Purification of two depressant insect neurotoxins and their gene cloning from the scorpion Buthus martensi Karsch.

Insect-specific neurotoxins are important components of scorpion venoms. In this study, two toxins from the scorpion Buthus martensi Karsch (BmK) were purified. They shared high sequence homology with other depressant insect toxins and were designated BmK ITa and BmK ITb, respectively. They were able to suppress the action potential of cockroach isolated axon, which is due to a decrease in the peak sodium current. Furthermore, the effect of BmK ITb was lower than that of BmK ITa, and some of the electrophysiological characteristics of BmK ITb even resemble that of excitatory insect toxins. Their primary structures were determined by N-terminal partial sequence determination and cDNA cloning. The differences in their structures, especially the 31st residues, may result in the unique activity of BmK ITb.

Predicting response to initial therapy with interferon plus ribavirin in chronic hepatitis C using serum HCV RNA results during therapy.

In patients with chronic hepatitis C, 48 weeks of therapy with interferon (IFN) plus ribavirin results in a sustained virologic response of 40%. Preliminary analysis suggests that measuring HCV RNA at week 24, rather than week 12, might provide the best prediction of treatment response. To assess the clinical utility of serum HCV RNA determinations at different times during therapy as a predictor of a sustained virologic response we evaluated 912 treatment-naïve patients. Patients were randomized to receive IFN-alpha2b, 3 million units (MU) three times weekly (tiw), for 24 or 48 weeks with either ribavirin or placebo, and then followed for 24 weeks. Serum HCV RNA was measured at weeks 4 and 12 in patients treated for 24 weeks; at 4, 12, and 24 weeks during therapy in those treated for 48 weeks, and week 24 post-therapy in all patients. Sustained response was defined as loss of serum HCV RNA at week 24 follow-up. Other patients were considered virologic nonresponders. For patients receiving 48 weeks of combination therapy, detectable serum HCV RNA at week 24 predicted nonresponse (positive predictive value) in 99% of patients compared to 89% at week 12. In patients treated for 24 weeks, testing at week 12 was more predictive of nonresponse than testing at week 4 in the combination-therapy group but not in the monotherapy group. Hence, for combination therapy, testing for serum HCV RNA as a predictor of nonresponse is most accurate at week 24 of therapy; a positive test correctly identified 99% of nonresponders.

Molecular characterization of an anti-epilepsy peptide from the scorpion Buthus martensi Karsch.

For a long time Asian scorpion Buthus martensi Karsch (BmK) has been used in Chinese traditional medicine to cure many diseases of nervous system. Here we report the purification and characterization of a pharmacologically active neurotoxin from the scorpion BmK. This toxin had little toxicity in mice and insects but was found to have an anti-epilepsy effect in rats, and is thus named as BmK anti-epilepsy peptide (BmK AEP). Its amino-acid sequence was determined by lysylendopeptidase digestion, Edman degradation and mass spectrographic analysis. Based on the determined sequence, the gene coding for this peptide was also cloned and sequenced by the 3' and 5' RACE methods. It encodes a precursor of 85 amino-acid residues including a signal peptide of 21 residues, a mature peptide of 61 residues and three additional residues Gly-Lys-Lys at the C-terminus. The additional Gly sometimes followed by one or two basic residues is prerequisite for the amidation of its C-terminus. C-terminal amidation was also verified by the molecular-mass determination of BmK AEP. This anti-epilepsy peptide toxin shares homology with other depressant insect toxins. The remarkable difference between them was mainly focused at residues 6, 7 and 39; these residues might relate to the unique action of BmK AEP.

Cost-effectiveness of 24 or 48 weeks of interferon alpha-2b alone or with ribavirin as initial treatment of chronic hepatitis C. International Hepatitis Interventional Therapy Group.

