Parasympathetic involvement in PAF-induced contraction in canine trachealis in vivo

We studied the contractile response elicited by platelet-activating factor (PAF) administered intra-arterially into the tracheal circulation of 34 dogs in vivo. A method that avoided tachyphylaxis encountered in prior investigations was developed for isometric measurement of multiple dose-response effects. PAF was a very potent contractile agent; active tension was elicited with 10(-11) mol ia PAF. To determine the mechanism by which contraction was induced, dose-response curves were generated in groups of five animals each treated with either 0.5 mg/kg (approximately 1.5 X 10(-5) mol) iv + 10(-3) mg/kg (3 X 10(-8) mol) ia atropine, 5 mg/kg iv indomethacin (INDO), or 7.5 mg/kg iv hexamethonium (HEX). After pretreatment with atropine, contraction still was elicited with 10(-11) mol ia PAF. However, maximal contraction was only 16.2 +/- 2.74 g/cm (vs. 35.7 +/- 5.74 g/cm for untreated controls; P less than 0.02). The dose at which maximal contraction was elicited after atropine was 10(-7) mol ia (vs. 1.9 X 10(-9) mol for controls; P less than 0.001). Pretreatment with INDO caused minimal attenuation, and HEX had no effect on the response elicited by ia PAF. We demonstrate a method for assessing the effects of PAF in central airways that avoids tachyphylaxis and permits dose-response studies in the same animal. We also demonstrate that PAF is an extremely potent mediator that elicits tracheal smooth muscle contraction at least in part by postganglionic activation of parasympathetic nerves. A direct contractile effect of PAF which is not related to secretion of products of the cyclooxygenase pathway is also suggested.     

Purification of Clostridium perfringens enterotoxins by high performance liquid chromatography

Abstract Treatment of Saccharomyces cerevisiae a cells with α-factor partially inhibits mannosylation of the high M _r mannoproteins, although there is an increase in the total amount of these molecules present in the wall. They show a similar mobility in SDS-acrylamide gels to those from untreated mnn2 cells. No other significant effects on wall mannoproteins have been observed, except a decrease in the amount of the 29 kDa species.     

Optimum autoregulator addition strategy for maximum virginiamycin production in batch culture of Streptomyces virginiae

Virginiae butanolides (VBs) are autoregulators of Streptomyces virginiae, which induce virginiamycin biosynthesis. Generally, autoregulators are synthesized by the microorganism itself during culture. Addition of chemically synthesized virginiae butanolide-C (VB-C), which is one of the VBs, can also control the induction time and the amount of virginiamycin production. The optimum concentration and shot-feeding time of VB-C for the maximum production of virginiamycins M and S were investigated in flasks and jar-fermentor batch cultures. VB-C addition later than 8 h from the start of culture induced not only virginiamycin M and S synthesis but also VB synthesis. Virginiamycin M and S production increased with the decrease of total VBs (produced VBs and added VB-C) concentration. That is, although VBs are needed to induce virginiamycin M and S synthesis, the amount of VB-C added should be such that as small an amount as possible of VBs is synthesized to achieve the maximum production of virginiamycins M and S. However, the VB-C addition earlier than 8 h from the start of culture showed no clear relationship between the amounts of VBs and virginiamycins M and S produced. In conclusion, the maximum production of virginiamycins M and S was attained by the shot addition of 5 mug/L VB-C at 8 h from the start of culture. The maximum value was about twofold that without VB-C addition. The optimum addition strategy of VB-C was confirmed by the jar-fermentor experiments. (c) 1995 John Wiley & Sons, Inc.     

