Experimental yolk sac dysfunction as a model for studying nutritional disturbances in the embryo during early organogenesis

Our investigations concerning the importance of cell surface macromolecules during embryonic development led us to the discovery in 1961 that heterologous anti-rat kidney serum produced teratogenesis, growth retardation and embryonic death when injected into the pregnant rat during early organogenesis. It was established that IgG was the teratogenic agent, primarily directed against the visceral yolk sac (VYS) but not the embryo. Heterologous anti-rat VYS serum was prepared which was teratogenic localized in the VYS and served as a model for producing VYS dysfunction and embryonic malnutrition. The role of the yolk sac placenta in histiotrophic nutrition is now recognized to be critical for normal embryonic development during early organogenesis in the rodent. VYS antiserum affects embryonic development primarily by inhibiting endocytosis of proteins by the VYS endoderm, resulting in a reduction in the amino acids supplied to the embryo. Our laboratory has recently developed teratogenic monoclonal yolk sac antibodies (MCA) which can be utilized; to study VYS plasma membrane synthesis and recycling, to compare yolk sac function among different species, and to identify components of the plasma membrane involved in pinocytosis. MCA prepared against certain VYS antigens provide an opportunity to study embryonic nutrition with minimal interference with the nutritional state of the mother. Recent developments in the study of the human yolk sac along with our laboratory's ability to isolate a spectrum of yolk sac antigens, prepare monoclonal antibodies, and perform functional studies, should provide information that will increase our understanding of yolk sac function and dysfunction in the human and determine the relative importance of various amino acids to normal development during mammalian organogenesis.     

Affinity of Alkylphosphocholines to Biological Membrane of Prostate Cancer: Studies in Natural and Model Systems

The effectiveness of two alkylphosphocholines (APCs), hexadecylphosphocholine (miltefosine) and erucylphosphocholine to combat prostate cancer has been studied in vitro with artificial cancerous membrane, modelled with the Langmuir monolayer technique, and on cell line (Du-145). Studies performed with the Langmuir method indicate that both the investigated drugs have the affinity to the monolayer mimicking prostate cancer membrane (composed of cholesterol:POPC = 0.428) and the drug-membrane interactions are stronger for erucylphosphocholine as compared to hexadecylphosphocholine. Moreover, both studied drugs were found to fluidize the model membrane, which may lead to apoptosis. Indeed, biological studies confirmed that in Du-145 cell line both investigated alkylphosphocholines cause cell death primarily by apoptosis while necrotic cells constitute only a small percentage of APC-treated cells.     

Differential lipid metabolism in monocytes and macrophages: influence of cholesterol loading

The influence of the hypercholesterolemia associated with atherosclerosis on monocytes is poorly understood. Monocytes are exposed to high concentrations of lipids, particularly cholesterol and lysophosphatidylcholine (lyso-PC). Indeed, in line with recent reports, we found that monocytes accumulate cholesteryl esters (CEs) in hypercholesterolemic mice, demonstrating the need for studies that analyze the effects of lipid accumulation on monocytes. Here we analyze the effects of cholesterol and lyso-PC loading in human monocytes and macrophages. We found that cholesterol acyltransferase and CE hydrolase activities are lower in monocytes. Monocytes also showed a different expression profile of cholesterol influx and efflux genes in response to lipid loading and a different pattern of lyso-PC metabolism. In monocytes, increased levels of CE slowed the conversion of lyso-PC into PC. Interestingly, although macrophages accumulated glycerophosphocholine, phosphocholine was the main water-soluble choline metabolite being generated in monocytes, suggesting a role for mono- and diacylglycerol in the chemoattractability of these cells. In summary, monocytes and macrophages show significant differences in lipid metabolism and gene expression profiles in response to lipid loading. These findings provide new insights into the mechanisms of atherosclerosis and suggest potentials for targeting monocyte chemotactic properties not only in atherosclerosis but also in other diseases.     

