Relation between plasma hormone profiles,symptoms, and response to oestrogen treatment in women approaching the menopause.

Out of a consecutive series of 300 patients seen at a menopause clinic, 82 complained of symptoms generally associated with the climacteric, although they were still menstruating. Vasomotor disturbances were absent in 42 of these patients (group 1) and present in 40 (group 2). Headaches, insomnia, and dyspareunia were the most common complaints among the women with vasomotor symptoms, whereas loss of libido and depression predominated in those without. Conjugated equine oestrogens (Premarin) 1.25 mg daily given for three weeks out of four relieved nearly all symptoms in group 2, but in group 1 the response was disappointing. The mean plasma oestradiol concentration in women with vasomotor symptoms was significantly lower than that observed during days 1-10 of the menstrual cycle, but plasma testosterone values were not significantly different from those observed in younger women. Plasma follicle-stimulating hormone (FSH) and luteinising hormone (LH) concentrations were similar to those seen after the menopause. Concentrations of these hormones in the women without vasomotor symptoms were similar to those in the younger, regularly menstruating women. After six months of oestrogen treatment patients in group 2 had a 2.1-fold increase in mean plasma oestradiol concentration, and plasma FSH and LH concentrations were reduced to 39% and 66% of their pretreatment values respectively; in group 1, however, no such pronounced changes occurred. High concentrations of FSH were present in patients with oestrogen-responsive symptoms, 15 U/1 being the diagnostic cut-off point. This measurement in the presence of characteristic symptoms therefore constitutes the best method of selecting patients for oestrogen-replacement therapy.     

Protein expression profiles in the epidermis of cyclooxygenase-2 transgenic mice by 2-dimensional gel electrophoresis and mass spectrometry

Exposure of murine skin to tumor-promoting agents such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) causes up-regulation of cyclooxygenase-2 (COX-2) and increased prostaglandin (PG) synthesis. Pharmacological inhibition of COX-2 significantly reduces skin tumor development. However, we previously demonstrated that K14.COX-2 transgenic (TG) mice that overexpressed COX-2 in the epidermis were unexpectedly resistant to tumor development under the classical 7,12-dimethylbenz[a]anthracene-TPA protocol. In the present study, we employed a proteomic approach of 2-dimensional gel electrophoresis (2-DE) and mass spectrometry to profile differentially expressed proteins in the epidermis of K14.COX-2 TG and wild-type control mice. Various 2-DE approaches were used to identify the maximum number of differentially expressed proteins: 20 for untreated samples, 3 for acetone-treated samples, and 22 for TPA-treated samples. These proteins include 14-3-3 sigma, numerous actin fragments, actin filament related proteins cofilin-1 and destrin, galectin-3, galectin-7, prohibitin, S100A6, S100A9, and many others. The differential expression of galectin-3, galectin-7, S100A9 was validated by Western blot analysis and/or immunohistochemical analysis. The current data suggest that some of the differentially expressed proteins might increase apoptosis and cell cycle arrest, which, in turn, may provide insight into the role of COX-2 in skin tumorigenesis.     

Apoptosis, cell proliferation and vitellogenesis during the folliculogenesis and follicular growth in teleost fish

Aiming to better understand folliculogenesis, this study evaluated cell death and proliferation of ovarian cells, besides cathepsin-D expression in Prochilodus argenteus captured in two sites of the São Francisco River downstream from the Três Marias Dam, Brazil. In the site immediately following the Dam (S1), low levels of dissolved oxygen were registered in the rainy period. The water temperature was higher in the São Francisco River immediately after the confluence with the Abaeté River (S2), regardless of the period. In S1, the ovaries showed smaller oocytes, high caspase-3 enzymatic activity and apoptosis, lower cells in proliferation and GSI, as well as a lesser quantity of cathepsin-D when compared to females captured from S2. Regarding relative frequency of ovarian structures, in the dry period, only oogonia and perinucleolar oocytes were found in fish ovaries from both sites. On the other hand, in the rainy period, the relative frequency of oogonia and perinucleolar oocytes decreased and the vitellogenic oocytes increased in S2. Postovulatory follicles were observed only in S2, whereas atretic follicles occurred at a higher frequency in S1. Our results showed that apoptosis, cell proliferation and cathepsina-D evaluation can be used as biomarkers of environmental impact.     

Structures of Receptor Complexes of a North American H7N2 Influenza Hemagglutinin with a Loop Deletion in the Receptor Binding Site

Human infections with subtype H7 avian influenza viruses have been reported as early as 1979. In 1996, a genetically stable 24-nucleotide deletion emerged in North American H7 influenza virus hemagglutinins, resulting in an eight amino acid deletion in the receptor-binding site. The continuous circulation of these viruses in live bird markets, as well as its documented ability to infect humans, raises the question of how these viruses achieve structural stability and functionality. Here we report a detailed molecular analysis of the receptor binding site of the North American lineage subtype H7N2 virus A/New York/107/2003 (NY107), including complexes with an avian receptor analog (3′-sialyl-N-acetyllactosamine, 3′SLN) and two human receptor analogs (6′-sialyl-N-acetyllactosamine, 6′SLN; sialyllacto-N-tetraose b, LSTb). Structural results suggest a novel mechanism by which residues Arg220 and Arg229 (H3 numbering) are used to compensate for the deletion of the 220-loop and form interactions with the receptor analogs. Glycan microarray results reveal that NY107 maintains an avian-type (α2-3) receptor binding profile, with only moderate binding to human-type (α2-6) receptor. Thus despite its dramatically altered receptor binding site, this HA maintains functionality and confirms a need for continued influenza virus surveillance of avian and other animal reservoirs to define their zoonotic potential.     

