The smoking habits of 82 patients with malignant-phase hypertension were compared with those of subjects in three control groups matched for age and sex. Sixty-seven (82%) of the patients with malignant-phase hypertension were smokers compared with 41 (50%) and 71 (43%) of the patients in two control groups with non-malignant hypertension, and 43 people (52%) in a general population survey. The excess of smokers in the malignant-phase group was significant for men and women, together and separately, for cigarette smoking alone, and for all forms of smoking. There were no significant differences between the control groups. The chance of a hypertensive patient who smoked having the malignant phase was five times that of a hypertensive patient who did not. Twelve patients in the malignant-phase group had never smoked. All were alive three and a half years on average after presentation (range 11 months to seven years). Twenty-four (36%) of the smokers with malignant-phase hypertension died during the same period. The mortality rate was significantly higher among patients with renal failure, as was the prevalence of smoking. Eighteen patients with malignant-phase hypertension had a serum creatinine concentration higher than 250 μmol/l (2·8 mg/100 ml); 17 were smokers and one an ex-smoker. Eleven of these 18 patients died.
It is concluded that hypertensive patients who smoke are much more likely to develop the malignant phase than those who do not, and that once the condition has developed it follows a particularly lethal course in smokers.
Pancreatic cancer is one of the deadliest of all human malignancies with limited options for therapy. Here, we report the development of an optimized targeted drug delivery system to inhibit advanced stage pancreatic tumor growth in an orthotopic mouse model.
Targeting specificity in vitro was confirmed by preincubation of the pancreatic cancer cells with C225 as well as Nitrobenzylthioinosine (NBMPR - nucleoside transporter (NT) inhibitor). Upon nanoconjugation functional activity of gemcitabine was retained as tested using a thymidine incorporation assay. Significant stability of the nanoconjugates was maintained, with only 12% release of gemcitabine over a 24-hour period in mouse plasma. Finally, an in vivo study demonstrated the inhibition of tumor growth through targeted delivery of a low dose of gemcitabine in an orthotopic model of pancreatic cancer, mimicking an advanced stage of the disease.
We demonstrated in this study that the gold nanoparticle-based therapeutic containing gemcitabine inhibited tumor growth in an advanced stage of the disease in an orthotopic model of pancreatic cancer. Future work would focus on understanding the pharmacokinetics and combining active targeting with passive targeting to further improve the therapeutic efficacy and increase survival.
Targeting; Epidermal Growth Factor Receptor (EGFR); Gold Nanoparticles; Pathway Switching; Lipid Microdomain; GTPases
Targeted radiotherapies maximize cytotoxicty to cancer cells. In vivo α-generator targeted radiotherapies can deliver multiple α particles to a receptor site dramatically amplifying the radiation dose delivered to the target. The major challenge with α-generator radiotherapies is that traditional chelating moieties are unable to sequester the radioactive daughters in the bioconjugate which is critical to minimize toxicity to healthy, non-target tissue. The recoil energy of the 225Ac daughters following α decay will sever any metal-ligand bond used to form the bioconjugate. This work demonstrates that an engineered multilayered nanoparticle-antibody conjugate can deliver multiple α radiations and contain the decay daughters of 225Ac while targeting biologically relevant receptors in a female BALB/c mouse model. These multi-shell nanoparticles combine the radiation resistance of lanthanide phosphate to contain 225Ac and its radioactive decay daughters, the magnetic properties of gadolinium phosphate for easy separation, and established gold chemistry for attachment of targeting moieties.
This chapter describes a method for generating yeast respiratory oscillations in continuous culture and monitoring rhythmic promoter activity of the culture by automated real-time recording of luminescence. These techniques chiefly require the use of a strain of Saccharomyces cerevisiae that has been genetically modified to express firefly luciferase under the control of a promoter of interest and a continuous culture bioreactor that incorporates a photomultiplier apparatus for detecting light emission. Additionally, this chapter describes a method for observing rhythmic (cell cycle-related) promoter activity in small batch cultures of yeast through luminescence monitoring.
