Vasoactive properties of dihomo-gamma-linolenic acid and series one prostaglandins in a freshwater teleost, Channa maculata

1. Prostaglandins A1, B1, E1 and F1 alpha (2-120 micrograms/kg), arachidonic acid and dihomo-gamma-linolenic acid (0.1-2 mg/kg) were injected intravenously into Channa maculata and changes in arterial blood pressure were recorded. 2. Injection of PGF1 alpha had no significant effect on arterial blood pressure. Injection of PGA1 and PGE1 was followed by dose-dependent hypotension whereas injection of PGB1 elicited significant dose-dependent increase in arterial blood pressure. 3. Both dihomo-gamma-linolenic acid and arachidonic acid were also depressor agents but dihomo-gamma-linolenic acid was more potent. 4. A single bolus intravenous injection of indomethacin (5 mg/kg) or 4 daily intraperitoneal injections (4 x 10 mg/kg) significantly lowered arterial blood pressure. One hour after pre-treatment of indomethacin, the vascular effects of both prostaglandin precursors were abolished. 5. It appears that the vascular effects of prostaglandins in Channa maculata are qualitatively different from those reported for mammals.     

Synteny on mouse chromosome 5 of homologs for human DNA loci linked to the Huntington disease gene

Comparative mapping in man and mouse has revealed frequent conservation of chromosomal segments, offering a potential approach to human disease genes via their murine homologs. Using DNA markers near the Huntington disease gene on the short arm of chromosome 4, we defined a conserved linkage group on mouse chromosome 5. Linkage analyses using recombinant inbred strains, a standard outcross, and an interspecific backcross were used to assign homologs for five human loci, D4S43, D4S62, QDPR, D4S76, and D4S80, to chromosome 5 and to determine their relationships with previously mapped markers for this autosome. The relative order of the conserved loci was preserved in a linkage group that spanned 13% recombination in the interspecific backcross analysis. The most proximal of the conserved markers on the mouse map, D4S43h, showed no recombination with Emv-1, an endogenous ecotropic virus, in 84 outcross progeny and 19 recombinant inbred strains. Hx, a dominant mutation that causes deformities in limb development, maps approximately 2 cM proximal to Emv-1. Since the human D4S43 locus is less than 1 cM proximal to HD near the telomere of chromosome 4, the murine counterpart of the HD gene might lie between Hx and Emv-1 or D4S43h. Cloning of the region between these markers could generate new probes for conserved human sequences in the vicinity of the HD gene or possibly candidates for the murine counterpart of this human disease locus.     

Multiple T and B cell epitopes in the S1 subunit ("A"-monomer) of the pertussis toxin molecule

The immunogenicity and reactogenicity of Bordetella pertussis vaccine are mediated in part by the S1 subunit of pertussis toxin (PT). To identify the immune epitopes in the S1 subunit of PT, synthetic peptides were prepared and tested for their capacity to induce antibodies in mice with different MHC genotypes. In BALB/c mice, peptides corresponding to sequences 1-17, 70-82 and 189-199 generate T cell proliferative responses, induce the production of antibodies capable of neutralization of the toxin in the Chinese hamster ovary-cell assay, and protect mice from a shock-like syndrome caused by alternate injections of BSA and PT. Protection and neutralization correlated with the ability of these peptides to elicit high anti-PT titers. Different B cell epitopes were detected in other inbred mouse strains. The antibody reactivity against synthetic peptides from two infants vaccinated with pertussis vaccine was tested. These infants had antibodies reactive to a variety of epitopes in the S1 subunit, including peptides 1-17, 70-82, 99-112, 135-145, and 189-199. Thus, it appears that there are multiple T and B cell epitopes in the S1 subunit of PT.     

