Parasite infections often result in a switch of the human body’s predominant immune reaction from T-helper 1 (Th1)-type to Th2-type. Hence, parasite infections are widely expected to accelerate the progression of human immunodeficiency virus (HIV) infections to acquired immunodeficiency syndrome (AIDS). In the People’s Republic of China, both parasitic diseases and AIDS are epidemic in certain rural areas, and co-infections are relatively common. However, no population-based studies have yet investigated the frequency of HIV and parasite co-infections, and its effects on immune responses. We studied (1) the immune status of an HIV-infected population, and (2) the effect of co-infection of HIV and intestinal parasites on selected parameters of the human immune system.
A total of 309 HIV-infected individuals were recruited and compared to an age-matched and sex-matched control group of 315 local HIV-negative individuals. Questionnaires were administered to all participants to obtain information on sociodemographic characteristics, sanitation habits, family income, and recent clinical manifestations. Two consecutive stool samples and 10 ml samples of venous blood were also collected from each individual for the diagnosis of parasite infections and quantitative measurements of selected cytokines and CD4+ T-lymphocytes, respectively.
During the study period, 79 HIV-infected individuals were not under highly active antiretroviral therapy (HAART) and were thus included in our analysis; the prevalence of intestinal helminth infections was 6.3% and that of protozoa was 22.8%. The most common protozoan infections were Blastocystis hominis (B. hominis) (13.9%) and Cryptosporidium spp. (10.1%). The prevalence of Cryptosporidium spp. in HIV-infected individuals was significantly higher than that in HIV negative individuals (P < 0.05). Compared to the non-co-infected population, no significant difference was found for any of the measured immunological indicators (P > 0.05). However, the following trends were observed: IFN-γ levels were lower, but the IL-4 level was higher, in the population co-infected with HIV and helminths. In the population co-infected with HIV and B. hominis, the IL-2 level was higher. The population co-infected with HIV and Cryptosporidium spp. had markedly lower CD4+ T-lymphocyte counts.
According to the immunologic profile, co-infection with helminths is disadvantageous to HIV-infected individuals. It was associated with a shift in the Th1/Th2 balance in the same direction as that caused by the virus itself, which might indicate an acceleration of the progress from an HIV infection to AIDS. Co-infection with Cryptosporidium spp. was not associated with a significant change in immune factors but co-infection with Cryptosporidium spp. was associated with a reduced level of CD4 + T-lymphocytes, confirming the opportunistic nature of such infections. Co-infection with B. hominis, on the other hand, was associated with an antagonistic shift in the immunological profile compared to an HIV infection.
HIV; Intestinal parasite; Co-infection; Immunological profile; People’s Republic of China
We investigated the prevalence of and risk factors for small airway obstruction (SAO) among Chinese island residents to establish means to prevent and treat SAO.
From October 17, 2011 to November 1, 2011, a total of 2,873 residents aged >20 years who lived on the Huangqi Peninsula of Fujian were recruited by random cluster sampling. They were asked to complete a Burden of Obstructive Lung Disease (BOLD) questionnaire and underwent physical examinations and lung function evaluations. SAO was defined as a forced expiratory flow at 50% of vital capacity, Vmax50%, of less than 70% of predicted. Risk factors for SAO were assessed from among demographic and anthropometric variables, blood chemistry results, and questionnaire response items.
A total of 216 (7.52%) Chinese island residents were identified as having SAO (95 males; 121 females). Their survey and test results were compared with 432 age and sex-matched healthy controls (192 males; 240 females) for SAO risk factors. Among numerous factors investigated, only diabetes mellitus (p = 0.039), smoking index (SI, p<0.001 for SI>600), second hand smoke (p = 0.002), and lack of regular exercise (p<0.001) were significant risk factors for SAO.
The risk factors for SAO among Chinese island residents appeared to be similar to those among people who live in high-density urban environments and impoverished rural areas. Public health policies and medical practices directed toward improving respiratory health for island residents should be comparable to those used for urban and rural dwellers.
To investigate the effects of Msx2 on lens epithelium cell cycle, and evaluate the changes of the proliferation, apoptosis of lens epithelium cells.
