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This randomised, double-blind, multicentric clinical study compared the efficacy and tolerability of the two vaginal antiseptics, 10 mg dequalinium chloride (CAS 522-51-0, Fluomycin N) and 200 mg povidone iodine (CAS 25655-41-8), in a parallel-group design. A total of 180 patients with vaginal infections of varying etiology participated in this study (bacterial vaginosis, fluor vaginalis, vulvo-vaginal candidiasis, trichomoniasis). Patients were randomly allocated to one of the two treatment groups and were treated once per day for 6 days. Control examinations took place 5 to 7 days after the end of treatment, and 3 to 4 weeks after the first control examination. The total symptoms score, a summary score for the clinical symptoms, discharge, burning, pruritus, redness of vulva/vagina, was defined as primary efficacy parameter. The treatments at the first control examination were compared in the full analysis set using the Wilcoxon-Mann-Whitney U-test, 2-sided, thereby proving equivalence of both treatments at the 5% level. Both treatments strongly improved the symptoms of vaginal infections both on short-term and long-term follow-up. Descriptive analysis of the secondary parameters, vaginal pH, degree of purity of the vaginal flora, and number of lactobacilli in the wet mounts, supported the comparable efficacy of both therapies to restore the vaginal milieu. Analysis of the diagnostic subgroups indicated that irrespective of the diagnosis, both treatments improved the efficacy criteria as observed for the entire population. The global assessment of the therapeutic efficacy by investigators and patients supported the results of the efficacy analysis with good to very good ratings in 70-90% of the cases. A good tolerability of both preparations was observed in this study with a low number of adverse events in the test group (5.8%).

Dequalinium chloride (CAS 522-51-0) and povidone iodine (CAS 25655-41-8) are known as antiseptic agents and used in the local treatment of vaginal infections. Clotrimazole (CAS 23593-75-1) is an anti-fungal drug and applied primarily in the therapy of vulvo-vaginal candidiasis and to a lesser extent in bacterial vaginosis and trichomoniasis. However, antimicrobial activities of those three agents in comparison to each other have not been reported so far. To address this issue the antimicrobial activities of these agents against 18 germs relevant to vaginal infections were determined. The tested species are representatives of the genera Staphylococcus, Streptococcus, Enterococcus, Listeria, Escherichia, Proteus, Gardnerella, Bacteroides, Prevotella, Porphyromonas, Candida, and Trichomonas. All micro-organisms were susceptible to dequalinium chloride with the exception of Proteus mirabilis. At a given dose, the activity of dequalinium chloride was higher as compared to the other substances. In view of its wide antimicrobial spectrum dequalinium chloride is an efficient alternative in the local therapy of vaginal infections such as fluor vaginalis, bacterial vaginosis, aerobic vaginitis, vulvo-vaginal candidiasis and trichomoniasis.

Background Sleeping sickness (human African trypanosomiasis [HAT]) is caused by protozoan parasites and characterized by a chronic progressive course, which may last up to several years before death. We conducted two Phase 2 studies to determine the efficacy and safety of oral pafuramidine in African patients with first stage HAT. Methods The Phase 2a study was an open-label, non-controlled, proof-of-concept study where 32 patients were treated with 100 mg of pafuramidine orally twice a day (BID) for 5 days at two trypanosomiasis reference centers (Angola and the Democratic Republic of the Congo [DRC]) between August 2001 and November 2004. The Phase 2b study compared pafuramidine in 41 patients versus standard pentamidine therapy in 40 patients. The Phase 2b study was open-label, parallel-group, controlled, randomized, and conducted at two sites in the DRC between April 2003 and February 2007. The Phase 2b study was then amended to add an open-label sequence (Phase 2b-2), where 30 patients received pafuramidine for 10 days. The primary efficacy endpoint was parasitologic cure at 24 hours (Phase 2a) or 3 months (Phase 2b) after treatment completion. The primary safety outcome was the rate of occurrence of World Health Organization Toxicity Scale Grade 3 or higher adverse events. All subjects provided written informed consent. Findings/Conclusion Pafuramidine for the treatment of first stage HAT was comparable in efficacy to pentamidine after 10 days of dosing. The cure rates 3 months post-treatment were 79% in the 5-day pafuramidine, 100% in the 7-day pentamidine, and 93% in the 10-day pafuramidine groups. In Phase 2b, the percentage of patients with at least 1 treatment-emergent adverse event was notably higher after pentamidine treatment (93%) than pafuramidine treatment for 5 days (25%) and 10 days (57%). These results support continuation of the development program for pafuramidine into Phase 3., Author Summary Sleeping sickness (human African trypanosomiasis [HAT]) is caused by parasites, and has a chronic progressive course that may last from several months to several years before death occurs. The present studies were done to assess the effectiveness and safety of oral pafuramidine versus intramuscular pentamidine (the standard treatment), in patients with first stage HAT. The results indicated that, several months after treatment, pafuramidine administered for 10 days was as effective as pentamidine administered for 7 days, and it had a better safety profile than pentamidine. With further study, pafuramidine could be a promising alternative for patients with first stage HAT. In addition, the design of the studies can be used a guide for future studies for identification and delivery of treatment to affected individuals in rural Africa.

