Breast-conserving treatment for ductal carcinoma in situ: Impact of boost and tamoxifen on local recurrences.

PURPOSE: Ductal carcinoma in situ represents 15 to 20% of all breast cancers. Breast-conserving surgery and whole breast irradiation was performed in about 60% of the cases. This study reports local recurrence rates in patients with ductal carcinoma in situ treated by breast-conserving surgery and whole breast irradiation with or without boost and/or tamoxifen and compares different therapeutic options in two European countries. PATIENTS AND METHODS: From 1998 to 2007, 819 patients with pure ductal carcinoma in situ were collected, both in France (266) and Italy (553). Median age was 56. All underwent breast-conserving surgery and whole breast irradiation; 391 (48%) received a boost (55% in France and 45% in Italy, P=0.017) and 173 (22.5%) tamoxifen (4.5% in France and 32% in Italy, P<0.0001). RESULTS: With a 90-month median follow-up, there were 51 local recurrences (6.2%), including 27 invasive (53%). The 5- and 10-year local recurrence rates were 4% and 8.6%. Two patients developed axillary recurrence and 12 (1.5%) metastases (seven after invasive local recurrence); 41 (5%) patients had contralateral breast cancer. In the multivariate analysis, high nuclear grade and lack of tamoxifen are the most powerful predictors of local recurrence, with 2.6 (95% confidence interval [95% CI]: 1.74-3.89, P=0.0012) and 2.85 (95% CI: 1.42-5.72, P=0.04) odds ratio (OR) estimates, respectively. Age, margin status and boost did not influence local recurrence rates. CONCLUSIONS: This study confirms the ductal carcinoma in situ treatment heterogeneity among countries and the unfavourable prognostic role of nuclear grade. Tamoxifen reduces local recurrence rates and might be considered for some subgroups of patients, but further confirmation is required. The boost usefulness still remains unclear.

High-dose radiotherapy plus prolonged hormone therapy in CT2-3 prostatic carcinoma: is it useful?

AIMS AND BACKGROUND: Clinical studies published in the last decade have shown the possible improvement in prognosis of patients with prostatic carcinoma undergoing radiation therapy with dose escalation or in combination with hormone therapy. However, in studies on hormone therapy, moderate doses of radiation therapy have been used, whereas in studies with high-dose radiotherapy, hormone therapy usually was not administered. Therefore, it is not clear whether the concomitant use of high doses and prolonged hormone therapy could determine an additional beneficial effect. The aim of the present study was therefore to evaluate the relative prognostic role of different dose levels (< 70 versus > or = 70 Gy) of external beam radiotherapy and of different hormone therapies (neoadjuvant only versus neoadjuvant + adjuvant). METHODS: A total of 426 patients (median age, 71 yrs; range, 51-87 yrs) underwent external beam radiotherapy (70 Gy median dose to prostate volume +/- 45 Gy to pelvic lymph nodes) and neoadjuvant hormone therapy (bicalutamide for 30 days; goserelin, 3.6 mg every 28 days starting two months before radiotherapy and for its entire duration). Dose to the prostate was < 70 Gy in 44.8% of patients and > or = 70 Gy in 55.2%. A total of 244 patients received adjuvant hormonal therapy. The distribution according to the clinical stage was 48.1% T2 and 51.9% T3. The distribution according to the Gleason score was 14.3% grades 2-4, 66.7% grades 5-7 and 19.0% grades 8-10. The distribution according to pretreatment prostate-specific antigen levels (in ng/mL) was 7.0% for 0-4, 29.3% for 4-10, 30.3% for 10-20, and 33.3% for > 20. RESULTS: With a median follow-up of 35 months (range, 1-151), 81 patients (19.0%) showed biochemical recurrence, 17 patients (4.0%) showed local disease progression, and 12 patients (2.8%) showed distant metastases. Overall, 23 patients (5.4%) showed disease progression. Four patients (0.9%) died. At the time of this writing, no patient has died from prostatic carcinoma. At univariate analysis, the radiation dose delivered to the tumor and the administration of adjuvant hormone therapy were shown to be significantly correlated with biochemical disease-free survival. At multivariate analysis, the single parameter significantly correlated with biochemical disease-free survival was the radiation dose delivered to the tumor. In the subset of patients not treated with adjuvant hormone therapy, there was a significant correlation between radiation dose and biochemical disease-free survival at univariate and multivariate analysis. A similar correlation between adjuvant hormone therapy and biochemical disease-free survival was observed in the subset of stage cT3 patients at univariate and multivariate analysis. In patients undergoing combined treatment without adjuvant hormone therapy, a significant correlation was observed between clinical stage and biochemical disease-free survival, at univariate and at multivariate analysis. CONCLUSIONS: The results of the study confirmed the positive impact of radiotherapy doses > 70 Gy and of adjuvant hormone therapy in patients with locally advanced prostatic carcinoma. Owing to the lack of evidence of a correlation between radiation dose and biochemical outcome in patients undergoing prolonged hormone therapy, the role of further dose escalation in patients undergoing combined hormone and radiation therapy is still unclear.

