Cloning and localization of two multigene receptor families in goldfish olfactory epithelium

Goldfish reproduction is coordinated by pheromones that are released by ovulating females and detected by males. Two highly potent pheromones, a dihydroxyprogesterone and a prostaglandin, previously have been identified, and their effects on goldfish behavior have been studied in depth. We have cloned goldfish olfactory epithelium cDNAs belonging to two multigene G-protein coupled receptor families as a step toward elucidating the molecular basis of pheromone recognition. One gene family (GFA) consists of homologs of putative odorant receptors (approximately 320 residues) found in the olfactory epithelium of other fish and mammals. The other family (GFB) consists of homologs of putative pheromone receptors found in the vomeronasal organ (VNO) of mammals and also in the nose of pufferfish. GFB receptors (approximately 840 residues) are akin to the V2R family of VNO receptors, which possess a large extracellular N-terminal domain and are homologs of calcium-sensing and metabotropic glutamate receptors. In situ hybridization showed that the two families of goldfish receptors are differentially expressed in the olfactory epithelium. GFB mRNA is abundant in rather compact cells whose nuclei are near the apical surface. In contrast, GFA mRNA is found in elongated cells whose nuclei are positioned deeper in the epithelium. Our findings support the hypothesis that the separate olfactory organ and VNO of terrestrial vertebrates arose in evolution by the segregation of distinct classes of neurons that were differentially positioned in the olfactory epithelium of a precursor aquatic vertebrate.     

Identification of a hereditary pancreatitis mutation in four West Virginia families

Hereditary pancreatitis (HP) is the second most common cause of chronic childhood pancreatitis in the United States. Mutations in the cationic trypsinogen gene on chromosome 7 are known to cause HP. We identified four families in West Virginia with symptoms consistent with HP. To determine whether members of these families had defects in the trypsinogen gene, we tested for linkage between the HP gene and simple tandem repeat markers on chromosome 7q and screened for a specific mutation in the cationic trypsinogen gene. Two-point linkage analysis indicated that the disease gene is closely linked to three 7q markers (D7S661, D7S2511, and D7S1805). Restriction fragment length polymorphism analysis showed that all clinically affected members and nonpenetrant carriers from the four families carried a G to A mutation in the third exon of the trypsinogen gene. These findings indicate that this mutation is the cause of HP in the families in our study. The observation that most individuals who carry the mutation have symptoms of HP is consistent with the high but incomplete penetrance of the trait. The presence of a single mutation and a common linked haplotype indicates that the defective allele arose in an ancestor common to all four families.     

Identification of high-affinity binding sites for the insulin sensitizer rosiglitazone (BRL-49653) in rodent and human adipocytes using a radioiodinated ligand for peroxisomal proliferator-activated receptor gamma

A radioiodinated ligand, [125I]SB-236636 [(S)-(-)3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]3-[125I]i odo phenyl]2-ethoxy propanoic acid], which is specific for the gamma isoform of the peroxisomal proliferator activated receptor (PPARgamma), was developed. [125I]SB-236636 binds with high affinity to full-length human recombinant PPARgamma1 and to a GST (glutathione S-transferase) fusion protein containing the ligand binding domain of human PPARgamma1 (KD = 70 nM). Using this ligand, we characterized binding sites in adipose-derived cells from rat, mouse and humans. In competition experiments, rosiglitazone (BRL-49653), a potent antihyperglycemic agent, binds with high affinity to sites in intact adipocytes (IC50 = 12, 4 and 9 nM for rat, 3T3-L1 and human adipocytes, respectively). Binding affinities (IC50) of other thiazolidinediones for the ligand binding domain of PPARgamma1 were comparable with those determined in adipocytes and reflected the rank order of potencies of these agents as stimulants of glucose transport in 3T3-L1 adipocytes and antihyperglycemic agents in vivo: rosiglitazone > pioglitazone > troglitazone. Competition of [125I]SB-236636 binding was stereoselective in that the IC50 value of SB-219994, the (S)-enantiomer of an alpha-trifluoroethoxy propanoic acid insulin sensitizer, was 770-fold lower than that of SB-219993 [(R)-enantiomer] at recombinant human PPARgamma1. The higher binding affinity of SB-219994 also was evident in intact adipocytes and reflected its 100-fold greater potency as an antidiabetic agent. The results strongly suggest that the high-affinity binding site for [125I]SB-236636 in intact adipocytes is PPARgamma and that the pharmacology of insulin-sensitizer binding in rodent and human adipocytes is very similar and, moreover, predictive of antihyperglycemic activity in vivo.     

