Cgl-SLT2 is required for appressorium formation, sporulation and pathogenicity in Colletotrichum gloeosporioide

The mitogen-activated protein (MAP) kinase pathways has been implicated in the pathogenicity of various pathogenic fungi and plays important roles in regulating pathogenicity-related morphogenesis. This work describes the isolation and characterization of MAP kinase gene, Cgl-SLT2, from Colletotrichum gloeosporioides. A DNA sequence, including 1,633 bp of Cgl-SLT2 open-reading frame and its promoter and terminator regions, was isolated via DNA walking and cloned. To analyze gene function, a gene disruption cassette containing hygromycin-resistant gene was constructed, and Cgl-SLT2 was inactivated via gene deletion. Analysis on Cgl-slt2 mutant revealed a defect in vegetative growth and sporulation as compared to the wild-type strain. When grown under nutrient-limiting conditions, hyperbranched hyphal morphology was observed in the mutant. Conidia induction for germination on rubber wax-coated hard surfaces revealed no differences in the percentage of conidial germination between the wild-type and Cgl-slt2 mutant. However, the percentage of appressorium formation in the mutant was greatly reduced. Bipolar germination in the mutant was higher than in the wild-type at 8-h post-induction. A pathogenicity assay revealed that the mutant was unable to infect either wounded or unwounded mangoes. These results suggest that the Cgl-SLT2 MAP kinase is required for C. gloeosporioides conidiation, polarized growth, appressorium formation and pathogenicity.

Cgl-SLT2 is required for appressorium formation, sporulation and pathogenicity in Colletotrichum gloeosporioides.

The mitogen-activated protein (MAP) kinase pathways has been implicated in the pathogenicity of various pathogenic fungi and plays important roles in regulating pathogenicity-related morphogenesis. This work describes the isolation and characterization of MAP kinase gene, Cgl-SLT2, from Colletotrichum gloeosporioides. A DNA sequence, including 1,633 bp of Cgl-SLT2 open-reading frame and its promoter and terminator regions, was isolated via DNA walking and cloned. To analyze gene function, a gene disruption cassette containing hygromycin-resistant gene was constructed, and Cgl-SLT2 was inactivated via gene deletion. Analysis on Cgl-slt2 mutant revealed a defect in vegetative growth and sporulation as compared to the wild-type strain. When grown under nutrient-limiting conditions, hyperbranched hyphal morphology was observed in the mutant. Conidia induction for germination on rubber wax-coated hard surfaces revealed no differences in the percentage of conidial germination between the wild-type and Cgl-slt2 mutant. However, the percentage of appressorium formation in the mutant was greatly reduced. Bipolar germination in the mutant was higher than in the wild-type at 8-h post-induction. A pathogenicity assay revealed that the mutant was unable to infect either wounded or unwounded mangoes. These results suggest that the Cgl-SLT2 MAP kinase is required for C. gloeosporioides conidiation, polarized growth, appressorium formation and pathogenicity.

Cgl-SLT2 is required for appressorium formation, sporulation and pathogenicity in Colletotrichum gloeosporioide

The mitogen-activated protein (MAP) kinase pathways has been implicated in the pathogenicity of various pathogenic fungi and plays important roles in regulating pathogenicity-related morphogenesis. This work describes the isolation and characterization of MAP kinase gene, Cgl-SLT2, from Colletotrichum gloeosporioides. A DNA sequence, including 1,633 bp of Cgl-SLT2 open-reading frame and its promoter and terminator regions, was isolated via DNA walking and cloned. To analyze gene function, a gene disruption cassette containing hygromycin-resistant gene was constructed, and Cgl-SLT2 was inactivated via gene deletion. Analysis on Cgl-slt2 mutant revealed a defect in vegetative growth and sporulation as compared to the wild-type strain. When grown under nutrient-limiting conditions, hyperbranched hyphal morphology was observed in the mutant. Conidia induction for germination on rubber wax-coated hard surfaces revealed no differences in the percentage of conidial germination between the wild-type and Cgl-slt2 mutant. However, the percentage of appressorium formation in the mutant was greatly reduced. Bipolar germination in the mutant was higher than in the wild-type at 8-h post-induction. A pathogenicity assay revealed that the mutant was unable to infect either wounded or unwounded mangoes. These results suggest that the Cgl-SLT2 MAP kinase is required for C. gloeosporioides conidiation, polarized growth, appressorium formation and pathogenicity.

