Identification and characterization of an enzyme involved in the elongation of n-6 and n-3 polyunsaturated fatty acids

The enzymes that are involved in the elongation of fatty acids differ in terms of the substrates on which they act. To date, the enzymes specifically involved in the biosynthesis of polyunsaturated fatty acids have not yet been identified. In an attempt to identify a gene(s) encoding an enzyme(s) specific for the elongation of gamma-linolenic acid (GLA) (18:3n-6), a cDNA expression library was made from the fungus Mortierella alpina. The cDNA library constructed in a yeast expression vector was screened by measuring the expressed elongase activity [conversion of GLA to dihomo-GLA (20:3n-6)] from an individual yeast clone. In this report, we demonstrate the isolation of a cDNA (GLELO) whose encoded protein (GLELOp) was involved in the conversion of GLA to dihomo-GLA in an efficient manner (60% conversion). This cDNA contains a 957-nucleotide ORF that encodes a protein of 318 amino acids. Substrate specificity analysis revealed that this fungal enzyme acted also on stearidonic acid (18:4n-3). This report identifies and characterizes an elongase subunit that acts specifically on the two Delta6-desaturation products, 18:3n-6 and 18:4n-3. When this GLELO cDNA was coexpressed with M. alpina Delta5-desaturase cDNA in yeast, it resulted in the conversion of GLA to arachidonic acid (20:4n-6) as well as the conversion of stearidonic acid to eicosopentaenoic acid (20:5n-3). Thus, this GLELO gene may play an critical role in the bio-production of both n-6 and n-3 polyunsaturated fatty acids.     

Histamine synthesis is required for granule maturation in murine mast cells

Mast cells are the major sources of histamine, which is released in response to immunological stimulations. The synthesis of histamine is catalyzed by histidine decarboxylase (HDC). Previous studies have shown that Hdc^?/? mast cells exhibit aberrant granule morphology with severely decreased granule content. Here, we investigated whether the histamine synthesized in mast cells regulates the granule maturation of murine mast cells. Several genes, including those encoding granule proteases and enzymes involved in heparin biosynthesis, were downregulated in Hdc^?/? peritoneal mast cells. Impaired granule maturation was also found in Hdc^?/? BM‐derived cultured mast cells when they were cocultured with fibroblasts in the presence of c‐kit ligand. Exogenous application of histamine and several H₄ receptor agonists restored the granule maturation of Hdc^?/? cultured mast cells. However, the maturation of granules was largely normal in Hrh4^?/? peritoneal mast cells. Depletion of cellular histamine with tetrabenazine, an inhibitor of vesicular monoamine transporter‐2, did not affect granule maturation. In vivo experiments with mast cell deficient Kit^W/Kit^W‐v mice indicated that the expression of the Hdc gene in mast cells is required for granule maturation. These results suggest that histamine promotes granule maturation in mast cells and acts as an proinflammatory mediator.     

RHAMM, a receptor for hyaluronan-mediated motility, compensates for CD44 in inflamed CD44-knockout mice: a different interpretation of redundancy

We report here that joint inflammation in collagen-induced arthritis is more aggravated in CD44-knockout mice than in WT mice, and we provide evidence for molecular redundancy as a causal factor. Furthermore, we show that under the inflammatory cascade, RHAMM (receptor for hyaluronan-mediated motility), a hyaluronan receptor distinct from CD44, compensates for the loss of CD44 in binding hyaluronic acid, supporting cell migration, up-regulating genes involved with inflammation (as assessed by microarrays containing 13,000 cDNA clones), and exacerbating collagen-induced arthritis. Interestingly, we further found that the compensation for loss of the CD44 gene does not occur because of enhanced expression of the redundant gene (RHAMM), but rather because the loss of CD44 allows increased accumulation of the hyaluronic acid substrate, with which both CD44 and RHAMM engage, thus enabling augmented signaling through RHAMM. This model enlightens several aspects of molecular redundancy, which is widely discussed in many scientific circles, but the processes are still ill defined.     

The good and bad effects of cysteine S-nitrosylation and tyrosine nitration upon insulin exocytosis: a balancing act

As understanding of the mechanisms driving and regulating insulin secretion from pancreatic beta cells grows, there is increasing and compelling evidence that nitric oxide (?NO) and other closely-related reactive nitrogen species (RNS) play important roles in this exocytic process. ?NO and associated RNS, in particular peroxynitrite, possess the capability to effect signals across both intracellular and extracellular compartments in rapid fashion, affording extraordinary signaling potential. It is well established that nitric oxide signals through activation of guanylate cyclase-mediated production of cyclic GMP. The intricate intracellular redox environment, however, lends credence to the possibility that ?NO and peroxynitrite could interact with a wider variety of biological targets, with two leading mechanisms involving 1) Snitrosylation of cysteine, and 2) nitration of tyrosine residues comprised within a variety of proteins. Efforts aimed at delineating the specific roles of ?NO and peroxynitrite in regulated insulin secretion indicate that a highly-complex and nuanced system exists, with evidence that ?NO and peroxynitrite can contribute in both positive and negative regulatory ways in beta cells. Furthermore, the ultimate biochemical outcome within beta cells, whether to compensate and recover from a given stress, or not, is likely a summation of contributory signals and redox status. Such seeming regulatory dichotomy provides ample opportunity for these mechanisms to serve both physiological and pathophysiologic roles in onset and progression of diabetes. This review focuses attention upon recent accumulating evidence pointing to roles for nitric oxide induced post-translational modifications in the normal regulation as well as the dysfunction of beta cell insulin exocytosis.     