Relationships of PPARgamma and PPARgamma2 mRNA levels to obesity, diabetes and hyperinsulinaemia in rhesus monkeys

OBJECTIVE: To examine the expression of peroxisome proliferator-activated receptor gamma (PPARgamma) together with CCAAT/enhancer binding protein alpha (C/EBPalpha), lipoprotein lipase (LPL) and glucose transporter (GLUT4) mRNA in adipose tissue of rhesus monkeys in relation to obesity. DESIGN: Cloning of the PPARgamma1 and gamma2 cDNAs and analysis of PPARgamma, C/EBPalpha, LPL and GLUT4 mRNA levels in the adipose tissue of lean and obese monkeys. SUBJECTS: 28 rhesus monkeys (Macaca mulatta) with a wide range of body weights (9.2-22.6 kg) and with or without type 2 diabetes. MEASUREMENTS: Sequence of PPARgamma1 and gamma2. Tissue distribution of PPARgamma1 and gamma2. The mRNA levels of PPARgamma, C/EBPalpha, LPL and GLUT4 in adipose tissue. The ratio of PPARgamma2 mRNA to total PPARgamma mRNA. RESULTS: The monkey PPARgamma2 protein showed 99% identity with the human protein. PPARgamma1 mRNA was shown to be expressed in various tissues and most abundantly in adipose tissue. PPARgamma2 existed mainly in adipose tissue. A significant correlation between the ratio of PPARgamma2 mRNA to total PPARgamma mRNA and obesity was observed, whereas total PPARgamma mRNA levels showed no significant relationships to obesity. There was also a significant relationship between the ratio of PPARgamma2 mRNA to total PPARgamma mRNA and fasting plasma insulin concentration. The mRNA levels of C/EBPalpha, LPL and GLUT4 were highly correlated to that of total PPARgamma mRNA. They were also significantly correlated to the mRNA levels of PPARgamma1 and PPARgamma2. CONCLUSIONS: The ratio of PPARgamma2 mRNA to total PPARgamma mRNA is related to obesity in the rhesus monkey and mRNA expression of PPARgamma1, PPARgamma2, C/EBPalpha, LPL and GLUT4 appear to be coordinated in vivo.     

Oxalogenesis in parenteral nutrition solution components

Oxalate has been implicated in the etiology of nephrocalcinosis in premature infants as well as in the formation of insoluble precipitates in total parenteral nutrition (TPN) intravenous tubing. Oxidation of ascorbate to oxalate, especially in the presence of catalysts such as copper and iron, has been implicated in formation of these precipitates. The purpose of this project was to measure oxalate formation in certain TPN components separately and in combination. Neonatal TPN solution components in combination were infused at 5 mL/h under simulated clinical conditions used in a neonatal intensive care unit. Aliquots were assayed at intervals for oxalate by capillary electrophoresis. Oxalate is present in one TPN mixture at concentrations up to 8 ppm. The addition of ascorbate to an aqueous solution of trace metals may promote oxalogenesis.     

