Bacterial β-lactamase is efficiently secreted in Saccharomyces cerevisiae under control of the invertase signal sequence

Summary The enzyme -lactamase, a secretory protein that is located in the Escherichia coli periplasmic space, can be highly expressed in Saccharomyces cerevisiae. Although the protein can cross eukaryotic membranes, it is only inefficiently secreted by yeast. To determine whether the lack of secretion in yeast is due to the nature of the bacterial signal sequence, it was replaced with the signal peptide of yeast invertase. The presence of the invertase signal peptide led to -lactamase secretion of up to 75%. The results indicate that the bacterial signal peptide is not functional in yeast, although cleavage can take place at the authentic processing site. The mature enzyme does not interfere with the yeast secretion pathway.     

Limited value of zinc protoporphyrin as a marker of iron status in chronic hemodialysis patients

BACKGROUND: In an attempt to find new parameters able to evaluate the actual iron availability by bone marrow cells, zinc protoporphyrin (ZnPP), a metabolic intermediate generated in the red blood cell by the incorporation of zinc instead of iron, has been proposed. ZnPP is a good marker of iron-deficiency anemia in non-uremic people, as red blood cell ZnPP concentration rises specifically (except for lead intoxication) in this condition. Existing data on ZnPP as a marker of iron deficiency in uremic patients comes mainly from cross sectional studies on chronic hemodialysis and has produced conflicting results. SUBJECTS AND METHODS: Therefore, we prospectively studied 42 HID patients, 28-88 years old, 13-346 months of dialysis age, beginning from a period of maximal iron deficiency, due to the lack of parenteral iron compounds (T0) up to the end of more than one year of follow-up with continuous parenteral iron supplementation (T4). ZnPP, hemoglobin, transferrin saturation and ferritin were serially determined before and after six weeks (T1), four months (T2), seven months (T3) and 14 months (T4) of parenteral iron supplementation at a maintenance dose of 0.5-1 mg/kg/week. RESULTS: In comparison with baseline values (95+/-37 micromol/mol heme) there were no significant changes in ZnPP levels at T1 and T2 despite a continuous increase in both transferrin saturation and ferritin values, while ZnPP significantly decreased at T4 (63+/-37 micromol/mol heme, p<0.001). There was no correlation between ZnPP and both transferrin saturation and ferritin at any time during the study, the same was true for ZnPP and zinc and lead serum concentration, fibrinogen and reactive C protein levels at T1 and T4, respectively. At T4, only 2/10 patients who still showed ZnPP levels >80 micromol/mol heme had absolute or functional iron deficiency, when the percentage of hypochromic red cells were measured. CONCLUSION: We conclude that ZnPP untimely parallels a change in iron balance in only a proportion of uremic people, in as much as confounding factors, such as chronic inflammation and uremia in itself may obscure its relationship with iron status. Therefore, ZnPP cannot be assumed to be a first-line diagnostic marker of iron balance in uremic patients.     

Development of fluridil,a topical suppressor of the androgen receptor in androgenetic alopecia

Nonsteroidal antiandrogens (AA) cannot be topically used for androgenetic alopecia (AGA) because of systemic resorption. A new class of androgen receptor (AR) suppressors designed for safe topical treatment of AGA was synthesized from (3‐amino‐2‐hydroxy‐2‐methyl‐N‐(4‐nitro‐3‐trifluoromethyl)phenyl) propanamide (BP‐34), to contain perfluoroalkyl moieties. The trifluoromethyl derivative (fluridil) at 10 μM decreased expression of the AR in LNCaP human cells by 95%, its serum half‐life was 6 h; it decomposes hydrolytically to BP‐34 and trifluoroacetic acid. Acute intraperitoneal maximum tolerated dose (MTD) of fluridil in mice is 270–300 mg/kg/d and the subacute MTD is 450 mg/kg/d. The oral LD₅₀ in mice was 2,872 mg/kg in males, 2,232 mg/kg in females, and >2,500 mg/kg in rats. Fluridil solution in isopropanol was not cutaneously absorbed in rabbits, did not sensitize or show any phototoxic or photoallergic effects on guinea pig skin, and demonstrated no skin irritation potential in rabbits and humans. Fluridil solid induced only slight and reversible eye irritancy in rabbits and displayed no cytotoxicity to rabbit corneal fibroblasts in vitro. Fluridil demonstrated no significant mutagenicity potential by Ames method. In a double‐blind study, 43 males with AGA, Norwood grade II to Va, used topical 2% fluridil in isopropanol or the vehicle daily for 12 months. Anagens (growing hairs) increased in the fluridil group from 76% to 89%. All hematological and biochemistry values remained within normal range, including testosterone, which varied but seasonally. No fluridil or its decomposition product (BP‐34) was detected in serum. No adverse side effects were reported. Drug Dev. Res. 59:292–306, 2003. © 2003 Wiley‐Liss, Inc.     

Chromosomal instability rather than p53 mutation is associated with response to neoadjuvant cisplatin-based chemotherapy in gastric carcinoma.

PURPOSE: The objective of the study was to evaluate microsatellite alterations [microsatellite instability (MSI) and loss of heterozygosity (LOH)] and mutation in the p53 gene in relation to response and patient survival to a cisplatin-based neoadjuvant chemotherapy in gastric cancer. EXPERIMENTAL DESIGN: Fifty-three pretherapeutic gastric carcinoma biopsies were analyzed with 11 microsatellite markers. The entire coding region of the p53 gene (exons 2-11) was analyzed for mutations by denaturing high-pressure liquid chromatography and sequencing. p53 protein expression was evaluated by immunohistochemistry. Patients were treated with a cisplatin-based, neoadjuvant chemotherapy regimen. Therapy response was evaluated by computed tomography scan, endoscopy, and endoluminal ultrasound. The median follow-up of the patients was 45.6 months. RESULTS: p53 mutations were identified in 19 of the 53 (36%) analyzed tumors. No significant association with response or survival was found for p53 mutation or for p53 protein expression. MSI (either high-grade MSI or low-grade MSI) did not show a correlation with response. With respect to LOH, LOH at chromosome 17p13 showed a significant association with therapy response (P = 0.022) but did not reach statistical significance in terms of patient survival. The global LOH rate, expressed as fractional allelic loss (FAL), was assessed, and tumors were classified into tumors with a high (>0.5), medium (>0.25-0.5), and low (0-0.25) FAL value. A statistically significant association of FAL with therapy response was found (P = 0.003), with a high FAL being related to therapy response. The sensitivity, specificity, positive predictive value, and negative predictive value for FAL > 0.5 were 45%, 93%, 82%, and 72%, respectively. CONCLUSIONS: A high level of chromosomal instability (high FAL value) defines a subset of patients who are more likely to benefit from cisplatin-based neoadjuvant chemotherapy. p53 mutation status is not significantly associated with therapy response and is not a useful marker for response prediction.