Phase memory relaxation times of spin labels in human carbonic anhydrase II: pulsed EPR to determine spin label location.

Phase memory relaxation times (T(M) or T(2)) of spin labels in human carbonic anhydrase II (HCA II) are reported. Spin labels (N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide, IPSL) were introduced at cysteines, by site-directed mutagenesis at seven different positions in the protein. By two pulse electron paramagnetic resonance (EPR), electron spin echo decays at 45 K are measured and fitted by stretched exponentials, resulting in relaxation parameters T(M) and x. T(M) values of seven positions are between 1.6 micros for the most buried residue (L79C) and 4.7 micros for a residue at the protein surface (W245C). In deuteriated buffer, longer T(M) are found for all but the most buried residues (L79C and W97C), and electron spin echo envelop modulation (ESEEM) of deuterium nuclei is observed. Different deuterium ESEEM patterns for W95C and W16C (surface residue) indicate differences in the local water concentration, or accessibility, of the spin label by deuterium. We propose T(M) as a parameter to determine the spin label location in proteins. Furthermore, these systems are interesting for studying the pertaining relaxation mechanism.     

Life history tradeoffs in relation to the degree of polyandry and developmental pathway in Pieris napi (Lepidoptera, Pieridae)

Pieris napi females have different heritable reproductive tactics. Polyandrous females have higher lifetime fecundity, whereas monandrous ones start to reproduce at a faster rate. Butterfly larvae are time‐constrained in seasonal environments. Thus, polyandry is expected to be associated with fast juvenile development, which may result in biased mortality due to physiological costs. We compared how females with varying degrees of polyandry allocate between duration of larval period and achievable size in directly developing and over‐wintering generations. Offspring survival and growth were monitored under a high density and low quality diet. Polyandrous females developed at a faster rate than monandrous ones, regardless of developmental pathway. The growth rate of female offspring correlated with their mothers’ degree of polyandry, which underpins polyandry and monandry as distinct strategies with life history differences reaching beyond mating frequency. The high growth rate of polyandrous females resulted in a short larval period among directly developing females, and in large size within an over‐wintering cohort. A change in either the duration of the larval period or pupal mass had no significant effect on the other, emphasising that growth rate is not necessarily a simple outcome of the tradeoff between development time and size at maturity. The correlation between the degree of polyandry and juvenile growth rate diminished when larvae were exposed to environmental stress, which offers an explanation why juvenile mortality was decoupled from mating tactic. We conclude that polyandry is a strategy that allows larvae to utilise optimal conditions in a more effective way than monandry. As a consequence, polyandrous females either achieve larger size or they mature faster. This gives them a double advantage over monandrous ones within an over‐wintering generation or diminishes the effects of asynchronous hatching of offspring within a directly developing generation. Possible costs of high growth rate are discussed.     

Exopolysaccharide-producing strains of thermophilic lactic acid bacteria cluster into groups according to their EPS structure

AIMS: To compare galactose-negative strains of Streptococcus thermophilus and Lactobacillus delbrueckii subspecies bulgaricus isolated from fermented milk products and known to produce exopolysaccharides (EPSs). METHODS AND RESULTS: The structures of the EPSs were determined using nuclear magnetic resonance (NMR) and their genetic relationships determined using restriction endonuclease analysis (REA) and random amplification of polymorphic DNA (RAPD). Similar groupings were apparent by REA and RAPD, and each group produced an EPS with a particular subunit structure. CONCLUSION: Although none of the strains assimilated galactose, all inserted a high proportion of galactose into their EPS when grown in skimmed milk, and fell into three distinct groups. Significance and Impact of the Study: This information should help in an understanding of genetic exchanges in lactic acid bacteria.     

