Long-term efficacy and safety of addition of carboplatin with or without veliparib to standard neoadjuvant chemotherapy in triple-negative breast cancer: 4-year follow-up data from BrighTNess, a randomized phase III trial.

BACKGROUND: Primary analyses of the phase III BrighTNess trial showed addition of carboplatin with/without veliparib to neoadjuvant chemotherapy significantly improved pathological complete response (pCR) rates with manageable acute toxicity in patients with triple-negative breast cancer (TNBC). Here, we report 4.5-year follow-up data from the trial. PATIENTS AND METHODS: Women with untreated stage II-III TNBC were randomized (2 : 1 : 1) to paclitaxel (weekly for 12 doses) plus: (i) carboplatin (every 3 weeks for four cycles) plus veliparib (twice daily); (ii) carboplatin plus veliparib placebo; or (iii) carboplatin placebo plus veliparib placebo. All patients then received doxorubicin and cyclophosphamide every 2-3 weeks for four cycles. The primary endpoint was pCR. Secondary endpoints included event-free survival (EFS), overall survival (OS), and safety. Since the co-primary endpoint of increased pCR with carboplatin plus veliparib with paclitaxel versus carboplatin with paclitaxel was not met, secondary analyses are descriptive. RESULTS: Of 634 patients, 316 were randomized to carboplatin plus veliparib with paclitaxel, 160 to carboplatin with paclitaxel, and 158 to paclitaxel. With median follow-up of 4.5 years, the hazard ratio for EFS for carboplatin plus veliparib with paclitaxel versus paclitaxel was 0.63 [95% confidence interval (CI) 0.43-0.92, P = 0.02], but 1.12 (95% CI 0.72-1.72, P = 0.62) for carboplatin plus veliparib with paclitaxel versus carboplatin with paclitaxel. In post hoc analysis, the hazard ratio for EFS was 0.57 (95% CI 0.36-0.91, P = 0.02) for carboplatin with paclitaxel versus paclitaxel. OS did not differ significantly between treatment arms, nor did rates of myelodysplastic syndromes, acute myeloid leukemia, or other secondary malignancies. CONCLUSIONS: Improvement in pCR with the addition of carboplatin was associated with long-term EFS benefit with a manageable safety profile, and without increasing the risk of second malignancies, whereas adding veliparib did not impact EFS. These findings support the addition of carboplatin to weekly paclitaxel followed by doxorubicin and cyclophosphamide neoadjuvant chemotherapy for early-stage TNBC.

The immune profile of small HER2-positive breast cancers: a secondary analysis from the APT trial.

BACKGROUND: Previous data suggest that the immune microenvironment plays a critical role in human epidermal growth factor receptor 2 (HER2) -positive breast cancer; however, there is little known about the immune profiles of small HER2-positive tumors. In this study, we aimed to characterize the immune microenvironment of small HER2-positive breast cancers included in the Adjuvant paclitaxel and trastuzumab for node-negative, HER2-positive breast cancer (APT) trial and to correlate the immune markers with pathological and molecular tumor characteristics. PATIENTS AND METHODS: The APT trial was a multicenter, single-arm, phase II study of paclitaxel and trastuzumab in patients with node-negative HER2-positive breast cancer. The study included 406 patients with HER2-positive, node-negative breast cancer, measuring up to 3 cm. Exploratory analysis of tumor infiltrating lymphocytes (TIL), programmed death-ligand 1 (PD-L1) expression (by immunohistochemistry), and immune gene signatures using data generated by nCounter PanCancer Pathways Panel (NanoString Technologies, Seattle, WA), and their association with pathological and molecular characteristics was carried out. RESULTS: Of the 406 patients, 328 (81%) had at least one immune assay carried out: 284 cases were evaluated for TIL, 266 for PD-L1, and 213 for immune gene signatures. High TIL (≥60%) were seen with greater frequency in hormone-receptor (HR) negative, histological grades 2 and 3, as well in HER2-enriched and basal-like tumors. Lower stromal PD-L1 (≤1%) expression was seen with greater frequency in HR-positive, histological grade 1, and in luminal tumors. Both TIL and stromal PD-L1 were positively correlated with 10 immune cell signatures, including Th1 and B cell signatures. Luminal B tumors were negatively correlated with those signatures. Significant correlation was seen among these immune markers; however, the magnitude of correlation did not indicate a monotonic relationship between them. CONCLUSION: Immune profiles of small HER2-positive breast cancers differ according to HR status, histological grade, and molecular subtype. Further work is needed to explore the implication of these findings on disease outcome. CLINICAL TRIAL REGISTRATION: clinicaltrials.gov identifier: NCT00542451.