Saccharomyces cerevisiae; Luciferase; Bioluminescence; Continuous culture; Bioreactor; Yeast respiratory oscillation
In this communication we describe a simple one step synthesis of a targeted drug delivery system with enhanced cytotoxicity to EGFR+cancer cells. The delivery system comprises of carboplatin as a chemotherapeutic, anti-EGFR antibody cetuximab as a targeting agent and gold nanoparticle as a delivery vehicle. Nanoconjugates were characterized using ICP-MS, TEM, INAA and by stability studies in mouse plasma. Stability study indicates that the nanoconjugates prepared using this simple mixing procedure is significantly stable as more than 50 % of carboplatin remains in gold bound form even after incubation in mouse plasma for 24 h. Targeting efficacy of the nanoconjugates determined by gold-content of EGFR+ lung and ovarian cancer cells demonstrated enhanced uptake of gold nanoconjugates when delivered in a targeted fashion as compared to its non-targeted counterpart. Cytoxicity of the Nano conjugates were tested by 3H-thymidine incorporation assay against EGFR+ lung and ovarian cancer cell lines. Cytoxicity data demonstrates that carboplatin is more effective to inhibit the proliferation of EGFR+ lung and ovarian cancer cells when delivered in a targeted fashion. Such strategies may be utilized in future for targeted delivery of cytotoxic drugs to cancer with enhanced efficacy and reduced side effects.
We report the development of a genetically encodable and ratiometic pH probe named “pHlash” that utilizes Bioluminescence Resonance Energy Transfer (BRET) rather than fluorescence excitation. The pHlash sensor–composed of a donor luciferase that is genetically fused to a Venus fluorophore–exhibits pH dependence of its spectral emission in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification in vivo. Its spectral ratio response is H+ specific; neither Ca++, Mg++, Na+, nor K+ changes the spectral form of its luminescence emission. Moreover, it can be used to image pH in single cells. This is the first BRET-based sensor of H+ ions, and it should allow the approximation of pH in cytosolic and organellar compartments in applications where current pH probes are inadequate.
This study examined the gene transfer efficiency and toxicity of 2-kDa polyethylenimine conjugated to gold nanoparticles (PEI2-GNP) in the human cornea in vitro and rabbit cornea in vivo. PEI2-GNP with nitrogen-to-phosphorus (N/P) ratios of up to 180 exhibited significant transgene delivery in the human cornea without altering the viability or phenotype of these cells. Similarly, PEI2-GNP applied to corneal tissues collected after 12 h, 72 h, or 7 days exhibited appreciable gold uptake throughout the rabbit stroma with gradual clearance of GNP over time. Transmission electron microscopy detected GNP in the keratocytes and the extracellular matrix of the rabbit corneas. Additionally, slitlamp biomicroscopy in live animals even 7 days after topical PEI2-GNP application to the cornea detected no inflammation, redness, or edema in rabbit eyes in vivo, with only moderate cell death and immune reactions. These results suggest that PEI2-GNP are safe for the cornea and can be potentially useful for corneal gene therapy in vivo.
gold nanoparticles; polyethylenimine; cornea; gene transfer; toxicity
Ecological opportunity is any change that allows populations to escape selection from competition and predation. After encountering ecological opportunity, populations may experience ecological release: enlarged population size, broadened resource use, and/or increased morphological variation. We identified ecological opportunity and tested for ecological release in three lizard colonists of White Sands, New Mexico (Sceloporus undulatus, Holbrookia maculata, and Aspidoscelis inornata). First, we provide evidence for ecological opportunity by demonstrating reduced species richness and abundance of potential competitors and predators at White Sands relative to nearby dark soils habitats. Second, we characterize ecological release at White Sands by demonstrating density compensation in the three White Sands lizard species and expanded resource use in White Sands S. undulatus. Contrary to predictions from ecological release models, we observed directional trait change but not increased trait variation in S. undulatus. Our results suggest that ecological opportunity and ecological release can be identified in natural populations, especially those that have recently colonized isolated ecosystems.
Adaptation; colonization; density compensation; ecological opportunity; ecological release; natural selection; reptiles; selection; speciation; White Sands
Inorganic nanoparticles provide promising tools for biomedical applications including detection, diagnosis and therapy. While surface properties such as charge are expected to play an important role in their in vivo behavior, very little is known how the surface chemistry of nanoparticles influences their pharmacokinetics, tumor uptake, and biodistribution.
