Cloning and Characterization of Two Human G Protein-Coupled Receptor Genes (GPR38 and GPR39) Related to the Growth Hormone Secretagogue and Neurotensin Receptors

The recent cloning of a growth hormone secretagogue receptor (GHS-R) from human pituitary gland and brain identified a third G protein-coupled receptor (GPC-R) involved in the control of growth hormone release. The nucleotide sequence of the GHS-R is most closely related to the neurotensin receptor-1 (NT-R1) (35% overall protein identity). Two human GPC-Rs related to both the type 1a GHS-R and NT-Rs were cloned and characterized. Hybridization at low posthybridizational stringency with restriction enzyme-digested human genomic DNA resulted in the identification of a genomic clone encoding a first GHS-R/NT-R family member (GPR38). A cDNA clone was identified encoding a second GHS-R-related gene (GPR39). GPR38 and GPR39 share significant amino acid sequence identity with the GHS-R and NT-Rs 1 and 2. An acidic residue (E124) in TM-3, essential for the binding and activation of the GHS-R by structurally dissimilar GHSs, was conserved in GPR38 and GPR39. GPR38 is encoded by a single gene expressed in thyroid gland, stomach, and bone marrow. GPR39 is encoded by a highly conserved single-copy gene, expressed in brain and other peripheral tissues. Fluorescencein situhybridization localized the genes for GPR38 and GPR39 to separate chromosomes, distinct from the gene encoding the GHS-R and NT-R type 1. The ligand-binding and functional properties of GPR38 and GPR39 remain to be determined.     

Numerical taxonomy of some non-saccharolytic and saccharolytic Bacteroides species

Various Bacteroides spp. were examined by physiological tests, presence of specific enzymes, antibiotic sensitivity, menaquinone composition and a few miscellaneous tests. The data matrix containing 58 strains and 55 unit characters was examined using Gower's similarity coefficients (S _G ) and included matching negative character states and multistate characters. The highly saccharolytic strains were separated from the less saccharolytic and non-fermentative strains at the 55% similarity level; while at the slightly higher level of 63% strains of Capnocytophaga (formerly Bact. ochraceus ) were recovered as a compact phenon distinct from other saccharolytic species. The phenogram was divided into 6 clusters at 72% similarity level. Most of the ' Bact. fragilis group' of species clustered in one phenon while Bact. melaninogenicus ssp. melaninogenicus, Bact. bivius and a new species, Bact. denticola , formed another group. Another phenon comprised the saccharolytic non-pigmented species closely related to Bact. oralis such as Bact. buccalis and Bact. pentosaceus. The less saccharolytic strains of Bact. melaninogenicus ssp. intermedius and Bact. disiens were recovered in a distinct phenon. The low affinity (less than 55% similarity) between the two subspecies of Bact. melaninogenicus emphasised the need for reclassifying these taxa into separate species. The non-fermentative and very weakly saccharolytic strains formed good taxospecies. The separation of this cluster into three subclusters is in excellent agreement with chemotaxonomic data now available.     

Effect of Lactacidosis on Pyridine Nucleotide Stability During Ischemia in Mouse Brain

Brain levels of NADH and NAD+ were measured in three models of cerebral ischemia to determine whether degradation of the pyridine nucleotides is enhanced in models that generate high concentrations of lactic acid. Complete ischemia (decapitation), in which lactate increased to 14 mmol/kg, caused a gradual decrease in the NAD pool to 50% of control by 2 h. During focal ischemia (occlusion of the middle cerebral artery), the decrease in the NAD pool was less pronounced (82% of control at 2 h) despite the accentuated accumulation of lactate to 33 mmol/kg. In a third model (unilateral hypoxia-ischemia), pretreatment of animals with glucose augmented the ischemic elevation of lactate from 30 mmol/kg to 40 mmol/kg and greatly impaired restoration of energy metabolites during recirculation. However, glucose pretreatment had no effect on the size of the NAD pool during ischemia or early recovery. These results, therefore, demonstrate that the pyridine nucleotide pool is not rapidly degraded during ischemic insults that accumulate high concentrations of lactic acid. The stability of the NAD pool may have been enhanced by the limited increase in brain levels of NADH that occurred in these models of incomplete ischemia.     

The effect of lowering the pH on the composition and metabolism of a community of nine oral bacteria grown in a chemostat

Nine oral bacteria, associated with both healthy and diseased sites in the mouth, were grown at D = 0.05 h-1 (mean generation time 13.9 h) in a glucose-limited chemostat. After an initial period of steady-state growth at pH 7.0, pH control was discontinued. The pH then decreased until it stabilized at pH 4.1 after 9 d (16 generations), while the Eh rose from -165 mV to +160 mV. The lowering in pH resulted in the composition and metabolism of the flora being altered and in increased bacterial aggregation. At pH 7.0, 'Streptococcus mitior', Veillonella alcalescens and S. sanguis were most numerous while at pH 4.1 the counts of all bacteria fell except for Lactobacillus casei, which became predominant. The proportions of S. mutans within the community also increased while S. sanguis was recovered only occasionally and Bacteroides intermedius was not detected below pH 4.6. The survival at pH 4.1 of several other species would not have been predicted from earlier pure culture studies. Relative to pH 7.0, the community growing at pH 4.1 produced more lactic acid, washed cells had a greater glycolytic activity over a wider pH range but amino acid metabolism decreased. In general, when pH control was restored, so were the original patterns of metabolism and bacterial counts, except for B. intermedius, which was still not detected. The inverse relationship between S. sanguis and S. mutans, and the increase in proportions of L. casei and S. mutans during growth in a low pH environment parallel observations made in vivo and suggest that the chemostat can be used as a model for microbial behaviour in dental plaque.     

