NaPi2b expression in a large surgical Non-Small Cell Lung Cancer (NSCLC) cohort.

BACKGROUND: NaPi2b is a multi-transmembrane sodium-dependent phosphate transporter expressed at normal levels in several organs, including lung. High expression levels have been reported in various tumors including breast, thyroid, ovarian and non-small cell lung cancer. To date evaluation of NaPi2b expression has mostly been restricted to smaller lung cancer cohorts. METHODS: Analyses were performed on archival formalin fixed paraffin embedded primary tumor specimens from patients who had undergone curative intent resection at an Australian tertiary hospital. Tissue microarrays were constructed and stained with the chimeric anti-NaPi2b antibody, MERS67. Semi-quantitative H-scores (range 0 - 300) were calculated for each core tissue sample (H-score = % tumor cells staining for NaPi2b multiplied by staining intensity). An overall average H-score was reported for each specimen, with a cut-off score of 50 considered positive. RESULTS: Of 438 cases, high NaPi2b expression was observed in 151 (34.5%) overall, high expression in 137 of 208 (65.9%) adenocarcinoma cases, and 5 of 179 (2.8%) squamous cases (P < .0001). High NaPi2b expression was associated with female sex, EGFR or KRAS mutation, and TTF1 positivity (adenocarcinoma cases only). High NaPi2b expression was associated with improved overall survival (median 54 vs. 35 months, P = .029). CONCLUSION: High NaPi2b expression was noted in a significant subset of adenocarcinoma cases, and in particular amongst those who were TTF1+, or exhibited EGFR or KRAS mutations. This agrees with earlier reports and highlights the significance that NaPi2b may have a role as a possible target for delivery of cytotoxic agents via antibody-drug conjugate models for some patients with lung adenocarcinoma.

Pharmacokinetics and tissue disposition of meloxicam in beef calves after repeated oral administration.

The objective of this study was to investigate the pharmacokinetics and tissue disposition of meloxicam after repeated oral administration in calves. Thirteen male British × Continental beef calves aged 4 to 6 months and weighing 297-392 kg received 0.5 mg/kg meloxicam per os once daily for 4 days. Plasma meloxicam concentrations were determined in 8 calves over 6 days after first treatment. Calves were randomly assigned to be euthanized at 5, 10, 15 (n = 3/timepoint), and 19 days (n = 4) after final administration. Meloxicam concentrations were determined in plasma (LOQ= 0.025 µg/mL) and muscle, liver, kidney, and fat samples (LOQ = 2 ng/g) after extraction using validated LC-MS-MS methods. The mean (± SD) Cmax , Cmin , and Caverage plasma meloxicam concentrations were 4.52 ± 0.87 µg/mL, 2.95 ± 0.77 µg/mL, and 3.84 ± 0.81 µg/mL, respectively. Mean (± SD) tissue meloxicam concentrations were highest in liver (226.67 ± 118.16 ng/g) and kidney samples (52.73 ± 39.01 ng/g) at 5 days after final treatment. Meloxicam concentrations were below the LOQ in all tissues at 15 days after treatment. These findings suggest that tissue from meloxicam-treated calves will have low residue concentrations by 21 days after repeated oral administration.

Impact of oral meloxicam administered alone or in combination with gabapentin on experimentally induced lameness in beef calves.

