Effect of cycling on final mixed culture fate

Cycling in feed substrate concentration and dilution rate is examined as a means of modifying the final fate of a mixed culture. It is shown for the case where the specific growth rate of one species is always greater than that of the second that no cycling strategy will provide the desired extinction of the faster growing species unless time delay is included in the modeling. To account for the time lag in adjusting organism metabolic activities to environmental changes, an adaptability parameter is introduced. Numerical simulations are carried out and an operating diagram indicating the conditions under which the desired extinction occurs is constructed. Cycling in feed substrate concentration and dilution rate are both found to produce the desired result.     

Production of toluene cis-glycol by Pseudomonas putida in glucose feb-batch culture

Toluene was oxidized by a mutant strain of Pseudomonas putida (strain NG1) to toluene Cis-Glycol (TCG). Product was accumulated in fed-batch cultures to concentrations (18-24 g/L) higher than hitherto achieved. In vitro activities of toluene dioxygenase from P. Putida NG1 were fivefold lower than that from the toluene-grown wild-type organism, whereas comparable activities of both catechol 2,3- and catechol 1,2-oxygenase were obtained; irreversible inhibition of toluene dioxygenase activity by TCG was shown in vitro. Ammonia deprivation during the production phase limited the growth of revertant organisms but had little effect on either the duration (25h) of the process or the final concentration of TCG achieved. The rate of glucose utilization decreased throughout the biotransformation and cell death accompanied the cessation of TCG accumulation in cultures. These changes were a consequence of TCG formation and a cooperative toxic effect was demonstrated for toluene and TCG. Adenylate energy charge values decreased from ca. 0.8 to 0.2 over the course of the biotransformation but were maintained above 0.5 in the absence of TCG. Similarly, cellular AMP levels increased dramatically during biotransformation, presumably as a consequence of RNA degradation, but were maintained at low levels in the absence of TCG. The results suggest that TCG is the mediate of a gradual deterioration in the state of the culture which leads to a loss of both in vivo and in vitro toluence dioxygenase activity and a marked decrease in culture viability.     

Glands of the eyelids of rhesus monkeys (Macaca mulatta)

The various glands of rhesus monkey eyelids and human eyelids are similar. Numerous modified sebaceous glands are located along the tarsus. These conform with the meibomian glands, while typical sebaceous glands associated with the hair follicles of the lashes are consistent with the glands of Zeis. Lobules of accessory lacrimal tissue, corresponding to the glands of Krause and Wolfring, are located in the conjunctiva of the fornix and along the orbital border of the tarsal plate. Goblet cells are plentiful in the mucosa of the palpebral and bulbar conjunctiva, and along the lid margin are the sweat glands of Moll.     

Southern blot analysis of HLA-DP gene polymorphisms in Caucasoid rheumatoid arthritis (RA) patients and controls

Despite extensive analysis of the incidence ofHLA-DR andHLA-DQ allele frequencies in defined autoimmune disease groups, there is very little information available onHLA-DP allele frequencies. This is largely becauseHLA-DP typing has until recently been restricted to primed lymphocyte typing (PLT). However, allelic polymorphism of theHLA-DP subregion can now be studied by Southern blot analysis or genotyping withDPA1 andDPB1 probes. By direct counting of allele-specific DNA fragments, we have analyzed the frequencies of five majorDP genotypes (DPw1, DPw2, DPw3/6, DPw4, andDPw5), in a large number of Caucasoid rheumatoid arthritis (RA) patients (n=74), and controls (n=91). The predicted frequency ofDP alleles in both patient and control groups was comparable to PLT-determinedDP allele frequencies in normal Caucasoids. However, the gene frequency ofDPw4 was increased in the RA patients, with 51% of the patients studied scoring asDPw4, 4 homozygotes. With the exception of one possible combination (DPw5 andDRw6) in the controls, no significant linkage disequilibrium was detected betweenDP andDR alleles in either patient or control groups. Thus the prevalence ofDPw4 in the RA patients is independent of any disease association with theDR loci, and may represent a new class II association with RA.     

The order of loci in the pericentric region of chromosome 17, based on evidence from physical and genetic breakpoints.