Co-culture of rabbit 2-cell embryos with rabbit oviduct epithelial cells and other somatic cells

Rabbit 2-cell embryos were co-cultured in Basel Synthetic Medium II + 10% fetal bovine serum with one of the following: primary cultures of rabbit oviduct epithelial cells (ROEC), a rabbit kidney epithelioid cell line (RK13), a rabbit epidermal epithelioid cell line (Sf1), or a rabbit skin fibroblast-like cell line (RAB9). Embryos cultured in medium alone served as controls. After 4 d of culture at 39 degrees C in 5% CO2 in air, 77-93% of the rabbit embryos which were co-cultured with somatic cells had reached the blastocyst stage, and 60-76% were hatching through their zonae pellucidae. These percentages, however, were not significantly different (P greater than .05) from those of embryos in medium alone, of which 90% had reached the blastocyst stage and 83% were hatching. Mean intrazonal embryo diameters also did not differ significantly among treatments (239-302 microns). Bovine 1-8-cell embryos were also co-cultured with ROEC. This stimulated 60% of these embryos to develop beyond the so-called "16-cell block" in vitro, whereas 0% of the embryos cultured in medium alone developed past this block. Evaluation of the ROEC cultures by light microscopy, immunocytochemistry, and gel electrophoretic analysis of conditioned medium, together with the positive results with bovine embryos, indicate that the ROEC culture partially simulates oviductal conditions in vivo. Therefore, our results suggest that oviduct epithelial cells may play a less pivotal role in regulating early development in the rabbit than in the cow.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a plasmalemma redox system in sugarcane

A plasmalemma-bound NADH-dependent redox system has been identified in protoplasts isolated from cell suspensions of sugarcane. This system oxidized NADH as well as NADPH, increased O₂ consumption 3-fold, and increased the pH of the external medium while the cytoplasmic pH was decreased. In the presence of NADH, ferricyanide was rapidly reduced and the external medium was acidified. The uptake rates of K⁺, 3-O-methylglucose, leucine, and arginine were all decreased in the presence of NADH.     

The effect of seasonality upon the development of lotic biofilms and microbial biostabilisation

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Metabolically engineered Escherichia coli for efficient production of glycosylated natural products

Significant achievements in polyketide gene expression have made Escherichia coli one of the most promising hosts for the heterologous production of pharmacologically important polyketides. However, attempts to produce glycosylated polyketides, by the expression of heterologous sugar pathways, have been hampered until now by the low levels of glycosylated compounds produced by the recombinant hosts. By carrying out metabolic engineering of three endogenous pathways that lead to the synthesis of TDP sugars in E. coli, we have greatly improved the intracellular levels of the common deoxysugar intermediate TDP‐4‐keto‐6‐deoxyglucose resulting in increased production of the heterologous sugars TDP‐L‐mycarose and TDP‐d ‐desosamine, both components of medically important polyketides. Bioconversion experiments carried out by feeding 6‐deoxyerythronolide B (6‐dEB) or 3‐α‐mycarosylerythronolide B (MEB) demonstrated that the genetically modified E. coli B strain was able to produce 60‐ and 25‐fold more erythromycin D (EryD) than the original strain K207‐3, respectively. Moreover, the additional knockout of the multidrug efflux pump AcrAB further improved the ability of the engineered strain to produce these glycosylated compounds. These results open the possibility of using E. coli as a generic host for the industrial scale production of glycosylated polyketides, and to combine the polyketide and deoxysugar combinatorial approaches with suitable glycosyltransferases to yield massive libraries of novel compounds with variations in both the aglycone and the tailoring sugars.     

Molecular analysis of the cyclic AMP-dependent protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene in patients with Carney complex and primary pigmented nodular adrenocortical disease (PPNAD) reveals novel mutations and clues for pathophysiology: augmented PKA signaling is associated with adrenal tumorigenesis in PPNAD

We studied 11 new kindreds with primary pigmented nodular adrenocortical disease (PPNAD) or Carney complex (CNC) and found that 82% of the kindreds had PRKAR1A gene defects (including seven novel inactivating mutations), most of which led to nonsense mRNA and, thus, were not expressed in patients' cells. However, a previously undescribed base substitution in intron 6 (exon 6 IVS +1G-->T) led to exon 6 skipping and an expressed shorter PRKAR1A protein. The mutant protein was present in patients' leukocytes and tumors, and in vitro studies indicated that the mutant PRKAR1A activated cAMP-dependent protein kinase A (PKA) signaling at the nuclear level. This is the first demonstration of an inactivating PRKAR1A mutation being expressed at the protein level and leading to stimulation of the PKA pathway in CNC patients. Along with the lack of allelic loss at the PRKAR1A locus in most of the tumors from this kindred, these data suggest that alteration of PRKAR1A function (not only its complete loss) is sufficient for augmenting PKA activity leading to tumorigenesis in tissues affected by CNC.     

fRFLP and fAFLP: medium-throughput genotyping by fluorescently post-labeling restriction digestion

Genome-scale studies of population structure and high-resolution mapping of genetically complex traits both require techniques for accurately and efficiently genotyping large numbers of polymorphic sites in multiple individuals. Many high-throughput genotyping technologies require the purchase of expensive equipment or consumables and are therefore out of reach of some individual research laboratories. Conversely, less expensive technologies are often labor intensive so that the effort involved in typing large numbers of samples or polymorphic sites is prohibitive. Here we present a method of fluorescently post-labeling restriction digestion using standard dye-terminator sequencing chemistry so that RFLP and AFLP products can be visualized on an automated sequencer This labeling method is efficient, inexpensive, easily multiplexed, and requires no unusual equipment or reagents, thus striking a balance between cost and throughput that should be appropriate for many research groups and core facilities.