This study examined the pharmacokinetics and analgesic effect of oral meloxicam (MEL) administered alone or in combination with gabapentin (GABA) in an experimental bovine lameness model. Eighteen male British × Continental beef calves aged 4 to 6 mo and weighing 297 to 392 kg were randomly assigned to receive either 1) 0.5 mg/kg lactose monohydrate placebo (PLBO; n = 6), 2) 0.5 mg/kg MEL (n = 6), or 3) 0.5 mg/kg MEL combined with 15 mg/kg GABA (MEL-GABA; n = 6) once daily for 4 d. The first treatment was administered 4 h after a chemical synovitis/arthritis was induced with injection of 15 mg amphotericin B into the left hind lateral distal interphalangeal joint. Changes in activity were evaluated continuously with pedometers. Contact force, contact area, contact pressure, impulse, and stride length were recorded once daily with a pressure mat and visual lameness scores were determined by a masked observer using a 5-point scale. Cortisol and drug concentrations were determined daily by immunoassay and HPLC-mass spectrometry, respectively. Outcomes were compared statistically using a random effects mixed model and analysis of covariance. There was a positive association between lameness scores and serum cortisol concentrations (P = 0.02) and a negative association between lameness score and step count (P < 0.0001), total force (P = 0.001), force applied to the lateral claw (P = 0.02), contact pressure (P = 0.005), and impulse of the lateral claw (P = 0.01). Step count was greater in MEL calves compared with PLBO (P = 0.008) and MEL-GABA (P = 0.04) calves. Impulse was greater in the MEL-GABA calves compared with the PLBO calves (P = 0.03). There was an inverse relationship between plasma MEL concentrations and lameness score (P = 0.02) and a positive association between MEL concentrations and force applied to the lateral claw (P = 0.03), total contact pressure (P = 0.03), and impulse on the lateral claw (P = 0.02). There was a tendency towards a positive association between GABA concentrations, total impulse, and impulse on the lateral claw (P = 0.08) and a negative associate between GABA concentrations and step count (P = 0.08). The results of this study suggest that MEL administered alone or in combination with GABA reduced the severity of lameness in calves following induction of lameness with amphotericin B. These findings have implications for developing analgesic protocols in lame calves that address both production and welfare concerns.

Comparative effects of castration and dehorning in series or concurrent castration and dehorning procedures on stress responses and production in Holstein calves.

The study objective was to compare serum cortisol as an acute stress measure, chute exit velocity as a behavioral measure, and ADG as an indicator of performance and well-being after castration, dehorning, or concurrent castration/dehorning of calves when performed in parallel and in series. Intact male Holstein calves, 3 to 4 mo, underwent sham handling before 2 procedures performed in series separated by 2 to 3 wk. In Period 1, calves were either dehorned by amputation, surgically castrated, concurrently castrated/dehorned, or served as nonsurgical controls (n = 10/treatment). In Period 2, calves that had been dehorned, castrated, or castrated/dehorned were then castrated, dehorned, or served as nonsurgical controls, respectively. Indicators of distress were measured after all procedures; ADG was assessed for 7 d after each procedure and over the 2 to 3 wk interim. Period 1 cortisol concentrations in dehorned calves were less than in castrated and castrated/dehorned calves at 120 min and from 50 to 240 min, respectively (P < 0.02). There was marginal evidence that cortisol concentrations were greater in castrated/dehorned than castrated calves at 60 min (P = 0.06). Period 2 cortisol concentrations were less in dehorned than castrated calves at 120 min (P = 0.005) but were greater from 360 to 480 min (P < 0.002). The Period 2 cortisol profile of control calves did not differ from the baseline obtained during sham handling, despite the intervening castration/dehorning in Period 1, suggesting that memory did not affect cortisol. The cortisol profile of castrated calves did not differ between periods except at 720 min, when Period 1 concentrations were greater than Period 2 (P = 0.02). Cortisol concentrations of calves dehorned in Period 2 were greater than those dehorned in Period 1 at 20 and 240 to 480 min (P < 0.05). In both periods, castrated calves exited the chute slower than dehorned calves (P < 0.05). The ADG did not differ between surgically treated calves in Period 1; in the interim, the ADG of castrated calves was greater than that of castrated/dehorned calves (difference ± SED, 1.4 ± 0.6 kg/d; P = 0.03), and in Period 2, the ADG of dehorned calves was less than castrated calves (1.8 ± 0.6 kg/d; P = 0.005). Our study supports both the common practice of concurrent castration/dehorning and the sequence of dehorning and castration. Delayed dehorning (vs. delayed castration) appeared to be more acutely stressful and more detrimental to ADG.

Effect of oral meloxicam on health and performance of beef steers relative to bulls castrated on arrival at the feedlot.