Mice lens epithelium cells were cultured and transfected with pEGFP-Msx2 and control. Msx2-deﬁcient mice (Msx2−/−) lens tissue were isolated. Lens tissue and transfected cells were prepared for mRNA extraction using Trizol reagent. CyclinD1 and Prox1 expression were evaluated by real-time RT-PCR. BrdU incorporation and apoptosis rate were investigated by immunofluorescence and flow cytometry analysis.
After transfected with pEGFP-Msx2, lens epithelium cells failed to incorporate BrdU and anti-phospho-histone-3 immunofluorescence failed to detect cell nuclei which GFP were positive. Msx2 over expression resulted in increasing apoptosis rate in lens epithelium cells. CyclinD1 and Prox1 expression increased significantly in Msx2 knockout mice by real-time RT-PCR quantization and CyclinD1 expression decreased significantly in Msx2 overexpressed cell.
Msx2 has the effect of inhibiting proliferation and differentiation, triggering apoptosis on mice lens epithelium cells.
Msx2; lens epithelium cell; cell cycle
The purpose of this study was to evaluate the relationship between preoperative inflammatory markers (neutrophil-lymphocyte ratio and platelet-lymphocyte ratio) and cervical stromal involvement in patients with endometrioid adenocarcinoma.
We studied 318 patients with endometrioid adenocarcinoma who underwent comprehensive surgical staging. We used univariate and multivariate analyses of cervical stromal involvement and receiver-operating curves to calculate optimal cutoff values for neutrophil-lymphocyte and platelet-lymphocyte ratios to predict cervical stromal involvement.
The presence of cervical stromal involvement was associated with neutrophil-lymphocyte ratio and platelet-lymphocyte ratio (P = 0.009 and P = 0.031, respectively). Multivariate analysis showed that higher neutrophil-lymphocyte and platelet-lymphocyte ratios independently predicted cervical stromal involvement (odds ratio 3.10, 95% confidence interval 1.10–8.76, P = 0.032, and odds ratio 5.27, 95% confidence interval 1.94–14.35, P = 0.001, respectively). At a threshold of 2.01, the neutrophil-lymphocyte ratio was 71.0% sensitive and 63.8% specific for stromal involvement; at a 172.24 threshold, the platelet-lymphocyte ratio was 48.4% sensitive and 88.9% specific.
Preoperative neutrophil-lymphocyte and platelet-lymphocyte ratios can help identify the risk of cervical stromal involvement in patients with endometrial cancer. Evaluating these ratios may help select patients who should be particularly watched and tested for cervical stromal involvement.
neutrophil-lymphocyte ratio; platelet-lymphocyte ratio; endometrioid adenocarcinoma; cervical stromal involvement
The GABAC receptor, a postsynaptic membrane receptor expressed prominently in the retina, is a ligand-gated ion channel that consists of a combination of ρ subunits. We report characterization of a novel guinea pig polyclonal antibody, termed GABAC Ab N-14, directed against a 14-mer peptide (N-14) of the extracellular domain of the human ρ1 subunit. The antibody exhibits high sensitivity for N-14 by ELISA. In Western blots, GABAC Ab N-14 shows reactivity with the ρ1 subunit of preparations obtained from ρ1 GABAC-expressing neuroblastoma cells, Xenopus oocytes, and mammalian retina and brain. Flow cytometry reveals a rightward shift in mean fluorescence intensity of GABAC-expressing neuroblastoma cells probed with GABAC Ab N-14. Immunostaining of neuroblastoma cells and oocytes with GABAC Ab N-14 yields fluorescence only with GABAC-expressing cells. Antibody binding has no effect on GABA-elicited membrane current responses. Immunostaining of human retinal sections shows preferential staining within the inner plexiform layer. GABAC Ab N-14 appears well suited for investigative studies of GABAC ρ1 subunit in retina and other neural tissues.
GABA receptor; GABAC; retina; ρ1 subunit; antibody
The title mononuclear complex, [Ni(C7H3NO4)(C9H10N2)3], shows a central NiII atom which is coordinated by two carboxylate O atoms and the N atom from a pyridine-2,6-dicarboxylate ligand and by three N atoms from different 5,6-dimethyl-1H-benzimidazole ligands in a distorted octahedral geometry. The crystal structure shows intermolecular N—H⋯O hydrogen bonds.
