Changes in Viability, Cell Composition, and Enzyme Levels During Starvation of Continuously Cultured (Ammonia-Limited) Selenomonas ruminantium

Under nitrogen (ammonia)-limited continuous culture conditions, the ruminal anaerobe Selenomonas ruminantium was grown at various dilution rates (D). The proportion of the population that was viable increased with D, being 91% at D = 0.5 h⁻¹. Washed cell suspensions were subjected to long-term nutrient starvation at 39°C. All populations exhibited logarithmic linear declines in viability that were related to the growth rate. Cells grown at D = 0.05, 0.20, and 0.50 lost about 50% viability after 8.1, 4.6, and 3.6 h, respectively. The linear rates of decline in total cell numbers were dramatically less and constant regardless of dilution rate. All major cell constituents declined during starvation, with the rates of decline being greatest with RNA, followed by DNA, carbohydrate, cell dry weight, and protein. The rates of RNA loss increased with cells grown at higher D values, whereas the opposite was observed for rates of carbohydrate losses. The majority of the degraded RNA was not catabolized but was excreted into the suspending buffer. At all D values, S. ruminantium produced mainly lactate and lesser amounts of acetate, propionate, and succinate during growth. With starvation, only small amounts of acetate were produced. Addition of glucose, vitamins, or both to the suspending buffer or starvation in the spent culture medium resulted in greater losses of viability than in buffer alone. Examination of extracts made from starving cells indicated that fructose diphosphate aldolase and lactate dehydrogenase activities remained relatively constant. Both urease and glutamate dehydrogenase activities declined gradually during starvation, whereas glutamine synthetase activity increased slightly. The data indicate that nitrogen (ammonia)-limited S. ruminantium cells have limited survival capacity, but this capacity is greater than that found previously with energy (glucose)-limited cells. Apparently no one cellular constituent serves as a catabolic substrate for endogenous metabolism. Relative to losses in viability, cellular enzymes are stable, indicating that nonviable cells maintain potential metabolic activity and that generalized, nonspecific enzyme degradation is not a major factor contributing to viability loss.     

The TMK1 gene from Arabidopsis codes for a protein with structural and biochemical characteristics of a receptor protein kinase.

Genomic and cDNA clones that code for a protein with structural and biochemical properties similar to the receptor protein kinases from animals were obtained from Arabidopsis. Structural features of the predicted polypeptide include an amino-terminal membrane targeting signal sequence, a region containing blocks of leucine-rich repeat elements, a single putative membrane spanning domain, and a characteristic serine/threonine-specific protein kinase domain. The gene coding for this receptor-like transmembrane kinase was designated TMK1. Portions of the TMK1 gene were expressed in Escherichia coli, and antibodies were raised against the recombinant polypeptides. These antibodies immunodecorated a 120-kD polypeptide present in crude extracts and membrane preparations. The immunodetectable band was present in extracts from leaf, stem, root, and floral tissues. The kinase domain of TMK1 was expressed as a fusion protein in E. coli, and the purified fusion protein was found capable of autophosphorylation on serine and threonine residues. The possible role of the TMK1 gene product in transmembrane signaling is discussed.     

Toxicity of abamectin to cockroaches (Dictyoptera: Blattellidae, Blattidae).

Abamectin was fed to German cockroaches, Blattella germanica (L.), in non-choice tests. LT50s and LC50s were estimated by probit analysis. The LT50s for the German cockroach ranged from 4.4 to 1.7 d for males, from 9.0 to 2.4 d for females, and from 4.4 to 1.6 d for nymphs for bait concentrations of abamectin between 0.0025 and 0.0500%. The LC50s of abamectin were 0.0110 and 0.0040% from males, 0.0240 and 0.0090% for females, and 0.0200 and 0.0080% for nymphs at 3 and 6 d, respectively. The LT50 values of 0.0550% abamectin bait were 3.4, 3.4, 2.4, 7.5, 2.9, and 4.5 d for Periplaneta americana (L.), P. fuliginosa (Serville), P. brunnea Burmeister, P. australasiae (F.), Blatta orientalis L., and Supella longipalpa (Serville). Although the bait was effective against various cockroach species, time to death for the larger species was longer than for the German cockroach. In preference tests in which male German cockroaches were allowed to feed on rat chow or abamectin bait, all died within 5 d of exposure to abamectin bait. Abamectin bait consumption was not significantly lower than that of untreated rat chow. Arena tests with 0.0550% abamectin bait resulted in 31-75% mortality of German cockroaches after 9 d, with most control being achieved by treating harborages with the bait. The hydramethylnon standard resulted in 65% mortality after 9 d.     

