High CO(2) Requiring Mutant of Anacystis nidulans R(2)

Some physiological characteristics of a mutant (E₁) of Anacystis nidulans R₂, incapable of growing at air level of CO₂, are described. E₁ is capable of accumulating inorganic carbon (C_i) internally as efficiently as the wild type (R₂). The apparent photosynthetic affinity for C_i in E₁, however, is some 1000 times lower than that of R₂. The kinetic parameters of ribulose 1,5-bisphosphate carboxylase/oxygenase from E₁ are similar to those observed in R₂. The mutant appears to be defective in its ability to utilize the intracellular C_i pool for photosynthesis and depends on extracellular supply of Ci in the form of CO₂. The very high apparent photosynthetic K_m (CO₂) of the mutant indicate a large diffusion resistance for CO₂. Data obtained here are used to calculate the permeability coefficient for CO₂ between the bulk medium and the carboxylation site of cyanobacteria.     

Behavioral treatment of irritable bowel syndrome: A 1-year follow-up study

Sixteen clients afflicted with irritable bowel syndrome (IBS) were reassessed 1 year following completion of a multicomponent treatment package incorporating progressive muscle relaxation, thermal biofeedback, cognitive therapy, and IBS education. For the 14 patients who kept a 2-week symptom diary, significant reductions in ratings of abdominal pain and tenderness, diarrhea, and flatulence were obtained comparing pretreatment and follow-up symptom-diary ratings. Eleven of 14 clients were improved over pretreatment levels, 57% met the criteria for clinical improvement of at least a 50% reduction in major symptom scores, and all but 1 of 16 rated themselves as subjectively improved.     

Desensitization of mouse Leydig cells in vivo: evidence for the depletion of cellular cholesterol

The study presents a characterization of the refractory state in purified mouse Leydig cells desensitized by a single injection of human chorionic gonadotropin (hCG) in vivo. The treatment of mice with 1 microgram hCG i.p. for 48 h followed by Leydig cell isolation and purification resulted in a decrease in the maxima of hCG-induced cAMP accumulation and testosterone production by approximately 70% and approximately 55%, respectively, when compared to cells of control mice. Despite a 55% reduction in 125I-hCG binding sites, the sensitivity of stimulation was not changed. The refractoriness in testosterone production in vitro was also present when the Leydig cells were stimulated with cholera toxin or dibutyryl cAMP; however, it was not observed when testosterone production was induced by the addition of pregnenolone or 20 alpha- and 22(R)-hydroxycholesterol. Mouse lipoproteins, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) in natural composition, were also able to overcome the steroidogenic block (although not always completely). On the basis of the cholesterol content of the lipoproteins, the two classes were similarly effective. They increased maximal hCG-induced testosterone production not only in desensitized cells, but also in control cells (by 80-100%), whereas their effect on basal testosterone production was negligible. In desensitized cells from hCG-treated mice (2 micrograms i.p., 48 h) cellular unesterified and esterified cholesterol were decreased by 21% and 81%, respectively, when compared to control cells. This loss occurred in the face of unchanged plasma cholesterol levels. In conclusion, our data indicate that the impaired steroidogenesis in mouse Leydig cells desensitized in vivo by a single injection of hCG is the result of a depletion in cellular cholesterol, rather than of an impaired conversion of cholesterol to testosterone.     

Amino acid sequence of Manduca sexta adipokinetic hormone elucidated by combined fast atom bombardment (FAB)/tandem mass spectrometry

Combination of Fast Atom Bombardment Tandem Mass Spectrometry with Amino Acid Analysis assigns the amino acid sequence of the Manduca sexta adipokinetic hormone as pGlu-Leu-Thr-Phe-Thr-Ser-Ser-Trp-GlyNH2. Similarities and differences with other invertebrate hormones and with mammalian glucagon are discussed.     

Microsome- and hepatocyte-mediated mutagenicity of hydroxyurea and related aliphatic hydroxamic acids in V79 Chinese hamster cells

The potential of N-hydroxyurea to induce gene mutations in V79 Chinese hamster cells was investigated. Upon metabolic activation by liver microsomes from phenobarbital-treated rats or by isolated rat hepatocytes co-cultured with the V79 cells, hydroxyurea caused a concentration-dependent increase in the frequency of HGPRT-deficient mutants. Hydroxyurea was not mutagenic in the absence of metabolic activation. Addition of catalase inhibited microsome-mediated mutagenicity, indicating that hydrogen peroxide was involved in the formation of the mutagenic DNA lesion. Acetohydroxamic acid and N-hydroxyurethane also induced hepatocyte-mediated mutagenicity, suggesting that the potential to elicit metabolism-dependent mutagenicity may be a common property of aliphatic hydroxamic acids.     