In the title complex, [Ni(C7H3NO4)(C7H6N2)3], the NiII ion is coordinated by two carboxylate O atoms and the N atom from a pyridine-2,6-dicarboxylate ligand and by three N atoms from three benzimidazole ligands to form a slightly distorted octahedral geometry. In the crystal, molecules are linked by N—H⋯O hydrogen bonds to form a three-dimensional network.
Homonymous quadrantanopsia results from retrochiasmal lesions in the visual pathway. Invasive mole is a benign tumor that arises from myometrial invasion of a hydatidiform mole via direct extension through tissue or venous channels. Cerebral metastasis of invasive mole is rare and there has been no report demonstrating homonymous quadrantanopsia as the first manifestation of metastasis in any trophoblastic neoplasms.
We report the case of a 31-year-old Asian woman who presented with right homonymous inferior quadrantanopsia from the mass effect of a solitary cerebral metastasis from an invasive mole. A magnetic resonance image (MRI) of the brain showed a metastatic tumor in the left occipital lobe. The visual field improved slightly after chemotherapy. There was a reduction in the tumor size and the surrounding edema. This is the first case report demonstrating that homonymous quadrantanopsia should be included in the manifestations of the metastasis of an invasive mole.
The presentation of homonymous quadrantanopsia must alert ophthalmologists to conduct a complete medical history and arrange specialist consultation.
Cerebral metastasis; Homonymous Quadrantanopsia; Invasive mole
Motivation: Immunoassays are primary diagnostic and research tools throughout the medical and life sciences. The common approach to the processing of immunoassay data involves estimation of the calibration curve followed by inversion of the calibration function to read off the concentration estimates. This approach, however, does not lend itself easily to acceptable estimation of confidence limits on the estimated concentrations. Such estimates must account for uncertainty in the calibration curve as well as uncertainty in the target measurement. Even point estimates can be problematic: because of the non-linearity of calibration curves and error heteroscedasticity, the neglect of components of measurement error can produce significant bias.
Methods: We have developed a Bayesian approach for the estimation of concentrations from immunoassay data that treats the propagation of measurement error appropriately. The method uses Markov Chain Monte Carlo (MCMC) to approximate the posterior distribution of the target concentrations and numerically compute the relevant summary statistics. Software implementing the method is freely available for public use.
Results: The new method was tested on both simulated and experimental datasets with different measurement error models. The method outperformed the common inverse method on samples with large measurement errors. Even in cases with extreme measurements where the common inverse method failed, our approach always generated reasonable estimates for the target concentrations.
Availability: Project name: Baecs; Project home page: www.computationalimmunology.org/utilities/; Operating systems: Linux, MacOS X and Windows; Programming language: C++; License: Free for Academic Use.
Supplementary data are available at Bioinformatics online.
Intestinal parasite infections (IPIs) are among the most significant causes of illness and disease of socially and economically disadvantaged populations in developing countries, including rural areas of the People's Republic of China. With the spread of the human immunodeficiency virus (HIV) among rural Chinese populations, there is ample scope for co-infections and there have been increasing fears about their effects. However, hardly any relevant epidemiological studies have been carried out in the country. The aim of the present survey was to assess the IPI infection status among a representative sample of HIV-positive Chinese in rural Anhui province, and compare the findings with those from a cohort of non-infected individuals.
A case control study was carried out in a rural village of Fuyang, Anhui province, China. Stool samples of all participants were examined for the presence of intestinal parasites. Blood examination was performed for the HIV infection detection and anemia test. A questionnaire was administered to all study participants.
A total of 302 HIV positive and 303 HIV negative individuals provided one stool sample for examination. The overall IPI prevalence of intestinal helminth infections among HIV positives was 4.3% (13/302) while it was 5.6% (17/303) among HIV negatives, a non-significant difference. The prevalence of protozoa infections among HIV positives was 23.2% while the rate was 25.8% among HIV negatives. The species-specific prevalences among HIV positives were as follows: 3.6% for hookworm, 0.7% for Trichuris trichiura, zero for Ascaris lumbricoides, 0.3% for Clonorchis sinensis, 1.3% for Giardia intestinalis, 16.2% for Blastocystis hominis, 1.7% for Entamoeba spp. and 8.3% for Cryptosporidium spp.. Cryptosporidium spp. infections were significantly more prevalent among HIV positives (8.3%) compared to the HIV negative group (3.0%; P < 0.05). Among people infected with HIV, Cryptosporidium spp. was significantly more prevalent among males (12.6%) than females (4.4%; P < 0.05). According to multivariate logistic regression, the factors significantly associated with parasite infections of the people who were HIV positive included sex (male: OR = 6.70, 95% CI: 2.030, 22.114), younger age (less than 42 years old: OR = 4.148, 95% CI: 1.348, 12.761), and poor personal hygiene habits (OR = 0.324, 95% CI: 0.105, 0.994).