Soybeans as bioreactors for biopharmaceuticals and industrial proteins.

Plants present various advantages for the production of biomolecules, including low risk of contamination with prions, viruses and other pathogens, scalability, low production costs, and available agronomical systems. Plants are also versatile vehicles for the production of recombinant molecules because they allow protein expression in various organs, such as tubers and seeds, which naturally accumulate large amounts of protein. Among crop plants, soybean is an excellent protein producer. Soybean plants are also a good source of abundant and cheap biomass and can be cultivated under controlled greenhouse conditions. Under containment, the plant cycle can be manipulated and the final seed yield can be maximized for large-scale protein production within a small and controlled area. Exploitation of specific regulatory sequences capable of directing and accumulating recombinant proteins in protein storage vacuoles in soybean seeds, associated with recently developed biological research tools and purification systems, has great potential to accelerate preliminary characterization of plant-derived biopharmaceuticals and industrial macromolecules. This is an important step in the development of genetically engineered products that are inexpensive and safe for medicinal, food and other uses.

Correct targeting of proinsulin in protein storage vacuoles of transgenic soybean seeds.

Soybean plants are promising bioreactors for the expression of biochemically complex proteins that cannot be produced in a safe and/or economically viable way in microorganisms, eukaryotic culture cells or secreted by transgenic animal glands. Soybeans present many desirable agronomic characteristics for high scale protein production, such as high productivity, short reproductive cycle, photoperiod sensitivity, and natural organs destined for protein accumulation in the seeds. The significant similarities between plant and human cells in terms of protein synthesis processes, folding, assembly, and post-translational processing are important for efficient accumulation of recombinant proteins. We obtained two transgenic lines using biolystics, incorporating the human proinsulin gene under control of the monocot tissue-specific promoter from sorghum gamma-kafirin seed storage protein gene and the alpha-coixin cotyledonary vacuolar signal peptide from Coix lacryma-jobi (Poaceae). Transgenic plants expressed the proinsulin gene and accumulated the polypeptide in mature seeds. Protein targeting to cotyledonary protein storage vacuoles was successfully achieved and confirmed with immunocytochemistry assays. The combination of different regulatory sequences was apparently responsible for high stability in protein accumulation, since human proinsulin was detected after seven years under room temperature storage conditions.

Phase II trial of the use of paclitaxel and gemcitabine as a salvage treatment in metastatic breast cancer.

The purpose of this study was to evaluate gemcitabine plus paclitaxel in heavily pretreated patients with metastatic breast cancer (MBC). Patients with MBC with second or third relapse to anthracycline-containing regimens received a 3-hour infusion of paclitaxel 175 mg/m2 on day 1, and gemcitabine 1.0 g/m2 on days 1, 8, and 15, every 28 days. Because of unacceptable thrombocytopenia seen in the first 5 patients, the gemcitabine schedule was changed to days 1 and 8 (G-1,8) for the remainder of the study, every 21 days. Twenty-nine patients (median age, 46 years; range, 32-68 years) received 137 cycles (median: 4 per patient). The regimen was well tolerated. World Health Organization grades III and IV thrombocytopenia were observed in 5 (18.5%) of the first 27 cycles (G-1,8,15), and in 6 (5.4%) of the 110 subsequent cycles (G-1,8)--p = 0.04 for the difference between schedules. Five patients had grade I and two had grade III neuropathy. Eight patients had grade III neutropenia, two had grade IV neutropenia associated with fever (G-1,8,15), and eight had grades I and II myalgia and fatigue. There were 16 (55%) objective responses (95% CI 36-73%); 5 (17%) complete responses, 11 (38%) partial responses (95% CI 3-30% and 19-56%, respectively), and 6 (20.5%) patients with stable disease. Median response duration was 8 months (range, 4-26 months). Median overall survival was 12 months (range, 4-28+ months), and 1-year and 2-year survival rates were 45% and 30%, respectively. This phase II study demonstrated a manageable toxicity profile with the gemcitabine day 1, 8 schedule in combination with paclitaxel and significant and promising activity in heavily pretreated patients with MBC. A confirmatory phase III trial is warranted.