Background Sleeping sickness (human African trypanosomiasis [HAT]) is a neglected tropical disease with limited treatment options that currently require parenteral administration. In previous studies, orally administered pafuramidine was well tolerated in healthy patients (for up to 21 days) and stage 1 HAT patients (for up to 10 days), and demonstrated efficacy comparable to pentamidine. Methods This was a Phase 3, multi-center, randomized, open-label, parallel-group, active control study where 273 male and female patients with first stage Trypanosoma brucei gambiense HAT were treated at six sites: one trypanosomiasis reference center in Angola, one hospital in South Sudan, and four hospitals in the Democratic Republic of the Congo between August 2005 and September 2009 to support the registration of pafuramidine for treatment of first stage HAT in collaboration with the United States Food and Drug Administration. Patients were treated with either 100 mg of pafuramidine orally twice a day for 10 days or 4 mg/kg pentamidine intramuscularly once daily for 7 days to assess the efficacy and safety of pafuramidine versus pentamidine. Pregnant and lactating women as well as adolescents were included. The primary efficacy endpoint was the combined rate of clinical and parasitological cure at 12 months. The primary safety outcome was the frequency and severity of adverse events. The study was registered on the International Clinical Trials Registry Platform at www.clinicaltrials.gov with the number ISRCTN85534673. Findings/Conclusions The overall cure rate at 12 months was 89% in the pafuramidine group and 95% in the pentamidine group; pafuramidine was non-inferior to pentamidine as the upper bound of the 95% confidence interval did not exceed 15%. The safety profile of pafuramidine was superior to pentamidine; however, 3 patients in the pafuramidine group had glomerulonephritis or nephropathy approximately 8 weeks post-treatment. Two of these events were judged as possibly related to pafuramidine. Despite good tolerability observed in preceding studies, the development program for pafuramidine was discontinued due to delayed post-treatment toxicity., Author Summary Sleeping sickness, or human African trypanosomiasis (HAT), is a neglected tropical disease. Because only 2 treatment options are available to treat persons with stage 1 disease, and both require parenteral administration, oral drugs would be of great benefit to the affected population. In this Phase 3, multi-center, randomized, open-label, parallel-group study, we compared oral pafuramidine with intramuscular pentamidine in persons in sub-Sahara Africa with first stage HAT. At 12 months, the overall cure rates (combined clinical and parasitological cure) were similar: 89% in the pafuramidine group and 95% in the pentamidine group. At 24 months, the cure rates continued to be high: 84% and 89%, respectively. Pafuramidine’s safety profile was superior to the comparator drug, and it was consistent with the overall safety profile seen in previous Phase 2 studies. Upon further analysis, however, a renal safety issue was identified as being possibly related to pafuramidine and further clinical development was halted. Nevertheless, the clinical studies conducted in the pafuramidine development program provide a model for future studies in rural Africa.