HIV positive individuals are more susceptible to co-infections with Cryptosporidium spp. than HIV negative people, particularly younger males with poor personal hygiene habits, indicating a need for targeted hygiene promotion, IPI surveillance and treatment.
HIV; Intestinal parasites; Helminths; Protozoa; Co-infection; China
Fatty acids and derivatives (FADs) are resources for natural antimicrobials. In order to screen for additional potent antimicrobial agents, the antimicrobial activities of FADs against Staphylococcus aureus were examined using a microplate assay. Monoglycerides of fatty acids were the most potent class of fatty acids, among which monotridecanoin possessed the most potent antimicrobial activity. The conventional quantitative structure-activity relationship (QSAR) and comparative molecular field analysis (CoMFA) were performed to establish two statistically reliable models (conventional QSAR: R
LOO=0.910; CoMFA: R
2=0.588, respectively). Improved forecasting can be achieved by the combination of these two models that provide a good insight into the structure-activity relationships of the FADs and that may be useful to design new FADs as antimicrobial agents.
Fatty acid derivatives; Quantitative structure-activity relationship; Comparative molecular field analysis; Antimicrobial activity
AIM: To compare and evaluate the appropriate prognostic indicators of lymph node basic staging in gastric cancer patients who underwent radical resection.
METHODS: A total of 1042 gastric cancer patients who underwent radical resection and D2 lymphadenectomy were staged using the 6th and 7th edition International Union Against Cancer (UICC) N staging methods and the metastatic lymph node ratio (MLNR) staging. Homogeneity, discriminatory ability, and gradient monotonicity of the various staging methods were compared using linear trend χ2, likelihood ratio χ2 statistics, and Akaike information criterion (AIC) calculations. The area under the curve (AUC) was calculated to compare the predictive ability of the aforementioned three staging methods.
RESULTS: Optimal cut-points of the MLNR were calculated as MLNR0 (0), MLNR1 (0.01-0.30), MLNR2 (0.31-0.50), and MLNR3 (0.51-1.00). In univariate, multivariate, and stratified analyses, MLNR staging was superior to the 6th and 7th edition UICC N staging methods. MLNR staging had a higher AUC, higher linear trend and likelihood ratio χ2 scores and lower AIC values than the other two staging methods.
CONCLUSION: MLNR staging predicts survival after gastric cancer more precisely than the 6th and 7th edition UICC N classifications and should be considered as an alternative to current pathological N staging.
Gastric cancer; Metastatic lymph node ratio; Prognosis
AIM: To identify and assess the novel makers for detection of Shiga toxin producing Escherichia coli (STEC) O157:H7 with an integrated computational and experimental approach.
METHODS: High-throughput NCBI blast (E-value cutoff e-5) was used to search homologous genes among all sequenced prokaryotic genomes of each gene encoded in each of the three strains of STEC O157:H7 with complete genomes, aiming to find unique genes in O157:H7 as its potential markers. To ensure that the identified markers from the three strains of STEC O157:H7 can serve as general markers for all the STEC O157:H7 strains, a genomic barcode approach was used to select the markers to minimize the possibility of choosing a marker gene as part of a transposable element. Effectiveness of the markers predicted was then validated by running polymerase chain reaction (PCR) on 18 strains of O157:H7 with 5 additional genomes used as negative controls.
RESULTS: The blast search identified 20, 16 and 20 genes, respectively, in the three sequenced strains of STEC O157:H7, which had no homologs in any of the other prokaryotic genomes. Three genes, wzy, Z0372 and Z0344, common to the three gene lists, were selected based on the genomic barcode approach. PCR showed an identification accuracy of 100% on the 18 tested strains and the 5 controls.
CONCLUSION: The three identified novel markers, wzy, Z0372 and Z0344, are highly promising for the detection of STEC O157:H7, in complementary to the known markers.