Gemcitabine and paclitaxel as salvage therapy in metastatic breast cancer.

In a phase II trial, 29 patients with anthracycline-pretreated or anthracycline-resistant metastatic breast cancer in whom anthracycline-containing first- or second-line chemotherapy failed received combination paclitaxel (Taxol)/gemcitabine (Gemzar). The initial regimen of paclitaxel at 175 mg/m2 on day 1 and gemcitabine at 1,000 mg/m2 on days 1, 8, and 15 of a 28-day cycle was given to five patients for a total of 27 cycles. The regimen resulted in excessive thrombocytopenia and was subsequently changed to gemcitabine at the same dose on days 1 and 8 of a 21-day cycle, with study treatment being given for a maximum of eight cycles. This regimen was well tolerated. Further evaluation of this regimen in minimally and heavily pretreated patients with advanced breast cancer is warranted.

Phase II trial of paclitaxel and ifosfamide as a salvage treatment in metastatic breast cancer.

PURPOSE: Treatment results in patients failing first-line chemotherapy in metastatic breast cancer (MBC) are still unsatisfactory, with patients exhibiting poor responses to salvage therapy and a short overall survival. Both paclitaxel and ifosfamide are able to produce objective tumor responses in this disease. Therefore, the antitumor effects and toxicity of their combined use could be worthwhile studying in patients progressing after doxorubicin-containing combinations. PATIENTS AND METHODS: This Phase II trial of paclitaxel/ifosfamide included patients with bi-dimensionally measurable metastatic breast cancer in second or third relapse, following anthracycline-containing regimens; ECOG PS < 2, and adequate hepatic, cardiac, renal, and hematological functions. Paclitaxel 175 mg/m2 was given on day 1, in a 3-hour infusion with appropriate antiallergic pre-medication; while ifosfamide 1.8 g/m2 was given on days 2, 3, 4 with mesna 360 mg/m2 i.v., 15 minutes before and 4 hours after ifosfamide administration, and 720 mg/m2 P.O. 8 hours later at home, also on days 2, 3, 4. The cycles were repeated every 21 days, on an outpatient basis. RESULTS: Twenty-four patients were accrued for the study and 23 were considered eligible for the evaluation of toxicity and response. Previous chemotherapy included: CMF/FAC (16 cases); CMF plus mitoxantrone/FAC/cisplatin, vinblastine, mitomycin C (2 cases): and FAC/mitomycin C, vinblastine, and etoposide (5 cases). There were 11 (48%) objective responses (95% C.I.:27-69%), including 2 (9%) CR and 9 (39%) PR (95% C.I.:0-21% and 19-61%, respectively). Five (22%) patients attained disease stabilization. Median response duration was 7+ months (range 4 to 20+), and the median overall survival was 12 months (range 4-23+). The regimen was well tolerated. WHO nausea/ vomiting grades 1-2, alopecia grade 3, and neutropenia grades 1-2 were seen in most patients. Four patients experienced mild neuropathy, while it was grade 3 in 1 case. Seven patients had grade 3 neutropenia. In addition, grade 4 neutropenia associated with fever was documented in other 4 cases. No hypersensitivity reactions were seen. One case of reversible tachycardia after drug administration was seen. Myalgia grades 1-2 was also reported in some patients. CONCLUSION: These results suggest that the present regimen has significant activity in heavily pretreated patients with a MBC, with a manageable toxicity profile. Further trials exploiting the above mentioned drug combination are warranted.