nab-Paclitaxel plus carboplatin or gemcitabine versus gemcitabine plus carboplatin as first-line treatment of patients with triple-negative metastatic breast cancer: results from the tnAcity trial.

Background: Metastatic triple-negative breast cancer (mTNBC) has a poor prognosis and aggressive clinical course. tnAcity evaluated the efficacy and safety of first-line nab-paclitaxel plus carboplatin (nab-P/C), nab-paclitaxel plus gemcitabine (nab-P/G), and gemcitabine plus carboplatin (G/C) in patients with mTNBC. Patients and methods: Patients with pathologically confirmed mTNBC and no prior chemotherapy for metastatic BC received (1 : 1 : 1) nab-P 125 mg/m2 plus C AUC 2, nab-P 125 mg/m2 plus G 1000 mg/m2, or G 1000 mg/m2 plus C AUC 2, all on days 1, 8 q3w. Phase II primary end point: investigator-assessed progression-free survival (PFS); secondary end points included overall response rate (ORR), overall survival (OS), percentage of patients initiating cycle 6 with doublet therapy, and safety. Results: In total, 191 patients were enrolled (nab-P/C, n = 64; nab-P/G, n = 61; G/C, n = 66). PFS was significantly longer with nab-P/C versus nab-P/G [median, 8.3 versus 5.5 months; hazard ratio (HR), 0.59 [95% CI, 0.38-0.92]; P = 0.02] or G/C (median, 8.3 versus 6.0 months; HR, 0.58 [95% CI, 0.37-0.90]; P = 0.02). OS was numerically longer with nab-P/C versus nab-P/G (median, 16.8 versus 12.1 months; HR, 0.73 [95% CI, 0.47-1.13]; P = 0.16) or G/C (median, 16.8 versus 12.6 months; HR, 0.80 [95% CI, 0.52-1.22]; P = 0.29). ORR was 73%, 39%, and 44%, respectively. In the nab-P/C, nab-P/G, and G/C groups, 64%, 56%, and 50% of patients initiated cycle 6 with a doublet. Grade ≥3 adverse events were mainly hematologic. Conclusions: First-line nab-P/C was active in mTNBC and resulted in a significantly longer PFS and improved risk/benefit profile versus nab-P/G or G/C.

Effect of denosumab versus zoledronic acid in preventing skeletal-related events in patients with bone metastases by baseline characteristics.

BACKGROUND: Analyses of phase III trials showed that denosumab was superior to zoledronic acid (ZA) in preventing skeletal-related events (SREs) irrespective of age, history of SREs, or baseline pain status. This analysis assessed the risk of SREs across additional baseline characteristics. PATIENTS AND METHODS: Patients (N = 5543) from three phase III trials who had breast cancer, prostate cancer, or other solid tumours and one or more bone metastasis were included. Superiority of denosumab versus ZA in reducing risk of first SRE and first and subsequent SREs was assessed in subgroups defined by the Eastern Cooperative Oncology Group performance status (ECOG PS), bone metastasis location, bone metastasis number, visceral metastasis presence/absence, and urinary N-telopeptide (uNTx) level using Cox proportional hazards and Anderson-Gill models. Subgroups except bone metastasis location were also assessed for each solid tumour type. RESULTS: Compared with ZA, denosumab significantly reduced the risk of first SRE across all subgroups (hazard ratio [HR] ranges: ECOG PS, 0.79-0.84; bone metastasis location, 0.78-0.83; bone metastasis number, 0.78-0.84; visceral metastasis presence/absence, 0.80-0.82; uNTx level, 0.73-0.86) and reduced the risk of first and subsequent SREs in all subgroups (HR ranges: ECOG PS, 0.76-0.83; bone metastasis location, 0.78-0.84; bone metastasis number, 0.79-0.81; visceral metastasis presence/absence, 0.79-0.81; uNTx level, 0.74-0.83). Similar results were observed in subgroups across tumour types. CONCLUSION: Denosumab was superior to ZA in preventing SREs in patients with bone metastases from advanced cancer, regardless of ECOG PS, bone metastasis number, baseline visceral metastasis presence/absence, and uNTx level.