Background: The microbiological basis of diaper dermatitis is not clearly elucidated, although a better knowledge of microbial colonisation can be of importance with regard to an adequate treatment. Objective: To investigate the relevance of Candida sp. and Staphylococcus aureus colonisation in diaper dermatitis and to determine the correlation between the extent of colonisation and the severity of disease. Methods: Growth of Candida sp. and S. aureus in the perianal, inguinal and oral regions was determined by positive/negative and semi-quantitative analysis in an open, multi-centre (n = 3) study. Forty-eight children with healthy skin and 28 with diaper dermatitis were analysed. The severity of diaper dermatitis was assessed using a total symptoms score. Results: Colonisation by Candida sp. was significantly more frequent in children with diaper dermatitis as compared to those with healthy skin (perianal 75 vs. 19%; inguinal 50 vs. 10%; oral 68 vs. 25%, p < 0.0003), whereas colonisation by S. aureus at the 3 swab locations was not different (p > 0.34). There was a highly significant, positive correlation between severity of disease and extent of Candida sp. colonisation at all swab locations. Conclusions: Limited microbial colonisation in diaper dermatitis is of questionable relevance, but extensive colonisation seems to aggravate the symptoms; therefore, we suggest that semi-quantitative evaluation should be preferred to the positive/negative assessment for a differential diagnosis.

Background: Hirustasin belongs to a class of serine protease inhibitors characterized by a well conserved pattern of cysteine residues. Unlike the closely related inhibitors, antistasin/ghilanten and guamerin, which are selective for coagulation factor Xa or neutrophil elastase, hirustasin binds specifically to tissue kallikrein. The conservation of the pattern of cysteine residues and the significant sequence homology suggest that these related inhibitors possess a similar three-dimensional structure to hirustasin. Results: The crystal structure of the complex between tissue kallikrein and hirustasin was analyzed at 2.4 A resolution. Hirustasin folds into a brick-like structure that is dominated by five disulfide bridges and is sparse in secondary structural elements. The cysteine residues are connected in an abab cdecde pattern that causes the polypeptide chain to fold into two similar motifs. As a hydrophobic core is absent from hirustasin the disulfide bridges maintain the tertiary structure and present the primary binding loop to the active site of the protease. The general structural topography and disulfide connectivity of hirustasin has not previously been described. Conclusions: The crystal structure of the kallikrein–hirustasin complex reveals that hirustasin differs from other serine protease inhibitors in its conformation and its disulfide bond connectivity, making it the prototype for a new class of inhibitor. The disulfide pattern shows that the structure consists of two domains, but only the C-terminal domain interacts with the protease. The disulfide pattern of the N-terminal domain is related to the pattern found in other proteins. Kallikrein recognizes hirustasin by the formation of an antiparallel β sheet between the protease and the inhibitor. The P1 arginine binds in a deep negatively charged pocket of the enzyme. An additional pocket at the periphery of the active site accommodates the sidechain of the P4 valine.

A synthetic gene coding for the 55-amino acid protein hirustasin, a novel tissue kallikrein inhibitor from the leech Hirudo medicinalis, was generated by polymerase chain reaction using overlapping oligonucleotides, fused to the yeast alpha-factor leader sequence and expressed in Saccharomyces cerevisiae. Recombinant hirustasin was secreted mainly as incompletely processed fusion protein, but could be processed in vitro using a soluble variant of the yeast yscF protease. The processed hirustasin was purified to better than 97% purity. N-terminal sequence analysis and electrospray ionization mass spectrometry confirmed a correctly processed N-terminus and the expected amino acid sequence and molecular mass. The biological activity of recombinant hirustasin was identical to that of the authentic leech protein. Crystallized hirustasin alone and in complex with tissue kallikrein diffracted beyond 1.4 A and 2.4 A, respectively. In order to define the reactive site of the inhibitor, the interaction of hirustasin with kallikrein, chymotrypsin, and trypsin was investigated by monitoring complex formation in solution as well as proteolytic cleavage of the inhibitor. During incubation with high, nearly equimolar concentration of tissue kallikrein, hirustasin was cleaved mainly at the peptide bond between Arg 30 and Ile 31, the putative reactive site, to yield a modified inhibitor. In the corresponding complex with chymotrypsin, mainly uncleaved hirustasin was found and cleaved hirustasin species accumulated only slowly. Incubation with trypsin led to several proteolytic cleavages in hirustasin with the primary scissile peptide bond located between Arg 30 and Ile 31. Hirustasin appears to fall into the class of protease inhibitors displaying temporary inhibition.

