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Biomedical subjects

J Heim

Publications and source records attributed to J Heim.

At least 19 recordsLinked to original sources
PubMed1992

Thrombolytic activity of two chimeric recombinant plasminogen activators (FK2tu-PA and K2tu-PA) in rabbits.

The aim of this study was to evaluate the thrombolytic activity of two hybrid plasminogen activators (HPAs) in a rabbit jugular vein thrombosis model. In the two HPAs the kringle-2 domain (K2tu-PA) or the finger and the kringle-2 domains (FK2tu-PA) of tissue-type plasminogen activator (t-PA) are linked to the catalytic protease domain of single chain urokinase type plasminogen activator (scu-PA). The two HPAs were compared with rt-PA and scu-PA on a weight/weight basis. K2tu-PA, FK2tu-PA, rt-PA and scu-PA were infused at doses of 0.4, 0.8 and 1.2 mg/kg over 3 h. Saline served as control. Saline produced 11 +/- 2% thrombolysis. The three doses of K2tu-PA led to 38 +/- 4%, 66 +/- 5% and 89 +/- 7% thrombolysis, respectively; the three doses of FK2tu-PA: 18 +/- 3%, 29 +/- 5% and 33 +/- 6%, respectively; the three doses of rt-PA 32 +/- 2%, 49 +/- 3% and 68 +/- 6%, respectively; the three doses of scu-PA 16 +/- 2%, 24 +/- 3% and 32 +/- 4%, respectively. K2tu-PA and rt-PA showed a statistically significant higher thrombolytic activity than FK2tu-PA and scu-PA at the three tested doses (p less than 0.01). The thrombolytic activity of K2tu-PA was significantly higher than rt-PA at the two higher doses (p less than 0.01). Both K2tu-PA and rt-PA produced a statistically significant reduction of fibrinogen, alpha 2-antiplasmin and plasminogen 3 h after the start of the infusions of the two higher doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Biogenesis of the yeast vacuole (lysosome). The precursor forms of the soluble hydrolase carboxypeptidase yscS are associated with the vacuolar membrane.

We have studied the structure, biosynthesis, intracellular routing, and vacuolar localization of carboxypeptidase ysCS in the yeast Saccharomyces cerevisiae. Nondenaturing polyacrylamide gel electrophoresis revealed two forms of carboxypeptidase yscS with different electrophoretic mobility. Antibodies specific for carboxypeptidase yscS recognized two glycoproteins of 77- and 74-kDa apparent molecular mass which differ by one N-linked carbohydrate residue. Both observations suggest that carboxypeptidase yscS exists in two catalytically active forms. The enzyme was found to be synthesized as two active high molecular mass precursor forms which are converted to the mature forms with a half-time of 20 min. The mature forms of carboxypeptidase yscS appeared soluble in the vacuolar lumen, while the precursor proteins accumulated tightly associated with the vacuolar membrane. The single hydrophobic domain present at the N terminus is believed to be responsible for the membrane association of the precursor molecules. Double mutants defective in proteinase yscA and proteinase yscB synthesize solely the carboxypeptidase yscS precursor forms. Correct proteolytic cleavage of the precursor forms was performed using purified proteinase yscB in vitro. Sec61, sec18, and sec7 mutants, conditionally defective in the secretory pathway, accumulate carboxypeptidase yscS precursor protein. Thus the carboxypeptidase yscS precursor molecules are delivered to the vacuole in a membrane bound form via the secretory pathway. After assembly into the vacuolar membrane, proteinase yscB presumably cleaves the precursor molecules to release soluble carboxypeptidase yscS forms into the lumen of the vacuole. The proposed mechanism is different from the delivery mechanism found for the other soluble vacuolar hydrolases in yeast.

Carboxypeptidases↗
PubMed1992

In-vitro cleavage of a fusion protein bound to cellulose using the soluble yscFs (Kex2) variant.

In order to show site-specific cleavage of fusion proteins with an engineered soluble yscF variant, we have constructed a fusion gene encoding eglinC from Hirudo medicinalis and the cellulose-binding domain from the Cellulomonas fimi exoglucanase (Cex). The two fusion partners were separated by a Lys-Arg-containing recognition sequence for the yeast endoprotease yscF (Kex2). The fusion protein (eglinC-Cex) was expressed intracellularly in Saccharomyces cerevisiae. After disruption of the cells eglin-C-Cex was shown to bind to cellulose when present in total crude cell lysates. This step efficiently removed the majority of contaminating host proteins. While immobilized on cellulose, eglin-C-Cex could be cleaved into the expected fragments. Upon cleavage the eglinC part was released from the cellulose, while the purification tag, i.e. the cellulose binding domain, stayed bound to the cellulose matrix.