Castration in weaned calves is stressful and affects profitability by reducing ADG and increasing susceptibility to disease. This study evaluated the effect of meloxicam, a nonsteroidal anti-inflammatory drug (NSAID), on performance and health of calves received as steers compared with bull calves surgically castrated on arrival at the feedlot. British × Continental bulls (n = 145) and steers (n = 113; BW = 193 to 285 kg) were transported for 12 h in 3 truckloads (d 0), weighed, and randomly assigned to receive either lactose placebo (CONT; 1 mg/kg) or meloxicam (MEL; 1 mg/kg) suspended in water and administered per os, 24 h before castration. On d 1, bulls were surgically castrated (CAST) and steers were processed without castration (STR). Combinations of CONT/MEL and CAST/STR were allocated to 24 pens (6 pens per treatment) of 8 to 14 calves each. Pen was the experimental unit. Plasma meloxicam concentrations at the time of castration (d 1) were determined by HPLC-mass spectroscopy. Pen-level ADG, DMI, and G:F were estimated using BW obtained on d 0, 14, and 28 and weigh-back of feed. Individual animals were classified as sick based on a depression score of ≥2 on a 5-point scale and a rectal temperature of ≥39.8°C. On d 0, 1, and 14, calf chute temperament was evaluated using a 4-point scale. Data were analyzed using generalized linear mixed models and survival curve analyses. Castration reduced pen ADG (P < 0.001) and G:F (P < 0.001) from d 1 to 14, yet no effects (P > 0.45) were apparent by d 28. For all treatment groups, DMI increased with days on feed (P < 0.0001) but was less in CAST compared with STR calves (P < 0.016) throughout the study. From d 15 to 28, ADG increased (P = 0.0011) in CAST but not STR calves, and G:F decreased (P = 0.0004) in STR but not CAST calves. In CAST calves only, MEL treatment reduced the pen-level first pull rate (P = 0.04) and reduced bovine respiratory disease morbidity rate (P = 0.03). The frequency of chute escape behavior was greater on arrival and at castration in CAST vs. STR calves (P < 0.01) but not significantly different at d 14 (P = 0.22). Mean MEL concentrations at castration were no different between treated STR and CAST calves (P = 0.70). Meloxicam administration before castration in postweaning calves reduced the incidence of respiratory disease at the feedlot. These findings have implications for developing NSAID protocols for use in calves at castration with respect to addressing animal health and welfare concerns.

Pharmacokinetics of oral meloxicam in ruminant and preruminant calves.

The pharmacokinetics of oral meloxicam has been studied in ruminant, but not preruminant calves. Oral meloxicam was administered at 0.5 mg/kg to six ruminant calves via gavage (RG); to six preruminant calves via gavage (PRG); and to six preruminant calves via suckling in milk replacer (PRF). Plasma drug concentrations, determined over 120-h postadministration, were analyzed by compartmental and noncompartmental methods. The rate of drug absorption was faster (P<0.01) in PRF (0.237±0.0478/h) than RG calves (0.0815±0.0188/h), while absorption in PRG calves (0.153±0.128/h) was not different from other groups. C(max) was lower (P=0.03) in PRF (1.27±0.430 µg/mL) than in PRG calves (2.20±0.467 µg/mL), while C(max) of RG calves (1.95±0.955 µg/mL) was not different from other groups. V/F was higher in PRF calves (365±57 mL/kg) than either PRG (177±63 mL/kg, P<0.01) or RG (232±83 mL/kg, P=0.01) calves. These observations were likely due to differences in bioavailability, physiological maturity, and timing of the drug delivery into different compartments of the ruminant gastrointestinal tract. Results suggest that an adjustment in meloxicam dose may be necessary when administered with milk replacer.

Effect of intravenous sodium salicylate administration prior to castration on plasma cortisol and electroencephalography parameters in calves.

Nociception is an unavoidable consequence of many routine management procedures such as castration in cattle. This study investigated electroencephalography (EEG) parameters and cortisol levels in calves receiving intravenous sodium salicylate in response to a castration model. Twelve Holstein calves were randomly assigned to the following groups: (i) castrated, untreated controls, (ii) 50 mg/kg sodium salicylate IV precastration, were blood sampled at 0, 5, 10, 20, 30, 45, 60, 90, 120, 150, 180, 240, 360, and 480 min postcastration. The EEG recording included baseline, castration, immediate recovery (0-5 min after castration), middle recovery (5-10 min after castration), and late recovery (10-20 min after castration). Samples were analyzed by competitive chemiluminescent immunoassay and fluorescence polarization immunoassay for cortisol and salicylate, respectively. EEG visual inspection and spectral analysis were performed. Statistical analyses included anova repeated measures and correlations between response variable. No treatment effect was noted between the two groups for cortisol and EEG measurements, namely an attenuation of acute cortisol response and EEG desynchronization in sodium salicylate group. Time effects were noted for EEG measurements, cortisol and salicylates levels. Significant correlations between cortisol and EEG parameters were noted. These findings have implications for designing effective analgesic regimens, and they suggest that EEG can be useful to monitor pain attributable to castration.