Salivary duct carcinoma. Clinicopathologic and immunohistochemical review of 26 cases

The diterpenoic compound steviol (ent-kaur-16-en-13-ol-19-oic acid) is the aglycone of sweet glycosides accumulated in Stevia rebaudiana Bertoni. This compound is the hydroxylated form of ent-kaurenoic acid (ent-kaur-16-en-19-oic acid; ent-KA). The hydroxylation of ent-KA to form steviol requiring NADPH and molecular oxygen was detected in stroma prepared from S. rebaudiana Bertoni. The enzyme was purified from leaf extract to apparent homogeneity with a molecular mass of 39 kDa. Taken together with the value of 160 kDa estimated for native enzyme, this suggested that the hydroxylating enzyme is a homotetramer. The N-terminal sequence was determined through 20 residues. The pH optimum was 7.5-7.8. Apparent Km values were 11.1 microM for ent-KA and 20.6 microM for NADPH. Its visible absorption spectrum suggested that the enzyme was flavoprotein. The stoichiometric relationship between the formation of steviol and the utilization of ent-KA and cofactors confirmed the equation ent-KA + NADPH + H(+) + O2-->steviol + NADPH(+) + H2O.     

Mitochondrial DNA variable number tandem repeats (VNTRs): utility and problems in molecular ecology

Analysis of mitochondrial (mt)DNA size polymorphism in the form of variable number tandem repeats (mtVNTRs) has become an increasingly popular methodology for addressing questions in molecular ecology. When detected by PCR, mtVNTR analysis can provide a sensitive, rapid, and cost-effective measure of genetic variability that may be exploited in studies of population differentiation and biogeography. Despite the emergence of this approach, there has been little critical evaluation of its success or utility as a practical tool. In this review, we identify problematic methodological, theoretical and interpretive factors that can influence the utility of mtVNTR analysis. The reliability of the procedure is considered in terms of both detection of alleles and scoring of intra-individual allele frequencies. While many of the potential technical problems of the technique do not raise serious practical concerns, this rapid and sensitive methodology is seriously compromised by the difficulty of reliably assessing allele frequencies, of assaying only germline tissue, and in our ignorance of the mechanisms generating mtVNTR diversity. Thus, although there is a considerable potential for mtVNTR pilot studies to assess genetic diversity, the utility of the technique to resolve broader questions in molecular ecology should be treated cautiously until such a time as the system is better understood.     

EPR evidence for the involvement of free radicals in the iron-catalysed decomposition of qinghaosu (artemisinin) and some derivatives; antimalarial action of some polycyclic endoperoxides

The role of pharmacies that specialize in the treatment of specific chronic diseases in the alternate-site health care setting is discussed. The optimal use of medications through disease management programs can improve patient outcomes and lower overall health care costs. The increase in disease management programs has spawned the growth of disease-specific pharmacies in the home care and other alternate-site health care settings. These pharmacies usually operate from a single location or are regionalized operations that deliver pharmaceutical products to patients throughout the United States. The pharmacies employ clinicians who specialize in a particular disease. These clinicians conduct comprehensive patient education programs, drug-use review, and compliance monitoring. Disease management pharmacies focus on chronic, expensive diseases; costs related to inventory, equipment, and storage can be very high. Many disease management pharmacies are involved in preferred-distribution or closed-distribution arrangements with pharmaceutical manufacturers. Pharmacists involved in disease management programs routinely send compliance information about their patients to pharmaceutical companies, managed care organizations, or prescribing physicians. Disease management pharmacies act as advocates for patients with particular chronic diseases. Various foundations and patient advocacy and research groups have created their own disease management pharmacies. Disease management has also reached the community pharmacy practice setting. Pharmacies specializing in the treatment of specific chronic diseases in the alternate-site health care setting can improve health care and promote efficient use of health care dollars.     

Does disparate occurrence of autoregulatory programmed frameshifting in decoding the release factor 2 gene reflect an ancient origin with loss in independent lineages?

In Escherichia coli an autoregulatory mechanism of programmed ribosomal frameshifting governs the level of polypeptide chain release factor 2. From an analysis of 20 sequences of genes encoding release factor 2, we infer that this frameshift mechanism was present in a common ancestor of a large group of bacteria and has subsequently been lost in three independent lineages.