Pituitary luteinizing hormone microheterogeneity in the afternoon of proestrus in rats: some new insights

OBJECTIVE : The aim of the present report was to determine the possible modifications in rat pituitary LH isoforms induced by the spontaneous increase in GnRH at the time of the preovulatory gonadotropin surge. DESIGN: The changes in the quantitative pattern and relative proportions of pituitary LH isoforms in rats on the afternoon of proestrus [INT-P(PM)] were evaluated by comparison with other stages of the estrous cycle (diestrus-1, diestrus-2 and estrus) and ovariectomized (7 and 30 days earlier) animals killed in the morning and in the afternoon of the corresponding day. METHODS: The chromatofocusing technique (pH gradient 11.00-7.00) was used to analyze the different molecular species of intrapituitary LH. RESULTS: Pituitary LH from diestrus-1 animals, considered as a baseline pattern in the cycling rat, eluted as 11 isoforms distributed in pH 9.62-8.82, with greater percentages in pH 9.50-9.01. Except for INT-P(PM) pituitaries, there were no major differences in the pattern of LH heterogeneity in the pituitaries of rats from various stages of the cycle. In contrast, significant changes in the charge distribution and relative abundance of LH isoforms were found in the pituitaries from INT-P(PM) rats. INT-P(PM) pituitaries resolved in 16 LH isoforms with a significant shift to less alkaline pIs (pH 9.62-8.11), the more abundant being focused within pH 9.00-8.51. Conversely, a shift to more basic isoforms resulted after ovariectomy, leading to the accumulation of less mature isoforms in the gonadotrope. CONCLUSIONS: Presumably, the use of animals on INT-P(PM), at the time of the preovulatory LH surge, made it possible to discriminate such changes in LH isoform distribution. That GnRH, released in association with the rising phase of the LH surge, induces these changes in pituitary LH polymorphism appears to be the most likely possibility. In a previous study we demonstrated that GnRH stimulated galactose incorporation into LH in vitro. In the case of pituitaries from INT-P(PM) rats, the shift toward less alkaline isoforms could potentially result from sialylation of increased terminal galactose.     

Accumulation of fumonisins B1 and B2 in freshly harvested Brazilian commercial maize at three locations during two nonconsecutive seasons

Fifty-six Brazilian commercial maize cultivars were examined for FB1 and FB2 accumulation after two non-consecutive growing seasons. During the 94/95 growing season 35 cultivars were planted at three locations in the state of S?o Paulo, Brazil. All samples (total of 105) were contaminated (0.10 micro/g-6.58 microg/g FB1 and 0.04 microg/g-2.15 microg/g FB2). During the 97/98 growing season, 8 of the cultivars used during 94/95 and 21 others were replanted at the same locations. All 87 samples were contaminated (1.15 microg/g-43.80 microg/g FB1 and 0.08 microg/g-11.65 microg/g FB2). One cultivar accumulated significantly less fumonisins in all locations during both growing seasons, indicating that some degree of selection may be possible even in climates that favor F. moniliforme (verticillioides) infection of maize. The presence of water surplus in soil from kernel maturity to harvest correlated with concentrations of FB1 in the grain for the 8 cultivars planted during both seasons at three locations. Observed trends indicated that water excesses and deficits from silking to harvest increased fumonisin levels. The difference in the incidence of FB1, FB2, and FB1 + FB2 was significant between growing seasons, planting locations and between cultivars. Neither the level of hybridization, nor the type of endosperm, nor the length of the vegetative cycle showed any effect on the FB1 contamination.     

Secondary cell wall deposition causes radial growth of fibre cells in the maturation zone of elongating tall fescue leaf blades

A gradient of development consisting of successive zones of cell division, cell elongation and cell maturation occurs along the longitudinal axis of elongating leaf blades of tall fescue (Festuca arundinacea Schreb.), a C3 grass. An increase in specific leaf weight (SLW; dry weight per unit leaf area) in the maturation region has been hypothesized to result from deposition of secondary cell walls in structural tissues. Our objective was to measure the transverse cell wall area (CWA) associated with the increase in SLW, which occurs following the cessation of leaf blade elongation at about 25 mm distal to the ligule. Digital image analysis of transverse sections at 5, 15, 45, 75 and 105 mm distal to the ligule was used to determine cell number, cell area and protoplast area of structural tissues, namely fibre bundles, mestome sheaths and xylem vessel elements, along the developmental gradient. Cell diameter, protoplast diameter and area, and cell wall thickness and area of fibre bundle cells were calculated from these data. CWA of structural tissues increased in sections up to 75 mm distal to the ligule, confirming the role of cell wall deposition in the increase in SLW (r2 = 0.924; P < or = 0.01). However, protoplast diameter of fibre cells did not decrease significantly as CWA increased, although mean thickness of fibre cell walls increased by 95 % between 15 and 105 mm distal to the ligule. Therefore, secondary cell wall deposition in fibre bundles of tall fescue leaf blades resulted in continued radial expansion of fibre cells rather than in a decrease in protoplast diameter.     

Instability of a premutation-sized CGG repeat in FMR1 YAC transgenic mice

Fragile X syndrome results from the massive expansion of a CGG repeat in the 5' untranslated region of the gene FMR1. Data suggest that the hyperexpansion properties of FMR1 CGG repeats may depend on flanking cis-acting elements. We have therefore used homologous recombination in yeast to introduce an in situ CGG expansion corresponding to a premutation-sized allele into a human YAC carrying the FMR1 locus. Several transgenic lines were generated that carried repeats of varying lengths and amounts of flanking sequence. Length-dependent instability in the form of small expansions and contractions was observed in both male and female transmissions over five generations. No parent-of-origin effect or somatic instability was observed. Alterations in tract length were found to occur exclusively in the 3' uninterrupted CGG tract. Large expansion events indicative of a transition from a premutation to a full mutation were not observed. Overall, our results indicate both similarities and differences between the behavior of a premutation-sized repeat in mouse and that in human.     

