Adipose tissue intracrinology: potential importance of local androgen/estrogen metabolism in the regulation of adiposity.

The present article summarizes some of the studies available on steroid hormone conversion through the specific expression of steroidogenic enzymes in adipose tissue (adipose tissue intracrinology) and discusses the potential impact of local adipose tissue steroid metabolism on the regulation of adipocyte function and other metabolic parameters. Several studies have demonstrated significant steroid hormone uptake and conversion by adipose tissues from various body sites and in various cell fractions. Activities and/or mRNAs of aromatase, 3beta-hydroxysteroid dehydrogenase (HSD), 3alpha-HSD, 11beta-HSD, 17beta-HSD, 7alpha-hydroxylase, 17alpha-hydroxylase, 5alpha-reductase and UDP-glucuronosyltransferase 2B15 have been detected in adipose tissue or adipose cells. These studies have demonstrated potentially important roles for these enzymes in obesity, central fat accumulation, and the metabolic syndrome. Future studies on adipose tissue intracrinology will contribute further to our understanding of steroid action in adipocytes.     

An efficient synthesis of GDP-hexanolamine,a key tool for the purification of fucosyltransferases

A new efficient synthesis of GDP-hexanolamine from hexanolamine is reported with an overall yield of 71%. The pyrophosphate formation, the key step of this preparation, was achieved through a sequential GMP activation procedure based on polytrifluoroacetylation of GMP followed by activation of the phosphate group by 1-methylimidazole.     

Altered storage of proteases in mast cells from mice lacking heparin: a possible role for heparin in carboxypeptidase A processing

Heparin-deficient mice, generated by gene targeting of N-deacetylase/N-sulfotransferase-2 (NDST-2), display severe mast cell defects, including an absence of stored mast cell proteases. However, the mechanism behind these observations is not clear. Here we show that NDST-2+/+ bone marrow-derived mast cells cultured in the presence of IL-3 synthesise, in addition to highly sulphated chondroitin sulphate (CS), small amounts of equally highly sulphated heparin-like polysaccharide. The corresponding NDST-2-/- cells produced highly sulphated CS only. Carboxypeptidase A (CPA) activity was detected in NDST+/+ cells but was almost absent in the NDST-/- cells, whereas tryptase (mouse mast cell protease 6; mMCP-6) activity and antigen was detected in both cell types. Antigen for the chymase mMCP-5 was detected in NDST-2+/+ cells but not in the heparin-deficient cells. Northern blot analysis revealed mRNA expression of CPA, mMCP-5 and mMCP-6 in both wild-type and NDST-2-/- cells. A approximately 36 kDa CPA band, corresponding to proteolytically processed active CPA, as well as a approximately 50 kDa pro-CPA band was present in NDST-2+/+ cells. The NDST-2-/- mast cells contained similar levels of pro-CPA as the wild-type mast cells, but the approximately 36 kDa band was totally absent. This indicates that the processing of pro-CPA to its active form may require the presence of heparin and provides the first insight into a mechanism by which the absence of heparin may cause disturbed secretory granule organisation in mast cells.     

Orderly pattern of development of the autoantibody response in (New Zealand White x BXSB)F1 lupus mice: characterization of target antigens and antigen spreading by two-dimensional gel electrophoresis and mass spectrometry