A mammalian-like lipase gene is expressed in the female reproductive accessory glands of the sand fly Phlebotomus papatasi (Diptera, Psychodidae).

A lipase-like protein (PhpaLIP) was identified as the major protein component in the secretion of the female reproductive accessory glands of the sand fly Phlebotomus papatasi. The full-length cDNA encoding this protein was isolated and its nucleotide sequence determined. The deduced translational product of the gene contains a GFSFG motif, consistent with a GXSXG consensus, which is shared by most bacterial and eukaryotic hydrolases. Transcriptional analysis of the PhpaLIP gene showed that its expression is female-specific, and is also detectable in districts other than accessory glands, suggesting that it might play different functions. Taken together with the observation of sequence similarity shared by PhpaLIP and mammalian lipases, the demonstration of the presence of lipase activity in the accessory gland secretion suggests a possible biological role of PhpaLIP gene product.

The ceratotoxin gene family in the medfly Ceratitis capitata and the Natal fruit fly Ceratitis rosa (Diptera: Tephritidae).

Ceratotoxins (Ctxs) are a family of antibacterial sex-specific peptides expressed in the female reproductive accessory glands of the Mediterranean fruit fly Ceratitis capitata. As a first step in the study of molecular evolution of Ctx genes in Ceratitis, partial genomic sequences encoding four distinct Ctx precursors have been determined. In addition, anti-Escherichia coli activity very similar to that of the accessory gland secretion from C. capitata was found in the accessory gland secretion from Ceratitis (Pterandrus) rosa. SDS-PAGE analysis of the female reproductive accessory glands from C. rosa showed a band with a molecular mass (3 kDa) compatible with that of Ctx peptides, also slightly reacting with an anti-Ctx serum. Four nucleotide sequences encoding Ctx-like precursors in C. rosa were determined. Sequence and phylogenetic analyses show that Ctxs from C. rosa fall into different groups as C. capitata Ctxs. Our results suggest that the evolution of the ceratotoxin gene family might be viewed as a combination of duplication events that occurred prior to and following the split between C. capitata and C. rosa. Genomic hybridization demonstrated the presence of multiple Ctx-like sequences in C. rosa, but low-stringency Southern blot analyses failed to recover members of this gene family in other tephritid flies.

Ten years of preoperative chemoradiation for extraperitoneal T3 rectal cancer: acute toxicity, tumor response, and sphincter preservation in three consecutive studies.