Randomized, phase II, placebo-controlled trial of onartuzumab and/or bevacizumab in combination with weekly paclitaxel in patients with metastatic triple-negative breast cancer.

BACKGROUND: Increased hepatocyte growth factor/MET signaling is associated with an aggressive phenotype and poor prognosis in triple-negative breast cancer (TNBC). We evaluated the benefit of adding onartuzumab, a monoclonal anti-MET antibody, to paclitaxel with/without bevacizumab in patients with TNBC. PATIENTS AND METHODS: Women with metastatic TNBC were randomized to receive onartuzumab plus placebo plus weekly paclitaxel (OP; n = 60) or onartuzumab plus bevacizumab plus paclitaxel (OBP; n = 63) or placebo plus bevacizumab plus paclitaxel (BP; n = 62). The primary end point was progression-free survival (PFS); additional end points included overall survival (OS), objective response rate (ORR), and safety. This trial was hypothesis generating and did not have power to detect minimum clinically meaningful differences between treatment arms. RESULTS: There was no improvement in PFS with the addition of onartuzumab to BP [hazard ratio (HR), 1.08; 95% confidence interval (CI) 0.69-1.70]; the risk of a PFS event was higher with OP than with BP (HR, 1.74; 95% CI 1.13-2.68). Most patients had MET-negative tumors (88%); PAM50 subtype analysis showed basal-like tumors in 68% of samples. ORR was higher in the bevacizumab arms (OBP: 42.2%; 95% CI 28.6-57.1; BP: 54.7%; 95% CI 41.0-68.4) compared with OP (27.5%; 95% CI 15.9-40.6). Median OS was shorter with OBP (HR, 1.36; 95% CI 0.75-2.46) and OP (HR, 1.92; 95% CI 1.03-3.59), than with BP. Peripheral edema was more frequent in the onartuzumab arms (OBP, 51.8%; OP, 58.6%) versus BP (17.7%). CONCLUSION: This study did not show a clinical benefit of the addition of onartuzumab to paclitaxel with/without bevacizumab in patients with predominantly MET-negative TNBC. CLINICALTRIALSGOV: NCT01186991.

Long-term survivor characteristics in HER2-positive metastatic breast cancer from registHER.

BACKGROUND: Data characterising long-term survivors (LTS) with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer (MBC) are limited. This analysis describes LTS using registHER observational study data. METHODS: A latent class modelling (LCM) approach was used to identify distinct homogenous patient groups (or classes) based on progression-free survival (PFS), overall survival, and complete response. Demographics, clinicopathologic factors, first-line treatment patterns, and clinical outcomes were described for each class. Class-associated factors were evaluated using logistic regression analysis. RESULTS: LCM identified two survivor groups labelled as LTS (n=244) and short-term survivors (STS; n=757). Baseline characteristics were similar between groups, although LTS were more likely to be white (83.6% vs 77.8%) with oestrogen receptor-positive (ER+) or progesterone receptor-positive (PgR+) disease (59.4% vs 50.9%). Median PFS in LTS was 37.2 (95% confidence interval (CI): 32.9-40.5) vs 7.3 months (95% CI: 6.8-8.0) in STS. Factors associated with long-term survival included ER+ or PgR+ disease, metastasis to node/local sites, first-line trastuzumab use, and first-line taxane use. CONCLUSIONS: Prognostic variables identified by LCM define a HER2-positive MBC patient profile and therapies that may be associated with more favourable long-term outcomes, enabling treatment selection appropriate to the patient's disease characteristics.

Weekly docetaxel in the treatment of elderly patients with advanced breast cancer: a Minnie Pearl Cancer Research Network phase II trial.