Aromatic diamidines are potent trypanocides. Pentamidine, a diamidine, has been used for more than 60 years to treat human African trypanosomiasis (HAT); however, the drug must be administered parenterally and is active against first-stage HAT only, prior to the parasites causing neurological deterioration through invasion of the CNS. A major research effort to design novel diamidines has led to the development of orally active prodrugs and, remarkably, a new generation of compounds that can penetrate the CNS. In this review, progress in the development of diamidines for the treatment of HAT is discussed.

Objectives: To evaluate the efficacy of lyophilized lactobacilli in combination with 0.03 mg estriol when compared to metronidazole in the treatment of bacterial vaginal infections. Setting: Multicenter, randomized, single-blind, active-controlled pilot study in 3 independent gynecological practices in Belgium. Methods: Forty-six, 18- to 50-year-old premenopausal women with a disrupted vaginal flora due to a bacterial vaginal infection (bacterial vaginosis, aerobic vaginitis) were included, provided that fresh phase-contrast microscopy of the vaginal fluid showed lactobacillary flora grade 2B or 3. Patients were given a blinded box with either 12 vaginal tablets of Gynoflor® (study medication) or 6 vaginal suppositories containing 500 mg metronidazole (control medication). Eight efficacy variables were studied to assess the status of the vaginal flora at entry, 3–7 days (control 1), 4–6 (control 2) weeks and 4 months after the end of therapy. Results: At control 1, the combined variables equally improved in the lactobacilli group as in the metronidazole group. At control 2, the lactobacillus preparation showed slightly inferior results when compared to metronidazole. At 4 months, this analysis could not be performed due to low numbers, but analysis of recurrence rate and extra medication needed was not different between both groups. Conclusion: Lyophilized lactobacilli in combination with low-dose estriol are equivalent to metronidazole in the short-term treatment of bacterial vaginal infections, but have less effect after 1 month. Further studies are required to evaluate the long-term efficacy of lactobacilli when applied repeatedly.

Background In Human African Trypanosomiasis, neurological symptoms dominate and cardiac involvement has been suggested. Because of increasing resistance to the available drugs for HAT, new compounds are desperately needed. Evaluation of cardiotoxicity is one parameter of drug safety, but without knowledge of the baseline heart involvement in HAT, cardiologic findings and drug-induced alterations will be difficult to interpret. The aims of the study were to assess the frequency and characteristics of electrocardiographic findings in the first stage of HAT, to compare these findings to those of second stage patients and healthy controls and to assess any potential effects of different therapeutic antiparasitic compounds with respect to ECG changes after treatment. Methods Four hundred and six patients with first stage HAT were recruited in the Democratic Republic of Congo, Angola and Sudan between 2002 and 2007 in a series of clinical trials comparing the efficacy and safety of the experimental treatment DB289 to the standard first stage treatment, pentamidine. These ECGs were compared to the ECGs of healthy volunteers (n = 61) and to those of second stage HAT patients (n = 56). Results In first and second stage HAT, a prolonged QTc interval, repolarization changes and low voltage were significantly more frequent than in healthy controls. Treatment in first stage was associated with repolarization changes in both the DB289 and the pentamidine group to a similar extent. The QTc interval did not change during treatment. Conclusions Cardiac involvement in HAT, as demonstrated by ECG alterations, appears early in the evolution of the disease. The prolongation of the QTC interval comprises a risk of fatal arrhythmias if new drugs with an additional potential of QTC prolongation will be used. During treatment ECG abnormalities such as repolarization changes consistent with peri-myocarditis occur frequently and appear to be associated with the disease stage, but not with a specific drug., Author Summary In Human African Trypanosomiasis (HAT), neurological symptoms dominate and cardiac involvement has been suggested. Because of increasing resistance to the available drugs for HAT, new compounds are desperately needed. Evaluation of cardiotoxicity is one parameter of drug safety, but without knowledge of the baseline heart involvement in HAT, cardiologic findings and drug-induced alterations will be difficult to interpret. The electrocardiogram (ECG) is a tool to evaluate cardiac involvement and the risk of arrythmias. We analysed the ECG of 465 HAT patients and compared them with the ECG of 61 healthy volunteers. In HAT patients the QTc interval was prolonged. This comprises a risk of fatal arrhythmias if new drugs with antiarrhythmic potential will be used. Further, repolarization changes and low voltage were more frequent than in healthy controls. This could be explained by an inflammation of the heart. Treatment of HAT was associated with appearance of repolarization changes but not with a QTc prolongation. These changes appear to be associated with the disease, but not with a specific drug. The main conclusion of this study is that heart involvement is frequent in HAT and mostly well tolerated. However, it can become relevant, if new compounds with antiarrhythmic potential will be used.