PURPOSE: To compare acute toxicity, tumor response, and sphincter preservation in three schedules of concurrent chemoradiation in resectable transmural and/or node-positive extraperitoneal rectal cancer. PATIENTS AND METHODS: Between 1990 and 1999, 163 consecutive patients were treated according to the following combined modalities: FUMIR: between 1990 and 1995, 83 patients were treated with bolus i.v. mitomycin C (MMC), 10 mg/m(2) day 1, plus 24-h continuous infusion i.v. 5-fluorouracil (5-FU) 1,000 mg/m(2) days 1-4, and concurrent external beam radiotherapy (37.8 Gy). PLAFUR-4: between 1995 and 1998, 40 patients were treated with cisplatin (c-DDP) 60 mg/m(2) given as slow infusion (1-4 h) on days 1 and 29, plus 24-h continuous infusion i.v. 5-FU 1,000 mg/m(2), days 1-4 and 29-32 with concurrent external-beam radiotherapy (50.4 Gy). PLAFUR-5: between 1998 and 1999, 40 patients were treated with c-DDP 60 mg/m(2) given as slow infusion (during 1-4 h) on days 1 and 29, plus 24-h continuous infusion i.v. 5-FU 1,000 mg/m(2), days 1-5 and 29-33 with concurrent external-beam radiotherapy (50.4 Gy). RESULTS: Grade > or = 3 acute toxicity occurred in 14%, 5%, and 17% of patients treated in the FUMIR, PLAFUR-4, and PLAFUR-5 studies, respectively (p = 0.201). In the FUMIR, PLAFUR-4, and PLAFUR-5 studies, clinical response rate was 77%, 70%, and 83%, respectively. Tumor downstaging occurred in 57%, 68%, and 58% of patients, respectively. Pathologic complete response was recorded in 9% (FUMIR), 23% (PLAFUR-4), and 20% (PLAFUR-5) of patients. Sphincter-preserving surgery was feasible in 44% (FUMIR), 40% (PLAFUR-4), and 61% (PLAFUR-5) of patients having a distance between the anal-rectal ring and the lower pole of the tumor of 0-30 mm, and in 95%, 100%, and 100%, respectively, in those having a distance of 31-50 mm. Comparing FUMIR vs. PLAFUR, the clinical response rate was similar in the two series: a partial response was observed in 62/81 (77%) patients with FUMIR treatment, and in 61/80 (76%) patients with PLAFUR treatment. Tumor downstaging was observed in 46/81 (57%) patients and in 50/80 (68%) patients, respectively. The pathologic complete response rate was statistically higher in the PLAFUR series: 7/81 (9%) patients with FUMIR treatment and 17/80 (21%) patients with PLAFUR treatment (p = 0.04). Major downstaging (pT0+ pTmic+ pT1) in the FUMIR group was reported in 12/81 (15%) patients versus 31/80 (39%) patients in the PLAFUR group (p = 0.0006). The anal sphincter was preserved in 63/81 (78%) patients with FUMIR treatment and in 69/80 (86%) patients with PLAFUR treatment. The perioperative morbidity was statistically lower with PLAFUR: a perioperative morbidity was experienced by 20/81 (25%) patients with FUMIR treatment and by 9/80 (11%) patients with PLAFUR treatment (p = 0.042). CONCLUSION: In our experience, higher radiation dose (50.4 Gy vs. 37.8 Gy), a second course of concurrent 5-FU, and the use of c-DDP instead of MMC improved the pathologic response rate without increasing acute toxicity and perioperative morbidity. The use of 5-FU 5-day infusion (PLAFUR-5) resulted in higher toxicity with a similar response rate compared to 4-day infusion (PLAFUR-4).

Molecular structure of dynein and motility of a giant sperm axoneme provided with only the outer dynein arm.

The peculiar sperm axoneme of the dipteran Asphondylia ruebsaameni is characterized by an extraordinarily high number of microtubule doublets (up to 2,500) arranged in double parallel spirals. Doublets of the inner row of each spiral are tilted, so that their outer arms point towards the B-tubule of the next doublet in the outer row. Doublets are provided with only the outer arm, and no structure related to the central pair/radial spoke complex is present. When analyzed by quick-freeze, deep-etch electron microscopy, the structure of the dynein arms was shown to share the same organization described in other organisms; however, it appears to be somewhat more complex than that previously found in a related dipteran species, Monarthropalpus flavus, since the foot region of the arms displays a globular extra-domain that is intercalated between adjacent arms. Treatment of demembranated sperm with ATP and vanadate induced conformational changes in the dynein arms. SDS-page suggested the presence of a single dynein high molecular weight band or, in the gels with the best electrophoretic resolution, of two very closely spaced bands. This polypeptide positively reacted with a polyclonal antibody raised against a specific amino acid sequence located in the phosphate-binding loop of the dynein catalytic site. Dynein heavy chain-related DNA sequences corresponding to the catalytic phosphate-binding region were amplified by RT-PCR. Two distinct fragments (Asph-ax1 and Asph-ax2) encoding axonemal dynein sequences were identified. Southern blot analysis performed on genomic DNA using these sequences as a probe showed that they are part of different genes. An intron was identified in the Asph-ax1 fragment at a position corresponding to the site of a nucleotide deletion in the putative pseudogene of Monarthropalpus. Asphondylia spermatozoa exhibited in vivo a whirling movement both in the deferent duct and in the spermatheca, but they were unable to undergo processive movement in vitro. They propagated a three-dimensional wave only when constrained in a bent configuration by some mechanical means. The phylogenetic relationships between the two dipteran species, Monarthopalpus and Asphondylia, based on these biochemical and molecular data are also discussed.

