Biological and structural properties of MIP-1 alpha expressed in yeast.

The murine macrophage inflammatory proteins-1 alpha (MIP-1 alpha) and MIP-1 beta are distinct but closely related cytokines. Partially purified mixtures of the two proteins affect neutrophil function and cause local inflammation and fever. The particular properties of MIP-1 alpha have not been well studied, although it has been identified as being identical to an inhibitor of haemopoietic stem cell growth. We have expressed MIP-1 alpha in yeast cells and purified it to sequence homogeneity. Structural analysis of this biologically active material by circular dichroism and fluorescence spectroscopy confirms that MIP-1 alpha has a very similar secondary and tertiary structure to platelet factor 4 and interleukin 8 with which it shares limited sequence homology. The in-vitro stem cell inhibitory properties have been confirmed using a range of murine progenitor cells including purified bone marrow progenitor cells (FACS-1), the FDCP-mix A4 cell line, and spleen colony forming unit (CFU-S) populations. Plateau levels of inhibition of stem cell growth were achieved using concentrations of 0.15 micrograms/ml MIP-1 alpha. We have also demonstrated that MIP-1 alpha is active in vivo: 5 micrograms of MIP-1 alpha per mouse given as a bolus injection, protects stem cells from subsequent in-vitro killing by tritiated thymidine. MIP-1 alpha was also shown to enhance the proliferation of more committed progenitor granulocyte macrophage-colony forming cells (GM-CFC) in response to granulocyte macrophage-colony stimulating factor (GM-CSF).     

Characterization and linkage mapping often sheep microsatellite markers derived from a sheep x hamster cell hybrid

A cosmid library has been constructed from a sheep x hamster cell hybrid containing sheep chromosome t₁, rob (6;24). Clones containing sheep DNA were identified by hybridizing to a total sheep genomic DNA probe. Small fragments (<500 bp) containing (AC)_n microsatellites were subcloned and sequenced. Ten microsatellite markers were characterized and six were mapped back to chromosomes 6 and 24. The remaining microsatellites mapped to chromosome 26, which was shown to be present in a small proportion of cells of the cell line.     

Formation of glycine and aminoacetone from L-threonine by rat liver mitochondria

Threonine is a precursor of glycine in the rat, but the metabolic pathway involved is unclear. To elucidate this pathway, the biosynthesis of glycine, and of aminoacetone, from L-threonine were studied in rat liver mitochondrial preparations of differing integrities. In the absence of added cofactors, intact mitochondria formed glycine and aminoacetone in approximately equal amounts from 20 mM L-threonine, but exogenous NAD+ decreased and CoA increased the ratio of glycine to aminoacetone formed. In intact and freeze-thawed mitochondria, the ratio of glycine to aminoacetone formed was markedly sensitive to the concentration of L-threonine, glycine being the major product at low L-threonine concentrations. Disruption of mitochondrial integrity by sonication (1 min) decreased the ratio of glycine to aminoacetone formed, and in 20000 X g supernatant fractions from sonicated (3 min) mitochondria, aminoacetone was the major product. The main non-nitogenous two-carbon compound detected when intact mitochondria catabolized L-threonine to glycine was acetate, which was probably derived from deacylation of acetyl-CoA. These results suggest that glycine formation from L-threonine in rat liver mitochondria occurred primarily by the coupled activities of threonine dehydrogenase and 2-amino-3-oxobutyrate CoA-ligase, the extent of coupling between the enzymes being dependent upon a close physical relationship and upon the flux through the dehydrogenase reaction. In vivo glycine synthesis would predominate, and aminoacetone would be a minor product.     

Asymptomatic bacteriuria in the Port Harcourt metropolis of Nigeria

The Port Harcourt metropolis of Nigeria was screened to establish the prevalence of asymptomatic bacteriuria (ABU) over a 3 year period. An occurrence rate of 15% was detected. The females presented a higher incidence rate (9%) than the males (6%). Previous history of urinary tract infection (UTI) was seen to be contributory to ABU; so also was sexual activity. Of the isolates obtained, Escherichia coli was found to be predominant (45%). An isolation rate of 3.7% was realised for Staphylococcus epidermidis. This emphasizes its role in UTI. The advantages of screening for ABU was examined and it is suggested that individuals should be screened for ABU at least twice a year.     

Population genetics of coagulant factor IX: frequencies of two DNA polymorphisms in five ethnic groups.

Two frequently used restriction-enzyme polymorphisms (RFLPs) of coagulant F.IX, TaqI and XmnI, have been examined in five ethnic groups: white Americans, black Americans, East Indians, Chinese, and Malays. There is a distinct "cline" in the frequencies of both polymorphisms, from white Americans to Malays. The rarer type 2 alleles of both polymorphisms, in which middle recognition sites are present--and which in our sample reach their highest frequencies in white Americans--are marginally higher in four groups of Europeans previously reported by others. The frequencies of the rarer alleles are significantly higher in Europeans than in black Americans and East Indians, and these alleles are essentially absent in Chinese and Malays. The frequency of heterozygosity diminishes in the same order, being zero in Malays for both polymorphisms. The polymorphisms are in strong linkage disequilibrium, and in all groups the type 1 allele for TaqI is disproportionately accompanied by the type 1 allele for XmnI. The paucity of type 2 alleles and the low rate of heterozygosity in four non-European groups suggest that the polymorphisms will be of little diagnostic value south of Gibraltar and east of Suez. This prediction is confirmed by the observed haplotype frequencies in the black American and the Oriental groups.     

