Comparison of 16S rRNA sequences from the family Pasteurellaceae: phylogenetic relatedness by cluster analysis

The taxonomy of the family Pasteurellaceae has remained controversial despite investigations of biochemistry, serology, and nucleic acid relatedness. In an attempt to resolve some of this confusion, we have partially sequenced the 16S rRNAs of seven members of the family, representing all three genera. The sequences were aligned, similarity scores calculated, and single, average and complete linkage cluster analysis of the resulting distance matrix performed. In this way, an evolutionary branching pattern of these closely related species was reconstructed, and the approximate phylogenetic position of the family determined. Actinobacillus (Haemophilus) actinomycetemcomitans clustered with Haemophilus instead of Actinobacillus, supporting transfer of this species to the genus Haemophilus. Thus cluster analysis of phylogenetic relatedness was found to be particularly useful for studying closely related organisms, and could be performed using a microcomputer.     

Recombinant human granulocyte-colony stimulating factor and recombinant human macrophage-colony stimulating factor synergize in vivo to enhance proliferation of granulocyte-macrophage, erythroid, and multipotential progenitor cells in mice

Combinations of low dosages of purified recombinant human (rh) macrophage-colony stimulating factor (M-CSF; also termed CSF-1) and rh granulocyte-colony stimulating factor (G-CSF) were compared alone and in combination for their influence on the cycling rates and numbers of bone marrow and splenic granulocyte-macrophage, erythroid, and multipotential progenitor cells in vivo in mice pretreated with iron-saturated human lactoferrin (LF). LF was used to enhance detection of the stimulating effects of exogenously added CSFs. Concentrations of each CSF that were not active in vivo when given alone were active when given together, with the other CSF. The concentrations of rhM-CSF and rhG-CSF needed to increase progenitor cell cycling in the marrow and spleen were reduced by factors of 40-200 when these CSFs were administered in combination with low dosages of the other CSF. At the concentrations of rhM-CSF and rhG-CSF tested, synergism was not noted on absolute numbers of progenitor cells or total nucleated cell counts per organ or circulating in the blood. These findings may have potential relevance when considered in a clinical setting where the CSFs might be used in combination with other biotherapy and/or chemotherapy.     

Anaerobic biotransformations of pollutant chemicals in aquifers

Summary Anaerobic microbial communities sampled from either a methanogenic or sulfate-reducing aquifer site have been tested for their ability to degrade a variety of groundwater pollutants, including halogenated aromatic compounds, simple alkyl phenols and tetrachloroethylene. The haloaromatic chemicals were biodegraded in methanogenic incubations but not under sulfate-reducing conditions. The primary degradative event was typically the reductive removal of the aryl halides. Complete dehalogenation of the aromatic moiety was required before substrate mineralization was observed. The lack of dehalogenation activity in sulfatereducing incubations was due, at least in part, to the high levels of sulfate rather than a lack of metabolic potential. In contrast, the degradation of cresol isomers occurred in both types of incubations but proved faster under sulfate-reducing conditions. The requisite microorganisms were enriched and the degradation pathway forp-cresol under the latter conditions involved the anaerobic oxidation of the aryl methyl group. Tetrachloroethylene was also degraded by reductive dehalogenation but under both incubation conditions. The initial conversion of this substrate to trichloroethylene was generally faster under methanogenic conditions. However, the transformation pathway slowed when dichloroethylene was produced and only trace concentrations of vinyl chloride were detected. These results illustrate that pollutant compounds can be biodegraded under anoxic conditions and a knowledge of the predominant ecological conditions is essential for accurate predictions of the transport and fate of such materials in aquifers.     

Chromosome-size variation in Giardia lamblia: the role of rDNA repeats.

Giardia lamblia trophozoites contain at least five sets of chromosomes that have been categorized by chromosome-specific probes. Pulsed field separations of G. lamblia chromosomes also demonstrated minor bands in some isolates which stained less intensely with ethidium than the major chromosomal bands. Two of the minor bands of the E11 clone of the ISR isolate, MBa and MBb, were similar to each other and to chromosomal band I by hybridization to total chromosomal DNA and by hybridization of specific probes. In order to determine the extent of this similarity, I have developed a panel of probes for many of the Pacl restriction fragments and have shown that most of the Pacl and Notl fragments found in MBa are also present in MBb. The differences are found in both telomeric regions. At one end, MBb contains a 300 kb region not found in MBa. At the other end of MBb is a 160 kb region containing the rDNA repeats which is bounded on one end by the telomeric repeat and on the other by sites for multiple enzymes that do not digest the rDNA repeats. The corresponding region of MBa is 23 kb in size. The size difference is consistent with the eightfold greater number of rDNA repeats in MBb than MBa and suggests that 30% of the size difference is accounted for by different numbers of copies of the rDNA repeat. MBa of another ISR clone (ISR G5) is 150 kb larger in size than MBa of ISR E11. The data suggest that MBa and MBb are homologous chromosomes of different sizes and that a portion of the size difference is accounted for by different copy numbers of the rDNA repeat.     