Previous genetic analyses of chromosome 17 markers and NF1 (Fain et al. 1987) were extended in an attempt to order marker loci that map physically to 17cen----17q12. Three additional markers (HHH202, CRI-L581, and CRI-L946) were included in the analyses. Recombinants within the cluster of seven unordered marker loci were identified by pairwise analyses for each family and by examining the within-sibship segregation patterns for different markers. Changes in the segregation pattern for different loci define genetic breakpoints. Given that interference is complete in the region, markers with the same segregation pattern lie on one side of the breakpoint, while markers with different segregation patterns lie on opposite sides of the breakpoint. If the order of boundary markers is known, markers on each side of a breakpoint can be oriented in relation to the centromere. The order cen-(HHH202/NF1)-(EW207)-(EW203/CRI-L581)- (CRI-L946)-(HOX-2/NGFR)-qter was inferred by combining information from physical breakpoints in a panel of mouse/human hybrids and information from genetic breakpoints found in 16 NF1 families.     

Inhibitors of sterol synthesis. 15-oxygenated steryl ester hydrolase activity of rat liver at neutral and acid pH

5 alpha-Cholest-8(14)-en-3 beta-ol-15-one (15 ketosterol) is a potent inhibitor of cholesterol biosynthesis with significant hypocholesterolemic activity. The results of a recent study (Schroepfer, G.J., Jr., Christophe, A., Chu, A.J., Izumi, A., Kisic, A. and Sherrill, B.C. (1988) Chem. Phys. Lipids 48, 29-58) have indicated that, after intragastric administration of the 15-ketosterol in triolein to rats, most of the compound in intestinal lymph occurs in the form of the oleate ester, which is associated with chylomicrons. Moreover, after intravenous administration of chylomicrons containing the oleate ester of 15-[2,4-3H]ketosterol, rapid and selective uptake of 3H by liver was observed, which was associated with the rapid and substantial appearance of labeled free 15-ketosterol in liver. The present study concerns the capabilities of rat liver fractions to catalyze the hydrolysis of 15-ketosteryl oleate. Efficient hydrolysis was observed at acid pH with a digitonin-solubilized extract of rat liver, with a rate similar to that for the hydrolysis of cholesteryl oleate. The distribution of acid 15-ketosteryl oleate hydrolase of whole liver homogenate on a metrizamide isopycnic density gradient was similar to that of acid cholesteryl oleate hydrolase and acid phosphatase, suggesting that the lysosomal acid lipase is the enzyme responsible for the hydrolysis of the 15-ketosteryl oleate at acid pH. At neutral pH, 15-ketosteryl oleate and cholesteryl oleate was hydrolyzed at similar rates by the microsomal fraction of liver homogenate, whereas the 15-ketosteryl oleate was hydrolyzed at a much lower rate than cholesteryl oleate by the cytosolic fraction. The distribution of neutral 15-ketosteryl oleate hydrolase activity of whole liver homogenate on a metrizamide isopycnic density gradient was most correlated to a microsomal esterase, whereas cholesteryl oleate hydrolase activity was most correlated to a cytosolic enzyme. Both 15-ketosteryl oleate and cholesteryl oleate hydrolase activities were correlated to a mitochondrial marker enzyme.     

Tired,weak, or in need of rest: fatigue among general practice attenders.

OBJECTIVES--To determine the prevalence and associations of symptoms of fatigue. DESIGN--Questionnaire survey. SETTING--London general practice. PARTICIPANTS--611 General practice attenders. MAIN OUTCOME MEASURES--Scores on a fatigue questionnaire and reasons given for fatigue. RESULTS--10.2% Of men (17/167) and 10.6% of women (47/444) had substantial fatigue for one month or more. Age, occupation, and marital status exerted minor effects. Subjects attributed fatigue equally to physical and non-physical causes. Physical ill health, including viral infection, was associated with more severe fatigue. Women rather than men blamed family responsibilities for their fatigue. The profile of persistent fatigue did not differ from that of short duration. Only one person met criteria for the chronic fatigue syndrome. CONCLUSIONS--Fatigue is a common complaint among general practice attenders and can be severe. Patients may attribute this to physical, psychological, and social stress.     

Synthesis of myo-inositol 1,3,4,5,6-pentakisphosphate from inositol phosphates generated by receptor activation.

