Solubilization of active and stable receptors for vasoactive intestinal peptide from rat liver

Vasoactive intestinal peptide (VIP) receptors were solubilized from rat liver using the zwitterionic detergent CHAPS. Optimal conditions of solubilization were obtained with 5 mM CHAPS and 2.5 mg protein/ml. The binding of 125I-VIP to CHAPS extracts was time- and pH-dependent, saturable and reversible. The following order of potency of unlabeled VIP-related peptides for inhibiting 125I-VIP binding was observed: VIP greater than helodermin greater than peptide histidine isoleucine amide (PHI) greater than rat growth hormone releasing factor (rGRF) greater than secretin. This peptide specificity is identical to that of rat liver membrane-bound receptors. VIP binding activity in the CHAPS extract was destroyed by trypsin or dithiothreitol in accordance with the known sensitivity of membrane-bound receptors to these agents. VIP receptors in CHAPS extracts were stable for at least 5 days at 4 degrees C. Scatchard analysis of equilibrium binding data indicated the presence in CHAPS extracts of high (H) and low (L) affinity binding sites with the following characteristics: KdH = 0.27 nM and BmH = 34 fmol/mg protein; KdL = 51 nM and BmL = 1078 fmol/mg protein. The guanine nucleotide GTP inhibited 125I-VIP binding to soluble receptors and enhanced the dissociation of soluble VIP-receptor complexes, suggesting that GTP-binding proteins were functionally associated with VIP receptors in solution. Gel filtration of solubilized VIP receptors on Sephacryl S-300 revealed a single binding component with a Stokes radius of 6.1 nm. It is concluded that active VIP receptors can be extracted from liver membranes by CHAPS. The availability of this CHAPS-soluble, stable and functional receptor from a tissue which can be obtained in large amounts represents a major step toward the purification of VIP receptors.     

Extracellular complementation of a developmental mutation implicates a small sporulation protein in aerial mycelium formation by S. coelicolor

The filamentous bacterium S. coelicolor differentiates by forming aerial hyphae, which protrude into the air and metamorphose into chains of spores. Aerial hyphae formation is associated with the production of a small, abundant protein, SapB, which is present in a zone around colonies of differentiating bacteria. Production of SapB is impaired in bld mutants, which are blocked in aerial hyphae formation, but not in whi mutants in which spore formation is prevented. We report that aerial hyphae formation by a newly identified bld mutant is restored by juxtaposition of the mutant near colonies of SapB-producing bacteria or by the application of the purified protein near mutant colonies. These observations implicate SapB in aerial mycelium formation and suggest that SapB is a morphogenetic protein that enables hyphae on the surface of colonies to grow into the air.     

Stimulation of the adenylyl cyclase activity in human endometrial membranes by VIP and related peptides

Vasoactive intestinal peptide (VIP) has been shown to stimulate adenylyl cyclase activity in human endometrial membranes. The effect was dependent on the time and temperature of incubation as well as on the concentration of endometrial membrane proteins in the medium. In the presence of 1 M GTP, half-maximal stimulation of adenylyl cyclase activity was observed at 25.0±7.0 nM VIP, whereas the maximal activity (at 1 M VIP)corresponded to an increase of about 140% with respect to basal values (7.5±0.6 pmol cyclic AMP/min/mg of protein). However, the maximal stimulation of adenylyl cyclase activity was obtained with helodermin (1 M) that increased the activity by 170% over the basal. The relative potency of VIP-related peptides upon the adenylyl cyclase activity was: helodermin (ED₅₀=1.8±1.4 nM)>VIP(ED₅₀=25.0±7.0 nM)>PHI (ED₅₀=725.0±127.2 nM). Secretin had a faint effect upon the adenylyl cyclase activity and glucagon was completely inefficient at this level. The presence of _s and _i subunits of G proteins in human endometrium was detected by immunoblot. Preliminary results showed the presence of two classes of¹²⁵I-VIP receptors in human endometrial membranes with the following stoichoimetric parameters: high affinity receptor (Kd=2.0 nM, binding capacity 0.1 pmol VIP/mg protein) and low affinity receptor (Kd=0.43 M, binding capacity 13.1 pmol VIP/mg protein). The present results together with the known presence of VIP in human uterus and the actions of this neuropeptide in the adjacent myometrial tissue support the idea that VIP and related peptides may have a role in human endometrium.     