Amino Acid Sequence↗
PubMed1992

[Therapeutic control of premedication with famotidine given on the evening before surgery for the prevention of pneumonitis in heart surgery patients].

Pulmonary damage after aspiration of acid gastric content is one of the major risks of general anaesthesia. Antisecretory therapy with different H2-antagonists can effectively decrease the risk of aspiration. The effective dosage and the method and timing of administration remain unclear. PATIENTS AND METHODS. A series of 38 patients scheduled for elective cardiac surgery (coronary artery bypass graft) were premedicated at random at 10:00 p.m. on the evening prior to surgery with famotidine 40 mg and flunitrazepam 0.5 mg p.o. or with flunitrazepam alone. Continuous intragastric pH monitoring (720 values/h) with a combined glass electrode was started immediately after induction and continued for at least 12 h after surgery, in most cases up to extubation. The time periods analyzed were the first 30 min after induction, time of surgery, 12 h after surgery, and time of extubation. For each period the number of pH values less than 2.5 and pH less than 4, mean and median, and significance of differences were calculated. STATISTICS. Test statistics were evaluated using the Wilcoxon test and the Mann-Whitney U-test. RESULTS. (see Table 1, Fig. 1). The mean time interval between oral premedication and induction of anaesthesia was 9.7 h. There were no differences between the groups concerning age and duration of surgery. The average age was 51 in the group of treated patients (n = 15) and 59 years in the control group (n = 23). The average duration of surgery was 3.36 h and 4.02 h. During induction and in the following 30 min, in the famotidine treatment group 28.7% of all pH values were pH less than 2.5, as against 45.4% in the control group (P = 0.08). Intraoperative values: 16.3% pH less than 2.5 against 24.7% (not significant); 12 h postoperative: 21.1% vs 17.8% and during extubation 40% vs 21% (not significant). During induction there were more pH values less than 2.5 in the control than in the treatment group (P = 0.08). Treated patients had nearly 30% pH values less than 2.5 and therefore, a persisting risk of acid aspiration. In both groups intragastric acidity decreased during surgery. Postoperatively there was no difference between the two groups, and during extubation more patients in the famotidine-treated group had values below pH 2.5. CONCLUSION. Prophylaxis of acid aspiration before, during and after cardiac surgery can be achieved by increasing the pH of the gastric content. The timing and the method of administration must be selected to a safe decrease in intragastric acidity before the induction of general anaesthesia. Famotidine 40 mg given orally at 10.00 p.m. on the evening before surgery is not a reliable means of decreasing intragastric acidity or, consequently, of preventing of acid aspiration syndrome.

Administration, Oral↗
PubMed1991

Alpha-factor-leader-directed secretion of recombinant human-insulin-like growth factor I from Saccharomyces cerevisiae. Precursor formation and processing in the yeast secretory pathway.

A synthetic gene coding for human-insulin-like growth factor I (IGFI) was fused to the leader sequence of yeast prepro-alpha-factor and expressed in Saccharomyces cerevisiae under the control of a glyceraldehyde-3-phosphate dehydrogenase promoter fragment. Recombinant IGFI was found inside yeast cells and secreted into the medium. The secreted IGFI migrated on SDS gels with the same electrophoretic mobility as authentic IGFI, i.e. at about 7.5 kDa. HPLC analysis of secreted IGFI revealed the presence of the correctly folded, genuine molecule as well as an isomeric byproduct of equal molecular mass but with two of the three disulfide bonds interchanged. Inside exponentially growing cells the 7.5-kDa IGFI was also found, along with up to four additional IGFI-related polypeptides of higher molecular mass. By endoglycosidase F treatment the three polypeptides between 19-26 kDa were converted to a single peptide of 17 kDa. Since this peptide also reacted with an anti-alpha-factor antibody, it represents most likely the unglycosylated alpha-factor--IGFI fusion precursor. Pulse-chase experiments established the precursor nature of the intracellular higher-molecular-mass IGFI species. Conversion of the primary translation product to the differently glycosylated IGFI precursor proteins and into the mature form occurred very rapidly, within 2 min. Rapid maturation was, however, not followed by an equally rapid secretion of the mature form into the medium: only after 30-40 min did IGFI appear outside the cells. We therefore postulate the presence of an as yet undefined Golgi or post-Golgi bottleneck representing a major obstacle in secretion of recombinant IGFI from S. cerevisiae cells.