This randomised, double-blind, multicentric clinical study compared the efficacy and tolerability of the two vaginal antiseptics, 10 mg dequalinium chloride (CAS 522-51-0, Fluomycin N) and 200 mg povidone iodine (CAS 25655-41-8), in a parallel-group design. A total of 180 patients with vaginal infections of varying etiology participated in this study (bacterial vaginosis, fluor vaginalis, vulvo-vaginal candidiasis, trichomoniasis). Patients were randomly allocated to one of the two treatment groups and were treated once per day for 6 days. Control examinations took place 5 to 7 days after the end of treatment, and 3 to 4 weeks after the first control examination. The total symptoms score, a summary score for the clinical symptoms, discharge, burning, pruritus, redness of vulva/vagina, was defined as primary efficacy parameter. The treatments at the first control examination were compared in the full analysis set using the Wilcoxon-Mann-Whitney U-test, 2-sided, thereby proving equivalence of both treatments at the 5% level. Both treatments strongly improved the symptoms of vaginal infections both on short-term and long-term follow-up. Descriptive analysis of the secondary parameters, vaginal pH, degree of purity of the vaginal flora, and number of lactobacilli in the wet mounts, supported the comparable efficacy of both therapies to restore the vaginal milieu. Analysis of the diagnostic subgroups indicated that irrespective of the diagnosis, both treatments improved the efficacy criteria as observed for the entire population. The global assessment of the therapeutic efficacy by investigators and patients supported the results of the efficacy analysis with good to very good ratings in 70-90% of the cases. A good tolerability of both preparations was observed in this study with a low number of adverse events in the test group (5.8%).

An efficient expression/purification procedure has been developed which allows the production of pure, biologically active recombinant leech-derived tryptase inhibitor (rLDTI), originally found in the leech Hirudo medicinalis. The gene for LDTI was generated synthetically from three overlapping oligonucleotides by PCR synthesis. LDTI was expressed in the yeast Saccharomyces cerevisiae under the control of the copper-inducible CUP1 promoter and fused to the invertase signal sequence (SUC2). The entire expression cassette was inserted into the yeast high-copy vector pDP34. Appropriate host strains transformed with the expression plasmid secreted rLDTI into the medium upon copper addition. Proteinchemical analysis of the secreted rLDTI revealed exclusively inhibitor with the correct N-terminal sequence. Up to 60% of the rLDTI, however, appeared to be modified by glycosylation and the unglycosylated material showed heterogeneity at the C-terminus. Besides full-length rLDTI, truncated rLDTI species lacking either the terminal Asn46 or in addition the penultimate Leu45 were isolated. The C-terminally truncated variants were eliminated using a S. cerevisiae host strain disrupted in the structural genes of carboxypeptidases yscY and ysca, thus identifying these proteases as being responsible for the degradation of rLDTI. Mature rLDTI was purified in high yields from the culture supernatant of the carboxypeptidase-deficient yeast strain by cation-exchange chromatography and reverse-phase HPLC. The recombinant protein is at least 98% pure, based on HPLC and capillary electrophoresis, and is fully biologically active. Structural identity with the authentic leech protein was confirmed by sequence analysis and molecular-mass determination. The purified protein was tested for its ability to inhibit tryptase and trypsin in vitro and to interfere with the tryptase-induced proliferation of human fibroblasts and keratinocytes. Recombinant LDTI appears to be as potent as the authentic leech protein, exhibiting Ki-values of approximately 1.5 nM and approximately 1.6 nM against human tryptase and bovine trypsin, respectively. The tryptase-induced proliferation of human fibroblasts and keratinocytes was inhibited with half-maximum values of approximately 0.1 nM and approximately 1 nM, respectively. The availability of the recombinant material will allow evaluation of the concept of tryptase inhibition in various disease models and to test the therapeutic potential of LDTI in mast-cell-related disorders.