OBJECTIVE: Initial therapy with ribavirin and interferon alpha-2b results in a higher sustained virological response than interferon alone, but this regimen is expensive. We aimed to examine the cost-effectiveness of 24- or 48-wk initial treatment with combination therapy versus interferon alone for patients who have chronic hepatitis C. METHODS: Data from recent randomized clinical trials comparing combination therapy to interferon alone were applied to a previously published computer cohort simulation to project lifelong clinical and economic outcomes. Natural history and economic estimates were based on published literature, expert panel estimates, and actual variable cost and reimbursement data. RESULTS: Using treatment stopping rules, sustained viral negative response rates would be 33.1% and 39.8% for patients receiving 24 versus 48 wk of ribavirin/interferon, compared with 14.3% for 48 wk of interferon alone. Compared to the interferon alone strategy, 24 or 48 wk of combination therapy should prolong life expectancy by 1.4 to 2.0 yr at marginal cost-effectiveness ratios of $4,400 to $5,400 per discounted quality-adjusted life-year (DQALY) gained. Compared to 24 wk of combination therapy, 48 wk of combination therapy should prolong life expectancy by 0.6 yr at a marginal cost-effectiveness ratio of $7,700 per DQALY gained. The results were robust, with 24 or 48 wk of combination therapy remaining preferred and cost-effective in sensitivity analysis compared with interferon alone. CONCLUSION: For patients with chronic hepatitis C, 24 or 48 wk of ribavirin and interferon should prolong life and be cost-effective when compared with 48 wk, of interferon alone.

Genomic organization of three novel toxins from the scorpion Buthus martensi Karsch that are active on potassium channels.

The cDNA and genomic DNA of three novel toxins from the scorpion Buthus martensi Karsch that are active on K(+) channels, designated BmKTX (where KTX is kaliotoxin), BmTX1 and BmTX2, were cloned and sequenced. On the basis of their known amino acid sequences, gene-specific primers for 3' and 5' rapid amplification of cDNA ends (RACE) were designed and synthesized. By overlapping the two partial cDNA sequences obtained by 3' and 5' RACE, their full-length cDNA sequences were completed. BmKTX encodes a signal peptide of 22 amino acid residues and a mature toxin of 38 residues, whereas BmTX1 and BmTX2 encode signal peptides of 20 and 21 residues respectively and a mature toxin of 38 residues for each. Their cDNA-deduced amino acid sequences were totally consistent with those determined except that the C-terminus of BmKTX had an additional Gly residue, which was removed during post-translational processing and was indispensable for the amidation of its C-terminal Lys residue. In addition, the first deduced amino acid for both BmTX1 and BmTX2 is Gln instead of pyro-Glu in the reported toxins, which obviously also undergoes post-translational processing. The genomic DNA species of these three toxins were also amplified by PCR, then cloned and sequenced. They all consisted of two exons disrupted by a small single intron. All of these introns were inserted within the signal peptides at position -6 for BmKTX and at position -5 for both BmTX1 and BmTX2 upstream of the mature toxins, and consisted of 87, 87 and 80 bp respectively.

Nonhistone protein purified from porcine kidney acts as a suicide substrate inhibitor on furin-like enzyme.

AIM: To search and purify a naturally occurring protein inhibitor of the furin-like enzyme from the porcine kidney. METHODS: Recombinant kexin, a furin-like enzyme, from the yeast secretion expression was used as a target enzyme. The inhibitor component was extracted and purified from the acetone powder of porcine kidney. The inhibitory activity was monitored using a fluorogenic peptide substrate Boc-Arg-Val-Arg-MCA at spectrofluorimeter. RESULTS: The purified inhibitor component is a basic protein with an isoelectric point over 9.5. Its partial N-terminal sequence of 22 residues was determined, showing a high homology with nonhistone chromosomal protein HMG-17 in which there are four sites composed of dibasic residues, susceptible to be cleaved by the furin-like enzyme. This nonhistone protein could strongly compete with the fluorogenic substrate. However, this nonhistone protein would be degraded as a substrate by kexin if it was incubated with the enzyme for long time before adding the fluorogenic substrate, and subsequently lost its temporary inhibitory activity. CONCLUSION: The nonhistone protein isolated from the porcine kidney functioned as a suicide substrate inhibitor for the furin-like enzyme.