Modeling of electroosmotic and electrophoretic mobilization in capillary and microchip isoelectric focusing.

Our dynamic capillary electrophoresis model which uses material specific input data for estimation of electroosmosis was applied to investigate fundamental aspects of isoelectric focusing (IEF) in capillaries or microchannels made from bare fused-silica (FS), FS coated with a sulfonated polymer, polymethylmethacrylate (PMMA) and poly(dimethylsiloxane) (PDMS). Input data were generated via determination of the electroosmotic flow (EOF) using buffers with varying pH and ionic strength. Two models are distinguished, one that neglects changes of ionic strength and one that includes the dependence between electroosmotic mobility and ionic strength. For each configuration, the models provide insight into the magnitude and dynamics of electroosmosis. The contribution of each electrophoretic zone to the net EOF is thereby visualized and the amount of EOF required for the detection of the zone structures at a particular location along the capillary, including at its end for MS detection, is predicted. For bare FS, PDMS and PMMA, simulations reveal that EOF is decreasing with time and that the entire IEF process is characterized by the asymptotic formation of a stationary steady-state zone configuration in which electrophoretic transport and electroosmotic zone displacement are opposite and of equal magnitude. The location of immobilization of the boundary between anolyte and most acidic carrier ampholyte is dependent on EOF, i.e. capillary material and anolyte. Overall time intervals for reaching this state in microchannels produced by PDMS and PMMA are predicted to be similar and about twice as long compared to uncoated FS. Additional mobilization for the detection of the entire pH gradient at the capillary end is required. Using concomitant electrophoretic mobilization with an acid as coanion in the catholyte is shown to provide sufficient additional cathodic transport for that purpose. FS capillaries dynamically double coated with polybrene and poly(vinylsulfonate) are predicted to provide sufficient electroosmotic pumping for detection of the entire IEF gradient at the cathodic column end.

Five additional genes are involved in clavulanic acid biosynthesis in Streptomyces clavuligerus.

An approximately 12.5-kbp region of DNA sequence from beyond the end of the previously described clavulanic acid gene cluster was analyzed and found to encode nine possible open reading frames (ORFs). Involvement of these ORFs in clavulanic acid biosynthesis was assessed by creating mutants with defects in each of the ORFs. orf12 and orf14 had been previously reported to be involved in clavulanic acid biosynthesis. Now five additional ORFs are shown to play a role, since their mutation results in a significant decrease or total absence of clavulanic acid production. Most of these newly described ORFs encode proteins with little similarity to others in the databases, and so their roles in clavulanic acid biosynthesis are unclear. Mutation of two of the ORFs, orf15 and orf16, results in the accumulation of a new metabolite, N-acetylglycylclavaminic acid, in place of clavulanic acid. orf18 and orf19 encode apparent penicillin binding proteins, and while mutations in these genes have minimal effects on clavulanic acid production, their normal roles as cell wall biosynthetic enzymes and as targets for beta-lactam antibiotics, together with their clustered location, suggest that they are part of the clavulanic acid gene cluster.

Multivariate matching and bias reduction in the surgical outcomes study.

BACKGROUND: Outcomes studies often need a level of detail that is not present in administrative data, therefore requiring abstraction of medical charts. Case-control methods may be used to improve statistical power and reduce abstraction costs, but limitations of exact matching often preclude the use of many covariates. Unlike exact matching, multivariate matching may allow cases to be matched simultaneously on hundreds of covariates. OBJECTIVES: To develop multivariate matched case-control pairs in a study of death after surgery in the Medicare population. RESEARCH DESIGN: Using 830 randomly selected index cases of patients who died within 60 days from admission, controls were found who did not die within that time period, matching on risk for death and other patient characteristics with up to 173 variables used simultaneously in the matching algorithms. SUBJECTS: General and orthopedic Medicare surgical cases in Pennsylvania from 1995 to 1996. Controls were either selected from across the entire state (108,765 possible subjects), or from within the same hospital as the case. MEASURES: Percent bias reduction and the average difference between cases and controls in units of standard deviations. RESULTS: Matched controls were far more similar to cases (deaths) upon admission to the hospital than typical patients, both in statewide and within hospital matches. Bias reduction was usually greater than 50% and often approached 100%. The difference between cases and matched controls for most variables was usually below 0.2 SD. CONCLUSIONS: Multivariate matching methods may aid in conducting studies with Medicare claims records by improving the quality of matches, thereby achieving a better understanding of the etiology of outcomes.

