The Pichia pastoris HIS4 gene: nucleotide sequence,creation of a non-reverting his4 deletion mutant,and development of HIS4-based replicating and integrating plasmids

We have obtained a clone of the Pichia pastoris HIS4 gene and have determined its nucleotide sequence. Based upon its deduced amino-acid sequence, the product of the P. pastoris HIS4 gene has the same structural organization as the Saccharomyces cerevisiae His4 protein and appears to encode a trifunctional enzyme catalyzing the second (phosphoribosyl-ATP pyrophosphohydrolase), third (phosphoribosyl-AMP cyclohydrolase), and tenth (histidinol dehydrogenase) steps in histidine biosynthesis. The chromosomal copy of the HIS4 gene was disrupted by homologous recombination, creating the strain SGY58. The his4 deletion mutation in this strain lacks the entire coding region of this gene and has a reversion rate that is undetectable. A set of complementary plasmids that carry the HIS4 gene was also developed. Among these are nine E. coli-P. pastoris shuttle vectors that transform the His4 deletion mutant at high efficiency and an integration vector for creating site-specific alterations of the P. pastoris genome.     

Ovulation triggering in clomiphene citrate-stimulated cycles: Human chorionic gonadotropin versus a gonadotropin releasing hormone agonist

Purpose To compare the use of human chorionic gonadotropin (hCG) to a gonadotropin releasing hormone (GnRH) agonist, nafarelin, in initiating ovulation and supporting the luteal phase after priming with clomiphene.Methods In 26 infertile women 50 mg clomiphene citrate produced a preovulatory-size follicle. Then, 11 women were randomized to receive two 400-g doses of nafarelin intranasally 16 h apart, and 15 women were injected intramuscularly with 5000 IU of hCG (luteal day 0 = LD0). Starting on LD6, 7 more 400-g doses of nafarelin were repeated on an every 16-h schedule or a single 2500 IU dose of hCG was given, respectively. Serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E₂), progesterone (P), and hCG were measured. On LD13, endometrium was evaluated with ultrasonography and biopsy in 19 nonpregnant women.Results As judged by a threefold rise in serum LH, an LH surge was detected on LD1 in all 11 nafarelin patients, but in only 8 hCG patients (P = 0.01). LH and FSH levels were significantly higher on LD1, 7, and 8 and were significantly suppressed on LD13 in the nafarelin group. All patients had mid-luteal P levels greater than 10 ng/ml and luteal phases longer than 13 days. Significantly different luteal E₂ or P levels were noted only on LD13, with lower values in the nafarelin group. Pregnancies were achieved in 3 of 11 nafarelin cycles and 2 of 15 hCG cycles. Luteal phase defects were also similar: 4 of 8 nafarelin patients and 7 of 11 hCG patients.Conclusion Nafarelin or hCG in conjunction with clomiphene can result in viable pregnancies, but is associated with low pregnancy rates and a high incidence of luteal phase defects.     

Self-Directed Treatment of Intermittent Heartburn: A Randomized, Multicenter, Double-Blind, Placebo-Controlled Evaluation of Antacid and Low Doses of an H(2)-Receptor Antagonist (Famotidine)

BACKGROUND: Heartburn, a common symptom, is self-treated with oral antacids. Efficacy of antacids has not been demonstrated for individual, spontaneous heartburn episodes. METHODS: We conducted a double-blind, randomized, placebo-controlled, parallel-group study of self-directed treatment for episodic heartburn comparing famotidine (FAM) 5, 10, or 20 mg and antacid (11 mEq ANC) to placebo (PBO) during a 4-week period. Twenty-nine US investigators enrolled a total of 565 outpatients, ages 18--81 years (mean 44.1 years) with heartburn but not seeking care for heartburn. Treatment of spontaneous heartburn episodes was permitted as needed, up to twice daily, with self-administered test drug. An open-label, backup antacid was provided to use if test drug did not provide adequate relief. Patients assessed heartburn relief hourly and recorded use of backup antacid. Relief was defined as complete relief of symptoms without the use of backup antacid. RESULTS: The media proportion of episodes relieved was: PBO, 41%; FAM 5 mg, 59%, 0.05 less-than-or-equal p < 0.10; FAM 10 mg, 70%, p < 0.001; FAM 20 mg, 69%, p < 0.001; antacid, 62%, p < 0.05 (p-values versus PBO). Supplemental analyses incorporating time to relief confirmed that famotidine and antacid provided more rapid and more frequent relief than placebo (odds ratio for relief relative to PBO: FAM 5 mg, 1.55, p = 0.003; FAM 10 mg, 1.94, p < 0.001; FAM 20 mg, 2.13, p < 0.001; antacid 1.57, p = 0.003). The tolerability profile was similar with famotidine, antacid, and placebo. CONCLUSIONS: The positive results with antacid demonstrated for the first time the efficacy of antacid in self-treatment of individual heartburn episodes and provided internal validation of this study paradigm. Patients in this study self-medicated effectively using low doses of famotidine on an as needed basis for spontaneous episodes of heartburn.     

