Modulatory role of substance P on gonadotropin and prolactin secretion in the rabbit.

Substance P (SP) is present in large quantities in the brainstem and hypophysiotropic areas of the brain, but its roles in gonadotropin and prolactin secretion are controversial. The aim of this study was to measure luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin (PRL) release from the pituitary after either intracerebroventricular (ICV) injection or infusion of SP or its C- and N-terminal fragments in intact (INT) and ovariectomized (OVX) conscious rabbits. A single injection of SP into the 3rd cerebral ventricle (3CVT) in INT and OVX rabbits augmented plasma LH concentrations, especially when SP was applied during the initial phase of an LH peak. Injection of SP during the declining phase of LH release was not effective. Injection of SP into the 3CVT was followed by increased plasma PRL concentrations in OVX but not in INT rabbits. Both SP 1-11 and SP 1-7 failed to alter LH, FSH, and PRL secretion when the peptides were slowly infused into the 3CVT, although ICV infusion of SP 6-11 did cause a delayed increase in LH release. The results support a stimulatory role of SP on LH and prolactin release. The results further indicate that although the stimulatory effect of SP on LH is ovarian steroid-independent, in the absence of ovarian steroids, SP is stimulatory only during the rising phase of an LH pulse. A dual role of SP-ergic transmission in modulating LH secretion is discussed.     

Multiple enzymatic pathways involved in the metabolism of glyceryl trinitrate in Phanerochaete chrysosporium.

A study of glyceryl trinitrate metabolism by a filamentous fungus, Phanerochaete chrysosporium, carried out with the 14C-labeled substrate, provides evidence for a multienzymatic system leading to di- and mononitrate derivatives. At least two independent enzymatic activities were detected in the cytosolic fraction: an aerobic glutathione S-transferase activity and an anaerobic NADPH-dependent soluble cytochrome P450-like activity. Other hemoproteins with enzymatic activities dependent upon the presence of NADPH or ferrous ions were also detected in the microsomal fraction. Electron paramagnetic resonance spectra characteristic of an interaction between a hemoprotein and nitric oxide appeared in these two subcellular fractions during the anaerobic metabolism of glyceryl trinitrate.     

Generation of 19 STS markers that can be anchored at specific sites on human chromosome 21.

Sequence-tagged sites (STSs) are short stretches of DNA that can be specifically detected by the polymerase chain reaction (PCR) and can be used to construct long-range physical maps of chromosomal DNA. These STSs can be detected by PCR assays developed by reference to data obtained from the sequencing of restriction fragment length polymorphism-DNA markers for chromosome 21, which were derived from recombinant lamba-phage and plasmid clones made from DNA of a human-hamster hybrid cell line. In this report, we describe the generation of 19 new STSs that are specific for human chromosome 21.     

Application of automated DNA sizing technology for genotyping microsatellite loci.

Highly polymorphic microsatellite loci offer great promise for gene mapping studies, but fulfillment of this potential will require substantial improvements in methods for accurate and efficient genotyping. Here, we report a genotyping method based on fluorescently labeled PCR primers and size characterization of PCR products using an automated DNA fragment analyzer. We capitalize on the availability of three distinct fluorescent dyes to label uniquely loci that overlap in size, and this innovation increases by threefold the number of loci that can be analyzed simultaneously. We label size standards with a fourth dye and combine these with the microsatellite PCR products in each gel lane. Computer programs provide very rapid and accurate sizing of microsatellite alleles and efficient data management. In addition, fluorescence signals are linear over a much greater range of intensity than conventional autoradiography. This facilitates multiplexing of loci (since signal intensities often vary greatly) and helps distinguish major peaks from artifacts, thereby improving genotyping accuracy.     

Evidence implicating heterozygous deletion of chromosome 7 in the pathogenesis of familial leukemia associated with monosomy 7.

Complete or partial monosomy 7 is a recurring cytogenetic abnormality in acute myelogenous leukemia (AML) and myeloproliferative syndromes (MPS) and is particularly common in patients with Fanconi's anemia and in secondary AML. A familial form of monosomy 7 has been recognized in which two or more siblings develop MPS or AML before age 20. We tested the hypothesis that a recessive cancer susceptibility locus on chromosome 7 was important in the pathogenesis of leukemia in familial monosomy 7 by determining the parental origins of the chromosome 7 retained in the bone marrows of three pairs of affected siblings. We found no overlapping region where all three pairs retained DNA derived from the same paternal or maternal chromosome. These data suggest that inactivation of a single allele of a putative tumor-suppressor gene may be sufficient to contribute to leukemic transformation in familial monosomy 7.     