Ceratotoxins: female-specific X-linked genes from the medfly, Ceratitis capitata.

In this paper, we report the chromosomal localization of ceratotoxins, a gene family encoding antibacterial female-specific peptides from the mediterranean fruit fly Ceratitis capitata. The analysis of both polytene and mitotic chromosomes by in situ hybridization shows that ceratotoxins are the first case of female-specific X-linked genes from the medfly C. capitata. Southern blot analysis reveals that the ceratotoxin gene family is not specifically amplified in the female reproductive accessory glands of C. capitata.

Morphogenesis of the giant sperm axoneme in Asphondylia ruebsaameni Kertesz (Diptera, Cecidomyiidae).

The formation of the sperm giant axoneme of the gall-midge fly Asphondylia ruebsaameni is described here. The axoneme consists of a great number of microtubular doublets (up to 2,500) arranged in a double spiral wrapping around an axial cluster of mitochondria. Each microtubular doublet is provided with an outer arm only. In the early spermatid the occurrence of a large system of curved multi-layered filamentous material associated with membranous cisternae has been observed in the perinuclear region. Such a system extends throughout the cytoplasm to contact the plasma membrane. The filamentous material appears to act as a nucleating centre for the assembly of the microtubular doublets, which initially have a submembranous location and later are distributed in the interior of the cell. After their assembly, microtubular doublets are associated pairwise and are arranged in a single microtubular row with a zig-zag configuration. This configuration changes during spermiogenesis as a consequence both of a rotation of the microtubular doublet pairs and a compaction of the axonemal complex due to the elimination of the excess cytoplasm. As a result of this process, a double parallel spiral of microtubular doublets is formed.

Preoperative chemoradiation with cisplatin and 5-fluorouracil for extraperitoneal T3 rectal cancer: acute toxicity, tumor response, sphincter preservation.

PURPOSE: To evaluate the impact of preoperative external radiation therapy intensified by systemic chemotherapy including bolus cisplatin (c-DDP) and 4-day infusional 5-fluorouracil (PLAFUR-4) on tumor response and sphincter preservation in patients with extraperitoneal T3 rectal cancer with acceptable toxicity, and to compare the results to our previous experience with bolus mitomycin c (MMC) and 4-day infusion 5-FU (FUMIR). METHODS AND MATERIALS: Between October 1995 and March 1998, 40 consecutive patients with resectable extraperitoneal adenocarcinoma of the rectum were treated with preoperative chemoradiation: slow infusion i.v. c-DDP, 60 mg/m2, day 1 and 29 plus 24-h continuous infusion i.v. 5-fluorouracil (5-FU) 1000 mg/m2, days 1-4 and 29-32, and concurrent external beam radiotherapy (45 Gy whole pelvis followed by 5.4 Gy boost). All but 3 patients had T3 disease. Surgery was performed 6-8 weeks after the end of chemoradiation. RESULTS: No patient had Grade 4 acute toxicity. Grade 3 hematological toxicity was observed only in 2 (5%) patients. No patient had major gastrointestinal, skin, or urological acute toxicity. All patients had radical surgery. There was no perioperative mortality; perioperative morbidity rate was 12%. Overall, 23% (9 of 40) of patients had a complete pathological response and 10% (4 of 40) of patients had rare isolated residual cancer cells (Tmic). Comparing the stage at the diagnostic workup with the pathological stage, tumor downstaging was observed in 27 (68%) patients; nodal status downstaging was detected in 24 (60%) patients. Thirty-four (85%) patients had a sphincter-saving surgical procedure. In 4 of 10 (40%) patients who were definitive candidates for an abdominoperineal resection (APR), the sphincter was preserved, as it was in 13 of 13 (100%) probable candidates. Lengthening of the distance between the anorectal ring and the lower pole of the tumor > or =20 mm was observed in 9 (23%) patients. None of the patients had soilage after the sphincter-saving procedure. In our previous experience with FUMIR the complete pathological response was 9%, the sphincter-saving surgical procedure was performed in 66% cases, and the Grade 3+ toxicity was observed in 13% of patients. CONCLUSIONS: The addition of c-DDP to 5-FU (PLAFUR-4) in a neoadjuvant radiochemotherapy schedule improved the pathological response rate in comparison with our previous experience. Toxicity was low indeed, thus we commenced another study adding one more day of 5-FU infusion (PLAFUR-5) to further improve our results.

