Sequence of a cDNA encoding chicken high-mobility-group protein-2.

There are several members of the high-mobility-group (HMG) of DNA-binding proteins, including HMG-1, HMG-2, HMG-14 and HMG-17 [Johns: The HMG Chromosomal Proteins. Academic Press, London, 1982]. We report here sequences encoding the chicken HMG-2 protein of 207 amino acids (aa). This assignment is made on the basis of available data which indicate 89% homology of the chicken aa sequence to porcine HMG-2. This compares with 78-81% homology to the HMG-1 proteins of rat, hamster, human, porcine, and bovine origin.     

Aminergic control of vasopressin secretion in the conscious rat.

The influence of aminergic pathways on basal and stimulated vasopressin (AVP) release was studied in conscious rats, the stimulus for hormone release being an intracerebroventricular (ICV) injection of 5 microliters 0.85M sodium chloride. The animals were treated with either phenoxybenzamine, propranolol or haloperidol prior to administration of the central hypertonic stimulus. Phenoxybenzamine elevated basal plasma vasopressin concentrations, while propranolol and haloperidol had no effect. The secretion of AVP in response to the hypertonic stimulus was potentiated by phenoxybenzamine and haloperidol, but the effect of propranolol was equivocal. The antagonists had no effect on basal arterial pressure at the time of hypertonic saline administration or the pressor response to ICV sodium chloride.     

A minisatellite and a microsatellite polymorphism within 1.5 kb at the human muscle glycogen phosphorylase (PYGM) locus can be amplified by PCR and have combined informativeness of PIC 0.95.

We sequenced a genomic clone (pMCMP1), previously reported to detect a VNTR polymorphism at the PYGM locus, and found a dinucleotide repeat segment (CA)14(GA)25 and a complex (AT)-repeat-rich segment containing 63 repeats spanning 160 bp. Resolution of PCR-amplified genomic DNA from the (CA)(GA) repeat region on DNA sequencing gels revealed a highly informative polymorphism with alleles differing by 2-bp intervals and ranging in size from 156 to 190 bp. Among three racial groups, a total of 18 alleles were observed. Fourteen alleles were observed in Caucasians (PIC 0.89), 12 alleles in American Blacks (PIC 0.89), and 9 alleles in Pima Indians (PIC 0.73). PCR amplification of the (AT) repeat region and resolution of the products on DNA sequencing gels revealed a complex variable length polymorphism with alleles distributed in size from 367 to 970 bp. Twenty-eight alleles were found in American Blacks (PIC 0.94), 6 alleles in Pima Indians (PIC 0.70), and 11 alleles in Caucasians (PIC 0.71). Comparison of the previously described VNTR RFLP alleles visualized by Southern hybridization to the PCR products described in this report demonstrated that the polymorphism described in both assays was identical. However, a larger number of alleles could be detected from the PCR-amplified products. Combined informativeness, PIC 0.95, for the two polymorphisms was determined from haplotype analysis of 100 Caucasian chromosomes. Therefore, for genotyping purposes, informativeness is maximized from using both polymorphisms.     

High resolution functional analysis of antibody-antigen interactions.

A comprehensive mutational analysis was used to analyze the side-chains on human growth hormone (hGH) important for binding 21 different anti-hGH mouse monoclonal antibodies (MAbs) whose equivalent concentrations for 50% binding (EC50) ranged from approximately 10(7) to 3 x 10(10) M-1. A combination of homolog- and alanine-scanning mutagenesis coupled with a robot-aided enzyme-linked immunosorbent assay were used to create high resolution "functional epitopes" for each MAb. Every functional epitope mapped to at least two polypeptide segments of hGH that were close together in the folded protein to form a patch. Although these patches sometimes overlapped, each was different indicating no two MAbs bound identically to hGH. The MAbs bound to determinants in loops and helices that were generally most accessible to a 9 A radius probe. Only a few side-chains dominated each functional epitope and these tended to be Arg greater than Pro greater than Glu approximately Asp approximately Phe approximately Ile (Ala, Cys and Trp were not tested). Our studies indicate that most of the accessible surface of hGH is potentially antigenic in the mouse and suggest that functional epitopes are dominated by fewer side-chains than may be in the contact epitope.     

Biosynthesis of platelet-activating factor by two-cell mouse embryos.

Incubation of two-cell mouse embryos with a range of radiolabelled compounds resulted in the incorporation of label into platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in the culture media. The demonstration that known precursors ([1-14C]hexadecanol, [1-3H]hexadecanol, 1-O-[alkyl-1'2'-3H]lyso-PAF, 1-O-[alkyl-1'2'-3H]acetyl-glycerol and [methyl-3H]choline chloride) were incorporated into PAF showed that embryo-derived PAF biosynthesis occurred via pathways present in other PAF-producing cells. The enzyme responsible for the formation of the ether linkage of the PAF molecule, alkyl-dihydroxyacetone-phosphate synthase, was present in the preimplantation embryo as [1-3H]hexadecanol was incorporated into PAF. Incorporation of label from alkylacetyl-glycerol and choline chloride into lyso-PAF was also observed, suggesting a role for lyso-PAF in the metabolism of embryo-derived PAF. Incubation of embryos with each of three [14C]carbohydrate energy substrates resulted in the incorporation of label into PAF in culture media, indicating that the composition of embryo culture media is important in the synthesis of PAF precursors. Incorporation of label from [2-14C]pyruvate was greatest and is consistent with the suggestion that pyruvate is the major energy source at the two-cell stage of development. L-[U-14C]Lactate was also incorporated into embryo-derived PAF, but the mean amount incorporated relative to the concentration of labelled substrate in the medium was 40 times less. The incorporation of D-[U-14C]glucose into PAF was 2405 times less than that from pyruvate, relative to the concentration in the medium.     