Is an "a la carte" combination interferon alfa-2b plus ribavirin regimen possible for the first line treatment in patients with chronic hepatitis C? The ALGOVIRC Project Group.

UNLABELLED: Randomized trials have shown the enhancement of efficacy with interferon alfa-2b and ribavirin (IFN-R) in comparison with interferon monotherapy (IFN) as first line treatment of chronic hepatitis C. Further definition of response based on disease, patient, and treatment characteristics is needed to determine the degree of benefit for the various patient subgroups. The aim of this study was to answer this question by analyzing the data from 1,744 naive patients included in trials that compared 24- or 48-week IFN-R treatment. Response factors were identified by logistic regression and receiver operating characteristics curves. Five independent characteristics were associated with a sustained loss of hepatitis C virus (HCV) RNA (<100 copies/mL) 24 weeks after the end of treatment: genotype 2 or 3, baseline viral load less than 3.5 million copies/mL, no or portal fibrosis, female gender, and age younger than 40 years. There was a significant advantage for IFN-R in comparison with IFN alone whatever the combination of factors. The most efficient strategy is to treat all patients for 24 weeks. If the 24-week polymerase chain reaction (PCR) is positive, treatment can be stopped. If the 24-week PCR is negative, patients with fewer than 4 favorable factors should be treated for an additional 24 weeks. CONCLUSION: The combination of IFN-R is better as first line treatment than IFN monotherapy. For patients who are PCR negative after 24 weeks of treatment, genotyping and baseline viral load, fibrosis stage, gender, and age are useful predictive factors in determining whether to continue an additional 24 weeks of treatment.

Expression and purification of the BmK M1 neurotoxin from the scorpion Buthus martensii Karsch.

The gene encoding a neurotoxin (BmK M1) from the scorpion Buthus martensii Karsch was expressed in Saccharomyces cerevisiae at a high level with the alcohol dehydrogenase promoter. SDS-PAGE of the culture confirmed expression and showed secretion into medium from yeast. Recombinant BmK M1 was purified rapidly and efficiently by ion exchange and gel filtration chromatography to homogeneity, produced a single band on tricine-SDS-PAGE, and processed the homologous N-terminus. Amino acid analysis and N-terminal sequencing demonstrated that the recombinant toxin was processed correctly from the alpha-mating factor leader sequence and was chemically identical to the native form. The expressed recombinant BmK M1 was toxic for mice, which indicated that it was biologically active. Quantitative estimation showed that recombinant BmK M1 had an LD(50) similar to that of the native toxin.

The cDNA sequence of an excitatory insect selective neurotoxin from the scorpion Buthus martensi Karsch.

The full-length cDNA of an excitatory insect selective neurotoxin was amplified from total cDNAs of venomous glands of the scorpion Buthus martensi Karsch (BmK) using the 3'RACE and 5'RACE (rapid amplification of cDNA ends, RACE) method and sequenced. The cDNA encoded a precursor of the insect toxin of 88 amino acid residues, including a signal peptide of 18 residues and a mature toxin of 70 residues. The cDNA deduced sequence of this toxin was homologous with the determined amino acid sequence of BmK IT1, an excitatory insect toxin purified from the scorpion venom, except for three different residues, two at the positions 24-25, and another in the COOH-terminus of the toxin. Among them the COO-terminal residue Gly in the cDNA deduced sequence was predominantly different from the conserved residue Asn found in other known scorpion excitatory insect toxins.

Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. Hepatitis Interventional Therapy Group.