Augmentation of respiratory mast cell secretion of histamine caused by vagus nerve stimulation during antigen challenge

We studied the effect of vagus nerve stimulation on the mast cell secretion of histamine after intraarterial (i.a.) administration of Ascaris suum antigen (AA) into the bronchial circulation of 10 randomly selected, natively allergic dogs in vivo. Respiratory mast cell response was measured as the arteriovenous difference (AVd) in histamine concentration across the bronchus. Plasma histamine concentration was determined simultaneously from right atrium, right ventricle, and femoral artery 60 and 15 sec before and 15, 30, 45, 60, 75, and 90 sec after i.a. injection of sham (Kreb-Henseleit) diluent, 1:100, and 1:30 concentrations of AA. The mean AVd in plasma histamine for five parasympathetically blocked animals (neural blockade with hexamethonium and beta-adrenergic blockade with propranolol) was 1.28 +/- 0.61 ng/ml (sham), 5.16 +/- 19.7 ng/ml (1:100 AA), and 36.6 +/- 11.1 ng/ml (1:30 AA). Substantial augmentation was obtained when AA was administered during parasympathetic stimulation in five other animals (beta-adrenergic blockade, no neural blockade), which was caused by continuous bilateral electrical stimulation of the vagus nerves. A mean AVd in plasma histamine of 110 +/- 27.6 ng/ml was obtained after 1:100 AA (p less than 0.001 vs parasympathetic blockade) and 166 +/- 32.4 ng/ml for 1:30 AA (p less than 0.001 vs parasympathetic blockade). Parasympathetic stimulation alone did not cause secretion of histamine. In contrast to the AVd response, parasympathetic stimulation did not augment nonrespiratory mast cell secretion after AA challenge. We conclude that vagus nerve stimulation augments secretion of histamine from respiratory mast cells during antigen challenge. We demonstrate that parasympathetic stimulation may potentiate the response to antigen challenge in central airways through augmented mast cell secretion of mediator.     

In vitro fertilization and development of frozen-thawed bovine oocytes.

Bovine oocytes were vitrified (V-oocytes) or frozen slowly (S-oocytes) at the germinal vesicle (GV) stage or after maturation in vitro (IVM) and their survival assessed morphologically and also by in vitro fertilization (IVF) and culture. The morphological survival of S-oocytes was 30.7% after freezing at the GV stage and 53.3% after IVM. The corresponding survival rates of V-oocytes were significantly lower, viz. 14.6 and 14.0%, respectively. The fertilization rate of S-oocytes frozen after IVM (51.0%) was lower than that of unfrozen controls (75.8%), but higher than after other treatments. Development continued in 16.0% of the fertilized S-oocytes, compared to 39.4% of control IVF zygotes and 1.6% developed into morulae or blastocysts (4.5% in controls). Only 0.8% of frozen-thawed GV stage oocytes and 4.6% of post-IVM V-oocytes cleaved after IVF and none formed morulae or blastocysts. Transfer of four embryos (two morulae and two blastocysts) derived from post-IVM S-oocytes into a recipient heifer resulted in pregnancy and the birth of twin calves.     

Sex ratio of early embryos fertilized in vitro with spermatozoa separated by percoll

Early bovine embryos were obtained by in vitro fertilization and sexing carried out by chromosome analysis. Separation of bovine X- and Y-bearing spermatozoa was performed using Percoll density gradient centrifugation and the enrichment of X-sperm proportion was investigated. Through treatment with vinblastin sulfate and podophyllotoxin, 880 (48.6%) of 1812 embryos at two- to seven-cell stages at 48 to 53 h after sperm-egg incubation produced metaphase spreads, and 399 (45.3%) of these were successfully sexed; the sexable rate reaching 53.4% for four-cell embryos. Sexing rates for embryos from the original sperm of two bulls were 69.6% (32/46) in Bull A and 54.2% (58/107) in Bull B. Embryos fertilized in vitro with sperm sedimented at the bottom of sperm centrifuged under conditions (I) 50 to 85% of Percoll, 15 °C; (II) 30 to 80%, 10 °C; (III) 30 to 80% 20 °C; (IV) 30 to 90%, 20 °C, gave rise to male sex ratios of (I) 58.3% in Bull A and 53.5% in Bull B, (II) 65.9% in Bull A, (III) 49.3% in Bull B and (IV)_66.7% in Bull B. In conclusion, Percoll density gradient centrifugation under these four conditions was unsuccessful in separating X- and Y-bearing bull spermatozoa.     