Human serum amyloid A protein. The assignment of the six major isoforms to three published gene sequences and evidence for two genetic loci

Serum amyloid A protein (apo-SAA) is an acute-phase reactant and an apolipoprotein of high density lipoproteins (HDL). Six major isoforms of apo-SAA occur in humans (pI 6.0, 6.4, 7.0, 7.4, 7.5, 8.0). In this report we have rationalized the phenotypic expression of apo-SAA isoforms with published apo-SAA structures predicted from apo-SAA cDNA's pA1 and pSAA82 and the genomic DNA SAAg9. The six apo-SAA isoforms fall into three pairs, pI 6.0/6.4, 7.0/7.5, and 7.4/8.0, which are products of cDNA pA1, cDNA pSAA82, and genomic DNA SAAg9, respectively. The second of each isoform pair (i.e. pI 6.4, 7.5, and 8.0) is the "primary" product: a 104-residue peptide with the NH2-terminal sequence Arg-Ser-Phe-Phe. Each primary product is processed either to a major 103-residue peptide with the NH2-terminal sequence Ser-Phe-Phe or processed to a minor 102-residue product which results from the loss of both an Arg and a Ser residue from the NH2 termini. These "secondary" products have the lower pI values of 6.0, 7.0, and 7.4, respectively. The isoelectric points of the SAAg9 products were confirmed by expression of SAAg9 in transfected mouse L-cells. Both the pI 8.0 and 7.4 isoforms were present in cellular extracts, suggesting that post-translational modification of apo-SAA may occur intracellularly. However, the greater relative abundance of the pI 7.4 isoform extracellularly suggests that the major conversion may occur after secretion. Whereas the gene corresponding to the pA1 cDNA sequence does not show allelic variation, the segregation characteristics of the pI 7.0/7.5 and 7.4/8.0 isoform pairs amongst individuals suggests that these isoforms are the products of genes (with sequences corresponding to pSAA82 and SAAg9, respectively) which are allelic variants at a single locus distinct from that for the pI 6.0/6.4 isoform pair.     

Long-acting nifedipine versus metoprolol as monotherapy for essential hypertension. A randomized,controlled crossover study.

We assessed the efficacy of long-acting nifedipine as monotherapy in 52 patients with mild to moderate essential hypertension in a randomized, controlled crossover study. Good blood pressure control was achieved in 34 of 40 patients (85%) receiving nifedipine (mean daily dose, 52 mg in 2 divided doses) compared with 23 of 40 patients (58%) receiving metoprolol (mean daily dose, 155 mg in 2 divided doses). After treatment for 4 weeks, the mean blood pressures with nifedipine (149.7 +/- 16.6/88.7 +/- 11.1 mm of mercury) and metoprolol administration (163.9 +/- 23.3/94.2 +/- 10.2 mm of mercury) were significantly lower than with placebo (176.7 +/- 17.3/100.9 +/- 7.1 mm of mercury) (P less than .05). The mean systolic pressure during nifedipine treatment was 14.2 mm of mercury lower (95% confidence interval [CI], 3.9 to 24.5 mm of mercury) and mean diastolic pressure 5.5 mm of mercury (95% CI, 0.3 to 10.7 mm of mercury) lower than with metoprolol therapy. Both drugs were reasonably well tolerated, and intolerance requiring withdrawal was encountered in 3 of 45 (7%) patients receiving nifedipine, compared with 1 of 45 (2%) of those taking metoprolol and placebo, respectively. Adverse effects of nifedipine, most of which were transient, included palpitations, headache, facial flushing, and ankle edema. Long-acting nifedipine is a promising agent when given alone for mild to moderate hypertension and can be safely administered in clinical practice.     

Structural and energetic consequences of disruptive mutations in a protein core.

We have characterized the properties of a set of variants of the N-terminal domain of lambda repressor bearing disruptive mutations in the hydrophobic core. These mutations include some that dramatically alter the total core residue volume (by up to six methylene groups) and some that place a single polar residue into the otherwise hydrophobic core. The structural properties of the purified proteins have been studied by CD spectroscopy, biological activity, recognition by conformation-specific monoclonal antibodies, and 1H NMR spectroscopy. The stabilities of the proteins have been measured by thermal and guanidine hydrochloride denaturation. Proteins with disruptive core mutations are found to display a continuum of increasingly nonnative properties. Large internal volume changes cause both significant conformational rearrangements and destabilization by up to 5 kcal/mol. Variants with polar substitutions at core positions no longer behave like well-folded proteins but rather display characteristics of molten globules. However, even proteins bearing some of the most disruptive mutations retain many of the crude secondary and tertiary structural features of the wild-type protein. These results indicate that primitive elements of native structure can form in the absence of normal core packing.     

Transforming growth factor alpha and a PC12-derived growth factor induce neurites in PC12 cells and enhance the survival of embryonic brain neurons.

