Assignment of the alpha and beta chains of human propionyl-CoA carboxylase to genetic complementation groups.

Propionicacidemia is a metabolic disorder resulting from a deficiency of propionyl-CoA carboxylase activity. The enzyme is composed of two polypeptides: a 72,000-dalton alpha chain which contains the biotin ligand and a 56,000-dalton beta chain. It has been suggested that the two major complementation groups in this disorder, pccA and pccBC (with subgroups pccB and pccC), correspond to the genes encoding these two chains. To correlate gene product with complementation groups, 15 mutant and four normal human fibroblast strains were analyzed by [35S]methionine and [3H]biotin labeling. Immunoprecipitation and gel electrophoresis of the polypeptides revealed that alpha chains are synthesized by mutants of pccBC and both subgroups but not in four out of five pccA mutants. On the other hand, beta chains were detected only in pccB mutants. We suggest that pccA encodes the alpha chain of PCC while pccBC encodes the beta chain, and furthermore predict that the beta chain is unstable in the absence of the alpha chain.     

Terpenoid compounds from different parts ofAchillea collina Becker inflorescences

Extracts of small amounts of inflorescence material (3 lingular flowers, 3 tubular flowers, 1 receptacle with involucral bracts), obtained withn-hexane, were analysed by gas-liquid chromatography using a capillary column. The presence of terpenoids was demonstrated and differences in composition between extracts from different inflorescence parts were found.     

Changes in internal pH associated with initiation of motility and acrosome reaction of sea urchin sperm

The changes in the intracellular pH (pH_i) of sea urchin sperm associated with motility initiation and acrosome reaction were investigated using uptake of two different probes; 9-aminoacridine and methylamine, as a qualitative index. Sperm suspended in Na⁺-free sea water were immotile and able to concentrate these amines 20-fold or greater indicating that pH_i is more acidic than the external medium (pH_o = 7.7). This uptake ratio was essentially constant over a wide range of probe and sperm concentrations. Discharge of the pH gradient with specific ionophores (nigericin, monensin, and tetrachlorosalicylanilide) or nonspecifically using low concentration of detergents (Triton X-100 and lysolecithin) all resulted in the release of the probes indicating they are indeed sensing the pH gradient across the sperm membrane. Addition of Na⁺ to sperm suspended in Na⁺-free sea water resulted in activation of motility with concomitant efflux of the probes indicating the alkalinization of pH_i by 0.4–0.5 pH units. That this pH_i change is the causal trigger of motility was suggested by experiments using NH₄Cl and nigericin, which increased the pH_i and resulted in activation of motility in the absence of Na⁺. When sperm were directly diluted into artificial sea water (motility activated), a slow reacidification of pH_i was observed in one species of sea urchin (L. pictus) but not in the other (S. purpuratus). This acidification could be blocked by mitochondrial inhibitors, verapamil, or the removal of external calcium suggesting that the increase in metabolic activity stimulated by the influx of Ca²⁺ is responsible for the reacidification. Induction of acrosome reaction further alkalinized the pH_i by about 0.16 pH units and was also followed by prolonged reacidification which correlated with the observed increase in Ca²⁺ uptake. Either mitochondrial agents or the removal of external Ca²⁺ could also block this pH_i change suggesting a similar mechanism is involved.     

Subcellular localization of gonadotropins and testosterone in the developing fetal rat testis.

Gonadotropins and testosterone were immunocytochemically localized in the fetal rat testes 16-18 days of gestation with the unlabeled antibody-peroxidase anti-peroxidase complex technique. Maximum staining for gonadotropins with antiserum to the beta chain of human chorionic gonadotropin (anti-hCGbeta) occurred at 16 days gestation in the seminferous tubule and 17 days gestation in interstitial (Leydig) cells. Anti hCGbeta sites were on the plasma membranes at the luminal aspects of Sertoli cells at 16 days gestation. In addition, intracellular hCGbeta sites were evident including the nucleus, nucleolus, ribosomes, some vesicles, lysosomes and centrioles. The stain for hCGbeta disappeared rapidly and by 17 days was limited to patches in the cytoplasm and nuclei. In the fetal testes, staining for anti-testosterone binding sites was most intense at 18 days of gestation either in lipid droplets or on nuclei of Leydig and Sertoli cells. Very little testosterone stain was observed before 18 days of gestation. These findings agree with physiologic data that suggest that gonadotropins bind to receptors and stimulate testicular development and the capacity for testosterone production.     