Using a family of structurally homologous nanoparticles we have investigated how pharmacological properties including tumor uptake and biodistribution are influenced by surface charge using neutral (TEGOH), zwitterionic (Tzwit), negative (TCOOH) and positive (TTMA) nanoparticles. Nanoparticles were injected into mice (normal and athymic) either in the tail vein or into the peritoneum.
Neutral and zwitterionic nanoparticles demonstrated longer circulation time via both IP and IV administration, whereas negatively and positively charged nanoparticles possessed relatively short half-lives. These pharmacological characteristics were reflected on the tumor uptake and biodistribution of the respective nanoparticles, with enhanced tumor uptake by neutral and zwitterionic nanoparticles via passive targeting.
Kalamchi and MacEwen (K&M) described a four-group scheme for classifying osteonecrosis (ON) following treatment for developmental dysplasia of the hip (DDH). However, the four groups can overlap in radiographic appearance, making assessment difficult.
We (1) describe a simplified K&M classification; (2) determined whether the simplified classification was reliable; and (3) assessed whether differences in the type of reduction or age at reduction resulted in different degrees of ON.
Patients and Methods
We retrospectively reviewed 300 patients with DDH treated with either open or closed reduction. We included 101 of these patients (133 involved hips). Intraobserver and interobserver reliability testing of the original and our simplified classification was performed. ON occurred in 64 hips (48%). Of these, 22 had original K&M Group I disease (classified as simplified Group A), and 42 had original K&M Groups II, III, or IV disease (classified as simplified Group B). The mean age of the patients at final followup was 12.4 years (range, 6–26.3 years).
The interobserver reliability of the simplified classification was greater than that of the K&M classification (0.51 vs 0.33, respectively). Closed reduction after skin traction resulted in a lower incidence of Group B ON than open reduction, regardless of age at reduction.
We propose a simplified and more reliable classification of ON after DDH. With the new classification we found type of reduction (closed with traction versus open without femoral shortening) but not age influenced the risk of ON.
Level of Evidence
Level IV, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.
Herein, we demonstrate that the surface charge of gold nanoparticles (AuNPs) plays a critical role in modulating membrane potential of different malignant and non-malignant cell types and subsequent downstream intracellular events. The findings presented here describe a novel mechanism for cell-nanoparticle interactions and AuNP uptake: modulation of membrane potential and its effect on intracellular events. These studies will help understand the biology of cell-nanoparticle interactions and facilitate the engineering of nanoparticles for specific intracellular targets.
Gold nanoparticles; surface properties; cancer; lung; ovary; plasma membrane; membrane potential; calcium; apoptosis; proliferation
Pancreatic cancer is the fourth leading cause of cancer related deaths in America. Monoclonal antibodies are a viable treatment option for inhibiting cancer growth. Tumor specific drug delivery could be achieved utilizing these monoclonal antibodies as targeting agents. This type of designer therapeutic is evolving and with the use of gold nanoparticles it is a promising approach to selectively deliver chemotherapeutics to malignant cells.
Gold nanoparticles (GNPs) are showing extreme promise in current medicinal research. GNPs have been shown to non-invasively kill tumor cells by hyperthermia using radiofrequency. They have also been implemented as early detection agents due to their unique X-ray contrast properties; success was revealed with clear delineation of blood capillaries in a preclinical model by CT (computer tomography). The fundamental parameters for intelligent design of nanoconjugates are on the forefront. The goal of this study is to define the necessary design parameters to successfully target pancreatic cancer cells.
The nanoconjugates described in this study were characterized with various physico-chemical techniques. We demonstrate that the number of cetuximab molecules (targeting agent) on a GNP, the hydrodynamic size of the nanoconjugates, available reactive surface area and the ability of the nanoconjugates to sequester EGFR (epidermal growth factor receptor), all play critical roles in effectively targeting tumor cells in vitro and in vivo in an orthotopic model of pancreatic cancer.
Our results suggest the specific targeting of tumor cells depends on a number of crucial components 1) targeting agent to nanoparticle ratio 2) availability of reactive surface area on the nanoparticle 3) ability of the nanoconjugate to bind the target and 4) hydrodynamic diameter of the nanoconjugate. We believe this study will help define the design parameters for formulating better strategies for specifically targeting tumors with nanoparticle conjugates.