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The treatment of colorectal cancer continues to pose major challenges for oncologists throughout the world. Uracil and tegafur (UFT), as an oral agent, represents a new patient-focused approach to managing a malignancy with few treatment alternatives other than an intravenous fluorouracil (5-FU)-based regimen. The ability of UFT to achieve equivalent clinical outcomes compared with continuous 5-FU infusion, along with its oral formulation and mild toxicity profile, provide a compelling backdrop for fiscal analysis. An economic assessment of therapy attributes and effects would, therefore, be prudent and necessary when deliberating the adoption of this chemotherapy regimen. We developed a pharmacoeconomic model in Brazil and Argentina identifying clinical practices associated with chemotherapy administration and adverse event management practices from a panel of physicians assembled in each country. Practice patterns and clinical events were then evaluated for resource utilisation trends. The perspective of this pharmacoeconomic analysis was that of the healthcare payor. Country-specific charge data were applied to the identified resources to arrive at an average cost per patient receiving a 6-cycle course of 5-FU with either levamisole and/or leucovorin as a modulator vs a modelled oral UFT/leucovorin regimen. As a comparator, the oral UFT/leucovorin regimen was modelled based on the expert panel's input. Adverse events and incidence data were derived from clinical trial data for both agents. Both agents were analysed in the treatment of metastatic disease and as adjuvant therapy. The principal findings of a cost-minimisation analysis in Brazil revealed approximately equivalent treatment costs for both regimens in the adjuvant setting. When analysing the metastatic treatment arm, costs diverged by $R335/per patient ($R = Reals - the currency of Brazil) in favour of a UFT regimen. The profile in Argentina yielded more dramatic differences, with a UFT regimen costing $P782/per patient ($P = Pesos - the currency of Argentina) less than a 5-FU regimen in the adjuvant setting. In the treatment of metastatic disease, a UFT regimen provided $P1188/per patient in savings over a 5-FU regimen. These differences are predominantly driven by the mild toxicity profile of UFT and its corresponding less severe adverse event management practice patterns. In addition, the oral formulation of UFT versus intravenous 5-FU provides for ease of administration, lowering the total cost of care as well as likely impacting on the patient's quality of life. The pharmacoeconomic results suggest that a UFT regimen is a useful and economical alternative to the standard 5-FU regimen in the treatment of colorectal cancer in Brazil and Argentina.

Phase II trial of isotretinoin and interferon alpha-2a in the treatment of advanced recurrent cervical carcinoma.

Background: A recent trial by Lippman et al.16 demonstrated the effectiveness and synergy of the combination of interferon alpha-2a and isotretinoin in the treatment of patients with locally advanced, previously untreated squamous cell carcinoma of the cervix. Purpose: In this phase II trial, we used this combination to treat patients with advanced and recurrent squamous cell cervical carcinoma, aiming to assess its efficacy in terms of objective responses as well as toxicity. Methods: Eighteen patients with advanced recurrent squamous cell carcinoma of the cervix were enrolled. All 18 had previously been irradiated. In addition, six had received chemotherapy following progression after radiation therapy. Treatment consisted of interferon alpha-2a, 6 MU m-2 subcutaneously daily, 5 days a week and isotretinoin, 1 mg kg-1 orally, daily, for at least 2 months, depending on the response or toxicity. Results: All 18 patients (16 stage IVA and 2 stage IVB) were considered eligible for response and toxicity evaluation. After a median of 3 months (maximum of 8, in responders), we observed partial responses in two patients (11%); 95% confidence interval 0-25%. Five (27.7%) patients had stable disease. Responses and disease stabilization were observed only in the group of patients who had received irradiation alone. The median time to progression was 6 months (range 3-8+ months). The toxicity was very acceptable in this group of women. Low fever occurred at the beginning of the treatment in five patients. Fatigue and weakness were observed in four patients and required temporary interferon dose reduction, bone pain in three patients, grade 1 leukopenia in two patients and more intensive dry skin in four patients. Two patients had slight hypertriglyceridemia. Conclusion: Objective responses were observed using the combination of interferon alpha-2a and isotretinoin in a group of patients with advanced recurrent squamous cell carcinoma of the cervix. We now test this combination in previously untreated patients in a neoadjuvant setting.

