Inoculum-dependent division lag of Bacillus cultures and its relation to an endogenous factor(s) ("schizokinen")

Lankford, C. E. (The University of Texas, Austin), James R. Walker, James B. Reeves, N. H. Nabbut, B. R. Byers, and R. J. Jones. Inoculum-dependent division lag of Bacillus cultures and its relation to an endogenous factor(s) ("schizokinen"). J. Bacteriol. 91:1070-1079. 1966.-When cells of Bacillus megaterium, grown on Brain Heart Infusion Agar, were inoculated into a chemically defined medium, they exhibited a division lag which was an inverse function of inoculum size. The addition of filtrates of cultures from the same medium eliminated the inoculum-dependent component of lag, but not an inoculum-independent residual lag of constant duration. Culture filtrate of B. subtilis var. niger not only eliminated its inoculum-dependent lag but also was required to sustain exponential division. Dose-response and "growth time" bioassays, developed to measure lag-reducing activity of filtrates, demonstrated accumulation of active filtrate factor to a "critical" concentration prior to division initiation. Addition of this concentration to cultures eliminated the inoculum-dependent lag. Accumulation of the factor ceased temporarily at onset of division, but excretion was resumed later during exponential growth. Accumulation of a lag-reducing, cell-associated factor followed a similar course. Chromatographic and bioautographic analyses of culture filtrates of B. megaterium indicated that a single substance was primarily responsible for their activity. Results of dose-response tests for reciprocal activities of filtrates of different Bacillus species and strains suggested production of different factors by some, and of different quantities of similar factors by others. It is proposed that such endogenous factors which are synthesized and accumulate to a population-dependent concentration as a requisite to initiation and maintenance of division be designated as "schizokinens."     

Comparison of α1-Adrenergic Receptor-Stimulated Inositol Phosphate Formation in Primary Neuronal and Glial Cultures

alpha 1-Adrenergic receptor binding sites and norepinephrine-stimulated 3H-inositol phosphate (3H-InsP) accumulation were measured in primary cultures of neurons and glia from 1-day-old rat brains. The density of alpha 1-adrenergic receptor binding sites was approximately three times higher in membranes from neurons compared to glia. Although norepinephrine was slightly more potent in stimulating 3H-InsP formation in neurons than in glia, the maximal response was greater in glial cells. Norepinephrine-stimulated 3H-InsP formation remained constant for [3H]inositol prelabelling periods of 1-14 days in neurons, whereas the response increased with time in glia and was maximal after 7-10 days of prelabelling. Both the incorporation of [3H]inositol into lipid and basal levels of 3H-InsPs were lower in glial cells than in neurons, which accounted for the greater percent stimulation in glia. Pretreatment with phenoxybenzamine decreased norepinephrine-stimulated 3H-InsP formation in a dose-dependent manner in both neurons and glia by decreasing the maximal response without altering potency. HPLC separation showed that similar types of 3H-InsPs were accumulated in neurons and glial cells. These results demonstrate that alpha 1-adrenergic receptors exist on both neurons and glial cells and activate 3H-InsP accumulation in both cell types. Although receptor density is higher in neurons than in glia, the 3H-InsP response is higher in glia. This difference does not appear to be due to different receptor reserves, but may be due to differential coupling mechanisms in the two cell types.     

Physical mapping of the von Recklinghausen neurofibromatosis region on chromosome 17

The von Recklinghausen neurofibromatosis (NF1) locus has been linked to chromosome 17, and recent linkage analyses place the gene on the proximal long arm. NF1 probably resides in 17q11.2, since two unrelated NF1 patients have been identified who possess constitutional reciprocal translocations involving 17q11.2 with chromosomes 1 and 22. We have used a somatic-cell hybrid from the t(17;22) individual, along with other hybrid cell lines, to order probes around the NF1 locus. An additional probe, 17L1, has been isolated from a NotI linking library made from flow-sorted chromosome 17 material and has been mapped to a region immediately proximal to the translocation breakpoint. While neither NF1 translocation breakpoint has yet been identified by pulse-field gel analysis, an overlap between two probes, EW206 and EW207, has been detected. Furthermore, we have identified the breakpoint in a non-NF1 translocation, SP-3, on the proximal side of the NF1 locus. This breakpoint has been helpful in creating a 1,000-kb pulsed-field map, which includes the closely linked NF1 probes HHH202 and TH17.19. The combined somatic-cell hybrid and pulsed-field gel analysis we report here favors the probe order D17Z1-HHH202-TH17.19-CRYB1-17L1-NF1- (EW206, EW207, EW203, L581, L946)-(ERBB2, ERBA1). The agreement in probe ordering between linkage analysis and physical mapping is excellent, and the availability of translocation breakpoints in NF1 should now greatly assist the cloning of this locus.     

