493 genes; 67%) were upregulated than the number of downregulated genes (244 genes; 33%). Functional analysis of the upregulated genes mapped to cell proliferation and cancer related canonical pathways such as ‘ERK/MAPK signaling’, ‘CDK5 signaling’, and ‘14-3-3-mediated signaling’. We also observed upregulation of breast cancer related canonical pathways such as ‘breast cancer regulation by Stathmin1’, and ‘HER-2 signaling in breast cancer’ in IPA. Interestingly, the downregulated genes mapped to fewer canonical pathways involved in cell proliferation. We also observed that a number of genes with tumor suppressor function (GPRC5A, ELF1, NAB2, Sema4D, ACPP, MAP2, RUNX1) persistently remained downregulated in response to radiation exposure. Results from qRT-PCR on five selected differentially expressed genes confirmed microarray data. The PCR data on PPP4c, ELF1, MAPK12, PLCG1, and E2F6 showed similar trend in up and downregulation as has been observed with the microarray.
Conclusions
Exposure to a clinically relevant radiation dose led to long-term activation of mammary gland genes involved in proliferative and metabolic pathways, which are known to have roles in carcinogenesis. When considered along with downregulation of a number of tumor suppressor genes, our study has implications for breast cancer initiation and progression after therapeutic radiation exposure.
doi:10.1186/1748-717X-7-205
PMCID: PMC3551737  PMID: 23216862
Radiation exposure; Mouse mammary gland; Gene expression; Persistent microarray changes.
Journal of medicinal chemistry  2011;54(21):7567-7578.
Membrane-associated serine protease matriptase has been implicated in human diseases, and might be a drug target. In the present study, a novel class of matriptase inhibitors targeting zymogen activation is developed by a combination of the screening of compound library using a cell-based matriptase activation assay and a computer-aided search of commercially available analogs of a selected compound. Four structurally related compounds are identified that can inhibit matriptase activation with IC50 at low μM in both intact-cell and cell-free systems, suggesting that these inhibitors target the matriptase autoactivation machinery rather than the intracellular signaling pathways. These activation inhibitors can also inhibit prostasin activation, a downstream event that occurs in lockstep with matriptase activation. In contrast, the matriptase catalytic inhibitor CVS-3983 at a concentration 300-fold higher than its Ki fails to inhibit activation of either protease. Our results suggest that inhibiting matriptase activation is an efficient way to control matriptase function.
doi:10.1021/jm200920s
PMCID: PMC3214968  PMID: 21966950
The pharmacogenomics journal  2010;11(5):348-358.
Single nucleotide polymorphisms (SNPs) can be assayed using DNA isolated from archival formalin-fixed paraffin-embedded (FFPE) samples, making retrospective pharmacogenetic studies possible. Here we describe methods that significantly increase the number of SNP determinations possible using FFPE samples. Quantifying the amount of DNA amenable to PCR (amplification-quality DNA, AQ-DNA) allows a significant reduction in the amount of sample required for Taqman-based SNP assays. Optimizing AQ-DNA input increases PCR amplification efficiency and SNP determination accuracy. DNA was extracted from thirty-nine FFPE tumor sections and matched tumor and stromal cores, of the type used to generate tissue microarrays. Sections and tumor cores yielded sufficient AQ-DNA for over 1,000 SNP determinations. Seven SNPs were assessed, following individual assay optimization for minimal AQ-DNA. Genotypes from tumor cores for single SNPs were 92.3-100% concordant with those obtained from sections. Using these methods, the number of SNP genotypes that can be determined from single FFPE samples is greatly increased expanding the genetic association studies possible from limited archival specimens. The use of tumor cores is of particular importance since the harvesting of tumor cores has minimal impact on the utility of the donor blocks for other purposes.
doi:10.1038/tpj.2010.50
PMCID: PMC2996486  PMID: 20548328
genotyping; paraffin-embedded; tumor samples; genetic polymorphism
PLoS ONE  2011;6(12):e29629.
PilY1 is a type IV pilus (tfp)-associated protein from the opportunistic pathogen Pseudomonas aeruginosa that shares functional similarity with related proteins in infectious Neisseria and Kingella species. Previous data have shown that PilY1 acts as a calcium-dependent pilus biogenesis factor necessary for twitching motility with a specific calcium binding site located at amino acids 850–859 in the 1,163 residue protein. In addition to motility, PilY1 is also thought to play an important role in the adhesion of P. aeruginosa tfp to host epithelial cells. Here, we show that PilY1 contains an integrin binding arginine-glycine-aspartic acid (RGD) motif located at residues 619–621 in the PilY1 from the PAK strain of P. aeruginosa; this motif is conserved in the PilY1s from the other P. aeruginosa strains of known sequence. We demonstrate that purified PilY1 binds integrin in vitro in an RGD-dependent manner. Furthermore, we identify a second calcium binding site (amino acids 600–608) located ten residues upstream of the RGD. Eliminating calcium binding from this site using a D608A mutation abolished integrin binding; in contrast, a calcium binding mimic (D608K) preserved integrin binding. Finally, we show that the previously established PilY1 calcium binding site at 851–859 also impacts the protein's association with integrin. Taken together, these data indicate that PilY1 binds to integrin in an RGD- and calcium-dependent manner in vitro. As such, P. aeruginosa may employ these interactions to mediate host epithelial cell binding in vivo.
doi:10.1371/journal.pone.0029629
PMCID: PMC3248442  PMID: 22242136

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