We have constructed 2-micron-based yeast expression vectors containing a copy of the metallothionein (CUP1) gene of Saccharomyces cerevisiae as a semi-dominant, selectable marker. When used for the expression of the thrombin inhibitor hirudin, originally derived from the leech Hirudo medicinalis, these vectors displayed the following characteristics. (1) In the presence of copper salts, they were mitotically more stable than similarly designed control vectors lacking the CUP1 gene. In copper-sensitive host strains, the apparent plasmid stability was 100%, even in complex media and during fed-batch fermentation for an extended period of time. (2) Use of the CUP1-stabilized plasmids improved the production of hirudin by both copper-sensitive and copper-resistant hosts. The highest hirudin titers were obtained with a delta CUP1 host. (3) Copper selection resulted in a moderate increase in average plasmid copy numbers (up to two-fold) as assessed by measuring hirudin expression from a constitutive promoter (GAPFL). This effect was most noticeable if the vector showed an asymmetric segregation pattern (i.e., high rates of plasmid loss in the absence of copper). (4) The CUP1 marker proved particularly useful in combination with a CUP1-promoter-controlled expression cassette on the same plasmid. In such a set-up, the rates of transcription of the heterologous protein and that of the selectable marker are tightly linked. Therefore, an increase in selective pressure directly provokes an increase in product yields. In a copper-sensitive host strain, this plasmid design allowed for the production of very high amounts of biologically active hirudin. Our results clearly establish the utility of the CUP1 marker in the construction of stable yeast expression vectors.

The potent thrombin inhibitor hirudin variant 1, originally isolated from the leech Hirudo medicinalis, was expressed in Saccharomyces cerevisiae under the control of a truncated glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter fragment. Fusion of the yeast acid phosphatase (PHO5) signal sequence to the hirudin gene led to quantitative secretion of recombinant desulfato-hirudin variant 1 (r-hirudin) into the extracellular medium in a growth-dependent manner. In comparison to the genuine molecule, r-hirudin lacks the sulfate group at the Tyr in position 63. Besides the full-length protein of 65 amino acids (hir65), chemical analysis revealed the presence mainly of two derivatives lacking the last amino acid Gln (hir64) or the penultimate Leu (hir63) in addition. When expressing r-hirudin in mutant strains defective in all but one of the three major known carboxypeptidases, it turned out that the vacuolar carboxypeptidase yscY as well as the alpha-factor precursor-processing carboxypeptidase, ysc alpha, participate in the C-terminal degradation of r-hirudin. Direct involvement of yscY and ysc alpha was confirmed by sequential disruption of their structural genes PRC1 and KEX1, respectively. Disruption of PRA1, coding for the yscY-processing proteinase yscA, also abolished yscY-mediated C-terminal r-hirudin degradation, but clearly reduced the overall expression yield. Since ysc alpha is described to be highly specific for basic amino acids which are not present at the C-terminus of r-hirudin, a series of r-hirudin mutants with changes in the C-terminal amino acids were constructed and analysed for ysc alpha-mediated and yscY-mediated degradation. Chromatographic analysis of the expression products confirmed the preference of ysc alpha for basic amino acids, although Tyr, Leu and Gln were also hydrolysed. It could further be concluded that ysc alpha might also be responsible for the C-terminal degradation of recombinant atrial natriuretic factor and epidermal growth factor expressed in yeast.

