Effect of sex hormones on human T cell activation by concanavalin A

The effect of sex hormones on concanavalin A (Con A)-activated human T cells was studied. We show that neither 17 beta-estradiol (E2) nor progesterone, in concentrations of up to 10(-6) M, alters the proliferative response of peripheral-blood mononuclear cells (PBMC) of healthy postmenopausal women. Furthermore, the hormones had no effect on the composition of T cell populations and on the expression of activation markers. We extended our study to a unique T cell population that is characterized by the ability to form rosettes with human erythrocytes, following Con A activation (designated autorosette-forming cells; ARFC) and known to manifest suppressive activity. Indeed, the in vitro addition of E2 (neither progesterone nor testosterone) to Con A-stimulated PBMC brought an about 2- to 4-fold increase in the frequency of ARFC. Tamoxifen, an antiestrogen drug, reduced the frequency of estrogen-stimulated ARFC to the original low level. Furthermore, the inhibitory effect of growth medium from ARFC cultures originally stimulated with Con A + E2 was found to be higher than that of ARFC cultures originally stimulated with Con A alone.     

Effect of polyethylene glycol-electrolyte lavage solution on intestinal microflora.

The bacterial flora of colonic aspirates taken after oral administration of a polyethylene glycol-electrolyte lavage solution to patients undergoing colonoscopy was analyzed by standard bacteriological methods and DNA hybridization techniques. The data were compared with those for normal feces collected from the same patients the day before treatment. The results indicate that the bacterial composition of feces differs from individual to individual but is not altered by the oral intake of a polyethylene glycol-electrolyte lavage solution. This pilot study demonstrates that routine colonoscopy aspirates taken after standard colonoscopy preparation accurately reflect the microflora of feces and therefore may be used in large-scale epidemiological studies.     

Shape determinants of McLeod acanthocytes

Summary We have sought to elucidate the spiculated shape of McLeod erythrocytes. Red cells from a normal donor and from a McLeod patient were incubated in phosphate-buffered saline containing 0, 0.05, or 0.1mm chlorpromazine at 0°C for 5 min. then glutaraldehyde-fixed, and examined by scanning electron microscopy. The normal red cells were biconcave disks in which chlorpromazine induced inward (negative) curvature: deep cupping (stomatocytosis) and multiple invaginations. The McLeod cells were mostly spiculated. Chlorpromazine at lower concentration converted them into biconcave disks and, at higher concentration, into stomatocytes. These results support the hypothesis that the spiculation of McLeod cells is the result of an imbalance of surface area between the two lipid leaflets of the membrane; that is, a bilayer couple effect.We determined the numerical density of intramembrane particles (IMP) in replicas of both fracture faces of red cells subjected to freeze fracture and rotary shadowing. These values were as follows (expressed per m² of membrane ±sd): the normal protoplasmic fracture face had 2200±306 and the McLeod had 2300±250. The normal exoplasmic fracture face had 388±75 and the McLeod had 330±59. We conclude that there is no evidence for derangement of band 3, the principal protein in theIMP, in McLeod erythrocytes.     

Selective binding of the TATA box-binding protein to the TATA box-containing promoter: analysis of structural and energetic factors.

We report the results of an energy-based exploration of the components of selective recognition of the TATA box-binding protein (TBP) to a TATA box sequence that includes 1) the interaction between the hydrophobic Leu, Pro, and Phe residues of TBP with the TA, AT, AA, TT, and CG steps, by ab initio quantum mechanical calculations; and 2) the free energy penalty, calculated from molecular dynamics/potential of mean force simulations, for the conformational transition from A-DNA and B-DNA into the TA-DNA form of DNA observed in a complex with TBP. The GTAT, GATT, GAAT, and GTTT tetramers were explored. The results show that 1) the discrimination of TA, AT, AA, TT, or CG steps by TBP cannot rest on their interaction with the inserting Phe side chains; 2) the steric clash between the bulky and hydrophobic Pro and Leu residues and the protruding -NH2 group of guanine is responsible for the observed selectivity against any Gua-containing basepair; 3) the Pro and Leu residues cannot selectively discriminate among TA, AT, AA, or TT steps; and 4) the calculated energy required to achieve the TA-DNA conformation of DNA that is observed in the complex with TBP appears to be a key determinant for the observed selectivity against the AT, AA, and TT steps. The simulations also indicate that only the TA step can form a very efficient interbase hydrogen bond network in the TA-DNA conformation. Such an energetically stabilizing network is not achievable in the AA and TT steps. While it is viable in the AT step, structural constraints render the hydrogen bonding network energetically ineffective there.     

TIT for TAT: the properties of inosine and adenosine in TATA box DNA

The sequence dependent conformation, flexibility and hydration properties of DNA molecules constitute selectivity determinants in the formation of protein-DNA complexes. TATA boxes in which AT basepairs (bp) have been substituted by IC bp (TITI box) allow for probing these selectivity determinants for the complexation with the TATA box-binding protein (TBP) with different sequences but identical chemical surfaces. The reference promoter Adenovirus 2 Major Late Promoter (mlp) is formed by the apposition of two sequences with very different dynamic properties: an alternating TATA sequence and an A-tract. For a comparative study, we carried out molecular dynamics simulations of two DNA oligomers, one containing the mlp sequence (2 ns), and the other an analog where AT basepairs were substituted by IC basepairs (1 ns). The simulations, carried out with explicit solvent and counterinons, yield straight purine tracts, the A-tract being stiffer than the I-tract, an alternating structure for the YRYR tracts, and hydration patterns that differ between the purine tracts and the alternating sequence tracts. A detailed analysis of the proposed interactions responsible for the stiffness of the purine tracts indicates that the stacking between the bases bears the strongest correlation to stiffness. The hydration properties of the minor groove in the two oligomers are distinctly different. Such differences are likely to be responsible for the stronger binding of TBP to mlp over the inosine-substituted variant. The calculations were made possible by the development, described here, of a new set of forcefield parameters for inosine that complement the published CHARMM all-hydrogen nucleic acid parametrization.     