Oral ondansetron decreases vomiting after tonsillectomy in children

Vomiting is a common, unpleasant aftermath of tonsillectomy in children. Intraoperative intravenous ondansetron (OND) reduces vomiting after this operation. Our doubleblind, placebocontrolled, randomized investigation studied the effect of the oral form of OND on vomiting after outpatient tonsillectomy in children. We studied 233 healthy children age 2–14 yr undergoing elective tonsillectomy. Subjects were given placebo (PLAC) or OND 0.1 mg · kg^?1 rounded off to the nearest 2 mg one hr before surgery. Anaesthesia was induced with either propofol or halothane/N₂O. Vecuronium 0.1 mg · kg^?1 was administered at the discretion of the anaesthetist. Anaesthesia was maintained with halothane/N₂O, 50 μg · kg^?1 midazolam iv and 1–1.5 mg · kg^?1 codeine im. At the end of surgery, residual neuromuscular blockade was reversed with neostigmine and atropine. All episodes of inhospital emesis were recorded by nursing staff. Rescue antiemetics in the hospital were 1 mg · kg^?1 dimenhydrinate ivfor vomiting × 2 and 50 μg · kg^?1 droperidol iv for vomiting × 4. Parents kept a diary of emesis after discharge. Postoperative pain was treated with morphine, codeine and/or acetaminophen. The two groups were similar with respect to demographic data, induction technique and anaesthesia time. Oral OND (n = 109) reduced postoperative emesis from 54% to 39%, P < 0.05. This effect was most dramatic inhospital, where 10% of the OND-patients and 30% of the PLAC-group vomited, P < 0.05. The OND-subjects required fewer rescue antiemetics, 7% vs 17%, P < 0.05. In conclusion, oral ondansetron decreased the incidence of vomiting after outpatient tonsillectomy in children.     

Comparison of the polyvinyl alcohol sponge and expanded polytetrafluoroethylene subcutaneous implants as models to evaluate wound healing potential in human beings

Our current understanding of the complex processes involved in wound healing is based mainly on studies of animal models. Although this information has been useful, it may not totally reflect the response found in human beings. For example, human beings have a tendency to either "overheal," as seen in keloids and hypertrophic scar formation, or have deficient healing, as seen in chronic ulcer formation. No animal models are available to analyze these human clinical pathologic conditions. Therefore the objective of this study was to analyze the wound healing response in a large population (n = 40) of normal healthy human beings as a first step to begin studies of abnormal human wound healing. Simultaneously, a comparison was made between the polyvinyl alcohol implant and the expanded polytetrafluoroethylene implant model. Under sterile conditions with the use of local anesthesia, two preweighed polyvinyl alcohol implants and two standard 6 cm expanded polytetrafluoroethylene implants were placed subcutaneously in the upper arm of each subject. High-performance liquid chromatography was used to quantitate isoleucine and hydroxy-l-proline in acid hydrolysates of each implant. Isoleucine was used as an indicator of protein content in the tissue sample, whereas hydroxyproline reflected collagen content. No infectious or hemorrhagic complications were found in the 40 volunteers included in the study. No significant difference was found in isoleucine or hydroxy-l-proline content between postoperative day 7 polyvinyl alcohol implants and day 14 polyvinyl alcohol implants. In contrast, both isoleucine and hydroxy-l-proline content were significantly increased in day 14 expanded polytetrafluoroethylene implants compared with day 7 implants (p < 0.005 and p < 0.001, respectively). In addition, the ratio of hydroxy-l-proline to isoleucine was significantly increased in day 14 expanded polytetrafluoroethylene implants compared with day 7 expanded polytetrafluoroethylene and both day 7 and day 14 polyvinyl alcohol implants (p < 0.001). This observation suggests that by 14 days implantation of expanded polytetrafluoroethylene stimulated an increased deposition of collagen. No significant differences were found in the hydroxy-l-proline to isoleucine ratios among day 7 expanded polytetrafluoroethylene, day 7 polyvinyl alcohol, and day 14 polyvinyl alcohol implants. Histologic analyses correlated with the biochemical findings. These results suggest that expanded polytetrafluoroethylene may be the preferred implant for studies designed to examine pathologic processes associated with retarded wound healing. In contrast, the polyvinyl alcohol implant may be better suited for studies where a low background response is required. Moreover, the extreme variability in normal healthy volunteers seen in this study correlates clinically with the finding that, among the normal adult human population, there is a heterogeneous wound healing response.     