cDNA cloning and localization of the mouse leukosialin gene (Ly48) to chromosome 7

Mouse leukosialin, previously known as the 3E8 antigen, is expressed primarily on cells of the hematopoietic and lymphoid lineages and is shown to be the mouse homologue to the human leukosialin/sialophorin and rat W3/13 molecules. A partial leukosialin cDNA clone was isolated via cross-species hybridization with a portion of a human leukosialin cDNA. This mouse cDNA clone was used to demonstrate that the leukosialin isoforms are encoded by a single mRNA species of approximately 4.2 kilobases (kb) and that the leukosialin gene is located on chromosome 7. Based on these results, mouse leukosialin is given the designation Ly48.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M30693.     

Microtubule Depolymerization Inhibits Ethanol-Induced Enhancement of GABAA Responses in Stably Transfected Cells

Abstract: We studied whether microtubule organization is important for actions of ethanol on GABA_A ergic responses by testing the effects of microtubule depolymerization on ethanol enhancement of GABA action in mouse L(tk⁻) cells stably transfected with GABA_A receptor α₁β₁γ_2L subunits. The microtubule-disrupting agents colchicine, taxol, and vinblastine completely blocked ethanol-induced enhancement of muscimol-stimulated chloride uptake. β-Lumicolchicine, a colchicine analogue that does not disrupt microtubules, had no effect on ethanol action. Colchicine did not alter the potentiating actions of flunitrazepam or pentobarbital on muscimol-stimulated chloride uptake. Thus, colchicine specifically inhibited the potentiating action of ethanol. From these findings, we conclude that intact microtubules are required for ethanol-induced enhancement of GABA_A responses and suggest that a mechanism involving microtubules produces posttranslational modifications that are necessary for ethanol sensitivity in this cell system.     

Comparing the spectroscopic and electrochemical properties of ruthenium and osmium complexes of the tridentate polyazine ligands 2,2′:6′,2″-terpyridine and 2,3,5,6-tetrakis(2-pyridyl)pyrazine

A number of osmium and ruthenium complexes of the tridentate ligands 2,2′:6′,2″-terpyridine (tpy) and 2,3,5,6-tetrakis(2-pyridyl)pyrazine (tpp) have been prepared and characterized by our laboratory. All these complexes possess metal based oxidations and ligand based reductions localized on each polyazine ligand. Polymetallic complexes bridged by the tpp ligand exhibit two sequential tpp based reductions prior to the reduction of other polyazine ligands in these complexes. The spectroscopy of these complexes is dominated by ligand based π-π^* transitions in the ultraviolet and MLCT (metal-to-ligand charge transfer) bands terminating on each polyzine ligand in the visible. For the complexes reported herein the lowest lying optical transitionis a M → BL CT band. For most of the complexes reported, occupation of this excited state gives rise to an observable emission at room temperature. For ruthenium complexes of these tridentate ligands, the presence of a low-lying LF state shortens the excited state lifetimes of these chromophores. This gives rise to ruthenium complexes that display shorter excited state lifetimes than the analogous osmium based systems. Incorporation of tpp based chromophores into polymetallic frameworks leads to the production of bimetallic species with long-lived excited states, 100 ns at room temperature. This makes these chromophores good candidates for the development of stereochemically defined supramolecular complexes. It is possible to measure an electrochemical HOMO-LUMO energy gap and a correlation between this electrochemically measured energy gap and the spectroscopic energy associated with this HOMO→LUMO transition are reported herein (HOMO== highest occupied molecular orbital, LUMO = lowest unoccupied molecular orbital).     