FST, a female-specific transcript from the medfly Ceratitis capitata (Diptera: Tephritidae).

We report here the sequence of a novel cDNA clone (FST, female-specific transcript), isolated by differential screening during a search for sexually mature accessory gland-specific transcripts from the medfly Ceratitis capitata. It contains an open reading frame (ORF) with a potential translational start site encoding a putative precursor peptide of 100 amino acids. The FST gene is expressed only in the female reproductive accessory glands. Like the accessory gland-specific antibacterial peptides ceratotoxins, its expression reaches the maximum level when sexual maturity is achieved. However, in contrast to ceratotoxins, it is expressed at a basal level in newly emerged females, and its expression does not increase after mating.

Structural and molecular characterization of dynein in a gall-midge insect having motile sperm with only the outer arm.

The dipteran Monarthropalpus flavus possesses a peculiar sperm axoneme, characterized by multiple rows of microtubular doublets linked by the outer dynein arms only, lacking any equivalent of the central pair/radial spoke complex. The structure of these dynein molecules was studied by electron microscopy (EM). Using the quick-freeze, deep-etch method of EM, they were found to be similar to outer dynein arms described previously. Two globular "heads," each subdivided by a cleft, are clearly discernible. "Stalks" extend from proximal head to contact the B-tubule of the adjacent doublet. Unlike the situation in vertebrate sperm, the stalks sometimes branch into two thinner strands that contact the B-tubule at different sites. Treatment of demembranated sperm cells with ATP and vanadate induces conformational changes in the dynein outer arms. These are interpreted as the result of rotation of the dynein head with respect to what is observed in axonemes in rigor condition (after ATP depletion). SDS-PAGE indicates that the high-molecular-weight complement of this molecule comprises a single heavy chain. Specific dynein heavy chain-related DNA sequences corresponding to the catalytic-phosphate binding region were amplified by RT-PCR. Only one axonemal dynein sequence was identified among all amplified fragments. Southern blot analysis performed on genomic DNA using this sequence as a probe identified two hybridizing genes, only one of which is able to encode a functional product. Thus, genetic analysis indicates that this axonemal outer arm dynein is a homodymer of a single heavy chain subunit. In vivo, spermatozoa of this species are stored in a rolled configuration in female spermatheca, where they move rapidly with a wave-like motion. This movement could not be reproduced in vitro, except when spermatozoa were constrained in a bent configuration by some mechanical impediment. We propose that, in the absence of both the central pair/radial spoke complex and the inner arms, a curvature-dependent activation acts to trigger motility in these spermatozoa.

Radiotherapy and local control in rectal cancer.

Recurrence is a stage in the natural history of rectal cancer. Preoperative radiotherapy or postoperative radiochemotherapy lower the rate of recurrence, improving local control. From 1980 to 1997, at the "Divisione di Radioterapia" of the "Università Cattolica del S. Cuore" of Rome 380 patients with rectal cancer of early clinical stage T2-3, candidates for surgery for cure, underwent radiation therapy. 119 patients underwent postoperative radiotherapy (45-50 Gy); 45 patients underwent "sandwich" radiotherapy (45 Gy:27 Gy before and 28 Gy after surgery), of whom 7 were treated with preoperative radiotherapy alone; 145 patients underwent preoperative concomitant radiochemotherapy according to 3 different protocols, radiotherapy (38 Gy) combined with mitomycin C and 5-FU; radiotherapy (50.4 Gy) combined with cisplatin and 5-FU; radiotherapy (45 Gy) combined with 5-FU and folinic acid. 71 patients were treated with preoperative radiotherapy (38 Gy) combined with IORT (10 Gy). Median follow-up was 6 years. Overall local control was 85% at 3 years, 83% at 5 years, 81% at 10 years. The rate of local control at 5 years was: 76% for postoperative radiotherapy, 83% for "sandwich" radiotherapy, 84% for preoperative radiochemotherapy and 93% for preoperative radiotherapy combined with IORT. Local control was shown to be significantly better with preoperative treatment as compared to postoperative treatment (p = 0.02). The incidence of metastases was 35% in the patients with local recurrence and 16% in those with local control. The difference in survival was highly significant in patients with local control as compared to those with local recurrence: at 5 years 87% and 32% respectively. Patients with local control showed a lower incidence of metastasis and a better survival.