Lack of influence of test meal fatty acid composition on the contribution of intestinally-derived lipoproteins to postprandial lipaemia.

The extent and duration of postprandial lipaemia have been linked to risk of CHD but the influence of dietary variables on, and the relative contributions of, exogenous (chylomicron) and endogenous (VLDL) triacylglycerols to the total lipaemic response have not been comprehensively evaluated. In the present study the triacylglycerol, apolipoprotein (apo) B-48 and retinyl ester (RE) responses to three test meals of varying monounsaturated (MUFA) and saturated fatty acid (SFA) content were measured in the triacylglycerol-rich lipoprotein (TRL) fraction of plasma (rho = 1.006 g/ml) for 9 h after meal consumption. Fifteen healthy normolipidaemic young men consumed, on separate occasions, three test meals which were identical apart from their MUFA and SFA contents. Expressed as a percentage of total energy the MUFA/SFA contents of the meals were: (1) 12%/17%; (2) 17%/12% and (3) 24%/5%. The contribution of the intestinally-derived lipoproteins (chylomicrons) to the lipaemic response was investigated by determining the time to reach peak concentration and the total and incremental areas under the time response curves (AUC and incremental AUC) for RE, apoB-48 and triacylglycerol in the TRL fraction. No significant differences in these measurements were observed for the three meals. However, visual comparison of the postprandial responses to the three meals suggested that as meal MUFA content increased there was a tendency for the triacylglycerol, apoB-48 and RE responses to become biphasic as opposed to the typical monophasic response seen with the 12% MUFA/17% SFA meal. Comparison of the apoB-48 and RE responses for the three test meals confirmed other workers' findings of delayed entry of RE relative to apoB-48 in TRL. The value of the two markers in investigating dietary fat absorption and metabolism is discussed.     

Antibiotic resistance in nosocomial isolates in Scotland

In this first multi-centre study in Scotland, 1028 consecutive Gram-negative and staphylococci strains were obtained from four major teaching hospitals. E. coli was the most common organism among both intensive care units (ICUs) (39%) and non-ICU strains (46.6%). The prevalence of antibiotic resistance among E. coli was always higher in isolates from ICUs than non-ICUs: ceftazidime (14.1%, 7.2%), ceftriaxone (12.7%, 6.1%), cefotaxime (15.5%, 8.7%), cefuroxime (28.8%, 20.8%), amoxicillin plus clavulanic acid (52.2%, 38.8%) and gentamicin (7.0%, 2.8%). The highest incidences of resistance were identified among Enterobacter/Citrobacter spp. from ICUs; 43.8%, 41.7%, 45.8%, 54.2%, 87.5% and 10.4% of these organisms were resistant to ceftazidime, ceftriaxone, cefotaxime, cefuroxime, amoxicillin plus clavulanic acid and gentamicin, respectively.     

Effect of vitamin C on glycosylation of proteins.

Twelve nondiabetic subjects consumed 1 g/day vitamin C for 3 mo. A fasting blood sample was taken at the start of the study and at the end of each month for the measurement of plasma and intraerythrocyte glucose, vitamin C, glycosylated hemoglobin (affinity chromatography and electrophoresis), and glycosylated albumin (affinity chromatography). Although there were no significant changes in fasting glycemia, glycosylated hemoglobin (affinity chromatography) decreased 18%, from 6.18 +/- 0.48% (mean +/- SD) at the start to 5.05 +/- 0.50% (P less than 0.0001) after 3 mo, whereas, HbA1 measured by electrophoresis increased 16%, from 6.17 +/- 0.61 to 7.16 +/- 0.59% (P less than 0.0001) in this period. Glycosylated albumin decreased 33%, from 1.56 +/- 0.24 to 1.04 +/- 1.01% (P less than 0.0001) after 3 mo. This discrepancy between glycosylated hemoglobin measured by electrophoresis and affinity chromatography was due to methodological differences between the two techniques, with affinity chromatography measuring "true" glycosylated hemoglobin. The greater decrease found with glycosylated albumin was probably due to the different distribution of vitamin C between plasma and within the erythrocyte, levels after 1 mo of supplementation being 109 +/- 19 and 59 +/- 9 microM, respectively (P less than 0.001). This indicates that administration of oral vitamin C may inhibit the glycosylation of proteins in vivo by a competitive mechanism.