Kininogen and Kinin in Experimental Spinal Cord Injury

Activation of the kallikrein-kinin system has been implicated in the pathogenesis of vasogenic brain edema and posttraumatic vascular injury. We determined the levels of kininogen and kinin in an experimental spinal cord injury model in the rat. Kininogen content in traumatized cord segments increased in a time-dependent manner. Western blot analysis showed that the kininogen in traumatized cord comigrates with 68K low-molecular-weight kininogen or T-kininogen. Trypsin treatment of the kininogen in traumatized cord released both bradykinin and T-kinin, which were separated by HPLC and quantified with a kinin radioimmunoassay. Endogenous kinin levels in the frozen spinal cord also increased up to 40-fold 2 h after injury as compared with controls. The results demonstrate an increased accumulation of kininogen and its conversion to vasoactive kinins in experimental spinal cord injury.     

Contrasting Neurochemical Interactions of Tiletamine, a Potent Phencyclidine (PCP) Receptor Ligand, with the N-Methyl-D-Aspartate-Coupled and -Uncoupled PCP Recognition Sites

Neurochemical interactions of tiletamine, a potent phencyclidine (PCP) receptor ligand, with the N-methyl-D-aspartate (NMDA)-coupled and -uncoupled PCP recognition sites were examined. Tiletamine potently displaced the binding of [3H]1-(2-thienyl)cyclohexylpiperidine with an IC50 of 79 nM without affecting sigma-, glycine, glutamate, kainate, quisqualate, or dopamine (DA) receptors. Like other PCP ligands acting via the NMDA-coupled PCP recognition sites, tiletamine decreased basal, harmaline-, and D-serine-mediated increases in cyclic cGMP levels and induced stereotypy and ataxia. Tiletamine was nearly five times more potent than PCP at inhibiting the binding of 3-hydroxy[3H]PCP to its high-affinity NMDA-uncoupled PCP recognition sites. However, following parenteral administration, dizocilpine maleate (MK-801), ketamine, PCP, dexoxadrol, and 1-(2-thienyl)cyclohexylpiperidine HCl, but not tiletamine, increased rat pyriform cortical DA metabolism and/or release, a response modulated by the NMDA-uncoupled PCP recognition sites. Pretreatment with tiletamine did not attenuate the MK-801-induced increases in rat pyriform cortical DA metabolism, a result suggesting that tiletamine is not a partial agonist of the NMDA-uncoupled PCP recognition sites in this region. However, following intracerebroventricular administration (100-500 micrograms/rat), tiletamine increased pyriform cortical DA metabolism with a bell-shaped dose-response curve. These data indicate a differential interaction of tiletamine with the NMDA-coupled and -uncoupled PCP recognition sites. The paradoxical effects of tiletamine suggest that tiletamine might activate receptor(s) or neuronal pathways of unknown pharmacology.     

Analysis of the oligosaccharides on androgen-binding proteins: Implications concerning their role in structure/function relationships

Rat androgen-binding protein (rABP), human testosterone-binding globulin (hTeBG) and rabbit (rb) TeBG are heterodimeric proteins. The source of the heterogeneity arises from the differential glycosylation of a common protein core. This glycosylation results in a heavy subunit (more glycosylation) and a light subunit (less glycosylation). Glycosylation is one factor responsible for multiple charged species seen when rABP, hTeBG, and rbTeBG are analyzed by two-dimensional gel electrophoresis. Enzymatic digestion with the endoglycosidase, peptide: N-glycosidase F indicated that all three proteins have asparagine (Asn)-linked oligosaccharides as their major glycan substituent. Treatment with exoglycosidases provided evidence for terminal sialic acid, galactose and mannose and N-acetylglucosamine residues. About 16–22% of the mass of the heavy subunit and about 8–14% of the mass of the light subunit is contributed by carbohydrate.