PURPOSE: To evaluate the efficacy and toxicity of docetaxel administered weekly to elderly or poor-performance status patients with advanced breast cancer. PATIENTS AND METHODS: Forty-one patients with advanced breast cancer who were either over the age of 65 or considered to be poor candidates for combination chemotherapy received docetaxel 36 mg/m2 weekly for 6 consecutive weeks, followed by 2 weeks without treatment. The median age of patients in this trial was 74 years, and 73% of patients had one or more visceral sites of metastases. Seventy-five percent of patients received weekly docetaxel as first-line treatment for metastatic breast cancer, and the other 25% received it as second-line treatment. Thirty-six patients were assessable for efficacy, and all patients were assessed for toxicity. RESULTS: A total of 448 doses of weekly docetaxel were administered to 41 patients. Thirteen patients (36%) had objective responses to treatment, and an additional 13 patients (36%) had stable disease or minor response. Median time to progression for responding and stable patients was 7 months (range, 3 to 27 months). Median survival for the entire group was 13 months, with 1- and 2-year actuarial survival rates of 61% and 29%, respectively. Severe neutropenia occurred in only 0.4% of courses, and no other hematologic toxicity was observed. Grade 3/4 fatigue was the most common toxicity, occurring in 20% of patients. CONCLUSION: Weekly docetaxel therapy is active and well tolerated by elderly and/or poor-performance status patients with advanced breast cancer. This treatment can be administered with minimal myelosuppression. Weekly docetaxel provides an additional option for treatment in this difficult subgroup of patients with metastatic breast cancer. Well-tolerated combination regimens containing weekly docetaxel merit evaluation for this patient population.

Phase I study of paclitaxel given by seven-week continuous infusion concurrent with radiation therapy for locally advanced squamous cell carcinoma of the head and neck.

PURPOSE: Paclitaxel is one of the most active agents for squamous cell carcinoma of the head and neck (SCCHN) and an in vitro radiosensitizer. The dose-response relationship for paclitaxel may depend more on exposure duration than on peak concentration. This National Cancer Institute-sponsored phase I trial was designed to determine the feasibility of combining continuous-infusion (CI) paclitaxel with concurrent radiation therapy (RT). PATIENTS AND METHODS: Patients with previously untreated stage IVA/B SCCHN were eligible. Primary end points were determination of the maximum-tolerated dose, dose-limiting toxicity, and pharmacokinetics for paclitaxel given by CI (24 hours a day, 7 days a week for 7 weeks) during RT (70 Gy/7 weeks). RESULTS: Twenty-seven patients were enrolled and assessable for toxicity. Nineteen of the patients who completed > or = 70 Gy were assessable for response. Grade 3 skin and mucosal acute reactions occurred at 10.5 mg/m(2)/d, but uninterrupted treatment was possible in five of six patients. At 17 mg/m(2)/d, skin toxicity required a 2-week treatment break for all three patients. The mean paclitaxel serum concentration at dose levels > or = 6.5 mg/m(2)/d exceeded that reported to achieve in vitro radiosensitization. Initial locoregional control was achieved in 14 (58%) of 24 of patients treated to 70 Gy, and control persisted in nine (38%). CONCLUSION: CI paclitaxel with concurrent RT is a feasible and tolerable regimen for patients with advanced SCCHN and good performance status. Preliminary response and survival data are encouraging and suggest that further study is indicated. The recommended phase II dose of paclitaxel by CI is 10.5 mg/m(2)/d with RT for SCCHN.

Clinical and laboratory features and sequelae of deficiency of folic acid (folate) and vitamin B12 (cobalamin) in pregnancy and gynecology.

Classically, deficiency of folic acid (folate) or vitamin B12 (cobalamin) was recognized by the presence of a macrocytic anemia resulting from megaloblastic changes in the bone marrow. A markedly changing paradigm has identified both new mechanisms for altered folate and cobalamin status and new sequelae and clinical interrelationships that include altered mechanisms of absorption, a changing pattern of neurologic deficits, an increased risk of vascular occlusive lesions, and an important relationship with the mechanisms of neoplastic transformation. Several of these newer characterizations relate to issues of neoplasia in the nonpregnant woman and to issues in pregnancy, such as the potential for developmental abnormalities of the fetal nervous system.