Angiogenesis defects and mesenchymal apoptosis in mice lacking SMAD5

The transforming growth factor-beta (TGF-beta) signals are mediated by a family of at least nine SMAD proteins, of which SMAD5 is thought to relay signals of the bone morphogenetic protein (BMP) pathway. To investigate the role of SMAD5 during vertebrate development and tumorigenesis, we disrupted the Smad5 gene by homologous recombination. We showed that Smad5 was expressed predominantly in mesenchyme and somites during embryogenesis, and in many tissues of the adult. Mice homozygous for the mutation died between days 10.5 and 11.5 of gestation due to defects in angiogenesis. The mutant yolk sacs lacked normal vasculature and had irregularly distributed blood cells, although they contained hematopoietic precursors capable of erythroid differentiation. Smad5 mutant embryos had enlarged blood vessels surrounded by decreased numbers of vascular smooth muscle cells, suffered massive apoptosis of mesenchymal cells, and were unable to direct angiogenesis in vitro. These data suggest that SMAD5 may regulate endothelium-mesenchyme interactions during angiogenesis and that it is essential for mesenchymal survival.     

Roles of cyclin D1 and related genes in growth inhibition, senescence and apoptosis

It is now apparent that apoptosis is closely linked to the control of cell cycle progression. During the G1 to S progression, cyclin D1, p53, and the cyclin dependent kinase inhibitors p21^WAF1 and p27^kip1 can play roles in induction of apoptosis. During the G2 and M phases, premature activation of Cdk1 can cause cells to enter mitotic catastrophe, which results in apoptosis. In this review we focus on factors acting during G1 and S, particularly cyclin D1, and their effects on cell growth, senescence and apoptosis. We emphasize that cyclin D1 can have diverse effects on cells depending on its level of expression, the specific cell type, the cell context and other factors. Possible mechanisms by which cyclin D1 exerts these diverse effects, via cyclin dependent kinase-dependent and -independent pathways, are discussed.     

Indirect utilization of the phytosiderophore mugineic acid as an iron source to rhizosphere fluorescent Pseudomonas

The phytosiderophore mugineic acid (MA) was studied as a source of iron for rhizosphere fluorescent pseudomonads. ⁵⁵Fe supplied as Fe-MA was taken up by Pseudomonas putida WCS358, B10 and St3 grown under iron deficient conditions. The uptake decreased when the bacteria were grown in the presence of iron. However, no differences in uptake were observed when a siderophore deficient mutant was tested. Since ligand exchange between pseudobactin and MA was shown to occur rapidly with a half-life of 2 h, MA mediated iron uptake probably proceeds through this indirect mechanism. The ecological implications of these findings are discussed.     

Control of the rootknot nematode meloidogyne javanica by Bacillus cereus

Exposure of Meloidogyne javanica second‐stage juveniles to the bacterium Bacillus cereus in soil inhibited the penetration of the juvenile nematodes into tomato roots. Culture filtrate of the bacterium grown on nutrient broth and tryptic soy broth revealed nematocidal activity on M. javanica juveniles and eggs. Loss of the nematocidal activity of the media by lowering pH, boiling or dialysis raised the possibility that the active ingredient in the culture filtrate was ammonia, released during the breakdown process of peptides in the media by bacterial activity. Free ammonia (NH₃) concentrations in the nutrient broth and tryptic soy broth culture filtrates measured after 48 h were 140 and 190 µg ml^?1 respectively. Exposure of second‐stage juveniles to 9.3 µg ml^?1 ammonia for 40 h in vitro was lethal to 95% of the nematode population. In a nitrate medium, nitrite accumulated up to 250 µg ml^?1 during the growth of the bacterium, and its culture filtrate revealed nematocidal activity. The nematocidal activity of the bacterium increased when the bacterium was applied with various proteinaceous supplements to soil. Soil treated with the bacteria and peptone showed an earlier nematocidal activity than either the bacteria or peptone applied alone, and also had a higher level of ammonia than the individual treatments. However, the level of ammonia was lower than the lethal level for second‐stage juveniles recorded in vitro. The nematocidal activity exhibited by the bacterium‐proteinaceous amendment combination is not fully understood; the ammonia released during protein degradation by the bacterium may contribute significantly to the recorded nematocidal activity.     

20alpha-hydroxysteroid dehydrogenase: a T lymphocyte-associated enzyme.

20alpha-Hydroxysteroid dehydrogenase (20alpha-SDH), an enzyme which reduces progesterone to 20alpha-dihydroprogesterone, was found to be associated with T lymphocytes. 20alphaSDH activity was present in spleen cells bearing theta antigen, spleen cells nonadherent to nylon wool (T lymphocyte-enriched population), and in thymocytes. Almost no enzymatic activity was found in bone marrow cells from normal mice and in spleen cells from neonatally thymectomized or athymic nude mice. T cell mitogens (PHA and Con A), but not the B cell mitogen LPS, induced high levels of enzymatic activity 48 hr after addition to spleen cell cultures. The level of 20alphaSDH activity in lymphocytes was age dependent. At the age of 4 weeks 20alphaSDH activity in thymocytes, spleen cells, and lymph node lymphocytes was 3 to 5 times higher than at 8 and 16 weeks. Progesterone (5.0 X 10(-7) M) was found to inhibit thymocyte proliferation after exposure to mitogens, but not 20alpha-dihydroprogesterone (10(-6) M). 20alpha SDH may protect the embryonic thymocytes against high concentrations of progesterone.