Human placental trophoblast as an in vitro model for tumor progression

The human placenta is a highly invasive tumor-like structure in which a subpopulation of placental trophoblast cells known as the "extravillous trophoblast" (EVT) invades the uterine decidua and its vasculature to establish adequate fetal-maternal exchange of molecules. By utilizing in vitro-propagated short-lived EVT cell lines we found that molecular mechanisms responsible for their invasiveness are identical to those of cancer cells; however, unlike cancer cells, their proliferation, migration, and invasiveness in situ are stringently controlled by decidua-derived transforming growth factor (TGF)-beta. By SV40T antigen transfection of normal EVT cells followed by a forced crisis regimen in culture we produced an immortalized premalignant derivative that is hyperproliferative, hyperinvasive, and deficient in gap-junctional intercellular communication. Both premalignant and malignant EVT (JAR and JEG-3 choriocarcinoma) cell lines were found to be TGF-beta-resistant. Using these cell lines, we investigated genetic changes responsible for transition of the normal EVT cells to premalignant and malignant phenotype. Hyperinvasiveness in both cases resulted from a downregulation of tissue inhibitor of metalloprotease (TIMP)-1 and plasminogen activator inhibitor (PAI)-1 genes. In contrast to normal EVT cells, both cell types failed to upregulate these genes in response to TGF-beta. Loss of TGF-beta response in malignant EVT cells was explained by the loss of expression of Smad3 gene. Differential mRNA display of normal and premalignant EVT cells identified up- and down-regulation of numerous known or novel genes in premalignant EVT cells, with potential oncogenic and (or) tumor-suppressor functions, e.g., loss of fibronectin and insulin-like growth factor binding protein (IGFBP-5). Premalignant EVT cells also lost IGF receptor type 2 (IGFR-II). IGFBP-5 was shown to be a negative regulator of IGF-1-induced proliferation of premalignant EVT cells, so that loss of IGFBP-5 as well as IGFR-II permitted their unrestricted proliferation in an IGF-I-rich microenvironment of the fetal-maternal interface. The present model may be a good prototype for identifying genetic changes underlying epithelial tumor progression.     

Stress related changes of cell surface hydrophilicity in Bacillus subtilis

The changes of cell surface hydrophilicity in Bacillus subtilis were analyzed in response to oxygen-limitation, heat shock, salt stress, pH-shock, phosphate- and carbon-limitation. Although cell surface hydrophilicity varied during growth phases, an increase of surface hydrophilicity was observed under several of these stress conditions. An observed drop in intracellular GTP and/or ATP may be an element of the signal transduction pathway leading to an increase in surface hydrophilicity in response to environmental stresses. Attachment of cells to soil particles under salt stress conditions is strongly influenced by the degS/degU two-component system, which thereby provides a mechanism for the bacteria to escape from the hostile environment.     

A modified procedure for replica plating of mammalian cells allowing selection of clones based on gene expression.

The polyester cloth replica-plating technique for selection of mammalian cell clones was modified by growing cells in colonies on a flexible polytetrafluoroethylene membrane and then transferring them completely to polyester cloth (27-microns mesh), from which a replica was made by allowing cells to transfer to a cloth of smaller pore size (17-microns mesh). Using this technique, two phenotype selection methods are demonstrated here: in situ hybridization for detection of a specific mRNA and a photographic film assay for detection of luciferase expression. Cells were transfected with pSV2AL-A delta 5' in which firefly luciferase cDNA is under the control of the simian virus 40 promoter. The luciferase assay was adapted for colonies on polyester cloth; cells were permeabilized with digitonin to allow access of ATP and luciferin to the cell without disruption of colonies. Clones selected for expression or nonexpression of luciferase by the photographic film assay were positive or negative for expression after isolation from the cloth replica and subsequent growth under conventional culture conditions. The replica-plating procedure described here should be generally applicable to most mammalian cell types. The ability to produce replicas of colonies, combined with in situ hybridization or assays that can be adapted to in situ detection, provides phenotype selection for clones based on gene expression independent of growth characteristics.     

Kinetic analysis of a split cell population following reinoculation of an Ehrlich ascites tumor

When Ehrlich ascites cells from old tumors are inoculated into fresh hosts, their cell cycle parameters show several unusual features when studied by means of four independent techniques. These experiments measure: Labeling Index (LI); Mitotic Index (MI); Percent Labeled Mitosis (PLM); Percent of population mitotic and initially G0, (Mu/P). The minimum modifications of the standard cell-cycle model that allow simultaneous simulation of the four sets of data were then determined. These modifications are: (1) sudden shortening of transit time for the S state immediately following reinoculation; (2) a more gradual change in the transit time for M; (3) an orderly reentry of G0 cells into the beginning of the cycle; (4) a shorter first cycle for the reentering G0 cells along with a smaller coefficient of variation for the first cycle of the G0 cells. These changes allow a simultaneous simulation of the four experiments with a single parameter set. This was not achievable without the modifications. Hence, resolution of a cell population into two components allows one to account for additional kinetic features.     