Fetal cells in maternal blood: recovery by charge flow separation

Fetal blood cells can be recovered from the maternal circulation by charge flow separation (CFS), a method that obviates the risks associated with amniocentesis and chorionic villus sampling. By CFS, we processed blood samples from 13 women carrying male fetuses, 2 carrying fetuses with trisomy 21, and 1 who had delivered a stillborn infant with trisomy 18. On average more than 2000 fetal nucleated red blood cells were recovered per 20-ml sample of maternal blood. Recovery of fetal cells was confirmed by fluorescence in situ hybridization with probes for chromosomes Y, 18 and 21. After culturing of CFS-processed cells, amplification by the polymerase chain reaction revealed Y-chromosomal DNA in clones from four of six women bearing male fetuses, but not in clones from three women bearing female fetuses. Received: 8 January 1996 / Revised: 22 March 1996     

Neuropeptide Y perfused in the preoptic area of rats shifts extracellular efflux of dopamine,norepinephrine and serotonin during hypothermia and feeding

This study examined the localized action of neuropeptide Y (NPY) on monoamine transmitter activity in the hypothalamus of the unrestrained rat as this peptide induced hypothermia, spontaneous feeding or both responses simultaneously. A guide tube was implanted in the anterior hypothalamic pre-optic area (AH/POA) of Sprague-Dawley rats. Then either control CSF vehicle or NPY in a dose of either 100 ng/μl or 250 ng/μl was perfused by push-pull cannulae in this structure in the fully sated, normothermic rat. Successive perfusions were carried out at a rate of 20 μl/min for 6.0 min with an interval of 6.0 min elapsing between each. Samples of perfusate were assayed by HPLC for their levels of dopamine (DA), norepinephrine (NE), serotonin (5-HT) and their respective metabolites. Whereas control CSF was without effect on body temperature (T_b) or feeding, repeated perfusions of NPY over 3.0 hr caused dose—dependent eating from 4 to 39 g of food, hypothermia of 0.9 to 2.3°C or both responses concurrently. As the rats consumed 11–39 g of food, the efflux of NE, MHPG, DOPAC and 5-HT was enhanced significantly, whereas during the fall in T_b the efflux of NE, DOPAC and 5-HIAA from the AH/POA increased. When the T_b of the rat declined simultaneously with eating behavior, the levels in perfusate of DOPAC and HVA increased significantly while MHPG declined. During perfusion of the AH/POA with NPY the turnover of NE declined while DA and 5-HT turnover increased during hypothermia alone or when accompanied by feeding. These results demonstrate that the sustained elevation in NPY within the AH/POA causes a selective alteration in the activity of the neurotransmitters implicated in thermoregulation, satiety and hunger. These findings suggest that both DA and NE comprise intermediary factors facilitating the action of NPY on neurons involved in thermoregulatory and ingestive processes. The local activity of NPY on hypothalamic neurons apparently shifts the functional balance of serotonergic and catecholaminergic neurons now thought to play a primary role in the control of energy metabolism and caloric intake.     

Genomic Analysis of a New Mammalian Distal-less Gene: Dlx7

We have cloned a new Dlx gene (Dlx7) from human and mouse that may represent the mammalian orthologue of the newt geneNvHBox-5.The homeodomains of these genes are highly similar to all other vertebrate Dlx genes, and regions of similarity also exist between mammalian Dlx7 and a subset of vertebrate Dlx genes downstream of the homeodomain. The sequence divergence between human and mouse Dlx7 in these regions is greater than that predicted from comparisons of other vertebrate Dlx genes, however, and there is little sequence similarity upstream of the homeodomain both between these two genes and with other Dlx genes. We present evidence for alternative splicing of mouseDlx7upstream of the homeodomain that may account for some of this divergence. We have mapped humanDLX7distal to the 5′ end of the HOXB cluster at an estimated distance of between 1 and 2 Mb by FISH. Both the human and the mouse Dlx7 are shown to be closely linked to Dlx3 in a convergently transcribed orientation. These mapping results support the possibility that vertebrate distal-less genes have been duplicated in concert with the Hox clusters.