We have identified and characterized a 5000-Da protein that induces neurite outgrowth from PC12 pheochromocytoma cells, enhances the survival of embryonic rat brain neurons in primary culture, and induces the multiplication of embryonic rat brain astrocytes in primary culture. The factor is produced by a flat cell PC12 variant that expresses the activated ras oncogene after transfection of the gene. The factor resembles transforming growth factor alpha (TGF alpha) and epidermal growth factor (EGF) in that it induces anchorage-independent colony formation of normal rat kidney cells in soft agar and competes with EGF for binding to the EGF receptor. Rat TGF alpha and human TGF alpha also induce neurite outgrowth from PC12 and enhance the survival of embryonic brain neurons. The PC12 variant-derived factor can be distinguished from TGF alpha and EGF immunologically and by migration rates on reversed-phase high-performance liquid chromatography.     

Nucleophilic Distribution of Metallothionein in Human Tumor Cells

Metallothionein (MT), a major zinc-binding intracellular protein thiol, has been associated with cytoprotection from heavy metals, antineoplastic drugs, mutagens, and cellular oxidants. Despite its small mass (7 kDa), nuclear partitioning of MT has been observed in both normal and malignant tissues. The factors controlling MT sequestration are unknown. Thus, we examined the regulation of MT subcellular distribution in human cancer cell lines that exhibit prominent nuclear MT. The nuclear disposition of MT was unaltered during cell cycle passage in synchronized cells. MT redistributed to the cytoplasm when cells were exposed to reduced temperature. Cytoplasmic redistribution was also seen in DU-145 and HPC36M prostatic cancer cells after ATP depletion, but not in PC3-MA2 and SCC25/CP cells. Pretreatment with 10 μMCdCl₂did not significantly alter MT distribution but did render all cells sensitive to cytoplasmic redistribution after either reduced temperature or ATP depletion. Thus, nuclear retention of MT is energy requiring and this ability of MT to accumulate in subcellular compartments against its concentration gradient may be important in the capacity of MT to supply Zn or other metals to target sites within the cell.     

Production of Androgenetic Zebrafish (Danio Rerio)

To help investigate the evolutionary origin of the imprinting (parent-of-origin mono-allelic expression) of paternal genes observed in mammals, we constructed haploid and diploid androgenetic zebrafish (Danio rerio). Haploid androgenotes were produced by fertilizing eggs that had been X-ray irradiated to eliminate the maternal genome. Subsequent inhibition of the first mitotic division of haploid androgenotes by heat shock produced diploid androgenotes. The lack of inheritance of maternal-specific DNA markers (RAPD and SSR) by putative diploid and haploid androgenotes confirmed the androgenetic origin of their genomes. Marker analysis was performed on 18 putative androgenotes (five diploids and 13 haploids) from six families. None of 157 maternal-specific RAPD markers analyzed, some of which were apparently homozygous, were passed on to any of these putative androgenotes. A mean of 7.7 maternal-specific markers were assessed per family. The survival of androgenetic zebrafish suggests that if paternal imprinting occurs in zebrafish, it does not result in essential genes being inactivated when their expression is required for development. Production of haploid androgenotes can be used to determine the meiotic recombination rate in male zebrafish. Androgenesis may also provide useful information about the mechanism of sex determination in zebrafish.     

Helper virus-free transfer of herpes simplex virus type 1 plasmid vectors into neural cells.

Herpes simplex virus type 1 (HSV-1) plasmid vectors have promise for genetic intervention in the brain, but several problems caused by the helper virus have compromised their utility. To develop a helper virus-free packaging system for these vectors, the DNA cleavage/packaging signals were deleted from a set of cosmids that represents the HSV-1 genome. Following cotransfection into cells, this modified cosmid set supported replication and packaging of vector DNA. However, in the absence of the DNA cleavage/packaging signals, the HSV-1 genome was not packaged, and consequently vector stocks were free of detectable helper virus. In the absence of helper virus, the vectors efficiently infected rat neural cells in culture or in the brain with minimal cytopathic effects. beta-galactosidase-positive cells were observed for at least 1 month in vivo, and vector DNA persisted for this period. This system may facilitate studies on neuronal physiology and potential therapeutic applications.