In vitro self-assembled HCV core virus-like particles induce a strong antibody immune response in sheep.

The in vitro self-assembly properties of the entire hepatitis C virus core protein (HCcAg) obtained from Pichia pastoris cells and the induction of specific antibody immune response were studied. HCcAg was purified as a low-molecular-weight species by electroelution under denaturing conditions for confirmation of its self-assembly properties. After renaturalization, electron microscopy showed that HCcAg assembled into spherical particles of 30 nm. HCcAg also showed homogeneity and was specifically recognized by serum from a chronic HCV carrier patient. The data indicated that in vitro assembly of HCcAg, into virus-like particles resembling HCV nucleocapsid particles at a mature stage, is an intrinsic quality of this protein. Finally, HCcAg generated a strong antibody immune response in sheep, suggesting its usefulness for stimulating the host immune response against HCV.     

Propionic acid side chain hydrogen bonding in the malaria pigment beta-hematin

Malaria pigment, or beta-hematin, the insoluble heme detoxification product resulting from the intraerythrocitic digestion of hemoglobin by young malaria trophozoites has been structurally characterized by X-ray powder diffraction and shown to contain chains of propionic acid linked dimers. Although there is considerable spectroscopic evidence for a monodentate propionate-iron interaction in this crystalline material, the spectroscopic characterization of the propionic acid dimer is limited. Herein we demonstrate the presence of the propionic acid dimer unit by H/D isotope substitution in carboxylic acid dimer. In the Raman spectrum of the deuterium substituted compound there is a circa 12 cm(-1) shift, H: 1629 cm(-1) vs. D: 1617 cm(-1) in the symmetric ring breathing mode for the propionic acid dimer. On the other hand, the IR active asymmetric stretch has a very small shift, <3 cm(-1), upon deuteration. These, and other vibrational data, are consistent with the presence of a planar carboxylic acid dimer in the structure of beta-hematin.     

Crystal structures of two potent nonamidine inhibitors bound to factor Xa

There has been intense interest in the development of factor Xa inhibitors for the treatment of thrombotic diseases. Our laboratory has developed a series of novel non-amidine inhibitors of factor Xa. This paper presents two crystal structures of compounds from this series bound to factor Xa. The first structure is derived from the complex formed between factor Xa and compound 1. Compound 1 was the first non-amidine factor Xa inhibitor from our lab that had measurable potency in an in vitro assay of anticoagulant activity. The second compound, 2, has a molar affinity for factor Xa (K(iapp)) of 7 pM and good bioavailability. The two inhibitors bind in an L-shaped conformation with a chloroaromatic ring buried deeply in the S1 pocket. The opposite end of these compounds contains a basic substituent that extends into the S4 binding site. A chlorinated phenyl ring bridges the substituents in the S1 and S4 pockets via amide linkers. The overall conformation is similar to the previously published structures for amidine-based inhibitors complexed with factor Xa. However, there are significant differences in the interactions between the inhibitor and the protein at the atomic level. Most notably, there is no group that forms a salt bridge with the carboxylic acid at the base of the S1 pocket (Asp189). Each inhibitor forms only one well-defined hydrogen bond to the protein. There are no direct charge-charge interactions. The results indicate that electrostatic interactions play a secondary role in the binding of these potent inhibitors.     

Stabilization of iron-sulfur cluster F(X) by intra-subunit interactions unraveled by suppressor and second site-directed mutations in PsaB of Photosystem I

Intra-subunit interactions in the environment of the iron-sulfur cluster F(X) in Photosystem I (PS I) of Synechocystis sp. PCC 6803 were studied by site-directed and second site suppressor mutations. In subunit PsaB, the cysteine ligand (C565) of F(X) and a conserved aspartate (D566) adjacent to C565 were modified. The resulting mutants D566E, C556S/D566E, C556H/D566E and C565H/D566E did not assemble PS I in the thylakoids of the cyanobacterium. Yet, this is the first report of cells of the second site-suppressor mutant (D566E/L416P) and of second site-directed mutant (C565S/D566E) in PsaB that could grow autotrophically in light and were found to assemble a stable functional PS I containing all three iron-sulfur centers, F(X) and F(A/B). The newly resolved structure of PS I (PDB 1JB0) was used to interpret the functional interactions among the amino acid residues. It is suggested that the stability of F(X) is supported by a salt bridge formed between D566, which is adjacent to the cysteine ligand C565 of the iron-sulfur cluster located on loop hi, and R703 located at the start of loop jk. Hydrogen bond between R703 and D571 at the start of loop hi further stabilizes the arginine. Lengthening of the side by 1.2 A chain in mutation D566E caused destabilization of F(X). The extended side-chain was compensated for by the Fe-O, which is 0.3 A shorter than the Fe-S bond resulting in stabilization of the F(X) in the double mutations C565S/D566E. The suppressor mutation D566E/L416P allowed greater freedom for the salt bridge E566-R703, thus relieving the pressure introduced by the D566E replacement and enabling the formation of F(X). F(X) and R703 are therefore stabilized through short- and long-range interactions of the inter-helical loops between h-i, j-k and f-g, respectively.