Presence of antibacterial peptides on the laid egg chorion of the medfly Ceratitis capitata.

Female reproductive accessory glands of the medfly Ceratitis capitata produce a secretion with antibacterial activity mainly ascribed to ceratotoxin peptides. To study whether the secretion from the accessory glands of the female protects the eggs and early larva from microbes, we examined whether ceratotoxins and other accessory gland components could be found on the egg surface. This was found to be the case; a water-soluble material with the same protein and antibacterial pattern as that of the accessory gland secretion was recovered from the laid egg surface and was observed as electrondense, clustered droplets over the outer exochorion. Such material showed the same electrophoretic pattern in both mated and virgin females. These findings indicate that the accessory gland secretion is spread, at oviposition, onto the eggs producing an antibacterial coating, irrespective of fertilization. This is the first report of antimicrobial components recovered from a material layered on insect laid eggs.

Organ preservation in rectal cancer.

Treatment of distal rectal cancer is aimed at sphincter preservation. Three trials were conducted to this purpose. They differed for T stage selection and therapy. Two of the trials are now completed, while patients are still recruited for the third trial. 21 T2 patients were selected for the first series and treated with local excision plus postoperative radiotherapy. External beam radiotherapy (ERT) was delivered at the dose of 45 Gy. Grade 3-4 (RTOG) acute toxicity was 5%; late toxicity was never observed. Sphincter function was rated as excellent or good in all patients. Sphincter preservation was achieved in 86% of cases. In the T3 FUMIR trial, 83 patients with extraperitoneal rectal cancer (T3) were treated with concomitant mitomycin C (10 mg/m2, day 1) plus 5FU (1000 mg/m2 days 1-4) plus ERT (38Gy). Grade 3-4 acute toxicity (RTOG) was 13%; late toxicity was never observed. Sphincter function was rated excellent or good in 96% of patients; sphincter preservation was achieved in 66% of patients with a lesion at less than 50 mm from the internal anal orifice. In the T3 PLAFUR trial, 19 patients with extraperitoneal rectal cancer (T3) were treated with concomitant cisplatin (60mg/m2, day 1 and 28) plus 5FU (1000 mg/m2 days 1-4 and 28-32) plus ERT (50.4 Gy). Grade 3-4 (RTOG) acute toxicity was 5%; late toxicity was never observed. Sphincter function was rated excellent or good in 93% of patients. Sphincter preservation was achieved in 73.7% of patients; in particular, in 55.6% of those with a lesion at less than 50 mm from the internal anal orifice. Combined modality therapies are showing the ability of sphincter preservation in patients with distal rectal cancer. Ongoing studies will identify the patients who are candidates for this therapeutic approach and the most suitable combined treatment.

Organ preservation in the treatment of brain tumors.

Within the many histological forms, the preservation of function in the central nervous system has always been predominant. However, the limited or null therapeutic interval for high grade gliomas enables organ preservation in small neoplasms only. In case of favorable histology (e.g. dysgerminoma, low grade small glioma), organ preservation is feasible with adequate techniques. When local control is predominant (e.g. neoplasms of eye) the techniques are long known but applied in very few Centers.

Juvenile hormone regulates the expression of the gene encoding ceratotoxin a, an antibacterial peptide from the female reproductive accessory glands of the medfly Ceratitis capitata.