Doc2b is a key effector of insulin secretion and skeletal muscle insulin sensitivity

Exocytosis of intracellular vesicles, such as insulin granules, is carried out by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins. An additional regulatory protein, Doc2b (double C2 domain), has recently been implicated in exocytosis from clonal β-cells and 3T3-L1 adipocytes. Here, we investigated the role of Doc2b in insulin secretion, insulin sensitivity, and the maintenance of whole-body glucose homeostasis. Doc2b heterozygous (Doc2b(+/-)) and homozygous (Doc2b(-/-)) knockout mice exhibited significant whole-body glucose intolerance and peripheral insulin resistance, compared with wild-type littermates. Correspondingly, Doc2b(+/-) and Doc2b(-/-) mice exhibited decreased responsiveness of pancreatic islets to glucose in vivo, with significant attenuation of both phases of insulin secretion ex vivo. Peripheral insulin resistance correlated with ablated insulin-stimulated glucose uptake and GLUT4 vesicle translocation in skeletal muscle from Doc2b-deficient mice, which was coupled to impairments in Munc18c-syntaxin 4 dissociation and in SNARE complex assembly. Hence, Doc2b is a key positive regulator of Munc18c-syntaxin 4-mediated insulin secretion as well as of insulin responsiveness in skeletal muscle, and thus a key effector for glucose homeostasis in vivo. Doc2b's actions in glucose homeostasis may be related to its ability to bind Munc18c and/or directly promote fusion of insulin granules and GLUT4 vesicles in a stimulus-dependent manner.     

Radiographic fallopian tube recanalization: absorbed ovarian radiation dose

Absorbed radiation dose to the ovaries during radiographic fallopian tube recanalization was estimated in 29 patients with use of thermoluminescent dosimeters placed in the vaginal fornix. With an average fluoroscopic time of 8.5 minutes +/- 5.5 and an average of 14 +/- 5 105-mm spot radiographs obtained, the average absorbed dose to the ovaries was 8.5 mGy +/- 5.6 (0.85 rad +/- 0.56). Technical guidelines for keeping patient radiation exposure to a minimum during this new interventional procedure are suggested.     

Selective salpingography and fallopian tube recanalization

Obstruction of the uterine (proximal) end of the fallopian tube is noted on up to 20% of hysterosalpingograms and has a variety of underlying causes. Definitive diagnosis and treatment in the past have required laparoscopy or laparotomy with tubal resection. Selective salpingography and fallopian tube recanalization with fluoroscopically guided catheters has emerged as an improved method both for diagnosis and treatment in these patients. Technical success rates for overcoming the obstruction and visualizing distal tubal anatomy range from 76% to 95%. Pregnancy rates after the procedure vary depending on the patient populations studied; however, early results indicate a greater than 50% intrauterine pregnancy rate by 1 year. The rate of ectopic pregnancy is approximately 10% and that of early tubal reocclusion is less than 30%. Selective salpingography and fallopian tube recanalization is recommended as the first intervention in patients with obstruction of the proximal fallopian tube.     

The effect of pre-enrichment on recovery of Streptococcus agalactiae, Staphylococcus aureus and mycoplasma from bovine milk

The study was conducted to determine whether pre-enrichment would increase sensitivity of detecting Streptococcus (Str.) agalactiae, Staphylococcus (S.) aureus, and mycoplasma in bovine milk. Two procedures were followed, one involving direct inoculation of milk on bovine blood agar, and the other involving preenrichment in broth followed by inoculation on agar. Logistic regression was used to predict the probability of isolation as a function of culture procedure and two additional covariates, the California Mastitis Test (CMT) score of the milk and the type of sample (indicating sample storage temperature and herd mastitis status). A total of 13778 milk samples was cultured for each of the three bacteria. By using results of both direct inoculation and pre-enrichment, the probability of isolation compared to use of direct inoculation only and adjusted for effects of other variables was increased 3.6-fold for Str. agalactiae, 1.6-fold for S. aureus and 1.7-fold for mycoplasma. The probability of isolation for all three bacteria increased as the CMT score increased. For Str. agalactiae, there was a statistical interaction predicting that enrichment improved the odds of isolation more from milk with high CMT scores than from milk with low scores. Results indicate that pre-enrichment can substantially increase the sensitivity of bacteriological screening of dairy cows for mastitis caused by Str. agalactiae, S. aureus, and mycoplasma.