In vitro fertilization and cleavage capability of bovine follicular oocytes classified by cumulus cells and matured in vitro

Bovine oocytes were collected from ovaries obtained from an abattoir. They were classified according to the character of the cumulus cells using a stereomicroscope, and cultured in 25 mM Hepes buffered Tissue Culture Medium 199 supplemented with 10% fetal calf serum at 39 degrees C and inseminated by capacitated sperm. Maturation rates of Class A oocytes, with compact, dense cumulus cells; Class B, partially naked oocytes with thin cumulus layers or small remnants of cumulus cells and Class C, naked oocytes were 97.4% (38/39), 89.8% (106/118) and 52.9% (45/85), respectively. The fertilization rates for the three classes were 86.8%, 85.8% and 53.3%, respectively. The naked oocytes had a significantly lower fertilization rate than oocytes of the other two classes. Significantly more Class A oocytes cleaved (63.7%, 232/364) than those of Class B (29.5%, 36/122) and Class C (17.7%, 28/158).     

Association of the insulin-like growth factor1 gene with myocardial infarction in Japanese subjects

During adult life, the insulin/insulin-like growth factor1 (IGF1) signaling pathway plays an important role in cardiovascular function. Several reports have suggested that low baseline levels of IGF1 increase the risk of fatal ischemic heart disease. Thus, IGF1 may be involved in cardiovascular disease. The aim of the present study was to investigate the relationship between the human IGF1 gene and myocardial infarction (MI) in the Japanese population via the use of single nucleotide polymorphisms (SNPs). After selecting six SNPs in the human IGF1 gene (rs2162679, rs7956547, rs2288378, rs2072592, rs978458 and rs6218), we performed a case-control study using each of the SNPs and haplotypes in 320 MI patients and 307 non-MI controls. Multiple logistic regression analysis demonstrated that the GG+GA variant of rs2162679 (p=0.009) and the AA+GA variant of rs2072592 (p=0.026) exhibited a resistant effect for MI. The haplotype-based case-control study revealed that the frequency of the A-T-G-G haplotype for rs2162679-rs7956547-rs2072592-rs978458 was significantly higher in the MI group (47.3%) as compared to the non-MI group (41.4%) (p=0.037, odds ratio=1.270). The frequency of the A-T-G-T haplotype for rs2162679-rs7956547-rs978458-rs6218 was also significantly higher in the MI group (47.3%) as compared to the non-MI group (41.3%) (p=0.033, odds ratio=1.276). The current results suggest that specific SNPs and haplotypes can be utilized as genetic markers for MI risk or MI resistance. In addition, IGF1 or a neighboring gene might be associated with increased or decreased susceptibility to MI.     

Characterization of modification enzyme NukM and engineering of a novel thioether bridge in lantibiotic nukacin ISK-1

The lantibiotic nukacin ISK-1 is an antimicrobial peptide containing unusual amino acids such as lanthionine and dehydrobutyrine. The nukacin ISK-1 prepeptide (NukA) undergoes posttranslational modifications, such as the dehydration and cyclization reactions required to form the unusual amino acids by the modification enzyme NukM. We have previously constructed a system for the introduction of unusual amino acids into NukA by coexpression of NukM in Escherichia coli. Using this system, we describe the substrate specificity of NukM by the coexpression of a series of NukA mutants. Our results revealed the following characteristics of NukM: (1) its dehydration activity is not coupled to its cyclization activity; (2) its dehydration activity is site-specific; (3) the length of the substrate is important for its dehydration activity. Furthermore, we succeeded in introducing a novel thioether bridge in NukA by replacing an unmodified Ser at position 27 with a Cys residue.     

Lactic acid bacteria display on the cell surface cytosolic proteins that recognize yeast mannan

Fluorescent-labeled invertase, a hyperglycosylated mannoprotein from Saccharomyces cerevisiae, was found to bind to Lactococcus lactis IL1403 at acidic pH. Proteins on the cell wall of the bacterium affinity-purified using invertase as a ligand were identified to be heat shock proteins such as DnaK and GroEL and glycolytic enzymes such as pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase. DnaK bound to both the bacterium and yeast at pH 4 and aggregated them at above 0.1 mg/ml, whereas no significant difference between the circular dichroism spectra of DnaK at pH 4 and 7 was observed. These results indicate that the cytosolic proteins, including DnaK displayed on the cell wall, cause the lactic acid bacterium to adhere to the yeast.