BACKGROUND: Only 15 to 20 percent of patients with chronic hepatitis C have a sustained virologic response to interferon therapy. We compared the efficacy and safety of recombinant interferon alfa-2b alone with those of a combination of interferon alfa-2b and ribavirin for the initial treatment of patients with chronic hepatitis C. METHODS: We randomly assigned 912 patients with chronic hepatitis C to receive standard-dose interferon alfa-2b alone or in combination with ribavirin (1000 or 1200 mg orally per day, depending on body weight) for 24 or 48 weeks. Efficacy was assessed by measurements of serum hepatitis C virus (HCV) RNA and serum aminotransferases and by liver biopsy. RESULTS: The rate of sustained virologic response (defined as an undetectable serum HCV RNA level 24 weeks after treatment was completed) was higher among patients who received combination therapy for either 24 weeks (70 of 228 patients, 31 percent) or 48 weeks (87 of 228 patients, 38 percent) than among patients who received interferon alone for either 24 weeks (13 of 231 patients, 6 percent) or 48 weeks (29 of 225 patients, 13 percent) (P<0.001 for the comparison of interferon alone with both 24 weeks and 48 weeks of combination treatment). Among patients with HCV genotype 1 infection, the best response occurred in those who were treated for 48 weeks with interferon and ribavirin. Histologic improvement was more common in patients who were treated with combination therapy for either 24 weeks (57 percent) or 48 weeks (61 percent) than in those who were treated with interferon alone for either 24 weeks (44 percent) or 48 weeks (41 percent). The drug doses had to be reduced and treatment discontinued more often in patients who were treated with combination therapy. CONCLUSIONS: In patients with chronic hepatitis C, initial therapy with interferon and ribavirin was more effective than treatment with interferon alone.

Interferon alfa-2b alone or in combination with ribavirin for the treatment of relapse of chronic hepatitis C. International Hepatitis Interventional Therapy Group.

BACKGROUND: Interferon alfa is the only effective treatment for patients with chronic hepatitis C. Forty percent of patients have an initial response to this therapy, but most subsequently relapse. We compared the effect of interferon alone with that of interferon plus oral ribavirin for relapses of chronic hepatitis C. METHODS: We studied 345 patients with chronic hepatitis C who relapsed after interferon treatment. A total of 173 patients were randomly assigned to receive standard-dose recombinant interferon alfa-2b concurrently with ribavirin (1000 to 1200 mg orally per day, depending on body weight) for six months, and 172 patients were assigned to receive interferon and placebo. RESULTS: At the completion of treatment, serum levels of hepatitis C virus (HCV) RNA were undetectable in 141 of the 173 patients who were treated with interferon and ribavirin and in 80 of the 172 patients who were treated with interferon alone (82 percent vs. 47 percent, P<0.001). Serum HCV RNA levels remained undetectable 24 weeks after the end of treatment in 84 patients (49 percent) in the combination-therapy group, but in only 8 patients (5 percent) in the interferon group (P<0.001). Sustained normalization of serum alanine aminotransferase concentrations and histologic improvement were highly correlated with virologic response. Base-line serum HCV RNA levels of 2 x 10(6) copies per milliliter or less were associated with higher rates of response in both treatment groups. Viral genotypes other than type 1 were associated with sustained responses only in the combination-therapy group. Combined therapy caused a predictable fall in hemoglobin concentrations but otherwise had a safety profile similar to that of interferon alone. CONCLUSIONS: In patients with chronic hepatitis C who relapse after treatment with interferon, therapy with interferon and oral ribavirin results in higher rates of sustained virologic, biochemical, and histologic response than treatment with interferon alone.

The CDNA and genomic DNA sequences of a mammalian neurotoxin from the scorpion Buthus martensii Karsch.

The cDNA library of venomous glands of the scorpion Buthus martensii Karsch (BmK) was constructed. A cDNA encoding a mammalian neurotoxin corresponding to the known alpha-type toxin, BmK M1, was amplified by polymerase chain reaction (PCR) and cloned, and its full-length sequence was determined. The open reading frame encoded the precursor of BmK M1 with 84 amino acid residues, including a signal peptide of 19 residues, a mature toxin of 64 residues and an additional C-terminal residue Arg which might be cleaved off by proteinase postprocessing immediately after protein synthesis. Based on the determined cDNA sequence and using the total DNA of the scorpion as a template, the gene of BmK M1 was also amplified by PCR and sequenced. The genomic DNA sequence revealed an intron of 408 base pairs present within the signal peptide region. Both the intron and exon of BmK M1 share about 75% similarity with those of AaH I' another alpha-type mammalian neurotoxin in the scorpion Androctonus australis Hector.