Physical mapping of a 950-kb region surrounding a locus (D10S102) tightly linked to the MEN2A gene.

We have constructed a long-range contig of cosmid and YAC clones around D10S102, a locus that is tightly linked to the gene responsible for multiple endocrine neoplasia type 2A (MEN2A). With D10S102 as a starting point, a 360-kb cosmid contig was constructed by bidirectional genomic walking, and at least six fragments from these cosmids showed high sequence homology to other species. Five YAC clones were also isolated at the D10S102 locus, and they formed a contig covering 950 kb of genomic DNA. Furthermore, we obtained six RFLP systems from the contig, which will serve as new resources for fine-scale genetic linkage mapping of the MEN2A locus.     

The hypervariable DXS255 locus contains a LINE-1 repetitive element with a CpG island that is extensively methylated only on the active X chromosome.

The DXS255 locus at Xp11.22 is highly polymorphic due to a 26-bp variable number of tandem repeats (VNTR) motif. In previous studies, one of the MspI sites flanking the VNTR manifested a correlation between methylation and X chromosome inactivation. Here we show, by DNA sequence analysis, that this MspI site is located within the CpG island at the 5' end of a LINE-1 element, which is 2.5 kb from the VNTR. The methylation status of the CpG island was assessed in Southern blotting experiments using the methylation-sensitive enzymes HpaII, HhaI, and BssHII. All these sites were completely methylated on active X chromosomes, consistent with previously reported findings of full methylation of LINE-1 elements throughout the genome. However, on inactive X chromosomes these sites were predominantly unmethylated, although patterns were found to be heterogeneous. The results suggest that LINE-1 elements on the inactive X chromosome are not suppressed by full methylation of their CpG islands. The differential methylation of the DXS255 CpG island provides the basis for a highly informative X inactivation analysis system.     

Studies on the role of amino acids 38-45 in the expression of a functional thyrotropin receptor.

We previously reported that deletion or substitution of a unique eight-amino acid tract (residues 38-45) in the extracellular domain of the human TSH receptor led to the loss of specific ligand binding to the surface of transfected cells. In the present study we analyzed this region in more detail. Using site-directed mutagenesis of the TSH receptor cDNA, we substituted amino acid residues 38-45, either in three overlapping groups of four amino acids each or individually. The resultant TSH receptor mutant cDNAs were stably transfected into Chinese hamster ovary cells, and the cells were tested for their TSH-binding ability. Our data demonstrate that amino acid residues 38-40 and 42-45 in this region of the human TSH receptor can be substituted without alteration in receptor function and are, therefore, not critical in forming or maintaining the TSH-binding site. However, substitution of Cys41, either alone or together with adjacent amino acids, leads to the loss of TSH binding to its receptor. These data suggest a central role for the amino acid in position 41 in preserving the biological function of the TSH receptor.     

A missense mutation (Asp250----Asn) in exon 6 of the human lipoprotein lipase gene causes chylomicronemia in patients of different ancestries.

We have previously reported two common lipoprotein lipase (LPL) gene mutations underlying LPL deficiency in the majority of 37 French Canadians (Monsalve et al., 1990. J. Clin. Invest. 86: 728-734; Ma et al., 1991. N. Engl. J. Med. 324: 1761-1766). By examining the 10 coding exons of the LPL gene in another French Canadian patient, we have identified a third missense mutation that is found in two of the three remaining patients for whom mutations are undefined. This is a G to A transition in exon 6 that results in a substitution of asparagine for aspartic acid at residue 250. Using in vitro site-directed mutagenesis, we have confirmed that this mutation causes a catalytically defective LPL protein. In addition, the Asp250----Asn mutation was also found on the same haplotype in an LPL-deficient patient of Dutch ancestry, suggesting a common origin. This mutation alters a TaqI restriction site in exon 6 and will allow for rapid screening in patients with LPL deficiency.     

Stabilities of hydroxyl radical spin adducts of PBN-type spin traps.

The stability of the hydroxyl spin adduct of nine different PBN-type spin traps has been examined in phosphate buffer solutions of various pH. The hydroxyl adduct is produced by short illumination of hydrogen peroxide with UV light in the presence of spin trap and the decay of its EPR signal followed. The stability measured by the half life of the first-order decay is strongly dependent on the pH of the solution and the structure of the aromatic ring used in the trap. All hydroxyl adducts are more stable in acidic media. tert-Butyl hydroaminoxyl is detected as a degradation product of the hydroxyl adduct from all spin traps.