Serial lectin chromatography indicated that rABP is glycosylated differently from hTeBG and rbTeBG. About 40% of the rABP contains tri and tetraantennary complex oligosaccharides, while only about 20% of the hTeBG and TeBG from pregnant rabbits contains these types of glycans. About 9% of the TeBG from male rabbits bears these types of oligosaccharides. All of the biantennary complex oligosaccharides on rABP are fucosylated on the chitobiose core, but only 8% of those on hTeBG and none of those on rbTeBG are fucosylated in this manner. All three proteins are glycosylated at more than one site. The data indicate that the proteins may have more than one type of oligosaccharide on them. It is likely that differences in glycosylation are responsible for different physiological roles of the proteins.     

Human immunodeficiency virus type 1 and type 2 protease monomers are functionally interchangeable in the dimeric enzymes.

Human immunodeficiency virus type 1 (HIV-1) and HIV-2 proteases are dimers of identical subunits. We made a construct for the expression of recombinant one-chain HIV-2 protease dimer, which, like the previously described one-chain HIV-1 protease dimer, is fully active. The constructs for the one-chain dimers of HIV-1 and HIV-2 proteases were modified to produce hybrid one-chain dimers consisting of both HIV-1 and HIV-2 protease monomers. Although the monomers share only 47.5% sequence identity, the hybrid one-chain dimers are fully active, suggesting that the folding of both HIV-1 and HIV-2 protease monomers is functionally similar.     

Effects of dietary fat on cholesterol movement between tissues in CBA/J and C57BR/cdJ mice.

Differences in dietary fats cause differences in cholesterol metabolism in mice. CBA/J mice are resistant to diet-induced hypercholesterolemia and atherosclerosis; they adjust hepatic hydroxymethyl-glutaryl-CoA reductase activity (HMGR) to maintain homeostasis; C57BR/cdJ mice are susceptible, but young animals are thought to maintain homeostasis by changing fecal excretion of sterols. Compartmental modelling of movement of [4-14C]cholesterol was used to analyze movement of cholesterol between serum and liver, heart, and carcass in mice fed 40 en% fat, polyunsaturated to saturated fatty acid ratio (P/S) = 0.24 (US74) or 30 en% fat, P/S = 1 (MOD). Dietary effects were quite pronounced, while strain effects were more subdued. The C57/cdJ animals appear to regulate the overall cholesterol balance by reducing synthesis, as do the CBA/J animals, even though synthesis is not reduced to the same degree as in the CBA/J animals. Both diet and strain influence the whole-animal turnover rate, with slower turnover occurring for C57BR/cdJ animals and animals fed the US74 diet.     

The effect of light fluence rate in photodynamic therapy of normal rat brain.

This paper reports the effect of incident light fluence rate on the depth to which necrotic lesions are produced by photodynamic therapy (PDT) in the brains of normal Fisher rats. The rats were injected intraperitoneally with Photofrin (12.5 mg kg-1) 48 h prior to PDT with a fixed incident fluence of 35 J cm-2. The treatment was performed at 10, 50, 100, and 200 mW cm-2 and also in a periodic manner (30 s "on" at 100 mW cm-2, 30 s "off"). The depth to which necrosis occurred was determined 24 h after treatment by microscopic examination of tissue sections. No differences were found in the depth to which necrosis was produced by any of the five irradiation schedules. This finding is discussed in the context of other published dose-rate experiments.     

In vivo analysis of plant RNA structure: soybean 18S ribosomal and ribulose-1,5-bisphosphate carboxylase small subunit RNAs

A method to investigate the structure of RNA molecules within intact plant tissues has been developed. The RNA structures are analyzed using dimethyl sulfate (DMS), which modifies substituents of adenine and cytosine residues within single-stranded regions of RNA molecules. Reactive sites are identified by primer extension analysis. Using this procedure, an analysis of the secondary structure of the cytoplasmic 18S ribosomal RNA in soybean seedling leaves has been completed. DMS modification data are in good agreement with the phylogenetic structure predicted for soybean 18S rRNA. However, there are a few notable exceptions where residues thought to be involved in double-stranded regions in all 18S rRNAs are strongly modified in soybean leaf samples. These data taken together with the phylogenetic structure suggest that alternate structures may exist in vivo.The further applicability of this technique is demonstrated by comparing the modification pattern obtained in vivo to that obtained in vitro for a particular mRNA molecule encoding the small subunit of ribulose-1,5-bisphosphate carboxylase. The results obtained are compared to a predicted minimum energy secondary structure. The data indicate that the conformation of RNA molecules within the cell may not be reflected in a structural analysis of purified mRNA molecules.