Phosphorylation from inorganic phosphate and ATP synthesis of sarcoplasmic membranes

The incorporation of inorganic phosphate in the fragmented sarcoplasmic membranes induced by the removal of calcium ions bound to high affinity binding sites at the cytoplasmic surface of the membranes gives rise to the formation of two species of phosphoenzyme. The properties of the phosphoproteins formed depend on the absence or the presence of a gradient of calcium ions across the membranes. The phosphoenzymes differ by the affinity of the protein for phosphate, the enthalpy of formation, the kinetics of phosphate incorporation, and by the sensitivity to ionophores and ADP. In the absence of a calcium gradient less than 0.5 nmol phosphoenzyme per mg protein are formed in media containing less than 5 mM phosphate at pH7 and 10 degrees C. Under the same conditions approximately 2 nmol of phosphoenzyme per mg protein are formed with an initial rate of 0.5 nmol mg-1-s-1 when a calcium gradient exists. When the gradient is abolished by the addition of the ionophore X537A, the level of phosphoprotein drops to the same value as observed in the absence of a gradient. On addition of ADP at concentrations increasing from 0.3 to 10 muM continuous ATP formation is activated to its maximum rate, and simultaneously, the level of phosphoprotein declines. These concentrations of ADP scarcely affect phosphoprotein formed in the absence of a gradient, the phosphoryl residue of which is displaced when the concentration of ADP exceeds 10 micrometer without the formation of an equivalent amount of ATP. Minimum mechanisms for the formation of gradient-independent and gradient-dependent phosphoprotein are discussed.     

A virus inhibitor circulating in the blood of chickens,induced byFrancisella tularensis andListeria monocytogenes

Following the intravenous inoculation of chickens with large doses of livingFrancisella tularensis andListeria monocytogenes organisms, a virus inhibitor appeared in their serum. The first traces of the inhibitor were found four hours and the maximum levels eight and 24 hours after inoculation with a bacterial suspension. The administration of heat-inactivated microorganisms did not induced formation of the inhibitor. The dynamics of formation of the inhibitor and its properties resembled those of virus interferon.     

Pharmacological characterization of receptor types mediating the dilator action of anandamide on blood vessels of the rat knee joint

This study investigates the actions of N-(2-hydroxyethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (anandamide) on blood flow of the rat knee joint. Topical bolus administration of anandamide (10-1000 nmol) onto the exposed knee joint capsules produced dose-dependent increases in the knee joint blood flow. Various antagonists were tested on the vasodilator response to 100 nmol anandamide. Capsazepine (N-[2-(4-chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide), an antagonist of the transient receptor potential vanilloid type 1 (TRPV1) receptor, given at 10 and 100 nmol, suppressed the response by a maximum of 71%. A cannabinoid CB(1) receptor antagonist AM281 (10 nmol) and a CB(2) receptor antagonist AM630 (10 nmol) shortened its duration from 15 min to 5 min. O-1918 (1 nmol), an antagonist of the putative endothelial anandamide/abnormal-cannabidiol receptor, on its own or combined with capsazepine and the two cannabinoid receptor antagonists produced 38% and 24% inhibition on the peak vasodilator response to anandamide, respectively. URB597 (1 nmol), an inhibitor of fatty acid amide hydrolase (FAAH) suppressed the response by 40%, and an anandamide transporter inhibitor [N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide] (AM404; 1 nmol) or a cyclo-oxygenase (COX) inhibitor flurbiprofen (20 nmol) abolished the response. These findings suggest the vasodilator action of anandamide in the rat knee joint involved hydrolysis of the compound by FAAH, production of COX-derived eicosanoid(s), activation of TRPV1 receptors, and a small component involved activation of endothelial anandamide/abnormal-cannabidiol receptors; a minor delayed dilator response was mediated by activation of conventional cannabinoid receptors.     

Occurrence of Toxorhynchites guadeloupensis (Dyar Knab) in oviposition trap of Aedes aegypti (L.) (Diptera: Culicidae)

Toxorhynchites guadeloupensis (Dyar Knab), a poorly known mosquito species, was observed preying upon Aedes aegypti (L.) larvae, in an oviposition trap placed for routine dengue entomological surveillance, during 2003-2004 in the urban area of Boa Vista, Roraima, Brazil. This is the first report for Tx. guadeloupensis using Ae. aegypti oviposition traps as breeding places. This finding may have important consequences in the epidemiology and local dengue control since Ae. aegypti density is a basic variable in dengue prediction. Whether predation of Ae aegypti by Tx. guadeloupensis in the Amazon is of significance, is a question to be examined. Also, larval predation may be a cause for underestimation of the actual Ae aegypti numbers. Together these hypotheses need to be better investigated as they are directly related to dengue epidemiology, to the success of any outbreak prediction and surveillance program.