Interactions of Zn(II) Ions with Humic Acids Isolated from Various Type of Soils. Effect of pH,Zn Concentrations and Humic Acids Chemical Properties

The main aim of this study was the analysis of the interaction between humic acids (HAs) from different soils and Zn(II) ions at wide concentration ranges and at two different pHs, 5 and 7, by using fluorescence and FTIR spectroscopy, as well as potentiometric measurements. The presence of a few areas of HAs structures responsible for Zn(II) complexing was revealed. Complexation at α-sites (low humified structures of low-molecular weight and aromatic polycondensation) and β-sites (weakly humified structures) was stronger at pH 7 than 5. This trend was not observed for γ-sites (structures with linearly-condensed aromatic rings, unsaturated bonds and large molecular weight). The amount of metal complexed at pH5 and 7 by α and γ-structures increased with a decrease in humification and aromaticity of HAs, contrary to β-areas where complexation increased with increasing content of carboxylic groups. The stability of complexes was higher at pH 7 and was the highest for γ-structures. At pH 5, stability decreased with C/N increase for α-areas and -COOH content increase for β-sites; stability increased with humification decrease for γ-structures. The stability of complexes at α and β-areas at pH 7 decreased with a drop in HAs humification. FTIR spectra at pH 5 revealed that the most-humified HAs tended to cause bidentate bridging coordination, while in the case of the least-humified HAs, Zn caused bidentate bridging coordination at low Zn additions and bidentate chelation at the highest Zn concentrations. Low Zn doses at pH 7 caused formation of unidentate complexes while higher Zn doses caused bidentate bridging. Such processes were noticed for HAs characterized by high oxidation degree and high oxygen functional group content; where these were low, HAs displayed bidentate bridging or even bidentate chelation. To summarize, the above studies have showed significant impact of Zn concentration, pH and some properties of HAs on complexation reactions of humic acids with zinc.     

Studies Designed to Increase the Stability and Antiviral Activity (HCMV) of the Active Benzimidazole Nucleoside,TCRB

Abstract

The potent activity of 2,5,6-trichloro-1-(ß-D-ribofuranosyl)benzimidazole (TCRB) against Human Cytomegalovirus with the concomitant low cellular toxicity at concentrations that inhibit viral growth prompted considerable interest in this research area. This interest was moderated by the pharmacokinetic studies of TCRB in rats and monkeys that revealed the instability of TCRB in vivo. These studies suggested that the instability was due to a cleavage of the glycosidic bond in vivo which released the heterocycle (2,5,6-trichlorobenzimidazole) into the bloodstream. This prompted us to initiate synthetic studies designed to increase the stability of the glycosidic bond of TCRB and BDCRB. Several synthetic approaches to address this and other problems are presented.     

Sodium Iodate Selectively Injuries the Posterior Pole of the Retina in a Dose-Dependent Manner: Morphological and Electrophysiological Study

Sequential morphological and functional features of retinal damage in mice exposed to different doses (40 vs. 20 mg/kg) of sodium iodate (NaIO₃) were analyzed. Retinal morphology, apoptosis (TUNEL assay), and function (electroretinography; ERG) were examined at several time points after NaIO₃ administration. The higher dose of NaIO₃ caused progressive degeneration of the whole retinal area and total suppression of scotopic and photopic ERG. In contrast, the lower dose induced much less severe degeneration in peripheral part of retina along with a moderate decline of b- and a-wave amplitudes in ERG, corroborating the presence of regions within retina that retain their function. The peak of photoreceptor apoptosis was found on the 3rd day, but the lower dose induced more intense reaction within the central retina than in its peripheral region. In conclusion, these results indicate that peripheral area of the retina reveals better resistance to NaIO₃ injury than its central part.     