Molecular cloning of two cysteine proteinases from paw-paw (Carica papaya).

Two cDNA clones for plant cysteine proteinases have been isolated from a Carica papaya (paw-paw, papaya) leaf tissue cDNA library by using a mixture of 16 synthetic oligodeoxyribonucleotides as a hybridization probe. The inserted regions are 311 and 440 base-pairs in length and have the potential to encode a region corresponding to the C-terminal region of two proteins which are homologous with the known plant cysteine proteinases and the mammalian thiol cathepsins. One of the sequences shows a high (greater than 77%) homology with the plant cysteine proteinase papain, the other is closely related to papaya chymopapain. One sequence contains all, and the other most, of the 3' untranslated region of the mRNA. The inserts were used as specific probes in Northern Blot analyses giving an estimated size for the two mRNA species of 1.45 kilobases.     

Studies of the biochemical basis of steroid sulphatase deficiency--III. Phospholipid composition of microsomes from normal and steroid sulphatase deficient placentas

The phospholipid composition has been determined for placental microsomes from 11 normal and eight pregnancies complicated by steroid sulphatase deficiency. Phosphatidylcholine, phosphatidylethanolamine and sphingomyelin were found to be the major phospholipids of normal placental microsomes, comprising respectively 41.6 +/- 4.6% (mean +/- SD). 30 +/- 5.7% and 22.5 +/- 4.9% of the total phospholipid content. There was no correlation between the steroid sulphatase activity of the microsomes and the content of any of the three phospholipids. Though their contents were significantly decreased. (P less than 0.001) phosphatidylcholine, phosphatidylethanolamine and sphingomyelin similarly constituted the major portion of the total phospholipids in sulphatase deficient microsomes, representing 36 +/- 4.2%, 34 +/- 6.1% and 22.4 +/- 6.7% respectively. Only the percentage of phosphatidylcholine was significantly different (P less than 0.02) from normal microsomes. The results show that the decreased phospholipid content of steroid sulphatase deficient placental microsomes reflects a lower content of all major classes of phospholipids, particularly phosphatidylcholine.     

Hypomethylation of DNA during differentiation of friend erythroleukemia cells

DNA from mammalian cells has been shown to contain significant amounts of 5-methyl cytosine resulting from enzymatic transfer of methyl groups from s-adenosylmethionine to cytosine residues in the DNA polymer. The function of this modification is not known. We have found that DNA synthesized during chemically induced differentiation of friend erythroleukemia cells is hypomethylated, as measured by its ability to accept methyl groups transferred by homologous DNA methyltransferases in vitro. The extent of hypomethylation detected by this sensitive method is small, a decrease of less than 1.6 percent in 5-methylcytosine content. Hypomethylated DNA can be isolated from friend erythroleukemia cells grown in the presence of dimethyl sulfoxide, butyrate, hexamethylene-bis- acetamide, pentamethylene-bis acetamide, and ethionine. However, hypomethylated DNA is found only under conditions where differentiation is actually induced. DNA isolated from cells of a dimethyl sulfoxide- resistant subclone grown in the presence of that agent is not hypomethylated, although DNA of these cells becomes hypomethylated after growth in the presence of inducers that can trigger their differentiation. We also find that the DNA of friend erythroleukemia cells does not become hypomethylated when the cells are exposed to inducing agents in the presence of substances that inhibit differentiation. These results suggest a close link between genome modification by methylation and differentiation of friend erythroleukemia cells.     

Reducing the incidence of short estrous cycles in beef cows with norgestomet

Twenty-nine anestrous cows suckling calves 27 to 67 days old were allotted to 3 treatment groups according to calving date. Calves were weaned from all cows; 9 cows received no further treatment, while 10 were implanted with 6 mg of norgestomet 9 days before calves were weaned and 10 were implanted when calves were weaned. All implants were removed after 9 days. Twenty-two cows exhibited estrus within 10 days after treatment, and all exhibited estrus within 25 days. In the cows exhibiting estrus in 10 days, norgestomet reduced the incidence of 8 to 12-day cycles from 83.3 percent in the control group to 30 percent when implanted before weaning and to zero when implanted at weaning. Conception rates at first service were increased from zero for cows which were not implanted to 33.3 and 80 percent for those implanted before and at weaning, respectively. These data demonstrate that the presence of a progestogen before the first postpartum estrus increases conception rates and reduces the percentage of previously anestrous cows exhibiting short estrous cycles after their calves are weaned.     

Induction of Hepatitis B Antibody in Experimental Animals by Immunization with A-2 Plaque Virus

A sero-conversion for hepatitis B antibody has resulted from immunization of patas monkeys, guinea pigs, and rabbits with the A-2 plaque virus. This agent was isolated in tissue culture from patient sera that were positive for hepatitis B antigen. The immune response was assayed with techniques of direct and indirect counter-immunoelectrophoresis, and immune electron microscopy.