Shiga toxin producing Escherichia coli O157:H7; Diagnosis; Marker genes; Infectious diseases
ErbB2 protein is essential for the development of Schwann cells and for the normal fiber growth and myelin formation of peripheral nerves. We have investigated the fate of the otocyst-derived inner ear sensory neurons in the absence of ErbB2 using ErbB2 null mutants. Afferent innervation of the ear sensory epithelia shows numerous fibers overshooting the organ of Corti, followed by a reduction of those fibers in near term embryos. This suggests that mature Schwann cells do not play a role in targeting or maintaining the inner ear innervation. Comparable to the overshooting of nerve fibers, sensory neurons migrate beyond their normal locations into unusual positions in the modiolus. They may miss a stop signal provided by the Schwann cells that are absent as revealed with detailed histology. Reduction of overshooting afferents may be enhanced by a reduction of the neurotrophin Ntf3 transcript to about 25% of wild type. Ntf3 transcript reductions are comparable to an adult model that uses a dominant negative form of ErbB4 expressed in the supporting cells and Schwann cells of the organ of Corti. ErbB2 null mice retain afferents to inner hair cells possibly because of the prominent expression of the neurotrophin Bdnf in developing hair cells. Despite the normal presence of Bdnf transcript, afferent fibers are disoriented near the organ of Corti. Efferent fibers do not form an intraganglionic spiral bundle in the absence of spiral ganglia and appear reduced and disorganized. This suggests that either ErbB2 mediated alterations in sensory neurons or the absence of Schwann cells affects efferent fiber growth to the organ of Corti.
Ear development; Auditory sensory neuron; Neurotrophin; Pathfinding; Neuronal migration
Both genetic and epigenetic alterations can control the progression of cancer. Genetic alterations are impossible to reverse, while epigenetic alterations are reversible. This advantage suggests that epigenetic modifications should be preferred in therapy applications. DNA methyltransferases and histone deacetylases have become the primary targets for studies in epigenetic therapy. Some DNA methylation inhibitors and histone deacetylation inhibitors are approved by the US Food and Drug Administration as anti-cancer drugs. Therefore, the uses of epigenetic targets are believed to have great potential as a lasting favorable approach in treating breast cancer.
breast cancer; epigenetic therapy; DNA methylation inhibitors; Histone deacetylation inhibitors
Fumarase catalyzes the reversible hydration of fumarate to L-malate and is a key enzyme in the tricarboxylic acid (TCA) cycle and in amino acid metabolism. Fumarase is also used for the industrial production of L-malate from the substrate fumarate. Thermostable and high-activity fumarases from organisms that inhabit extreme environments may have great potential in industry, biotechnology, and basic research. The marine environment is highly complex and considered one of the main reservoirs of microbial diversity on the planet. However, most of the microorganisms are inaccessible in nature and are not easily cultivated in the laboratory. Metagenomic approaches provide a powerful tool to isolate and identify enzymes with novel biocatalytic activities for various biotechnological applications.
A plasmid metagenomic library was constructed from uncultivated marine microorganisms within marine water samples. Through sequence-based screening of the DNA library, a gene encoding a novel fumarase (named FumF) was isolated. Amino acid sequence analysis revealed that the FumF protein shared the greatest homology with Class II fumarate hydratases from Bacteroides sp. 2_1_33B and Parabacteroides distasonis ATCC 8503 (26% identical and 43% similar). The putative fumarase gene was subcloned into pETBlue-2 vector and expressed in E. coli BL21(DE3)pLysS. The recombinant protein was purified to homogeneity. Functional characterization by high performance liquid chromatography confirmed that the recombinant FumF protein catalyzed the hydration of fumarate to form L-malate. The maximum activity for FumF protein occurred at pH 8.5 and 55°C in 5 mM Mg2+. The enzyme showed higher affinity and catalytic efficiency under optimal reaction conditions: Km= 0.48 mM, Vmax = 827 μM/min/mg, and kcat/Km = 1900 mM/s.
We isolated a novel fumarase gene, fumF, from a sequence-based screen of a plasmid metagenomic library from uncultivated marine microorganisms. The properties of FumF protein may be ideal for the industrial production of L-malate under higher temperature conditions. The identification of FumF underscores the potential of marine metagenome screening for novel biomolecules.