Response surface optimization of effective medium constituents for the production of alkaline protease from a newly isolated strain of Pseudomonas aeruginosa

Optimization of the fermentation medium for maximum alkaline protease production was carried out with a new strain of Pseudomonas aeruginosa (B-2). Replacing the protein source/inducer (albumin in place of casein) brought about significant increase in yield after 48 hr of inoculation. Three most effective medium constituents identified by initial screening method of Plackett-Burman were albumin, (NH4)2SO4 and glucose. Central Composite Design (CCD) and Response Surface Methodology (RSM) were used in the design of the experiment and in the analysis of the results. Optimum levels of the effective medium constituents were albumin (6.586%); (NH4)2SO4, 0.164%; and glucose, 6.72%. The alkaline protease production increased from 533460 to 793492 Ul(-1).     

Acquired immune response to defined Plasmodium vivax antigens in individuals residing in northern India

In this investigation, we evaluated the naturally acquired immune response to Plasmodium vivax stage-specific antigens in individuals of different age groups belonging to malaria endemic areas of northern India.Four synthetic peptides containing both B- and T-cell epitopes from P. vivax circumsporozoite protein, merozoite surface protein-1, apical membrane antigen-1 and gametocyte surface antigen-1 were used to determine both humoral and cellular immune responses.Immunity, in terms of antibody response and T-cell proliferation against these stage-specific peptides, has been observed in the study subjects. The results demonstrated age-dependent antibody response in this population. Forty two patients were diagnosed with P. vivax. There was a significant association (P = 0.013) between number of antibody responders and recognition of stage-specific epitopes by antibodies. The antibody response to B-epitopes of P. vivax CSP, MSP1, AMA1 and GAM1 was associated with age; adults responded more frequently to these antigens than did younger children. In this population, 66% (201/304) cases showed seropositivity to all peptides and 13% (41/304) showed negative response. Peripheral blood mononuclear cells of more than 75% of individuals proliferated in response to stimulation by all four epitopes.In conclusion, the results demonstrated immunogenicity of the epitopes to P. vivax in population of this endemic zone.     

Regulatory mechanisms across networks of the circadian clock and senescence pathways

Journal of Plant Biochemistry and Biotechnology - The circadian clock serves the fitness of higher plants by controlling various aspects of plant growth and development ranging from photosynthesis...     

Depth-dependent analysis of membranes using benzophenone-based phospholipids

Any attempt to probe the membrane hydrophobic core with chemical reagents necessitates the use of reactive intermediates like carbenes and nitrenes, which can insert into C-H bonds. Several photoactivable reagents based on carbenes and nitrenes have been reported. However, the high reactivity of these reagents, often leads to very low insertion yields. We report here a high degree of cross-linking (35-40%) achieved with three benzophenone-based phospholipids and analyze the carbon functionalization data using a multiple Gaussian function. These phospholipids are so designed so as to permit depth-dependent labeling in membranes. Single bilayer vesicles were prepared from these phospholipids and dimyristoylphosphatidylcholine. The cross-linked product was isolated and characterized by mass spectroscopy. The results obtained indicated that the cross-linked product was dominated by dimeric product formed by intermolecular cross-linking. The Gaussian analysis used here provides insight into the relative depths of the probes inside the membrane.     

Algorithms for sequence analysis via mutagenesis

MOTIVATION: Despite many successes of conventional DNA sequencing methods, some DNAs remain difficult or impossible to sequence. Unsequenceable regions occur in the genomes of many biologically important organisms, including the human genome. Such regions range in length from tens to millions of bases, and may contain valuable information such as the sequences of important genes. The authors have recently developed a technique that renders a wide range of problematic DNAs amenable to sequencing. The technique is known as sequence analysis via mutagenesis (SAM). This paper presents a number of algorithms for analysing and interpreting data generated by this technique. RESULTS: The essential idea of SAM is to infer the target sequence using the sequences of mutants derived from the target. We describe three algorithms used in this process. The first algorithm predicts the number of mutants that will be required to infer the target sequence with a desired level of accuracy. The second algorithm infers the target sequence itself, using the mutant sequences. The third algorithm assigns quality values to each inferred base. The algorithms are illustrated using mutant sequences generated in the laboratory.