Detection of sugar-binding proteins in membrane-depleted nuclei

Nuclear sugar-binding proteins were detected in membrane-depleted nuclei isolated from hamster BHK cells and mouse L 1210 leukemia cells by means of fluorescein-labelled neoglycoproteins. In fluorescence microscopy, the fluorescence was seen throughout the nucleus but was generally brighter over the nucleoli than over the rest of the nucleus. Flow cytofluorometry analysis demonstrated the presence of nuclear sugar-binding proteins for synthetic glycoproteins associated with different sugar residues. Among the nine neoglycoproteins used, four neoglycoproteins (namely alpha-rhamnosylated, alpha-glucosylated, N-acetyl-beta-glucosaminylated and alpha-mannosylated-6P-serum albumin) strongly labelled nuclei. Various controls strongly argue for the specificity of the nuclear labelling. The possibility that some of the sugar-binding proteins might correspond to endogenous nuclear lectins is considered.     

Irreversible binding to the receptor of bacteriophages T5 and BF23 does not occur with the tip of the tail.

Treatment of purified tails of bacteriophage T5 with 0.05% sodium dodecyl sulfate specifically removed pb2, a protein of 108,000 molecular weight (108K), from the tail. Although these tails were devoid of the single straight tail fiber, they still inhibited adsorption of T5 to Escherichia coli cells. Reconstitution of these tails with pb2 increased the efficiency of inhibition of T5 adsorption. Treatment of tails with 0.1% sodium dodecyl sulfate removed, in addition to pb2, a protein of 67K from phage T5 and one of 60K from phage BF23. These tails failed to inhibit phage adsorption, and no reconstitution was achieved. Reconstitution of T5 tails with pb2 from BF23, and of BF23 tails with pb2 from T5, did not alter the host receptor specificity of the tails. Binding of untreated T5 tails to small FhuA receptor particles revealed that binding occurred with the conical part of the tail and that pb2 was most likely released from the tail upon binding. From these results and from recent observations with T5-BF23 hybrid phages (K.J. Heller, Virology 139:11-21, 1984), we conclude that the receptor-binding proteins of T5 and BF23 are the 67K and 60K proteins, respectively, and that they are not located at the tip of the tail but rather at or near the site where the straight tail fiber is attached to the conical part of the tail.     

Structural aspects of intranuclear matrix disintegration upon RNase digestion of HeLa cell nuclei

The organization of the intranuclear elements observed in histone-depleted (2 M NaCl-extracted) HeLa cell nuclei was investigated by means of electron microscopy and two-dimensional gel electrophoresis. This work was mainly aimed at verifying whether or not an intranuclear skeleton or matrix existed, which could explain the stable attachment of RNA to the residual nuclear structure after high-salt extraction, and its three-dimensional organization. We compared the ultrastructure and the polypeptide composition of RNA-containing and RNA-depleted (RNase-treated) nuclear residues, and we visualized intermediate stages of RNase action on the intranuclear material. We showed that this material was made of two types (fibrillar and granular) of salt-resistant RNP components equally sensitive to RNase when the enzyme was used prior to high-salt extraction. At least in our material and under our experimental conditions, no intranuclear matrix could be distinguished from the residual RNP material. Our results further suggest that formation of such a matrix is a path-dependent phenomenon.     

Isolation of a cAMP receptor protein from yeast mitochondria (Mr 45000) and comparison with mitochondrial RNA polymerase (Mr 45000)

We have isolated a cAMP-binding protein from highly purified yeast mitochondria by affinity chromatography. It is a lipophilic protein of molecular mass 45 000 Da, which is tightly membrane-bound and localized on the outer surface of the inner membrane. It can be solubilized in active form under mild conditions. The cAMP receptor resembles mitochondrial RNA polymerase prepared as described by Levens et al. [(1981) J. Biol. Chem. 256, 1474] in a surprisingly large number of properties including molecular mass. Comparison of the two proteins revealed that the polypeptide previously considered as RNA polymerase is, in fact, a mitochondrial cAMP receptor protein.     

Cloning and expression of Klebsiella pneumoniae genes coding for citrate transport and fermentation.

Three Escherichia coli clones (DH1/Cit1, DH1/Cit2 and DH1/Cit3) capable of utilizing citrate as a sole carbon source were isolated from a cosmid bank of Klebsiella pneumoniae wild-type DNA. Two of these clones (DH1/Cit1 and DH1/Cit2) only grew aerobically on citrate minimal medium, the third clone (DH1/Cit3) could also be cultured under fermentative conditions. The aerobic as well as the anaerobic generation times of the three clones were from 4.5 to 7 h. Whereas clone DH1/Cit3 showed a pronounced lag phase on citrate when the cells were pre-grown in medium without citrate, clone DH1/Cit1 immediately started growth, while with clone DH1/Cit2 a short lag phase could be observed upon transfer to citrate minimal medium. Restriction analyses of the three plasmids showed that no common fragments had been cloned. The length of the inserts were 13 and 6 kb for the aerobic Cit+ clones and 27 kb (10 kb) for the anaerobic one. Cultures of the anaerobic Cit+ clone were analyzed by immunoblotting techniques and shown to contain oxaloacetate decarboxylase, which confers citrate utilization under anaerobic conditions to K. pneumoniae. Enzyme assays demonstrated the active state of this biotin-containing membrane protein. The specific activity in vesicle preparations from the E. coli clone was 30% of the wild-type K. pneumoniae vesicles. Citrate acts as an inducer of enzyme protein synthesis in the E. coli clone as it does in K. pneumoniae.