Induction of interleukin-2 receptor by tumor necrosis factor α on cultured ovarian tumor-associated lymphocytes

Summary We have recently reported that autologous tumor-specific cytotoxic T lymphocyte (CTL) lines and clones can be developed from lymphocytes infiltrating ovarian malignant ascites (TAL). In this study, we investigated the biological effects of tumor necrosis factor (TNF) in the induction, expansion, long-term proliferation and lytic function of CD8⁺ TAL. TNF up-regulated the IL-2 receptor (IL-2R) chain (Tac antigen) on the surface of CD3⁺ CD8⁺ CD4^– TAL, enhanced the proliferation of autologous tumor-specific CTL, and potentiated their lytic function in long-term cultures. Furthermore, in the induction and expansion phase of CD8⁺ TAL, the presence of TNF was associated with a selective increase in CD8⁺ IL-2R⁺ (Tac⁺) cells, and subsequent decrease in CD4⁺ IL-2R⁺ (Tac⁺) cells. These results suggest that the observed facilitation of the outgrowth of CD8⁺ cells in TAL cultures may be due, at least in part, to the up-regulation of IL-2R, and indicate the usefulness of TNF in the analysis of signalling in autologous tumor-reactive CTL.     

Ammonia regulation of phosphate-activated glutaminase displays regional variation and impairment in the brain of aged rats

The regulation of PAG by ammonia in whole brain (Sprague-Dawley) and regional (Fischer-344) synaptosomal preparations from adult and aged animals was assessed. Whole brain synaptosomal preparations from both age groups displayed a significant decrease in PAG activity with increasing ammonium chloride concentrations, however, the aged rats exhibited a significant attenuation in ammonia-induced PAG inhibition. PAG activity measured in synaptosomes prepared from the striatum (STR), temporal cortex (TCX) and hippocampus (HIPP) was also inhibited by ammonium chloride. The STR showed the greatest degree of ammonia-induced PAG inhibition (55%) followed by the HIPP (30–35%) and the TCX (25–30%). This reduction in PAG activity was significantly attenuated in STR from aged rats at ammonium chloride concentrations greater than 50 M and in the TCX, PAG activity was significantly attenuated in the aged rats at ammonia concentrations of 0.5 and 1.0 mM. Ammonia regulation of PAG activity in the HIPP appeared to be unaffected by age. Ammonium chloride concentrations up to 5 mM had no effect on GLU release from cortical slices, although GLN efflux was significantly enhanced. These findings suggest that isozymes of PAG may exist in different brain regions based on their differential sensitivity to ammonia. The attenuation of ammonia-induced PAG inhibition seen in aged rats may have deleterious effects in the aged brain.Abbreviations PAG phosphate-activated glutaminase: L-glutamine amidohydrolase; EC 3.5.1.2 - STR striatum - TCX temporal cortex - HIPP hippocampus     

A free-radical hypothesis for the instability and evolution of genotype and phenotypein vitro

It has been known for several decades that cultured murine cells undergo a defined series of changes, i.e., anin vitro evolution, which includes crisis, spontaneous transformation (immortalization), aneuploidy, and spontaneous neoplastic transformation. These changes have been shown to be caused by thein vitro environment rather than an inherent instability of the murine phenotype or genotype. Serum amine oxidases were recently identified as a predominant cause of crisis. These enzymes generate hydrogen peroxide from polyamine substrates that enter the extracellular milieu. This finding implicates free-radical toxicity as the underlying cause ofin vitro evolution. We propose an oxyradical hypothesis to explain each of the stages ofin vitro evolution and discuss its significance for cytotechnology and long-term cultivation of mammalian cell types.ORR, CDER, FDA Mod-1, Room 2023, 8301 Muirkirk Road, Laurel MD 20708, USA     

Triterpene glycosides from Schefflera octophylla.

In addition to 3-epi-betulinic acid, three triterpene glycosides were isolated from leaves of Schefflera octophylla. The structures of the glycosides have been determined as 28-O-[alpha-L-rhamnopyranosyl(1----4)-O-beta-D-glucopyranosyl(1----6)-be ta-D- glucopyranosides of 3 alpha-hydroxy-lup-20(29)-ene-23,28-dioic acid, 3 alpha,11 alpha- dihydroxy-lup20(29)-ene-23,28-dioic acid and 3-epi-betulinic acid by spectroscopic data and chemical transformations. The last two compounds were found for the first time in the plant kingdom.