Osteoporosis in survivors of acute lymphoblastic leukemia.

Osteoporosis is currently receiving increasing attention as an important late effect in survivors of childhood cancer and its treatment because of their quality of life and its negative effect on the survivors' ability to perform developmentally appropriate activities. Survivors of childhood cancer are especially vulnerable because they are affected during childhood and adolescence, a time when peak bone mass should be achieved. This paper reviews decreased bone density in acute lymphoblastic leukemia (ALL), which is the most common childhood cancer and has a cure rate approaching 80%. Osteopenia/osteoporosis has been observed in all phases of the disease: at diagnosis, during treatment, and throughout the post-treatment period for as long as 20 years. Among the findings that have been described are musculoskeletal pain, disturbed gait, fractures, kyphosis, lordosis, and growth failure. Risk factors not specifically related to ALL include smoking, ingestion of carbonated beverages, and family history of "brittle bone" or fractures. Patients should be counseled in regard to diet, exercise, smoking cessation, and avoidance of carbonated beverages. There are a number of options for specific drug therapy; however, the administration of bisphosponates to children and adolescents must be approached with caution. Research is needed to determine how extensive the problem is and how to best prevent and treat the osteopenia/osteoporosis associated with ALL.

Applications of gene replacement technology to Streptomyces clavuligerus strain development for clavulanic acid production.

Cephamycin C production was blocked in wild-type cultures of the clavulanic acid-producing organism Streptomyces clavuligerus by targeted disruption of the gene (lat) encoding lysine epsilon-aminotransferase. Specific production of clavulanic acid increased in the lat mutants derived from the wild-type strain by 2- to 2.5-fold. Similar beneficial effects on clavulanic acid production were noted in previous studies when gene disruption was used to block the production of the non-clavulanic acid clavams produced by S. clavuligerus. Therefore, mutations in lat and in cvm1, a gene involved in clavam production, were introduced into a high-titer industrial strain of S. clavuligerus to create a double mutant with defects in production of both cephamycin C and clavams. Production of both cephamycin C and non-clavulanic acid clavams was eliminated in the double mutant, and clavulanic acid titers increased about 10% relative to those of the parental strain. This represents the first report of the successful use of genetic engineering to eliminate undesirable metabolic pathways in an industrial strain used for the production of an antibiotic important in human medicine.

Stromal-epithelial interactions in the progression of ovarian cancer: influence and source of tumor stromal cells.

Stromal cells are essential for the progression of many cancers including ovarian tumors. Stromal cell-epithelial cell interactions are important for tumor development, growth, angiogenesis, and metastasis. In the current study, the effects of normal ovarian bovine stromal cells on ovarian tumor progression was investigated. The hypothesis tested is that ovarian stromal cells will alter the onset and progression of ovarian tumors. Conditioned medium from normal bovine ovarian surface stromal cells was found to stimulate the growth of normal ovarian surface epithelium and had no effect on the growth of human tumor cell lines SKOV3 and OCC1. Human ovarian cancer cell lines, SKOV3 and OCC1, were injected subcutaneously into nude mice to examine tumor progression. Tumor growth in the nude mice was dramatically reduced when normal ovarian surface stromal cells were co-injected with SKOV3 or OCC1 cells. Similar results were obtained with normal bovine or human ovarian stromal cells. In contrast, irrelevant testicular stromal cells and epithelial cells had no effect on tumor growth in the nude mouse. Histological examination of these tumors revealed a characteristic stromal cell component adjacent to epithelial cell colonies. Sections of these tumors were hybridized with species specific genomic probes using fluorescence in situ hybridization to identify cell populations. Epithelial cells were shown to be of human origin (i.e. SKOV3 or OCC1), but stromal cells were found to be primarily murine in origin (i.e. host tissue). No detectable bovine cells were observed in the tumors after one week post-injection. Results suggest that stromal cells are an essential component of ovarian tumors. Interestingly, normal ovarian stromal cells had the ability to inhibit tumor growth, but were not able to survive long-term incubation at the tumor site. The developing tumor appears to recruit host (i.e. murine) stromal cells to invade the tumor and support its growth. In summary, normal ovarian stromal cells can inhibit ovarian tumor progression and the developing tumors recruit adjacent host stroma to become "tumor stroma". The tumor stroma likely develop an altered phenotype that cooperates with the tumorigenic epithelial cells to help promote the progression of ovarian cancer.