Relationship between intestinal microecology and the translocation of intestinal bacteria

It is now well known that endogenous bacteria can translocate from the intestinal tract and cause many of the complicating infections seen in severely ill, hospitalized patients. Of the hundreds of bacterial species in the intestinal tract, relatively few aerobic/facultative species appear to translocate with any frequency. Van der Waaij and colleagues (1971, 1972a, 1972b) originally proposed that, by a process termed colonization resistance, strictly anaerobic bacteria prevented the intestinal overgrowth and subsequent translocation of these potentially pathogenic aerobic/facultative bacteria. Selective antimicrobial decontamination, designed to maintain colonization resistance, has been effective in reducing the incidence of infectious morbidity in high risk patients. However, the mechanisms controlling bacterial translocation remain unclear, but appear to depend on host factors, as well as on factors inherent in the microbe itself. There is both clinical and experimental evidence supporting the concept that strictly anaerobic bacteria do not readily translocate. Bacteria that are able to survive within macrophages (e.g., Salmonella species and Listeria monocytogenes) translocate easier than others, and there is recent experimental evidence that normal intestinal bacteria may translocate to the draining mesenteric lymph node within host phagocytes. There is also evidence that anaerobic bacteria translocate along with facultative species in situations associated with intestinal epithelial damage, i.e., burn trauma, oral ricinoleic acid, and acute mesenteric ischemia. In contrast, recent experimental evidence demonstrates that facultative bacteria can translocate across a histologically intact intestinal epithelium, and that the ileal absorptive cell may be at least one portal of entry prior to transport into deeper tissues. It is anticipated that further clarification of the routes and mechanisms involved in bacterial translocation will provide new insights into the treatment and prevention of a significant proportion of the infectious morbidity seen in severely ill, hospitalized patients. Antoni van Leeuwenhoek Lecture presented at the Annual Meeting of the Netherlands Society of Microbiology, Utrecht, 23 November, 1989.     

Increased glucose transporter (GLUT4) protein expression in hyperthyroidism

We have studied skeletal muscle glucose uptake by perfused hindquarter preparations from rats treated with thyroxine. Basal glucose uptake (in the absence of insulin) was approximately 2 fold higher in muscle of hyperthyroid rats compared to controls. Insulin (10(-7) M) stimulated glucose uptake 4.0 and 6.8 fold in the 10 day and 30 day controls rats, respectively. Maximal glucose uptake (10(-7) M insulin) was not different in control and hyperthyroid rats and thus insulin responsiveness in the hyperthyroid animals was reduced to 2.5 fold stimulation. The abundance of the insulin-sensitive glucose transporter protein (muscle/fat, GLUT-4), measured by Western blot analysis using polyclonal antisera, was higher in skeletal muscle from both groups of hyperthyroid rats. These studies indicate that thyroid hormones increase basal glucose uptake in skeletal muscle and this is due, at least in part, to an increment of GLUT-4 isoform. Increased expression of muscle glucose transporter proteins may be responsible for the increased peripheral glucose utilization seen in hyperthyroidism.     

Multiple non-B-DNA conformations of polypurine.polypyrimidine sequences in plasmids

A polypurine.polypyrimidine (Pur.Pyr) sequence with a central interruption in a plasmid can adopt multiple non-B-DNA conformations depending on the conditions as revealed by specific chemical probes (OsO4, diethyl pyrocarbonate, and dimethyl sulfate) and two-dimensional electrophoresis. The relatively long mirror repeat Pur.Pyr sequences (GAA)9TTC(GAA)8 and (GGA)9TCC(GGA)8 form single canonical intramolecular triplexes at pH 7.0-6.0 in negatively supercoiled plasmids as isolated from Escherichia coli. With a lowering of the pH and/or an increase in the degree of negative supercoiling, these sequences undergo a novel conformational change as revealed by diethyl pyrocarbonate hypermodification of adenines in the middle of the polypurine strand and OsO4 reaction with thymines in the center and the quarter points of the polypyrimidine strand. To evaluate this structure, a family of related Pur.Pyr sequences were cloned and studied. The non mirror repeat sequence (GGA)9TCC(GAA)8 forms a non-B conformation only under acidic pH conditions, but the structural properties are different from those of the mirror repeat sequences. Furthermore, when the central interruptions of a mirror repeat sequence were increased from 3 to 9 bp, two canonical triplexes formed independently at pH 5.0 [at the (GAA)9 and (GAA)8 regions in the sequence (GAA)9TTAATTCGC(GAA)8]. Thus, if an interruption is sufficiently long, the two halves of the Pur.Pyr sequence do not interact with each other. Novel types of folded DNA geometries which explain these results are described.     

Bovine dopamine beta-hydroxylase, primary structure determined by cDNA cloning and amino acid sequencing

A cDNA clone encoding bovine dopamine beta-hydroxylase (DBH) has been isolated from bovine adrenal glands. The clone hybridizes to two oligonucleotide probes, one based on a previously reported active site peptide [DeWolf, W. E., Jr., et al. (1988) Biochemistry 27, 9093-9101] and the other based on the human DBH sequence [Lamouroux, A., et al. (1987) EMBO J. 6, 3931-3937]. The clone contains a 1.9-kb open reading frame that codes for the soluble form of bovine DBH, with the exception of the first six amino acids. Direct confirmation of 93% of the cDNA-derived sequence was obtained from cleavage peptides by protein sequencing and mass spectrometry. Differences were found between these two sequences at only two positions. Of the four potential N-linked carbohydrate attachment sites, two, Asn-170 and Asn-552, were shown to be partially and fully glycosylated, respectively. Within the 69% of the protein sequence confirmed by mass spectrometry, no other covalent modifications were detected.