To evaluate the epidemiology and to investigate the impact of respiratory viral infections (RVI) on chronic allograft rejection after pediatric lung transplantation, a retrospective study of pediatric lung transplant recipients from 2002 to 2007 was conducted. Association between RVI and continuous and categorical risk factors was assessed using Wilcoxon rank-sum tests and Fisher’s exact tests, respectively. Association between risk factors and outcomes were assessed using Cox proportional hazards models.
Fifty-five subjects were followed for a mean of 674 days (range 14–1790). Twenty-eight (51%) developed 51 RVI at a median of 144 days posttransplant (mean 246; range 1–1276); 41% of infections were diagnosed within 90 days. 25 subjects developed 39 lower respiratory infections, and eight subjects had 11 upper respiratory infections (URI). Organisms recovered included rhinovirus(n=14), adenovirus(n=10), parainfluenza(n=10), influenza(n=5) and RSV(n=4). Three subjects expired secondary to their RVI (2 adenovirus, 1 RSV). Younger age and prior CMV infection were risks for RVI (HR 2.4 95% CI 1.1–5.3 and 17.0; 3.0–96.2, respectively). RVI was not associated with the development of chronic allograft rejection (P=0.25) or death during the study period.
RVI occur in the majority of pediatric lung transplant recipients, but were not associated with mortality or chronic allograft rejection.
Lung transplantation; Respiratory virus infection; Pediatrics
Zinc is an essential trace element involved in a wide range of biological
processes and human diseases. Zinc excess is deleterious, and animals require
mechanisms to protect against zinc toxicity. To identify genes that modulate
zinc tolerance, we performed a forward genetic screen for Caenorhabditis
elegans mutants that were resistant to zinc toxicity. Here we
demonstrate that mutations of the C. elegans histidine ammonia
lyase (haly-1) gene promote zinc tolerance. C. elegans
haly-1 encodes a protein that is homologous to vertebrate HAL, an
enzyme that converts histidine to urocanic acid. haly-1 mutant
animals displayed elevated levels of histidine, indicating that C.
elegans HALY-1 protein is an enzyme involved in histidine
catabolism. These results suggest the model that elevated histidine chelates
zinc and thereby reduces zinc toxicity. Supporting this hypothesis, we
demonstrated that dietary histidine promotes zinc tolerance. Nickel is another
metal that binds histidine with high affinity. We demonstrated that
haly-1 mutant animals are resistant to nickel toxicity and
dietary histidine promotes nickel tolerance in wild-type animals. These studies
identify a novel role for haly-1 and histidine in zinc
metabolism and may be relevant for other animals.
Zinc is an essential nutrient that is critical for human health. However, excess
zinc can cause toxicity, indicating that regulatory mechanisms are necessary to
maintain homeostasis. The analysis of mechanisms that promote zinc homeostasis
can elucidate fundamental regulatory processes and suggest new approaches for
treating disorders of zinc metabolism. To discover genes that modulate zinc
tolerance, we screened for C. elegans mutants that were
resistant to zinc toxicity. Here we demonstrate that mutations of the histidine
ammonia lyase (haly-1) gene promote zinc tolerance.
haly-1 encodes a protein that is similar to vertebrate HAL,
an enzyme that converts histidine to urocanic acid. Mutations in the human HAL
gene cause elevated levels of serum histidine and abnormal zinc metabolism.
Mutations in C. elegans haly-1 cause elevated levels of
histidine, suggesting that histidine causes resistance to excess zinc.
Consistent with this hypothesis, we demonstrated that dietary histidine promoted
tolerance to excess zinc in wild-type worms. Mutations in
haly-1 and supplemental dietary histidine also caused
resistance to nickel, another metal that can bind histidine. A likely mechanism
of protection is chelation of zinc and nickel by histidine. These studies
suggest that histidine plays a physiological role in zinc metabolism.