Chromatography, High Pressure Liquid↗
PubMed1991

Carboxypeptidase yscS: gene structure and function of the vacuolar enzyme.

The gene encoding carboxypeptidase yscS in Saccharomyces cerevisiae, CPS1, was cloned by complementation of the cps1-3 mutation. The cloned CPS1 gene, which again enabled a leucine auxotrophic cps1-3 mutant to grow on the modified dipeptide Cbz-Gly-Leu (Cbz, benzyloxycarbonyl) as sole leucine source, was sequenced and found to consist of an open reading frame of 1728 bp encoding a protein of 576 amino acids. The putative protein contains a hydrophobic stretch of 20 amino acids and a putative signal sequence cleavage site. Five putative N-glycosylation sites are also in the protein sequence. This data is consistent with the previous finding of carboxypeptidase yscS being a vacuolar peptidase. Chromosomal disruption of the CPS1 gene completely abolishes carboxypeptidase yscS activity. This protein is yet another member of the peptidases in S. cerevisiae involved in nitrogen metabolism.

Amino Acid Sequence↗
PubMed1991

In-vitro processing of yeast alpha-factor leader fusion proteins using a soluble yscF (Kex2) variant.

The Saccharomyces cerevisiae KEX2 gene encodes the membrane-bound endoprotease yscF, which is responsible for the site-specific endoproteolytic cleavages at pairs of basic amino acid residues in the alpha-factor precursor. In order to obtain soluble yscF activity, a mutant KEX2 gene lacking 600 bp coding for the C-terminal 200 amino acids was constructed. Expression of the truncated KEX2 gene in yeast led to the secretion of an active soluble yscF protein (yscFs). The soluble yscF protein is able to efficiently cleave heterologous protein precursors in-vitro, as demonstrated for alpha-factor leader-hIGF1 and alpha-factor leader-hirudin fusion proteins.

Colorimetry↗
PubMed1991

Influence of yeast proteases on hirudin expression in Saccharomyces cerevisiae.

Recombinant desulfatohirudin variant 1 is efficiently expressed and secreted from Saccharomyces cerevisiae. Chemical analysis of the secreted hirudin compounds revealed the presence of the full-length hirudin molecule as well as two degradation products that lack the C-terminal and in addition the penultimate amino acid, respectively. To eliminate the yeast proteases possibly involved in C-terminal hirudin proteolysis, we disrupted either the structural gene for endoprotease yscA (PRA1) or the gene encoding carboxypeptidase yscY (PRC1). Both isogenic mutant strains secreted significantly higher amounts of full-length hirudin as compared to the parental strain. This suggests an involvement of carboxypeptidase yscY in hirudin proteolysis, since both protease disruptions lead to a lack in yscY activity; a yscA mutant accumulates the inactive yscY precursor. However, the strain devoid of protease yscA yielded significantly lower titers of total hirudin than the strain lacking yscY, but containing yscA.

Animals↗
PubMed1990

Isolation and structure of the pectin lyase D-encoding gene from Aspergillus niger.

The filamentous fungus, Aspergillus niger, produces a number of extracellular pectin-degrading enzymes. We present here the isolation and the complete nucleotide sequence of the gene, pelD, coding for a pectin lyase D (PLD), which was previously described as pectin lyase I (Van Houdenhoven, Ph.D. Thesis, Wageningen, 1975). The deduced amino acid (aa) sequence corresponds to 373 aa residues including a signal peptide of 19 aa. The coding region is interrupted by four short introns (57-65 bp). The nucleotide sequence of the 5'- and 3'-flanking regions is also presented and shows no unusual features. By comparing the deduced aa sequences of the A. niger PLD and a number of bacterial pectate lyases, short regions of homology were found despite the different substrate specificities (high methoxyl-pectin versus low methoxyl-pectin or polygalacturonate) of these enzymes.

Amino Acid Sequence↗
PubMed1989

Effect of Wy-45,727 on 24 h intragastric acidity in twenty normal volunteers.

1. The effects of single daily doses of 20, 50 and 150 mg of WY-45,727, a novel H2-receptor antagonist, and placebo were compared using long term pH-monitoring in 20 male volunteers. 2. Intragastric acidity was measured using combined Ingold glass electrodes. Subjects underwent four studies each under identical dietary conditions. Medication was taken after the evening meal. 3. Median 24 h pH rose from 1.3 (1.2-1.4 interquartile range) on placebo to 1.9 (1.6-2.8) on 20 mg WY-45,727, to 3.1 (2.3-3.7) on 50 mg WY-45,727 and to 4.5 (3.7-4.7) on 150 mg WY-45,727. All three doses increased 24 h and night-time pH significantly compared with placebo (P less than 0.0001). 4. The PAGE test for order effects confirmed clear dose dependent inhibition of acidity (P less than 0.0001). 5. Highly consistent individual responses were found during the night following 150 mg WY-45,727.

