In vivo studies involved monitoring the effect of morphine administration on catecholamine biosynthesis by the brain while in vitro studies involved studying the effect of morphine on the uptake of tritiated tyrosine by synaptosomes and its subsequent incorporation into the catecholamines. The extremely low levels of these endogenous compounds required the use of High Performance Liquid Chromatography with electrochemical detection. Intra-peritoneal injection of morphine at a dosage of 10 mg/kg did not produce appreciable changes in the catecholamine levels but a dosage of 30 mg/kg morphine was found to elevate dihydroxy phenylacetic acid content. At a dosage of 60 mg/kg, dopamine levels were elevated while noradrenaline was depleted. Morphine, at a concentration of 1 x 10(-5)M increases the incorporation of tritiated tyrosine into dopamine and dihydroxy phenylacetic acid in synaptosomal preparations.
Calmodulin, an activator protein in most calcium-dependent processes, was isolated to apparent homogeneity from the femurs of 1-day old chicks using phenyl-Sepharose and high performance liquid chromatography. The purified calmodulin was found to produce a 6-fold increase in the activity of alkaline phosphatase isolated from the same source. A Ca2+ concentration of 10(-5) M was required for the activation. Purification of alkaline phosphatase involved acetone precipitation, DEAE-Sephacel and Sephadex G-200 column chromatography. The enzyme was purified to 540-fold and had a specific activity of 10.75 U/mg protein.
We have analyzed 16 Y-STR loci (DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635 or Y-GATA C4, DYS392, Y-GATA H4, DYS437, DYS438 and DYS448) from the non-recombining region of the human Y-chromosome in 980 male individuals from three main ethnic populations in Malaysia (Malay, Chinese, Indian) using the AmpFlSTR((R)) Y-filertrade mark (Applied Biosystems, Foster City, CA). The observed 17-loci haplotypes and the individual allele frequencies for each locus were estimated, whilst the locus diversity, haplotype diversity and discrimination capacity were calculated in the three ethnic populations. Analysis of molecular variance indicated that 88.7% of the haplotypic variation is found within population and 11.3% is between populations (fixation index F(ST)=0.113, p=0.000). This study has revealed Y-chromosomes with null alleles at several Y-loci, namely DYS458, DYS392, DYS389I, DYS389II, DYS439, DYS448 and Y-GATA H4; and several occurrences of duplications at the highly polymorphic DYS385 loci. Some of these deleted loci were in regions of the Y(q) arm that have been implicated in the occurrence of male infertility.
The metabolism of varying quantities of pregnenolone has been studied in nuclei-free homogenates from Macaca fascicularis testes by using capillary gas chromatography, after derivatization of metabolites as O-methyl oximes/trimethylsilyl ethers. Evidence was obtained indicating that both pathways for testosterone biosynthesis were operating. 5-Androstene-3 beta, 17 beta-diol was formed in especially high quantities. Two 16-androstenes, namely 5,16-androstadien-3 beta-ol and 5 alpha-androst-16-en-3 beta-ol, were also quantitatively important as metabolites. Co-incubation of stored homogenates with relaxin resulted in 80-100% reduction of the formation of all metabolites quantified except for 5 alpha-androst-16-en-3-one, which was stimulated. Freezing the homogenates at -10 degrees C for 3 weeks resulted in marked 4- to 6-fold reduction in the yields of testosterone and of the 5-ene and 4-ene metabolites from pregnenolone.
The metabolism of pregnenolone in subcellular fractions of the testes of the macaque (Macaca fascicularis) has been studied using capillary gas chromatography to characterize and quantify the metabolites, after their conversion into the O-methyloxime and/or trimethylsilyl ether derivatives. The microsomal incubations yielded the greatest quantities of metabolites, with lesser amounts in the mitochondrial fraction. The cytosolic fraction contained no significant quantity of metabolites after incubation, except for 5alpha-androst-16-en-3 beta-ol. This, and other odorous androst-16-enes, found in the microsomal fraction, are of particular interest in the context of animal communication because of their possible pheromonal role. Pregnenolone was converted into androst-5-ene-3 beta,17 beta-diol, androst-4-ene-3,17-dione and testosterone, suggesting that both classical pathways for testosterone synthesis were operating. Testosterone was further converted into 5 alpha-reduced androstanediols, especially in the microsomal fraction.
The use of STR multiplexes with the incorporated gender marker Amelogenin is common practice in forensic DNA analysis. However, when a known male sample shows a dropout of the Amelogenin Y-allele, the STR system falsely genotypes it as a female. To date, our laboratory has observed 18 such cases: 12 from our Y-STR database and six from casework. A study on 980 male individuals in the Malaysian population using the AmpFlSTR Y-filer has revealed a distinct Y-chromosome haplotype associated with the Amelogenin nulls. Our results showed that whilst the Amelogenin nulls were noticeably absent among the Chinese, both the Indians and Malays exhibited such mutations at 3.2 and 0.6%, respectively. It was also found that the Amelogenin negative individuals predominantly belonged to the J2e lineage, suggesting the possibility of a common ancestor for at least some of these chromosomes. The null frequencies showed concordance with the data published in Chang et al. [Higher failures of Amelogenin sex test in an Indian population group, J. Forensic Sci. 48 (2003) 1309-1313] on a smaller Malaysian population of 338 males which used a Y-STR triplex. In the current study, apart from the absence of the Amelogenin Y-locus, a complete absence of the DYS458 locus in all the nulls was also observed. This study together with the 2003 study has indicated a similar deletion region exists on the Y(p)11.2 band in all the 18 Y-chromosomes. Using bioinformatics, this deletion has been mapped to a region of at least 1.13 Mb on the Y(p)11.2 encompassing the Amelogenin, MSY1 minisatellite and DYS458 locus. Further, the Y-filer haplotypes revealed an additional null at Y-GATA H4 in two of the Indian males presented here.