Semi-supervised analysis of gene expression profiles for lineage-specific development in the Caenorhabditis elegans embryo

MOTIVATION: Gene expression profiling is a powerful approach to identify genes that may be involved in a specific biological process on a global scale. For example, gene expression profiling of mutant animals that lack or contain an excess of certain cell types is a common way to identify genes that are important for the development and maintenance of given cell types. However, it is difficult for traditional computational methods, including unsupervised and supervised learning methods, to detect relevant genes from a large collection of expression profiles with high sensitivity and specificity. Unsupervised methods group similar gene expressions together while ignoring important prior biological knowledge. Supervised methods utilize training data from prior biological knowledge to classify gene expression. However, for many biological problems, little prior knowledge is available, which limits the prediction performance of most supervised methods. RESULTS: We present a Bayesian semi-supervised learning method, called BGEN, that improves upon supervised and unsupervised methods by both capturing relevant expression profiles and using prior biological knowledge from literature and experimental validation. Unlike currently available semi-supervised learning methods, this new method trains a kernel classifier based on labeled and unlabeled gene expression examples. The semi-supervised trained classifier can then be used to efficiently classify the remaining genes in the dataset. Moreover, we model the confidence of microarray probes and probabilistically combine multiple probe predictions into gene predictions. We apply BGEN to identify genes involved in the development of a specific cell lineage in the C. elegans embryo, and to further identify the tissues in which these genes are enriched. Compared to K-means clustering and SVM classification, BGEN achieves higher sensitivity and specificity. We confirm certain predictions by biological experiments. AVAILABILITY: The results are available at http://www.csail.mit.edu/~alanqi/projects/BGEN.html.     

Oospore dimensions and wall ornamentation in <Emphasis Type="Italic">Chara braunii</Emphasis>

Morphological features of oospores of a rarely noted species Chara braunii Gmel. were examined in a local population (fishpond near Tuplice village, Ziemia Lubuska region, mid-western Poland). The largest polar axis (LPA, length), largest equatorial diameter (LED, width), isopolarity index (ISI = LPA/LED* 100), and the number of ridges and width of fossa of 90 oospores were measured. Additionally, the oospore wall ornamentation pattern was examined by means of scanning electron microscopy (SEM). As a result, a cylindrical shape and the lack of so-called “shoulder” were found. Oospore length and width ranged between 466.8–600.1 μm and 250.1–366.7 μm, respectively, with the ISI index values between 148 and 213. The number of ridges was 8–10 and the width of fossa varied between 50.0 and 66.7 μm. The oospore width appeared to be the most changeable feature, whereas, by contrast, the number of ridges was the least variable character. Wall ornamentation can be described as tuberculate and a ribbon was present on the ridges. As a comparative material for the study performed, 9 oospores from Professor Izabela Dąmbska’s Collection of Charophytes of Poland were measured. It was evidenced that variation ranges of most of the features of Chara braunii oospores from Tuplice fishpond are similar to those of Professor Dąmbska’s herbarium materials and to data reported by authors from other countries as well. The morphology of reproductive structures does not follow the variation of thalli characteristics.     

Human immunodeficiency virus type 1 nef expression prevents AP-2-mediated internalization of the major histocompatibility complex class II-associated invariant chain

The lentiviral Nef protein has been studied extensively for its ability to induce the downregulation of several immunoreceptors on the surfaces of infected cells. However, Nef expression is unique in inducing highly effective upregulation of the major histocompatibility complex class II-associated chaperone invariant (Ii) chain complexes in different cell types. Under normal conditions, endocytosis of the Ii chain and other molecules, like the transferrin receptor and CD4, is rapid and AP-2 dependent. Human immunodeficiency virus type 1 (HIV-1) Nef expression strongly reduces the internalization of the Ii chain, enhances that of CD4, and does not modify transferrin uptake. The mutation of AP-2 binding motifs LL164 and DD174 in Nef leads to the inhibition of Ii chain upregulation. In AP-2-depleted cells, surface levels of the Ii chain are high and remain unmodified by Nef expression, further indicating that Nef regulates Ii chain internalization via the AP-2 pathway. Immunoprecipitation experiments revealed that the Ii chain can interact with Nef in a dileucine-dependent manner. Importantly, we have shown that Nef-induced CD4 downregulation and Ii chain upregulation are genetically distinguishable. We have identified natural nef alleles that have lost one of the two functions but not the other one. Moreover, we have characterized Nef mutant forms possessing a similar phenotype in the context of HIV-1 infection. Therefore, the Nef-induced accumulation of Ii chain complexes at the cell surface probably results from a complex mechanism leading to the impairment of AP-2-mediated endocytosis rather than from direct competition between Nef and the Ii chain for binding AP-2.