Adult↗
PubMed1988

Day-to-day variation of 24-hour intragastric acidity.

Twenty-four-hour intragastric acidity was measured continuously using an intragastric electrode in 13 normal volunteers studied four times. Subjects were studied twice in the hospital and twice as outpatients. The dietary conditions were strictly controlled and the replicate studies were compared to assess the variability of such recordings of acidity. The accuracy of the technique was assessed, and a detection limit for differences was calculated for commonly used time periods. Over 24 h, during the night, during the day, and during the evening the technique is able to detect consistent changes of pH of greater than 0.1 units. During separate 5-min periods the limits of detection were considerably greater. This study demonstrates the variability of 24-h intragastric acidity and confirms that continuous monitoring is able to detect important changes of acidity under both hospitalized and ambulant conditions.

Adult↗
PubMed1988

Double blind comparison of the effects of cimetidine, ranitidine, famotidine, and placebo on intragastric acidity in 30 normal volunteers.

Continuous measurement of 24 hour intragastric acidity was carried out in 30 normal volunteers during treatment with placebo, cimetidine 800 mg, ranitidine 300 mg, and famotidine 40 mg in a double blind study. Medication was taken after the evening meal (post cenam nocte, PCN). Median 24 hour acidity decreased with all H2-receptor antagonists from 25.1 mmol/l on placebo to 10 mmol/l (-60.1%) during cimetidine, to 3.2 mmol/l (-87.25%) during ranitidine and to 2.5 mmol/l (-90.0%) during famotidine treatment (p less than 0.0005). All drugs significantly inhibited night time acidity but only famotidine decreased acidity during the late morning compared with placebo. Significantly greater acid reduction was seen with famotidine and ranitidine compared with cimetidine but no difference was found between famotidine and ranitidine.

Cimetidine↗
PubMed1988

Antibiotics from basidiomycetes. XXIX: Pilatin, a new antibiotically active marasmane derivative from cultures of Flagelloscypha pilatii agerer.

Pilatin, a new marasmane derivative, was isolated from fermentations of the cyphelloid fungus Flagelloscypha pilatii. Its structure was determined by chemical and physical methods. Pilatin inhibits the growth of bacteria and fungi at concentrations of 5-50 micrograms/ml. The compound is highly cytotoxic. The incorporation of thymidine and uridine into DNA and RNA in Ehrlich carcinoma ascitic cells is strongly inhibited by pilatin. Like marasmic acid pilatin causes frameshift mutations in Salmonella typhimurium TA98.

Antibiotics, Antineoplastic↗
PubMed1988

Early evening ranitidine administration promotes faster duodenal ulcer healing.

In a prospective double-blind clinical trial, 141 patients with endoscopically diagnosed duodenal ulcer were randomly assigned to treatment with ranitidine 300 mg, taken either at 6 PM or at 10 PM. After 2 wk of treatment, 52 of 70 patients (74%) in the 6 PM treatment group had healed, compared with 32 of 64 patients (50%) taking ranitidine at 10 PM (p less than 0.01). After 4 wk, the cumulative healing rates were 100% and 94%, respectively, for the 6 PM and 10 PM treatment regimens. These results suggest that ranitidine, taken as a single daily 300-mg dose at 6 PM after dinner, provides more rapid duodenal ulcer healing than the same dose of the drug taken at 10 PM.

Adolescent↗
PubMed1987

Comparison of ranitidine 300 mg twice daily, 300 mg at night and placebo on 24-hour intragastric acidity of duodenal ulcer patients.

Continuous measurement of 24-hour intragastric acidity was performed in 12 duodenal ulcer patients in remission during treatment with placebo, ranitidine 300 mg nocte and ranitidine 300 mg b.d. Median 24-hour acidity was 79.4 mmol litre-1 during placebo treatment; it decreased to 28.2 mmol litre-1 during treatment with ranitidine 300 mg nocte and to 3.6 mmol litre-1 during treatment with ranitidine 300 mg b.d. The two regimens decreased intragastric acidity to a similar degree during the night, but significantly greater inhibition of daytime and 24-hour acidity followed use of ranitidine 300 mg b.d.

Adult↗