Human lymphoblastoid cell lines secreting antibodies with restricted HLA specificity

Peripheral B lymphocytes obtained from three healthy individuals who had been immunized against peripheral blood lymphocytes from appropriate HLA-incompatible donors were transformed by the use of Epstein-Barr virus. The transformed blastoid B cells were repeatedly subcultured by means of cluster picking, and the HLA antibody-producing cultures were identified by testing the culture supernatants by means of the cytotoxicity assay, using the corresponding donor cells. Thus far, four cell lines that secrete cytotoxic HLA antibodies (MP1, 3, 4, and 5) have been established. Specific immunoabsorption experiments revealed that the antibody activity is carried by lambda-type IgM for MP1, by kappa-type IgM for MP3 and MP5, and by both for MP4. Specificity analysis of a panel of HLA-pretyped cells indicated that MP1 detects DQw2, whereas MP5 recognizes B7. The specificity of MP3 was similar to a DQ specificity termed DC5 (probably equivalent to TA10) but not the same. In the case of MP4, both of the lambda-type and kappa-type antibodies appeared to be directed toward new HLA class 11 determinants.Abbreviations used in this paper HLA human major histocompatibility - EBV Epstein-Barr virus - B-LCL Blymphoblastoid cell line - NA not absorbed - PBS phosphate-buffered saline - SPA Sepharose protein A - NRS normal rabbit serum     

Septal deafferentation produces continuous rhythmic slow activity (theta) in the rat hippocampus

Hippocampal EEG was recorded in behaving rats in which the brain stem afferents to the septal region were previously damaged. In these animals rhythmic slow activity (RSA or theta) was continuously present, including drinking and immobility. The average frequency of RSA, however, was significantly higher during running (8-9 Hz) than during drinking or awake motionless state (6-8 Hz). In normal rats irregular sharp waves, rather than RSA were present during drinking and immobility. The results suggest that brain stem afferents are necessary to suppress the rhythmic firing of septal "pacemaker" cells.     

Evidence for a sex pheromone metabolizing cytochrome P-450 mono-oxygenase in the housefly

Direct evidence is presented for the role of a cytochrome P-450 monooxygenase (called mixed-function oxidase, or polysubstrate mono-oxygenase, PSMO) in the metabolism of the sex pheromone (Z)-9-tricosene to its corresponding epoxide and ketone in the housefly. A secondary alcohol, most likely an intermediate in the conversion of the alkene to the ketone, was also tentatively identified. The results of in vivo and in vitro experiments showed that the PSMO inhibitors, piperonyl butoxide (PB) and carbon monoxide, markedly inhibited the formation of epoxide and ketone from (9,10-³H) (Z)-9-tricosene. An examination of the relative rates of (Z)-9-tricosene metabolism showed that males exhibited a higher rate of metabolism than females with the antennae of males showing the highest activity of any tissue/organ examined. The major product from all tissues/organs was the epoxide. Data from experiments with subcellular fractions showed that the microsomal fraction had the majority of enzyme activity, which was strongly inhibited by PB and CO and required NADPH and O₂ for activity. A carbon monoxide difference spectrum with reduced cytochrome showed maximal absorbance at 450 nm and allowed quantification of the cytochrome P-450 in the microsomal fraction of 0.410-nmol cytochrome P-450 mg^?1 protein. Interaction of (Z)-9-tricosene with the cytochrome P-450 resulted in a type I spectrum, indicating that the pheromone binds to a hydrophobic site adjacent to the heme moiety of the oxidized cytochrome P-450.     