Ceratotoxin A is an antibacterial peptide produced by the reproductive female accessory glands of the medfly Ceratitis capitata. To investigate whether ceratotoxin A gene expression was affected by juvenile hormone, which has gonadotropic functions in adult insects, newly emerged female medflies were treated with precocene II, an antiallotropin compound capable of inhibiting juvenile hormone biosynthesis. Daily treatment of newly emerged flies with precocene II blocked ceratotoxin A gene expression in a dose-dependent manner. Ceratotoxin A gene expression could be recovered after withdrawl of precocene II treatment. Moreover, the effect of precocene II on ceratotoxin A gene expression could be countered by simultaneous treatment with methoprene, a juvenile hormone analogue. The effects of precocene II and methoprene treatments on the growth of both ovaries and accessory glands was also investigated. Our data suggest that ceratotoxin A gene expression is modulated by juvenile hormone.

The genes encoding the antibacterial sex-specific peptides ceratotoxins are clustered in the genome of the medfly Ceratitis capitata.

Ceratotoxins are antibacterial peptides produced in the female reproductive accessory glands of the medfly Ceratitis capitata. Their expression is not affected by bacterial infection, but is enhanced after mating and is modulated by juvenile hormone. Three different peptides, named ceratotoxins A, B and C, have been previously purified from the female accessory gland secretion and their amino acid and cDNA sequences have been determined. We report here the complete nucleotide sequences of four genes encoding closely related ceratotoxin peptides. One of them encodes a novel peptide, which we named ceratotoxin D. Restriction and nucleotide sequence analysis indicate that these ceratotoxin genes are organized in a large cluster spanning more than 26 kilobases of DNA. All ceratotoxin genes are coordinately expressed. Ceratotoxin transcripts appear in 2-3 day old adult females, and they reach a maximum in 6-7 day old females. The presence of highly conserved motifs in the upstream regions of all the sequenced ceratotoxin genes suggests the presence of common regulatory elements for all ceratotoxins.

Molecular characterization of ceratotoxin C, a novel antibacterial female-specific peptide of the ceratotoxin family from the medfly Ceratitis capitata.

Ceratotoxins A and B are antibacterial peptides produced by the sexually mature females of Ceratitis capitata. The gene expression is restricted to the female reproductive accessory glands, and is not affected by bacterial infection, but is enhanced by mating. We report here the purification and the amino acid sequence of ceratotoxin C, a novel member of the ceratotoxin family, the cloning of its cDNA and the analysis of its expression. Ceratotoxin C is coordinately expressed with the other members of the ceratotoxin family. Its antibacterial activity is directed against both Gram-negative and Gram-positive bacterial strains but it is lower than that of ceratotoxin A. We demonstrate in the genome of C. capitata the presence of at least three ceratotoxin genes which express, in the female accessory glands, a set of peptides presumably involved in the protection of the genital tract during fertilization.

Signals from the IL-1 receptor homolog, Toll, can activate an immune response in a Drosophila hemocyte cell line.

The Toll gene encodes an interleukin 1 receptor-like protein that mediates dorsoventral polarity in the Drosophila embryo. The possible involvement of Toll or Toll-like proteins also in the Drosophila immune response was investigated by overexpressing Toll10B, a constitutively active mutant protein, in the Drosophila blood cell line mbn-2. Induction of the Cecropin A1 (CecA1) gene, coding for a bactericidal peptide, was used as an indicator for the immune response. Toll10B was found to increase CecA1 transcription, as detected with a cotransfected CecA1-lacZ reporter gene construct. This effect depends on the presence of a kappa B-like site in the CecA1 promoter. The endogenous Toll gene is expressed in mbn-2 cells, indicating that this gene may normally play a role in Drosophila blood cells.

cDNA sequence and expression of the ceratotoxin gene encoding an antibacterial sex-specific peptide from the medfly Ceratitis capitata (diptera).

Ceratotoxins are antibacterial 3-kDa molecular mass amphiphilic peptides isolated from the female reproductive accessory glands of the medfly Ceratitis capitata. They are physiologically related to bee melittin and show amino acid sequence homology with magainin peptides. In this paper, we report the complete sequence of cDNA coding for ceratotoxin A and the expression of the gene during the life cycle of the insect. Experimental data show that the ceratotoxin is a gene expressed exclusively in the imaginal stages and that it is female-specific, related to sexual maturity, and stimulated by mating. Differently from most antibacterial insect hemolymph peptides, it is not induced by microbial infection. Western blot analysis using an anti-ceratotoxin antibody indicates the female accessory glands as the only site where the production of the ceratotoxin peptide occurs.