Seven-week continuous-infusion paclitaxel concurrent with radiation therapy for locally advanced non-small cell lung and head and neck cancers.

The goal of these National Cancer Institute-sponsored phase I trials is to determine the feasibility, toxicity, and pharmacokinetics of continuous-infusion (24 hr/d, 7 d/wk, 7 weeks total) intravenous paclitaxel combined with standard, curative-intent thoracic radiation therapy (XRT) for previously untreated, locally advanced non-small cell lung cancer and squamous cell cancer of the head and neck (HNSCC). Eligible patients have locally advanced (T4NXM0 or TXN2-3M0) non-small cell lung cancer ineligible for potentially curative surgical resection or locally advanced HNSCC with an expected 5-year survival rate of less than 25%, as well as a good performance status, adequate hematologic, hepatic, and renal function, and no distant metastases. Non-small cell lung cancer patients receive a total tumor dose of 64.8 Gy megavoltage XRT in 7 weeks at 1.8 Gy once daily, 5 d/wk. Patients with HNSCC receive 70 Gy megavoltage XRT in 7 weeks at 2 Gy once daily, 5 d/wk. Paclitaxel is delivered by continuous intravenous infusion starting 48 hours before XRT and continuing for its duration. The dose of paclitaxel is escalated in cohorts of three patients in a standard phase I design. To date, 49 patients have been entered on both studies and 43 are evaluable for toxicity. Paclitaxel dose is currently at the 17 mg/m2/d dose level, with no dose-limiting toxicity thus far. Clinical outcomes suggest significant activity for this combination. This therapy is feasible and has been well-tolerated through current dose levels. Dose escalation is ongoing.

Early and late quantitative angiographic results of vein graft lesions treated by excimer laser with adjunctive balloon angioplasty.

BACKGROUND: Percutaneous excimer laser coronary angioplasty (PELCA) has been approved for treatment of diseased saphenous vein bypass grafts. However, detailed and complete quantitative angiographic analysis of immediate procedural and late follow-up results has not been performed. METHODS AND RESULTS: PELCA using the CVX-300 excimer laser system was performed in 125 bypass lesions (mean graft age, 96 +/- 53 months; range, 2 to 240 months) in 106 consecutive patients at eight centers. Quantitative analyses of the procedural and follow-up angiograms were done with the Cardiac Measurement System. Stand-alone PELCA was done in 21 lesions (17%). Lesions were located at the ostium (20%), body (67%), or distal anastomosis (13%). The graft reference diameter was 3.26 +/- 0.79 mm (mean +/- SD). Minimal lumen diameter increased from 1.09 +/- 0.52 mm before treatment to 1.61 +/- 0.69 mm after laser and 2.18 +/- 0.63 mm after adjunctive balloon dilation (P < .001) but had declined at follow-up to 1.40 +/- 1.17 mm. Dissections were evident in 45% of lesions after laser treatment (types A and B, 27%; types C through F, 18%), including 7% occlusions. Angiographic success (< or = 50% diameter stenosis [% DS]) was 54% after laser and 91% after adjunctive PTCA, with an overall clinical success rate of 89%. In-hospital complications were death, 0.9%; myocardial infarction (Q-wave and non-Q-wave), 4.5%; and bypass surgery, 0.9%. Independent predictors of % DS after laser were reference diameter, lesion length, and minimal lumen diameter before laser. At angiographic follow-up in 83% of eligible patients, the restenosis rate per lesion (DS > 50%) was 52%, including 23 occlusions (24%). The only independent predictor of increased % DS at follow-up was lesion symmetry. Logistic regression indicated that smaller reference diameter was an independent predictor of late occlusion. Overall 1-year mortality was 8.6%. Actuarial event-free survival (freedom from death, myocardial infarction, bypass surgery, or target vessel percutaneous transluminal coronary angioplasty) was 48.2% at 1 year. CONCLUSIONS: Excimer laser angioplasty with adjunctive balloon angioplasty can be safely and successfully performed in diseased, old saphenous vein bypass graft lesions considered at high risk for reintervention. The extent of laser ablation remains limited by the diameter and effectiveness of the catheters. Late restenosis and, in particular, total occlusion mitigate the early benefits of the procedure. Other approaches such as the routine use of additional anticoagulation (eg, warfarin) should be considered to reduce the risk of late occlusions and restenosis after laser angioplasty of bypass grafts.