Using the anticoagulant, hirudin, from the leech Hirudo medicinalis as a secreted reporter protein, the influence of physiological parameters on activity and regulation of the yeast (Saccharomyces cerevisiae) metallothionein (CUP1) promoter was studied. Induction of CUP1-directed hirudin expression from 2 mu-based vectors was possible at any time point during diauxic batch growth, even in cells approaching stationary phase. The highest titers of hirudin were obtained when the CUP1 promoter was activated immediately following inoculation of the cultures. If such a pseudo-constitutive fermentation strategy was adopted, the promoter was superior to an optimized variant (GAPFL) of the strong, constitutive GAPDH promoter. This superiority was primarily due to the relative independence of CUP1 promoter activity of the physiological status of host cells: whilst the maximal strength of the CUP1 and GAPFL promoters was comparable, CUP1-directed hirudin expression was high in all phases of diauxic batch growth, whereas hirudin production from the GAPFL promoter declined in post-diauxic cultures. High activity of the CUP1 promoter was observed on both a fermentable (glucose) and a non-fermentable (ethanol) carbon source. Hirudin expression could be adjusted to different levels by varying the amount of inducer (cupric sulphate) added to cultures. The copper concentrations required for maximal promoter induction had no negative effects on host growth and interfered with neither hirudin secretion nor with the biological activity of the peptide. Overexpression of the transcriptional activator, ACE1, resulted in increased levels of hirudin mRNA. Hirudin titers increased in parallel to mRNA concentrations in cultures grown in the presence of low concentrations of copper. In contrast, at high copper doses, elevated levels of the ACE1 protein resulted in inferior hirudin production. Cells overexpressing ACE1 while harbouring a CUP1-drived hirudin expression cassette showed slow growth and poor plasmid maintenance. It was tested whether this might be the result of a block in the secretory pathway; however, measurements of intracellular hirudin did not support this hypothesis. The data rather indicated that hirudin production was limited by a metabolic constraint downstream of transcription but upstream of the secretory pathway.

The rapid phase of fructose-1,6-bisphosphatase (FBPase) inactivation following glucose addition to starved yeast cells [reported previously] is inhibited on addition of 10 mM chloroquine (CQ) at about pH 8. This inhibition of inactivation was shown to be due to the prevention of phosphorylation of the enzyme. CQ was also found to inhibit general protein phosphorylation in the yeast cells. Glycolysis, as observed by changes in intracellular glucose-6-phosphate and extracellular glucose and ethanol concentrations, was shown to be significantly inhibited in cells treated with CQ. Similarly, a decrease in ATP concentrations was observed. However, during the early stages of phosphorylation of FBPase, levels of ATP were similar in cells containing CQ as in those without CQ. Thus, decrease in ATP levels is not thought to be significantly responsible for the inhibition of protein phosphorylation. However, the phosphorylating activity of cyclic AMP-dependent protein kinases is inhibited in vitro by relatively low concentrations of CQ. Thus, prevention of protein phosphorylation by CQ is believed to be due to inhibition of protein kinases in yeast cells.

Publisher Summary Phosphorylation of purified FBPase a can be demonstrated in vitro by incubation with purified cyclic AMP-dependent protein kinase from yeast in the presence of ATP, Mg2+, and cyclic AMP. The final loss of catalytic activity is about 60%, a value very similar to that found in intact cells 1–3 minutes after addition of glucose to starved yeast. The in vitro phosphorylated enzyme shows characteristic kinetic properties similar to the enzyme assayed in a crude extract from glucose-treated yeast and to FBPase-P b purified from such an extract. Incubation of FBPase a with a lysate from yeast vacuoles, containing the bulk of the proteolytic activity of yeast, shows inactivation kinetics similar to those observed after incubation of purified FBPase a with cyclic AMP-dependent protein kinase. Chymostatin and the endogenous proteinase inhibitor IB from yeast, both known as specific inhibitors of proteinase B, completely prevent the limited proteolysis of FBPase a by the vacuole lysate. Chymostatin and the endogenous proteinase inhibitor IB from yeast, both known as specific inhibitors of proteinase B, completely prevent the limited proteolysis of FBPase a by the vacuole lysate. Studies in the laboratory of Earl Stadtman have pointed to a possible biological significance of oxidative inactivation of glutamine synthetase from Escherichia coli by mixed function oxidation reactions. Interestingly, oxidative modification of one histidine residue per subunit of glutamine synthetase parallels the loss of its activity.

Phosphorylation of purified yeast fructose-1,6-bisphosphatase was studied using purified preparations from yeast of two different cyclic AMP-independent protein kinases and a cyclic AMP-dependent protein kinase. Incorporation of 32P into fructose-1,6-bisphosphatase could be demonstrated only with the cyclic AMP-dependent protein kinase. Phosphorylation of fructose-1,6-bisphosphatase was stimulated by 3 microM fructose-2,6-bisphosphate and inhibited by 1 mM 5'-AMP.