L-641,953 (R-8-fluoro-dibenzo[b, f]thiepin-3-carboxylic acid-5-oxide): a novel thromboxane-prostaglandin endoperoxide antagonist

The effects of L-641,953 (R-8-fluoro-dibenzo[b, f]thiepin-3-carboxylic acid-5-oxide) have been studied on pulmonary and other smooth muscle preparations in vitro and in vivo. When studied in vitro on guinea-pig tracheal chains, L-641,933 produced significant shifts in the dose-response curves to the prostaglandin endoperoxide analogues, U-44069 (pA2 7.06) and U-46619 (pA2 7.14), and prostaglandin (PG) F2 alpha (pA2 6.33) had minimal activity against contractions induced by histamine (pA2 4.38), 5-hydroxytryptamine (pA2 4.63), and acetylcholine (pA2 4.56) and slightly enhanced relaxation induced by PGE2. When tested on the guinea-pig gall bladder strip in vitro, L-641,953 antagonized contractions induced by U-44069 (pA2 7.03) but was less active against those induced by PGF2 alpha (pA2 6.03), PGE1 (pA2 5.62), and histamine (pA2 4.84). When tested in vitro on the guinea-pig pulmonary artery, L-651-953 significantly antagonized contractions induced by U-44069 (pA2 7.04), U-46619 (pA2 7.14), and PGF2 alpha (pA2 7.16) but was less effective against contractions induced by histamine (pA2 4.19). Schild analysis indicated that L-641,953 was fully competitive against contractions of either the guinea-pig tracheal chain induced by U-46619 or the guinea-pig pulmonary artery induced by U-44069 and U-46619. When tested on human platelets in vitro L-641,953 inhibited aggregation induced by U-44069 (IC50 1.3 X 10(-6) M) but not ADP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Requirement for proliferating cell nuclear antigen expression during stages of the Chinese hamster ovary cell cycle

Proliferating cell nuclear antigen (PCNA/cyclin) is a nuclear protein that can stimulate purified DNA polymerase delta in vitro, and its synthesis correlates with the proliferation rate of cells. We have attempted to determine whether synthesis of PCNA/cyclin in Chinese hamster ovary cells is necessary to regulate entry into S phase. We have measured cellular PCNA/cyclin concentration of the mRNA or protein throughout the cell cycle. Cells were separated by centrifugal elutriation into populations enriched for G-1, S, and G-2/M phases. Quantitative Northern hybridization analysis was performed on RNA isolated from each cell population by using a cDNA clone of PCNA/cyclin as a probe. Results demonstrated that although intact PCNA/cyclin mRNA is present during all phases of the cell cycle, an induction of about 3-fold occurs during S phase. Two-parameter staining for PCNA/cyclin and DNA, and analysis by flow cytometry, confirmed that the quantity of PCNA/cyclin protein in the cells increases severalfold in G-1 or early S phase but generally is invariant in S and G-2/M phases. This cell cycle dependence of PCNA/cyclin expression suggests that the observed synthesis is a prerequisite for initiation of DNA replication. Introduction of an antisense oligonucleotide complementary to the PCNA/cyclin mRNA to inhibit PCNA/cyclin synthesis effectively prevented entry of G-1 phase cells into S phase. A complementary sense oligonucleotide used as a control did not have an inhibitory effect. This result suggests that a threshold concentration of PCNA/cyclin is necessary for entry into S phase.     

Virulence factors from Aeromonas species

Isolates (116) of Aeromonas were obtained from various sources and subjected to tests to establish their virulence factors. A high number of the isolates (69.8%) were found to be enterotoxigenic. The isolates from snails had more enterotoxigenic strains (73.3%), while those from cattle faeces had the lowest (33.3%). Haemolysin production was found to be high (60.3%) amongst the isolates, and human isolates gave the highest number of haemolysin producing strains (70.6%), while the least number (33.3%) was obtained from cattle strains. About 50% of the strains produced both enterotoxin and haemolysin. The enzyme profile of the isolates included amylase, lecithinase, lipase and protease. There was no definite pattern in the elaboration of these enzymes and the production of haemolysin and enterotoxin, thus inferring that the production of these factors is not specific to the source. Two isolates were seen to produce none of these enzymes, and one was positive for enterotoxin and haemolysin production, leaving only one isolate which yielded none of these factors. The work adds more support to the pathogenicity of Aeromonas species, and indicates the existence of non-pathogenic strains.