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

Reproductive performance of lactating dairy cows treated with cloprostenol at the time of insemination

The effect of intravenous cloprostenol treatment at the time of insemination on reproductive performance was consecutively evaluated in three different subpopulations of high producing lactating dairy cows: Study (1) early postpartum synchronized and fixed-time inseminated (about 50 days in milk) cows (n = 379: 187 control and 192 treated cows); Study (2) presumed high fertility cows first inseminated between 90 and 120 days postpartum (n = 248: 124 control and 124 treated cows); and Study (3) heat stressed repeat breeder cows (n = 183: 93 control and 90 treated cows). Data were analyzed using multiple regression methods. Study 1: Parity (primiparous versus multiparous), milk production, body condition score at AI, insemination season (cool versus warm period) and treatment were included in the analysis as potential factors affecting ovulation, double ovulation, return to estrus, and pregnancy to first AI and to second AI (first AI plus return AI) rates. Logistic regression analysis indicated that the final model for ovulation rate only included the interaction (P = 0.002) between insemination season and treatment. Cloprostenol treatment at insemination led to a 4.2-fold increase in the ovulation rate in cows inseminated during the warm period. There were no significant effects of treatment, parity, milk production, body score or the insemination season on the return to estrus rate. The only variables included in the final logistic model for double ovulation and pregnancy to first AI rates were treatment and season, respectively. Treatment led to a 2.6-fold increase (P = 0.001) in the double ovulation rate, whereas cows inseminated in the warm period were 2.1 times less likely (P = 0.007) to become pregnant at first AI compared to those inseminated in the cool season. The variables included in the final logistic model for the pregnancy rate to second AI were treatment and season. Cloprostenol given at AI increased the risk of pregnancy 1.9 times (P = 0.002), and cows inseminated during the warm season were two times less likely to become pregnant (P = 0.003). No significant interactions were found among these three dependent variables (double ovulation and pregnancy to first and to second AI rates). Study 2: Logistic regression analysis of all the dependent variables: return to estrus, and pregnancy to first and to second AI (first AI plus return to AI) rates indicated no significant effects of treatment, parity, days in milk, milk production or body score at AI. No significant interactions were found. Study 3: The final model for the pregnancy rate only included the interaction between parity (primiparous versus multiparous) and treatment. Days in milk, milk production and insemination number showed no significant effect on pregnancy rate. Cloprostenol treatment at insemination increased the pregnancy rate in primiparous repeat breeder cows (odds ratio: 3.6). The treatment group and parity showed significant (P < 0.0001) interaction. This interaction suggests that cloprostenol treatment of primiparous cows at insemination might enhance pregnancy yet have no effect in multiparous cows. Our findings indicate that cloprostenol administered at insemination promotes ovulation and double ovulation in lactating dairy cows. Cloprostenol treatment showed no benefit in cows with acceptable reproductive performance, suggesting that cloprostenol treatment at AI may only be useful in cows in which stress factors affect ovulation and in repeat breeder cows.     

Development of genetic techniques for the psychrotrophic fish pathogen Flavobacterium psychrophilum

Flavobacterium psychrophilum, a member of the Cytophaga-Flavobacterium-Bacteroides group, is an important pathogen of salmonid fish. Previous attempts to develop genetic techniques for this fastidious, psychrotrophic bacterium have met with failure. Here we describe the development of techniques for the genetic manipulation of F. psychrophilum and the identification of plasmids, selectable markers, a reporter system, and a transposon that function in several isolates of this fish pathogen. The antibiotic resistance genes ermF, cfxA, and tetQ function in F. psychrophilum. Cloning vectors based on the F. psychrophilum cryptic plasmid pCP1 which carried these selectable markers were introduced by conjugation from E. coli, resulting in antibiotic-resistant colonies of F. psychrophilum. Conjugative transfer of DNA into F. psychrophilum was strain dependent. Efficient transfer was observed for two of the seven strains tested (THC02-90 and THC04-90). E. coli lacZY functioned in F. psychrophilum when expressed from a pCP1 promoter, allowing its development as a reporter for studies of gene expression. Plasmids isolated from F. psychrophilum were efficiently introduced into F. psychrophilum by electroporation, but plasmids isolated from E. coli were not suitable for transfer by this route, suggesting the presence of a restriction barrier. DNA isolated from F. psychrophilum was resistant to digestion by Sau3AI and BamHI, indicating that a Sau3AI-like restriction modification system may constitute part of this barrier. Tn4351 was introduced into F. psychrophilum from E. coli and transposed with apparent randomness, resulting in erythromycin-resistant colonies. The techniques developed in this study allow for genetic manipulation and analysis of this important fish pathogen.     