Increased production of biomass crops in North America will require new agricultural land, intensify the cultivation of land already under production and introduce new types of biomass crops. Assessing the potential biodiversity impacts of novel agricultural systems is fundamental to the maintenance of biodiversity in agricultural landscapes, yet the consequences of expanded biomass production remain unclear. We evaluate the ability of two candidate second generation biomass feedstocks (switchgrass, Panicum virgatum, and mixed-grass prairie) not currently managed as crops to act as post-breeding and fall migratory stopover habitat for birds. In total, we detected 41 bird species, including grassland specialists and species of state and national conservation concern (e.g. Henslow's Sparrow, Ammodramus henslowii). Avian species richness was generally comparable in switchgrass and prairie and increased with patch size in both patch types. Grassland specialists were less abundant and less likely to occur in patches within highly forested landscapes and were more common and likely to occur in larger patches, indicating that this group is also area-sensitive outside of the breeding season. Variation in the biomass and richness of arthropod food within patches was generally unrelated to richness and abundance metrics. Total bird abundance and that of grassland specialists was higher in patches with greater vegetation structural heterogeneity. Collectively, we find that perennial biomass feedstocks have potential to provide post-breeding and migratory stopover habitat for birds, but that the placement and management of crops will be critical factors in determining their suitability for species of conservation concern. Industrialization of cellulosic bioenergy production that results in reduced crop structural heterogeneity is likely to dramatically reduce the suitability of perennial biomass crops for birds.
Protein phosphatases, in coordination with protein kinases, play crucial roles in regulation of signaling pathways. To identify protein tyrosine phosphatases (PTPs) and serine–threonine (ser–thr) phosphatases in the Strongylocentrotus purpuratus genome, 179 annotated sequences were studied (122 PTPs, 57 ser–thr phosphatases). Sequence analysis identified 91 phosphatases (33 conventional PTPs, 31 dual specificity phosphatases, 1 Class III Cysteine-based PTP, 1 Asp-based PTP, and 25 ser–thr phosphatases). Using catalytic sites, levels of conservation and constraint in amino acid sequence were examined. Nine of 25 receptor PTPs (RPTPs) corresponded to human, nematode, or fly homologues. Domain structure revealed that sea urchin-specific RPTPs including two, PTPRLec and PTPRscav, may act in immune defense. Embryonic transcription of each phosphatase was recorded from a high-density oligonucleotide tiling microarray experiment. Most RPTPs are expressed at very low levels, whereas nonreceptor PTPs (NRPTPs) are generally expressed at moderate levels. High expression was detected in MAP kinase phosphatases (MKPs) and numerous ser–thr phosphatases. For several expressed NRPTPs, MKPs, and ser–thr phosphatases, morpholino antisense-mediated knockdowns were performed and phenotypes obtained. Finally, to assess roles of annotated phosphatases in endomesoderm formation, a literature review of phosphatase functions in model organisms was superimposed on sea urchin developmental pathways to predict areas of functional activity.
Phosphatase; PTP; DSP; MKP; Serine-threonine phosphatase; PPP; PPM; Genome; Strongylocentrotus; Urchin
Although in vitro breast cancer models have demonstrated a role for protein kinase C (PKC) α and δ isoforms in endocrine insensitivity and resistance respectively, there is currently little clinical evidence to support these observations.
To define the pattern of PKC α and δ expression using breast cancer cell lines, with and without endocrine resistance, and also breast cancer samples, where expression can be correlated with clinicopathological and endocrine therapy outcome data.
PKC isoform expression was examined in tamoxifen responsive, oestrogen receptor positive (ER+), ER+ acquired tamoxifen resistant (TAM‐R) and oestrogen receptor negative (ER−) cell lines by western blotting and immunocytochemical analysis. PKC isoform expression was then examined by immunohistochemistry in archival breast cancer specimens from primary breast cancer patients with known clinical outcome in relation to endocrine response and survival on therapy.
ER+ breast cancer cell lines expressed considerable PKC‐δ but barely detectable levels of PKC‐α, whereas ER− cell lines expressed PKC‐α but little PKC‐δ. ER+ acquired TAM‐R cell lines expressed substantial levels of both PKC‐α and δ. In clinical samples, high PKC‐δ expression correlated to endocrine responsiveness whereas PKC‐α expression correlated to ER negativity. PKC‐δ was an independent predictor of duration of response to therapy. Patients showing a PKC‐δ+/PKC‐α− phenotype had a six times longer endocrine response than patients with the PKC‐δ+/ PKC‐α+ phenotype (equating to tamoxifen resistance in vitro).