Activation of mercuric reductase by the substrate NADPH

Preincubation of the oxidized form of the flavoenzyme mercuric reductase with the reducing substrate, NADPH, or with a high concentration of cysteine (30 mM) results in a substantial increase of the catalytic activity as measured in a standard spectrophotometric assay. Also NADH has some activating effect but NADP+ or EDTA have no effect. In the presence of 1 mM cysteine only one equivalent of NADPH per FAD seems to be required for full activation which occurs after an incubation time of about 10 min. Activated mercuric reductase appears to be stable under anaerobic conditions but eventually returns to the original level of activity in the presence of oxygen. The activated state seems to be stabilized by 1 mM cysteine. Activation of mercuric reductase does not seem to be correlated with a change in the number of reactive thiol groups. The chemical nature of the activation process is not yet understood. Stopped-flow studies have shown that the nonactivated enzyme is practically inactive prior to contact with the substrates. The enzyme is gradually activated during the assay. The kinetics of activation of the 'native' enzyme is biphasic but 'clipped' enzyme, lacking an 85-residue N-terminal domain, is activated in a single first-order process. The progress curves obtained with preactivated enzyme are approximately exponential even at saturating concentrations of NADPH (Km = 0.4 microM at 25 degrees C, pH 7.3) and Hg2+ (Km = 3.2 microM in the presence of 1 mM cysteine). The initial rates yield kcat values of about 13 s-1 per FAD molecule (25 degrees C, pH 7.3). We find no evidence for a thiol-dependent change from a rapid to a slow kinetic phase. The shape of the progress curves presumably depends on product inhibition, but NADP+ is not a sufficiently effective inhibitor to explain the effect fully.     

Peptides and neurotransmission in the central nervous system

Radioimmunoassays of brain extracts have shown that several peptides occur in high concentrations in the CNS. The releasing-factor peptides TRF, LRF, somatostatin, CRF and GRF have the highest concentration in the hypothalamic extracts. High levels of somatostatin, CCK octapeptide, neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) are found in cortical extracts. Substance P, CCK, NPY, and enkephalins are present in high concentrations in basal ganglia and mesolimbic areas. Pharmacological doses of these peptides result in several behavioural and vegetative effects. Immunocytochemical studies show that the CNS peptides are localised in neurones and in synaptic vesicles. In vitro studies with brain tissues show that peptides are capable of modifying the ongoing classical neurotransmission. In depressive patients several neuropeptides (CCK, CRF and NPY) have been shown to have low CSF levels. Patients dying of senile dementia have low cortical levels of somatostatin, CRF and substance P. In schizophrenic patients CCK peptides have shown to improve some symptoms. At present the therapeutic potentials of peptides are poorly known. More studies are required to understand their role in neurotransmission and related pathological states.     

Proton-metal distance determination in cobalt(II) stellacyanin by 1H nuclear magnetic resonance relaxation measurements including Curie-spin effects: a proposed structure of the metal-binding region

1H nuclear magnetic resonance (1H NMR) experiments on Co(II)-substituted stellacyanin have been performed. Large paramagnetic hyperfine shifts are observed, the whole spectrum covering a range of 190 ppm. Experiments were mainly performed at 270 MHz from which temperature and pH* dependencies of the out-shifted resonances were reported, as well as determinations of the longitudinal (T1) and transverse (T2) relaxation times. These relaxation times are among other things, dependent on the individual proton-metal distance, and the aim of this work has been to determine these distances, by use of the Solomon-Bloembergen equations modified to include the so-called "Curie spin". The application of this method to a protein has not been reported earlier. Experiments were also performed at 100, 400, and 500 MHz in order to estimate the size of the Curie spin from the field dependence of the line widths. Furthermore, determination of the values for the rotational correlation time, tau r, and the effective magnetic moment, mu eff, was necessary for the present approach. With apostellacyanin, tau r was found to be (6.0 +/- 0.4) X 10-8 s. From the paramagnetic susceptibility of Co(II) stellacyanin, the value (4.53 +/- 0.03)beta was determined for mu eff. The proposed assignments of several paramagnetically out-shifted resonances. the proton-metal distances obtained, and the known peptide sequence of stellacyanin have allowed us to build a three-dimensional model of the metal site and its surrounding structure consistent with all the experimental data. It is revealed that both histidine ligands bind the metal with their 3-nitrogens. Also we find strong indications that a second sulfur atom is actually binding the metal, this being the long-sought-after fourth ligand. The model suggests that this sulfur belongs to Cys-59, which together with Cys-93 constitutes the disulfide bridge known to be present in the structure. A potential fifth ligand, an amide oxygen from Asn-47, is also found.