Predictors of outcome of percutaneous excimer laser coronary angioplasty of saphenous vein bypass graft lesions. The Percutaneous Excimer Laser Coronary Angioplasty Registry.

A total of 495 patients underwent treatment with excimer laser angioplasty for 545 saphenous vein graft stenoses. Clinical success was achieved in 455 of 495 patients (92%), as indicated by < or = 50% residual stenosis at every target lesion and no complication during hospitalization. At least 1 in-hospital complication occurred in 30 of 495 patients (6.1%): death (1.0%), bypass surgery (0.6%), and Q-wave (2.4%) or non-Q-wave (2.2%) myocardial infarction. Relative risk analysis showed that ostial lesions (n = 65) tended to have higher clinical success (success rate = 95%, adjusted odds ratio [OR] = 2.1 [95% confidence interval (CI) 0.62, 6.88]; p = 0.24) and lower complications (complication rate = 0%, OR = 0.10 [CI 0.01, 0.79]; p = 0.03) than lesions in the body of the vein graft. Lesions > 10 mm (n = 131) had lower success (success rate = 84%, OR = 0.30 [CI 0.16, 0.56]; p = 0.001) and higher complications (complication rate = 12%, OR = 3.3 [CI 1.6, 6.6]; p = 0.004) than discrete lesions. Lesions in small vein grafts < 3.0 mm (n = 76) tended to have increased success (success rate = 94%, OR = 1.55 [CI 0.70, 3.44]; p = 0.39) and lower complications (complication rate = 2.2%, OR = 0.31 [CI 0.10, 0.94]; p = 0.03). Thus, excimer laser-facilitated angioplasty has the most favorable outcome for discrete lesions located at the ostium of all grafts and in the body of smaller saphenous vein grafts.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of amylase genotypes on growth rate and feed conversion of chickens.

Chickens from two breeds were screened for amylase alleles designated as AmyF and AmyS to establish breeder flocks homozygous for each. Offspring from these flocks were then used to test the hypothesis that AmyF and AmyS amylases differ in their ability to digest cornstarch and wheat starch. The amylase allozymes were found to affect growth and feed conversion performance of the chickens, and the effects were more pronounced in one breed. However, these effects seemed to be more related to specific activity of the amylases than to starch source in the diet. The results indicate that in some breeds of chickens selection for AmyS may improve growth and feed efficiency performance.

Effects of diet on amylase expression in the mosquitofish.

Diets high in various carbohydrates were fed to mosquitofish,Gambusia affinis holbrooki, to determine the effects on amylase expression. Both amylase activity and amount of amylase protein were used as measures of amylase expression. Fish were fed for 21 days in one experiment, seven days in a second experiment and 24 h in a third. The first experiment compared responses of fish fed on a high-starch diet relative to a control diet. The second and third experiments compared responses on four diets relative to the control diet: maltose, starch, glucose, and glucose + cyclic adenosine monophosphate (cAMP). In the first two experiments whole visceral extracts were used. In the third experiment, gut and hepatopancreatic extracts were examined separately. Diet had a significant affect on the amount of amylase in all three experiments but affected amylase activity only in the 24 h experiment. Generally, glucose decreased amylase expression while maltose or cAMP + glucose increased it. Length of feeding period and tissue type also had significant effects on amylase expression.

The amylase gene-enzyme system of chickens. II. Biochemical characterization of allozymes.