Levels of PKC‐α and δ expression appear to be indicative of response to anti‐oestrogen therapy and could be useful in predicting a patient's suitability for endocrine therapy.
To evaluate the epidemiology and investigate the impact of colonization and pulmonary fungal infections (PFI), we performed a retrospective analysis of 55 pediatric lung transplant recipients from 2002–2007 at a single institution. While 29 had positive pretransplant colonization, thirty-three (60%) were colonized post-transplant and 20% (11 subjects) developed proven or probable PFI. In a multivariable model, posttransplant fungal colonization was associated with older age (HR 2.9; 95% CI 1.1–7.6), CMV prophylaxis (5.6; 1.3–24.6) and respiratory viral infection prior to fungal colonization (2.9; 1.0–8.3). Neither fungal colonization nor PFI were associated with the development of chronic allograft rejection or death.
Lung transplantation; Fungal infection; Pediatrics; Aspergillus
Light-emitting diodes (LEDs) are becoming more commonly used as light sources for fluorescence microscopy. We describe the adaptation of a commercially available LED flashlight for use as a source for fluorescence excitation. This light source is long-lived, inexpensive, and is effective for excitation in the range of 440–600 nm.
fluorescence microscopy; light-emitting diode; LED; flashlight
A lipid-encapsulated perfluorocarbon nanoparticle molecular imaging contrast agent utilizing a PARAmagnetic Chemical Exchange Saturation Transfer (PARACEST) chelate is presented. PARACEST agents are ideally suited for molecular imaging applications because the contrast can be switched on and off at will simply by adjusting pulse sequence parameters. This obviates the need for pre- and post-injection images in order to define contrast agent binding. Spectroscopy (4.7 T) of PARACEST nanoparticles revealed a bound water peak at 52 ppm, in agreement with results from the water-soluble chelate. Imaging of control nanoparticles showed no appreciable contrast, while PARACEST nanoparticles produced >10% signal enhancement. PARACEST nanoparticles were targeted to clots via anti-fibrin antibodies and produced a contrast-to-noise ratio of 10 at the clot surface.
MRI; contrast agent; nanoparticle; fibrin; molecular imaging
Background: Autoantibodies may be present in a variety of underlying cancers several years before tumours can be detected and testing for their presence may allow earlier diagnosis. We report the clinical validation of an autoantibody panel in newly diagnosed patients with lung cancer (LC).
Patients and methods: Three cohorts of patients with newly diagnosed LC were identified: group 1 (n = 145), group 2 (n = 241) and group 3 (n = 269). Patients were individually matched by gender, age and smoking history to a control individual with no history of malignant disease. Serum samples were obtained after diagnosis but before any anticancer treatment. Autoantibody levels were measured against a panel of six tumour-related antigens (p53, NY-ESO-1, CAGE, GBU4-5, Annexin 1 and SOX2). Assay sensitivity was tested in relation to demographic variables and cancer type/stage.
Results: The autoantibody panel demonstrated a sensitivity/specificity of 36%/91%, 39%/89% and 37%/90% in groups 1, 2 and 3, respectively, with good reproducibility. There was no significant difference between different LC stages, indicating that the antigens included covered the different types of LC well.
Conclusion: This assay confirms the value of an autoantibody panel as a diagnostic tool and offers a potential system for monitoring patients at high risk of LC.