Statistical mixture modeling provides an opportunity for automated identification and resolution of cell subtypes in flow cytometric data. The configuration of cells as represented by multiple markers simultaneously can be modeled arbitrarily well as a mixture of Gaussian distributions in the dimension of the number of markers. Cellular subtypes may be related to one or multiple components of such mixtures, and fitted mixture models can be evaluated in the full set of markers as an alternative, or adjunct, to traditional subjective gating methods that rely on choosing one or two dimensions.
Four color flow data from human blood cells labeled with FITC-conjugated anti-CD3, PE-conjugated anti-CD8, PE-Cy5-conjugated anti-CD4 and APC-conjugated anti-CD19 Abs was acquired on a FACSCalibur. Cells from four murine cell lines, JAWS II, RAW 264.7, CTLL-2 and A20, were also stained with FITC-conjugated anti-CD11c, PE-conjugated anti-CD11b, PE-Cy5-conjugated anti-CD8a and PE-Cy7-conjugated-CD45R/B220 Abs respectively, and single color flow data were collected on an LSRII. The data was fitted with a mixture of multivariate Gaussians using standard Bayesian statistical approaches and Markov chain Monte Carlo computations.
Statistical mixture models were able to identify and purify major cell subsets in human peripheral blood, using an automated process that can be generalized to an arbitrary number of markers. Validation against both traditional expert gating and synthetic mixtures of murine cell lines with known mixing proportions was also performed.
This paper describes studies of statistical mixture modeling of flow cytometric data, and demonstrates their utility in examples with four-color flow data from human peripheral blood samples and synthetic mixtures of murine cell lines.
Statistics; Mixture models; Markov chain Monte Carlo; Bayesian analysis; Identification; Automation; Gating
In the title salt, [Cu(NCS)(C6H6N4)2]Cl, the CuII atom adopts a five-coordinated square-pyramidal geometry consisting of an N atom from a thiocyanate anion and four N atoms from two chealting biimidazole ligands. The thiocyanate ligand occupies the axial position and is, like the CuII centre, located on a mirror plane. The cation and anion are linked into a linear chain by N—H⋯S and N—H⋯Cl hydrogen bonds.
The title compound, C13H16N2O3, adopts an E configuration with respect to the C=N bond and an intramolecular N—H⋯N hydrogen bond results in the formation of a six-membered ring. In the crystal, intermolecular O—H⋯O hydrogen bonds link the molecules into a chain propagating along the b axis. Very weak π–π stacking interactions [centroid–centroid distance = 4.18 (2) Å] may further consolidate the packing, forming a two-dimensional supramolecular network.
Computer simulations are of increasing importance in modeling biological phenomena. Their purpose is to predict behavior and guide future experiments. The aim of this project is to model the early immune response to vaccination by an agent based immune response simulation that incorporates realistic biophysics and intracellular dynamics, and which is sufficiently flexible to accurately model the multi-scale nature and complexity of the immune system, while maintaining the high performance critical to scientific computing.
The Multiscale Systems Immunology (MSI) simulation framework is an object-oriented, modular simulation framework written in C++ and Python. The software implements a modular design that allows for flexible configuration of components and initialization of parameters, thus allowing simulations to be run that model processes occurring over different temporal and spatial scales.
MSI addresses the need for a flexible and high-performing agent based model of the immune system.
We describe here diffusion and imaging properties of three new lipophilic tracers, NeuroVue ™ Maroon (near infrared), NeuroVue ™ Red and NeuroVue ™ Green. Using pair wise comparisons between the new dyes and existing dyes (DiI, DiA, DiD, DiO, PKH2, PKH26) applied to the left and the right side of fixed spinal cord preparations, we show that NeuroVue Maroon (excitation max 647 nm) surpasses all other dyes in this study in signal to noise ratio. We also present data showing the utility of these new dyes for both double labeling and triple labeling in combination with each other or existing lipophilic tracers. Using mice bearing the PLP-eGFP transgene, we demonstrate that either NeuroVue Maroon or NeuroVue Red can readily be combined with eGFP labeling. Double labeling experiments using NeuroVue Red and eGFP allowed us to demonstrate that every fiber in the neonatal ear is surrounded by developing Schwann cells.
lipophilic tracers; diffusion rates; imaging; confocal microscopy
To determine whether Notch-1, a ligand-activated transmembrane receptor known to maintain cells in an undifferentiated state, primarily progenitor cells in other systems, could be used as a stem cell marker in human limbal epithelium.