The chicken amylase allozymes, AmyF and AmyS, were extracted from pancreatic tissues of AmyF/F and AmyS/S individuals and purified. Activities were measured under various reaction conditions (= treatments) to assess whether the allozymes were functionally different. The amylases had properties typical of alpha-amylases, i.e., both were inhibited by ethylenediaminetetraacetate and alpha-amylase inhibitor from wheat, had pH optima between 7.0 and 8.0, and could utilize a variety of substrates containing alpha 1,4 linkages. The amylases were also found to be inhibited by potassium phosphate buffer and p-chloromercuribenzoate. In terms of substrate specificity, both amylases could utilize all of the substrates tested with activity observed in the following order: amylopectin greater than potato starch greater than dextrin greater than glycogen greater than amylose. Statistical analysis indicated significant functional differences between the two allozymes in terms of specific activities, substrate specificities, and inhibitor sensitivities. AmyF had a significantly lower specific activity than did AmyS. The amylases responded differently to the substrate amylose, with AmyF better able to digest this substrate. AmyS was less sensitive than AmyF to alpha-amylase inhibitor from wheat.

Tissue-specific distribution of amylase in the mosquitofish (Gambusia affinis holbrooki).

Tissue-specific expression of alpha-amylase (E.C.3.2.1; α 1,4-glucan-4-glucano-hydrolase) was studied in the mosquitofish (Gambusia affinis holbrooki). Gut activity patterns, immunohistochemical distribution in the hepatopancreas and tissue-specific activities and amount of amylase protein were characterized. In the gut, activity was observed all along the gut and no variation in this pattern was observed in individuals from two natural populations. In the hepatopancreas, amylase is found in pancreatic tissue. Highest amylase activity was observed in gut tissue but highest amount of amylase protein was found in the hepatopancreas.

The amylase gene-enzyme system of chickens. I. Allozymic and activity variation.

Amylase allozymic and activity variation was studied in three flocks of chickens (Gallus domesticus). Individuals from one flock were studied to assess the effects of sex, tissue, and genotype on amylase activity. Additionally, the allozymes were purified and their specific activities compared. Variation was observed within and among the flocks. Two alleles were found to be segregating in the flocks, one flock being polymorphic and the other two monomorphic. Mean amylase activities among the three flocks were significantly different. The relationship of this activity variation to regulatory variation is discussed. There were no significant effects of sex or genotype on amylase activity and, in most cases, no correlation between activities in the various tissues. However, in heterozygotes one of the alloamylases had much lower activity than the other.

The amylase gene-enzyme system of fishes. I. Developmental expression of amylase in the mosquitofish.

The objective of this study is to examine the effects of developmental stage on amylase expression in the mosquitofish (Gambusia affinis holbrooki, Poeciliidae). Expression was characterized in terms of amylase activity and amount of amylase protein in five developmental stages: early, mid, and late embryos, 5-day-old juveniles, and adults. Two measures of activity were used: a starch-iodine assay, which measures the change in substrate, and the dinitrosalicyclic acid (DNSA) assay, which measures the change in product. To measure changes in the amount of amylase, the enzyme-linked immunosorbent assay (ELISA) was used. Highest activity and amount was observed in the adult stage. In general, both increased with development. While the two activity measures were positively correlated, these were only weakly correlated with the amount of amylase. Possible reasons for these weak correlations between amylase activity and amount are discussed.

An amylase gene from Drosophila pseudoobscura is expressed in Escherichia coli. Functional selection and biochemical comparisons of the fly- and clone-produced amylases.

An amylase gene from Drosophila pseudoobscura was isolated from a genomic library constructed in pBR322 and cloned in Escherichia coli by selecting for the ability of its product to hydrolyze starch, a carbon source not normally utilized by E. coli. Hybridization of pAMY17F to D. pseudoobscura polytene chromosomes shows a positive signal at the amylase pseudogene locus (bank 78, chromosome 3). The chimeric plasmid pAMY17F, has been altered in such a way as to increase amylase expression. Southern and Northern hybridizations to the cloned amylase DNA indicate that the source of the gene is from D. pseudoobscura. Biochemical properties such as pH optima, substrate specificities, electrophoretic analyses, inhibitor sensitivities, heat stabilities, temperature responsiveness and molecular weights indicate that the amylases produced by the fly and bacterial clone are similar and have similar properties. It appears that E. coli/pAMY17F is producing an amylase like that found in D. pseudoobscura.