autoantibodies; clinical validation; lung cancer; newly diagnosed patients
Surface-layer (S-layer) supported lipid membranes on solid substrates are interfacial architectures mimicking the supramolecular principle of cell envelopes which have been optimized for billions of years of evolution in most extreme habitats. The authors implement this biological construction principle in a variety of layered supramolecular architectures consisting of a stabilizing protein monolayer and a functional phospholipid bilayer for the design and development of new types of solid-supported biomimetic membranes with a considerably extended stability and lifetime—compared to existing platforms—as required for novel types of bioanalytical sensors. First, Langmuir monolayers of lipids at the water/air interface are used as test beds for the characterization of different types of molecules which all interact with the lipid layers in various ways and, hence, are relevant for the control of the structure, stability, and function of supported membranes. As an example, the interaction of S-layer proteins from the bulk phase with a monolayer of a phospholipid synthetically conjugated with a secondary cell wall polymer (SCWP) was studied as a function of the packing density of the lipids in the monolayer. Furthermore, SCWPs were used as a new molecular construction element. The exploitation of a specific lectin-type bond between the N-terminal part of selected S-layer proteins and a variety of glycans allowed for the buildup of supramolecular assemblies and thus functional membranes with a further increased stability. Next, S-layer proteins were self-assembled and characterized by the surface-sensitive techniques, surface plasmon resonance spectroscopy and quartz crystal microbalance with dissipation monitoring. The substrates were either planar gold or silicon dioxide sensor surfaces. The assembly of S-layer proteins from solution to solid substrates could nicely be followed in-situ and in real time. As a next step toward S-layer supported bilayer membranes, the authors characterized various architectures based on lipid molecules that were modified by a flexible spacer separating the amphiphiles from the anchor group that allows for a covalent coupling of the lipid to a solid support, e.g., using thiols for Au substrates. Impedance spectroscopy confirmed the excellent charge barrier properties of these constructs with a high electrical resistance. Structural details of various types of these tethered bimolecular lipid membranes were studied by using neutron reflectometry. Finally, first attempts are reported to develop a code based on a SPICE network analysis program which is suitable for the quantitative analysis of the transient and steady-state currents passing through these membranes upon the application of a potential gradient.
CMF1 protein is expressed in developing striated muscle before the expression of contractile proteins, and depletion of CMF1 in myoblasts results in inability to express muscle-specific proteins. Previous studies of CMF1 identify a functional Rb-binding domain, which is conserved in the murine and human homologues. Here, we show that CMF1 binds Rb family members, while a CMF1 protein with deletion of the Rb-binding domain (Rb-del CMF1) does not. Myogenic cell lines over-expressing Rb-del CMF1 proliferate normally, but exhibit markedly impaired differentiation, including dramatically reduced contractile proteins gene expression and failure to fuse into myotubes. Furthermore, by quantitative real-time polymerase chain reaction, MyoD and Myf5 mRNA levels are comparable to wild-type, while myogenin and contractile protein mRNA levels are significantly attenuated. These data demonstrate that CMF1 regulates myocyte differentiation by interaction with Rb family members to induce expression of myogenic regulatory factors.
CMF1; LEK1; LEK proteins; Rb; myocyte differentiation
About 20% of familial amyotrophic lateral sclerosis (ALS) is caused by mutations in SOD1 and is typically transmitted as an autosomal dominant trait. However, due to reduced mutation penetrance, the disease may present in a recessive or sporadic manner.
To determine the factors responsible for the low penetrance of the SOD1 mutation.
Twelve members of a Canadian ALS family of Filipino origin were recruited for the study. SOD1 was sequenced in the proband. SOD1 expression was assessed by real-time-PCR and immunoblotting.
The proband was a homozygous carrier of a novel 6 bp deletion in exon 2 (ΔG27/P28), the pathologic significance of which was confirmed by immunohistochemistry. Eight living family members are heterozygotes and remain unaffected at ages ranging between 48 and 85 years. Haplotype analysis showed that the deletion is a single founder mutation likely common in the Cagayan province (Philippines). The low penetrance of the mutation is explained by the fact that it enhances the naturally occurring alternative splicing of exon 2 of the SOD1 mRNA, leading to reduced transcription of the mutant allele. Indeed, Western blot analysis demonstrated the low level of SOD1 protein in carriers of the ΔG27/P28 compared to wild-type individuals or a carrier of the A4V SOD1 mutation.
The enhanced splicing of exon 2 acts as a natural knock-down of the mutant SOD1 allele in the Filipino amyotrophic lateral sclerosis (ALS) family. There is a need for careful investigation of splicing isoforms of SOD1 and other ALS genes as factors influencing the severity of disease.
= Alzheimer disease;
= amyotrophic lateral sclerosis;
= corticobasal syndrome;
= hyaline conglomerate inclusions;
= reverse transcriptase–polymerase chain reaction;
= sporadic amyotrophic lateral sclerosis;
= SOD1-exposed-dimer-interface antibody.