Multigenic control of Listeria monocytogenes susceptibility in mice.

We have used a novel quantitative trait locus model to study the genetics of survival of F2 progeny of susceptible BALB/cByJ and resistant C57BL/6ByJ mice that have been infected with Listeria monocytogenes. This allowed us to map modifiers of L. monocytogenes susceptibility to chromosomes 5 and 13.

Expression and actions of both the follicle stimulating hormone receptor and the luteinizing hormone receptor in normal ovarian surface epithelium and ovarian cancer.

The ability of gonadotropins to act on and regulate normal ovarian surface epithelial (OSE) cells and ovarian cancer cells was investigated. Bovine OSE was used as a model to study normal OSE. Results demonstrate that follicle stimulating hormone (FSH) and the luteinizing hormone (LH) like molecule, human chorionic gonadotropin (hCG), can both stimulate (3H)-thymidine incorporation into DNA in normal OSE cells. Similar results were obtained using either purified hormones or recombinant human hormones. A human ovarian cancer cell-line OCC1 was also stimulated to grow in response to FSH and hCG, but the growth of a different human ovarian cancer cell-line SKOV3 was not affected. In addition to effects on cell growth, gonadotropins also stimulated growth factor expression. Both FSH and hCG stimulated steady state levels of keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and kit ligand (KL) mRNA in OSE cells. Previously, KGF, HGF, and KL have been shown to stimulate OSE growth. Both follicle stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) were observed in OSE cells by Northern blot analysis. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed on fresh and cultured OSE cells. Normal OSE was found to express FSHR and LHR both in vivo and in vitro. The PCR reaction products were sequenced and found to provide a 100% homology with the bovine gonadotropin receptor sequences previously reported. FSHR and LHR transcripts were also detected in gonadotropin responsive OCC1 cells, but not in the gonadotropin insensitive SKOV3 cells. Observations support the hypothesis that gonadotropins may influence some ovarian cancers. In summary, the current study demonstrates the novel observation that both the FSHR and LHR are expressed by bovine OSE and selected ovarian cancers. Interestingly, the actions of FSH and LH to promote OSE growth may in part be mediated indirectly through an elevation in the expression of autocrine growth factors (KGF, HGF, and KL). Ovarian cancer is more common in conditions with elevated gonadotropins such as post-menopausal women. Therefore, gonadotropin actions on the OSE are postulated to be a potential factor in the onset and progression of some ovarian cancers.

Combined effects of Brassica napus seed meal and Trichoderma harzianum on two soilborne plant pathogens.

The effects of soil amendment with rapeseed meal from Brassica napus cv. 'Dwarf Essex' (high glucosinolate concentrations) and 'Stonewall' (low glucosinolate concentrations) on the biological control activity of Trichoderma harzianum towards Sclerotinia sclerotiorum and Aphanomyces euteiches were evaluated. Trichoderma harzianum added to soil reduced myceliogenic germination of S. sclerotiorum by 94%, but did not affect carpogenic germination. In contrast, 100% reduction in carpogenic germination was observed in soil amended with Dwarf Essex meal, along with a 33% reduction in myceliogenic germination. With Stonewall meal as soil amendment, carpogenic germination was reduced by 44% and myceliogenic germination was not affected. Both Dwarf Essex and Stonewall meals inhibited colonization of sclerotia in soil by T. harzianum, from 100% to 0% and 8%, respectively, so that biocontrol activity of T. harzianum was reduced in the presence of either meal. Aphanomyces euteiches root rot of pea was significantly reduced by T. harzianum alone (100%), by amendment with Dwarf Essex meal alone (77%), and by T. harzianum in combination with Dwarf Essex meal (100%). Amendment with Stonewall meal alone did not control root rot, and combination of Stonewall meal with T. harzianum reduced the biocontrol efficacy of T. harzianum.

Expression and action of keratinocyte growth factor (KGF) in normal ovarian surface epithelium and ovarian cancer.