Assistance of maltose binding protein to the in vivo folding of the disulfide-rich C-terminal fragment from Plasmodium falciparum merozoite surface protein 1 expressed in Escherichia coli

The C-terminal fragment of Plasmodium falciparum merozoite surface protein 1 (F19) is a leading candidate for the development of a malaria vaccine. Successful vaccination trials on primates, immunochemistry, and structural studies have shown the importance of its native conformation for its protective role against infection. F19 is a disulfide-rich protein, and the correct pairing of its 12 half-cystines is required for the native state of the protein. F19 has been produced in the Escherichia coli periplasm, which has an oxidative environment favorable for the formation of disulfide bonds. F19 was either expressed as a fusion with the maltose binding protein (MBP) or directly addressed to the periplasm by fusing it with the MBP signal peptide. Direct expression of F19 in the periplasm led to a misfolded protein with a heterogeneous distribution of disulfide bridges. On the contrary, when produced as a fusion protein with E. coli MBP, the F19 moiety was natively folded. Indeed, after proteolysis of the fusion protein, the resulting F19 possesses the structural characteristics and the immunochemical reactivity of the analogous fragment produced either in baculovirus-infected insect cells or in yeast. These results demonstrate that the positive effect of MBP in assisting the folding of passenger proteins extends to the correct formation of disulfide bridges in vivo. Although proteins or protein fragments fused to MBP have been frequently expressed with success, our comparative study evidences for the first time the helping property of MBP in the oxidative folding of a disulfide-rich protein.     

Simultaneous detection of Aeromonas salmonicida,Flavobacterium psychrophilum,and Yersinia ruckeri,three major fish pathogens,by multiplex PCR

A multiplex PCR assay based on the 16S rRNA genes was developed for the simultaneous detection of three major fish pathogens, Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri. The assay proved to be specific and as sensitive as each single PCR assay, with detection limits in the range of 6, 0.6, and 27 CFU for A. salmonicida, F. psychrophilum, and Y. ruckeri, respectively. The assay was useful for the detection of the bacteria in artificially infected fish as well as in fish farm outbreaks. Results revealed that this multiplex PCR system permits a specific, sensitive, reproducible, and rapid method for the routine laboratory diagnosis of infections produced by these three bacteria.     

Purification and properties of a new psychrophilic metalloprotease (Fpp2) in the fish pathogen Flavobacterium psychrophilum

To go further into the characterization of the proteolysis exocellular system of the salmonid pathogen Flavobacterium psychrophilum, the purification and characterization of a novel protease designated Fpp2 (F. psychrophilum protease 2) was undertaken. A protease (Fpp2) hydrolyzing azocasein was purified. The Fpp2 can be defined as a metalloprotease, it had an estimated molecular mass of 62 kDa with calcium playing an important role in the thermostability of the enzyme. Proteolytic activity was optimal at pH 6.0-7.0 and 24 degrees C and activation energy for the hydrolysis of azocasein was determined to be 5.4 kcal mol(-1), being inactive at temperatures above 42 degrees C. All these results are characteristic of 'cold adapted enzymes'. Fpp2 proved to be a broad range hydrolytic enzyme because in optimal conditions it was able to hydrolyze matrix and muscular proteins. It can be concluded that the Fpp1, a previously characterized 55 kDa metalloprotease, and the Fpp2 protease were produced under different physiological conditions and were immunologically as well as biochemically different.