Immunoblots of a two-dimensional PAGE-separated HL-60 cell proteomic map and mass spectrometry were combined to characterize proteins targeted by autoantibodies produced by male (New Zealand White x BXSB)F(1) (WB) mice that develop lupus and anti-phospholipid syndrome. Analysis of sera sequentially obtained from seven individual mice at different ages showed that six proteins, vimentin, heat shock protein 60, UV excision-repair protein RAD23, alpha-enolase, heterogeneous nuclear ribonucleoprotein L, and nucleophosmin, were the targets of the B cell autoimmune response, and that autoantibodies to them were synthesized sequentially in an orderly pattern that recurred in all the male WB mice analyzed: anti-vimentin first and anti-nucleophosmin last, with anti-RAD23 and anti-heat shock protein 60, then anti-alpha-enolase and anti-heterogeneous nuclear ribonucleoprotein L Abs occuring concomitantly. Anti-vimentin reactivity always appeared before anti-cardiolipin and anti-DNA Abs, suggesting that vimentin is the immunogen initiating the autoimmune process. The pattern of HL-60 proteins recognized by female WB sera differed from that of male sera, indicating that the Y chromosome-linked autoimmune acceleration gene is not an accelerator but a strong modifier of the autoimmune response. Thus, 1) combining two-dimensional PAGE and mass spectrometry constitutes a powerful tool to identify the set of Ags bound by autoantibodies present in a single serum and the whole autoantibody pattern of an autoimmune disease; 2) the diversification of the autoimmune response in male WB mice occurs in a predetermined pattern consistent with Ag spreading, and thus provides a useful model to further our understanding of the development of the autoantibody response in lupus.     

Risk factors for postpartum ovarian cysts and their spontaneous recovery or persistence in lactating dairy cows

Cystic ovarian disease is a major cause of reproductive failure and economic loss for the dairy industry. Many cysts that develop during the early postpartum period regress spontaneously. However, it is difficult to decide at what point it would be more cost effective to treat ovarian cysts than to wait for spontaneous recovery. The objective of this study was to analyze risk factors for the development of the ovarian cystic condition during early and late postpartum, and for its persistence or recovery during the pre-service period in lactating dairy cows. Using multiple logistic regression, we analyzed data derived from 873 lactating dairy cows from a single herd. An ovarian cyst was diagnosed if it was possible to observe a single follicular structure with an antrum diameter > or = 25 mm in the absence of a corpus luteum in three sonograms performed at 7-day intervals. The cystic condition was denoted as early if the cyst was diagnosed 43-49 days postpartum, and late if detected 57-63-day postpartum. Spontaneous cyst regression before 60-day postpartum was regarded as early cystic recovery. For the early cystic group, there were no significant effects of lactation number, body condition score on prepartum Day 60, at parturition or on postpartum Day 30, or of body condition loss from parturition to 30-day postpartum. Cows calving in summer were 2.6 times more likely to develop ovarian cysts than those giving birth in winter. The risk of having a cyst was 1.9 times higher in cows with an abnormal puerperium. A 1-kg increase in milk yield raised the risk of cysts by a factor of 1.05. A 1-unit increase in body condition score (scale from 1 to 5) from prepartum Day 60 to parturition increased the risk of cyst development 8.4 times. Milk production and lactation number were negatively correlated with spontaneous early cyst recovery. A 1-kg decrease in milk production increased the probability of cyst recovery by a factor of 1.06, and a 1-unit drop in lactation number was associated with a 1.4-fold increased probability of cyst recovery. For the late cystic group, there were no significant effects of abnormal puerperium and body score data, except for a prepartum change in body score. Calving season (Odds ratio: 2.3), lactation number (Odds ratio: 1.36), increased milk production (Odds ratio: 1.05) and increased body condition score during the prepartum period (Odds ratio: 4.3) were all related to an increased risk of ovarian cysts. The probability of having a late cyst was 36.6 times greater in cows with early cysts. These findings suggest that it would be profitable to treat multiparous cows having cysts very early in the postpartum period, while treatment of primiparous cows should be delayed, at least until the end of the pre-service period, to provide the opportunity for spontaneous recovery.     

"Light" epithelial cells of swine and bovine oviducts

The ultrastructure of oviduct epithelium of clinically healthy cows and 15 sows was investigated using scanning and transmission electron microscopy. In all parts of the oviduct, ciliated and non-ciliated epithelial cells are present, but their number varies in both the investigated animals in different regions of the oviduct, depending on the phase of the estrous cycle. In addition to ciliated cells with numerous cilia on their luminal surface, so-called pale ciliary cells were found in all parts of the oviduct of cows and sows. The cytoplasm of these cells is electron-lucent, their luminal surface carries few cilia and short microvilli. The apical cytoplasm contains species specific secretory granules, which means that these cells have features characteristic of both secretory and ciliated cells. It is suggested that the pale ciliated and non-ciliated secretory cells are functional stages of the same tubar epithelium cell, and that the transformation between these two cell types is regulated by functional requirements of the organ in different phases of the estrous cycle.     