Protein, cDNA, and genomic DNA sequences of the towel gourd trypsin inhibitor. A squash family inhibitor.

Two trypsin inhibitor components of the squash family were isolated and purified from the juice of the towel gourd (Luffa cylindrica) using anhydrotrypsin affinity chromatography followed by high pressure liquid chromatography. The inhibitors were sequenced and found to consist of 28 and 29 amino acid residues. The determined sequences show high similarity to other inhibitors of the squash family, especially in the location of disulfide bonds and the reactive site and also in the COOH-terminal region. A cDNA library of towel gourd was constructed and used as a template for polymerase chain reaction amplification of two cDNA fragments of the inhibitor with an overlapping sequence. A full-length cDNA sequence coding for the inhibitor was then completed. The open reading frame codes for a prepro-inhibitor protein with the pre- and pro-peptides consisting of 21 and 13 residues, respectively. The deduced amino acid sequence of 29 residues for the inhibitor is consistent with that determined by primary structure analysis. The genomic sequence of the mature inhibitor was also ascertained using the total DNA of the towel gourd as a polymerase chain reaction template. The genomic sequence is completely identical with that of the cDNA, showing no intervening sequence.

[The features of the 3' terminal sequences of the 18S rRNA gene from silkworm Attacus ricini].

The 3' terminal sequence of the gene for 18S rRNA of silkworm Attacus ricini have been sequenced. Comparison of this sequence with the 18S rDNA of silkworm Bombyx mori, Drosophila melanogaster, rat and the 16S rDNA of E. coli has shown that there is a remarkable homology between them. Moreover, the stem and loop formation of 3' regions of these rDNAs are very similar. There is a conservative EcoR1 site in the 3' region of these rDNAs. These results may contribute to the understanding of the functions of the 3' end of 18S rDNA in protein synthesis, in proceeding of rRNA precursor, and to the understanding of the evolutionary relation of rDNAs.

Side effects of corticosteroid therapy. Psychiatric aspects.

We reviewed the literature to determine the characteristics of corticosteroid-induced mental disturbances. We conclude that (1) while dosage may be correlated to the risk of developing mental disturbances, neither dosage nor duration of treatment seems to affect the time of onset, duration, severity, or type of mental disturbances; (2) euphoria, depression, and psychotic reactions are the common manifestations of corticosteroid-induced mental disturbances; (3) females seem to be more prone to these disturbances than males; (4) patients with past mental illness are not necessarily predisposed to such disturbances; and (5) corticosteroid-induced mental disturbances are usually reversible on dose reduction or discontinuation of the drug. At present there are no simple models to explain the psychotic reactions, anxiety, or agitation seen in corticosteroid-induced mental disturbances.

Factors associated with creatinine clearance changes following gentamicin therapy.

The relationship between creatinine clearance changes during gentamicin therapy and several patient and therapeutic variables was studied in a prospective, multicenter trial. Adult patients in three hospitals who were receiving gentamicin in doses based on lean body weight and creatinine clearance were studied. Pre- and post-therapy serum creatinine measurements were obtained for 62 patients (Group 1); only 45 of the patients had both pre- and post-therapy creatinine clearance measurements (Group 2). Other data collected for correlation with creatinine clearance changes were: body temperature, age, sex, blood pressure, albumin level, previous and concomitant therapy with nephrotoxic drugs, hematocrit, peak and trough gentamicin levels, and duration of therapy. In Group 2 patients, only peak gentamicin level, concomitant therapy, sex, and previous furosemide therapy correlated significantly (p less than or equal to 0.05) with change in creatinine clearance during gentamicin therapy. The first two variables decreased creatinine clearance, the last variable increased creatinine clearance, and women tended to have more of a decrease in clearance than did men. Sex, peak gentamicin level, prior furosemide therapy and concomitant cephalothin therapy explain about 50% of the renal function changes during gentamicin therapy, and should be considered in monitoring gentamicin therapy.