Human corneoscleral tissues obtained from the Doheny Eye & Tissue Transplant Bank were prepared for cross section and whole mount analysis. Tissue for whole mount was incubated in dispase; the epithelial sheet was removed and fixed in 4% paraformaldehyde. Sections and whole mount were stained with antibodies against Notch-1, Notch-2, β-1 integrin, α-6, and the G2 subtype member of the ATP binding cassette transporter (ABCG2). Specificity of the Notch-1 antibody was determined by western blot analysis with Cos-7 cells transfected with Notch-1. Explant culture was performed and only primary cultures were used in this experiment.
Notch-1 was found to be expressed in the limbal basal region where stem cells reside. Notch-1 antigenicity was more pronounced in cell clusters, mainly in the palisades of Vogt. The central cornea was almost devoid of Notch-1. The intensity of Notch-1 staining in cultured cells from the limbal explants was high in only a few cells. The Notch-1 signal was diminished in dividing cells. Expression in cultured cells was more cytoplasmic; few cells showed additional nuclear staining. The Notch-1-stained whole mount showed only a few cells in the limbal region. A 300 kDa and a 110 kDa band confirmed the specificity of the antibody in Cos-7 cells transfected with Notch-1. Double staining for ABCG2 and Notch-1 showed some ABCG2-positive cells co-expressing Notch-1 in the limbal basal epithelium, indicating that Notch-1-expressing cells might be a unique subpopulation of cells with stem cell properties.
Immunofluorescence data shows that Notch-1 could be a possible marker for the stem cells in the limbal basal epithelium. Further studies and characterization of the Notch pathway in corneal development will provide valuable clues for the identification of stem cells.
The transactivation of enhanced growth factor receptor (EGFR) by G protein–coupled receptor (GPCR) ligands is recognized as an important signaling mechanism in the regulation of complex biological processes, such as cancer development. Estrogen (E2), which is a steroid hormone that is intimately implicated in breast cancer, has also been suggested to function via EGFR transactivation. In this study, we demonstrate that E2-induced EGFR transactivation in human breast cancer cells is driven via a novel signaling system controlled by the lipid kinase sphingosine kinase-1 (SphK1). We show that E2 stimulates SphK1 activation and the release of sphingosine 1-phosphate (S1P), by which E2 is capable of activating the S1P receptor Edg-3, resulting in the EGFR transactivation in a matrix metalloprotease–dependent manner. Thus, these findings reveal a key role for SphK1 in the coupling of the signals between three membrane-spanning events induced by E2, S1P, and EGF. They also suggest a new signal transduction model across three individual ligand-receptor systems, i.e., “criss-cross” transactivation.
Virion infectivity factor (Vif) protein of human immunodeficiency virus type 1 (HIV-1) is essential for the productive infection of primary human CD4 T lymphocytes and macrophages. Vif overcomes the HIV-inhibitory effects of cellular factor APOBEC3G, which has cytidine deaminase activity. We previously reported the isolation of a Vif-interacting ring finger protein, Triad 3, from a human leukocyte cDNA library, using the yeast two-hybrid system. The full-length cellular protein homologue of Triad 3 has been recently identified as the zinc finger protein inhibiting NF-κB (ZIN). Sequence analysis indicates that Triad 3 protein contains all four major ring-like motifs of ZIN. We report here that ZIN binds to purified Vif in vitro and that Triad 3/ZIN interacts with HIV-1 Vif in transfected human 293T cells, as demonstrated by coimmunoprecipitation. To test the biological relevance of this interaction, we produced infectious HIV-1 NL4.3 in the presence or absence of cotransfected ZIN. HIV-1 NL4.3 virus stocks produced in the presence of exogenously expressed ZIN were twofold less infectious in a single-cycle infectivity assay than virus produced in the absence of exogenous ZIN. It was further shown that cells infected with HIV NL4.3 virus stocks produced in the presence of exogenously expressed ZIN were impaired in viral DNA synthesis by twofold. The impairment in viral reverse transcription and the reduction in single-cycle viral infectivity were both shown to be dependent on the presence of Vif in the virus producer cells. The possible mechanisms by which ZIN interferes with the early events of HIV-1 replication are discussed.