Mapping of quantitative trait loci controlling broomrape (Orobanche crenata Forsk.) resistance in faba bean (Vicia faba L.).

Orobanche crenata Forsk. is a root parasite that produces devastating effects on many crop legumes and has become a limiting factor for faba bean production in the Mediterranean region. The efficacy of available control methods is minimal and breeding for broomrape resistance remains the most promising method of control. Resistance seems to be scarce and complex in nature, being a quantitative characteristic difficult to manage in breeding programmes. To identify and map the QTLs (quantitative trait loci) controlling the trait, 196 F2 plants derived from the cross between a susceptible and a resistant parent were analysed using isozymes, RAPD, seed protein genes, and microsatellites. F2-derived F3 lines were studied for broomrape resistance under field conditions. Of the 130 marker loci segregating in the F2 population, 121 could be mapped into 16 linkage groups. Simple interval mapping (SIM) and composite interval mapping (CIM) were performed using QTL Cartographer. Composite interval mapping using the maximum number of markers as cofactors was clearly the most efficient way to locate putative QTLs. Three QTLs for broomrape resistance were detected. One of the three QTLs explained more than 35% of the phenotypic variance, whereas the others accounted for 11.2 and 25.5%, respectively. This result suggests that broomrape resistance in faba bean can be considered a polygenic trait with major effects of a few single genes.     

Prostaglandin profiles in nervous tissue and blood vessels of the brain of various animals

The endogenous formation of prostaglandin (PG) D2, E2, F2 alpha, and 6-keto-PGF1 alpha was determined in homogenates of mouse, rat, and rabbit brain, and of rat cerebral blood vessels, using gas chromatography mass spectrometry. In all species tested, 6-keto-PGF1 alpha could be identified in the brain homogenates, but was a minor component in relation to other PGs. In contrast 6-keto-PGF1 alpha was the most abundant PG in the blood vessels, being present in about 40-fold higher levels than in the brain tissue. PGD2 was the most abundant PG in rat and mouse brains, but was below detection limits in the analyzed blood vessels. These studies indicating differential metabolism of PG endoperoxides in nervous and vascular tissue, provide a biochemical basis for further studies on the role of the PGs in brain circulation and neuronal activity.     

Effect of p-chlorophenylalanine on the reproduction of the rat (author's transl)]

The effect of p-chlorophenylalanie (p-CPA) --300 mg/kg-- on reproduction has been studied in the female rat. Groups of animals were injected with a dose of 300 mg/kg of p-CPA 48 hours before proper copulation conditions at different moments along the ovarian cycle. Presence of spermatozoa in the vaginal frotis was negative in treated rats; in the control groups however, positivity was found in variable proportions according to the phase of the ovarian cycle: 30 ad 90% in diestrus and proestrus respectively. Treated animals showed continuous diestrus phases and diffuse luteinitation of the ovary. The results may indicate that a decrease of cerebral 5-HT, caused by p-CPA, lessens the reproductive behaviour of the female rat through mechanisms depending probably on the liberation of gonadrotrophins.     

Carbocyclic analogues of dTTP and UTP: properties in polymerase enzyme-catalyzed reactions

Racemic carbocyclic analogues of dTTP [(+/-)-C-dTTP] and its ribo counterpart, 5-methyl-UTP [(+/-)-C-m5UTP] were synthesized and examined, in comparison with dTTP and UTP (and m5UTP), as potential substrates of E. coli DNA and RNA polymerases, respectively. Unexpectedly, only a very low (terminal) incorporation of C-dTMP into DNAs of different structure was observed, C-dTTP did not serve as a substrate for chain elongation by the Klenow DNA polymerase. Inhibition of DNA replication was, however, observed in the presence of (+/-)-C-dTTP. The UTP analogue, (+/-)-C-m5UTP proved neither a substrate nor an inhibitor of the RNA polymerase enzyme.