myo-[3H]Inositol 1,3,4,5,6-pentakisphosphate can be made from myo-[3H]inositol 1,4,5-trisphosphate in a rat brain homogenate or soluble fraction. Although D-myo-inositol 3,4,5,6-tetrakisphosphate can be phosphorylated by a soluble rat brain enzyme to give myo-inositol 1,3,4,5,6-pentakisphosphate, it is not an intermediate in the pathway from myo-inositol 1,4,5-trisphosphate. The intermediates in the above pathway are myo-inositol 1,3,4,5-tetrakisphosphate, myo-inositol 1,3,4-trisphosphate and myo-inositol 1,3,4,6-tetrakisphosphate [Shears, Parry, Tang, Irvine, Michell & Kirk (1987) Biochem. J. 246, 139-147; Balla, Guillemette, Baukal & Catt (1987) J. Biol. Chem. 262, 9952-9955], and it is catalysed by soluble kinase activities of similar anion-exchange mobility and Mr value. Compounds with chromatographic and chemical properties consistent with the structures myo-inositol 1,3,4,5-tetrakisphosphate, myo-inositol 1,3,4,6-tetrakisphosphate and myo-inositol 3,4,5,6-tetrakisphosphate are present in avian erythrocytes, human 1321 N1 astrocytoma cells and primary-cultured murine bone-marrow-derived macrophages. The amounts of these inositol tetrakisphosphates rise upon muscarinic cholinergic stimulation of the astrocytoma cells or stimulation of macrophages with platelet-activating factor.     

Receptor and G-protein-dependent regulation of turkey erythrocyte phosphoinositidase C

Several lines of experimental evidence indicate the involvement of a guanine nucleotide-dependent protein (G-protein) in the hormone-stimulated hydrolysis of phosphatidylinositol(4,5)-bisphosphate (PtdIns(4,5)P2). However, the shortcomings of available procedures for cell-free assay of hormone-stimulated phosphoinositidase C (PIC) have limited our current understanding of the molecular and mechanistic details of PIC regulation. We recently have proposed that turkey erythrocyte membranes may provide a valuable model system for studies of G-protein-dependent PtdIns(4,5)P2 hydrolysis. The membranes can be simply prepared from [3H]inositol-labelled erythrocytes and they contain a PIC activity that hydrolyses endogenous phosphoinositides and is exquisitively sensitive to guanine nucleotides. PtdIns(4,5)P2 is the principal substrate for this enzyme, there being relatively little direct hydrolysis of phosphatidylinositol 4-phosphate and no detectable hydrolysis of PtdIns. The membranes also contain a purinoceptor of the P2y subclass that is efficiently coupled to PtdIns(4,5)P2 hydrolysis both in intact cells and in the isolated membranes. 2-Methylthioadenosine trisphosphate (2-methyl-S-ATP), a specific P2y receptor agonist, has no effect upon PtdIns(4,5)P2 hydrolysis in the absence of guanine nucleotides, but greatly enhances both the potency and efficacy of PIC activation by guanine nucleotides such as GTP gamma S. GTP gamma S alone stimulates PIC activity only after a prolonged time-lag; the effect of increasing doses of 2-methyl-S-ATP is progressively to shorten this lag phase. These results suggest that the mechanism of G-protein activation involves acceleration of a nucleotide exchange reaction as has been demonstrated for the activation of adenylate cyclase in the same membrane preparation. As well as contributing valuable information on the substrate specificity of PIC and its mode of regulation by hormones, turkey erythrocytes provide a plentiful source of plasma membranes and may be useful for purification of the appropriate G-protein and PIC activities.     

95-kilodalton B-Raf serine/threonine kinase: identification of the protein and its major autophosphorylation site.

B-Raf, a member of the Raf family of serine/threonine kinases, is expressed primarily in the brain and in the nervous system. In this study, the biochemical properties of the B-Raf protein were investigated in nerve growth factor (NGF)-responsive cell lines and in brain tissues. B-Raf was identified by using phosphopeptide mapping analysis and cDNA analysis as a 95-kDa protein which is primarily localized in the cytosol. NGF rapidly stimulated both serine and threonine phosphorylation in vivo and autophosphorylation activity in vitro of the B-Raf protein. In PC12 cells, B-Raf autokinase activity was induced by both differentiation factors and mitogens, with maximal activity observed after 5 min of factor addition. B-Raf kinase activity was also observed following NGF treatment of SH-SY5Y neuroblastoma cells and in adult mouse brain and hippocampus. Induction of B-Raf kinase activity in NGF-treated PC12 cells required expression of kinase-active trk receptors. Exogenous substrates or a peptide containing the autophosphorylation site became phosphorylated when added to immune complex kinase assays and reduced the in vitro autophosphorylation activity of B-Raf, suggesting that in vitro autophosphorylation sites and exogenous substrates compete for active sites of the B-Raf kinase. Finally, the major in vitro autophosphorylation site of B-Raf was identified as threonine 372 in the conserved region 2 domain. A threonine residue is present at similar positions in all three mammalian Raf family members and may represent a regulatory site for these proteins.