Phase II trial of bleomycin, ifosfamide, and carboplatin in metastatic cervical cancer.

PURPOSE: A bleomycin, carboplatin, and ifosfamide (BIC) chemotherapy protocol was designed to evaluate tumor response and palliation in patients with advanced cervical cancer. PATIENTS AND METHODS: Forty patients with stage IV primary or recurrent squamous cell carcinoma of the cervix (19 previously irradiated and 21 nonirradiated) were assigned to treatment with six cycles of BIC: bleomycin, 30 mg bolus on day 1; carboplatin, 200 mg/m2 bolus on day 1; and ifosfamide, 2g/m2 for 3 consecutive days, infused over 2 hours. Mesna was administered as a bolus 15 minutes, and 4 and 8 hours after ifosfamide at 20% (intravenous [IV]), and 40% (orally, at home) of the ifosfamide dose, respectively. RESULTS: Thirty-five patients (27 stage IVA and eight stage IVB) were considered eligible for response and toxicity evaluation. After a median of four cycles (maximum of six in responders), we observed objective responses in 21 patients (60%), with eight complete responses (CRs; 23%), including two histologically documented by laparotomy, and 13 (37%) partial responses (PRs) (95% confidence limits, 44% to 76%, 9% to 37%, and 21% to 53%, respectively). Median overall survival duration was 11 months (range, 3 to 24+). Median overall survival duration in the nonirradiated group was 17 months versus 4 months in the previously irradiated group (P = .005). The median progression-free survival duration of the responders was 12 months, and the median disease-free survival duration of the complete responders was 14 months. Toxicity was acceptable and included manageable alopecia, vomiting, and neutropenia. There was one toxic death due to febrile neutropenia and sepsis. CONCLUSION: BIC can be administered on an outpatient basis and seems to be effective in inducing tumor response and palliation in patients with disseminated squamous cell carcinoma of cervix, with a possible survival benefit for previously nonirradiated patients, with an acceptable toxicity profile.

Modified therapy with 5-fluorouracil, doxorubicin, and methotrexate in advanced gastric cancer.

BACKGROUND: In an attempt to decrease the toxic effects of fluorouracil, doxorubicin, and methotrexate (FAMTX) by reducing the dose of methotrexate from 1500 mg/m2, according to the original regimen, to 1000 mg/m2, the authors designed the modified FAMTX treatment that was evaluated in a prospective Phase II-III randomized trial. METHODS: Patients with advanced gastric cancer were randomized to receive modified FAMTX treatment or supportive measures only (control group). In the middle of the study, the randomization was interrupted because of strong evidence of benefit in terms of tumor reduction and projected survival in the treatment arm receiving chemotherapy. By the end of the study, 30 assessable patients had received chemotherapy and 10 had received supportive treatment. RESULTS: The overall response rate was 50% (15 patients); 12 patients (40%) had partial responses and 3 (10%) had complete responses (CR). One patient with extensive peritoneal carcinomatosis attained a CR pathologically documented by laparoscopic examination and peritoneal biopsy. The median overall survival time of the treated group was 9 months, whereas that of the control group was only 3 months (P = 0.001). The median overall survival time of the responders was 16 months, and their median remission duration was 8 months. The regimen was well tolerated, with a very acceptable toxicity profile. There was one toxic death resulting from neutropenia and sepsis in a patient who did not receive adequate leucovorin rescue. CONCLUSIONS: This regimen appears to prolong survival in patients with advanced gastric cancer, and the reduction of the methotrexate dose does not seem to compromise its efficacy.