The current study investigates the expression and action of keratinocyte growth factor (KGF) in normal ovarian surface epithelium (OSE) and ovarian cancer tissues. Ovarian tumors are primarily derived from the OSE. KGF is a mesenchymal cell-derived growth factor that mediates stromal cell-epithelial cell interactions in a variety of different tissues. Human ovarian tumors from borderline, stage I and stage III cases were found to express KGF protein in the epithelial cell component by immunocytochemical analysis. The stromal cell component of human ovarian tumors contained little or no KGF immunostaining. Normal bovine ovaries have similarities to human ovaries and are used as a model system to investigate normal OSE functions. KGF protein was detected in the OSE from normal human and bovine ovaries by immunocytochemistry. Ovarian stromal tissue contained light but positive KGF immunostaining. RNA was collected from normal bovine OSE and ovarian stromal cells to examine KGF gene expression. KGF transcripts were detected in cultured OSE and stromal cells by Northern blot analysis. In order to examine and quantitate KGF gene expression in freshly isolated versus cultured tissues, a sensitive quantitative RT-PCR assay for KGF was utilized. KGF gene expression was found to be high in freshly isolated OSE, but very low in freshly isolated stroma. Levels of KGF gene expression after culture of OSE and stromal cells increased. Observations indicate that normal OSE express high levels of KGF in vivo and in vitro. Expression of KGF by normal epithelial cells versus stromal cells was unexpected and suggests KGF may be an important autocrine stimulator of OSE. KGF actions on bovine OSE cells were investigated. KGF was found to stimulate the growth of normal OSE cells to the same level as epidermal growth factor. Two ovarian cancer cell lines, SKOV3 and OCC1, were also stimulated to proliferate in response to KGF. Current results demonstrate the production and action of KGF on normal OSE cells and ovarian cancer cells. Observations can be interpreted to suggest that KGF may in part be involved in the growth of ovarian tumors. This appears to be one of the first reports of KGF production by an epithelial cell. The autocrine stimulation of OSE growth by the local production and action of KGF provides insight into how the OSE may develop abnormal growth characteristics involved in the onset and progression of ovarian cancer.

Role of winged helix transcription factor (WIN) in the regulation of Sertoli cell differentiated functions: WIN acts as an early event gene for follicle-stimulating hormone.

Members of the winged helix transcription factor family are known to regulate epithelial cell differentiation by regulating cell-specific gene expression. rWIN is a newly discovered member of the winged helix family shown to be present in the adult rat testis. In the testis the human homolog of rWIN, HFH-11, was localized to the germ cells (i.e. spermatocytes and spermatids) undergoing spermatogenesis. In the present study we show that rWIN is also expressed in testicular Sertoli cells. Sertoli cells are the epithelial component of the seminiferous tubule and provide both the cytoarchitectural support and the microenvironment for developing germ cells. The presence of rWIN in Sertoli cells was confirmed by Northern blot and RT-PCR analysis. The rWIN transcript size in the Sertoli cells was different from the germ cell transcript that is probably due to alternative splicing or modifications of the 3'-untranslated region. At least two spliced variants of rWIN were observed in the Sertoli cells corresponding to the deletion of an exon in the DNA-binding region. Long term stimulation of cultured Sertoli cells with the gonadotropin FSH down-regulated rWIN expression. In contrast, short-term stimulation (2 h) transiently up-regulated rWIN expression. The FSH-induced transient stimulation of rWIN precedes expression of the transferrin gene that is a marker of Sertoli cell differentiation. FSH-induced transferrin promoter activity was inhibited when cultured Sertoli cells were treated with an antisense oligonucleotide to rWIN. Interestingly, the constitutive overexpression of the DNA-binding domain of rWIN also down-regulated transferrin promoter activity. Analysis of the transferrin promoter with various deletion mutations suggested that rWIN acts at an upstream gene of the transferrin promoter. The results indicate that a transient up-regulation of rWIN in part mediates the ability of FSH to activate the transferrin promoter, which can be inhibited with a rWIN antisense oligonucleotide or constitutive expression of the rWIN DNA-binding domain. The current study demonstrates that rWIN acts as an early event gene for FSH actions on Sertoli cells and that rWIN appears to have a role in the regulation of Sertoli cell differentiated functions.

Autocrine interactions of keratinocyte growth factor, hepatocyte growth factor, and kit-ligand in the regulation of normal ovarian surface epithelial cells.

Ovarian tumors are primarily derived from the layer of epithelium surrounding the ovary termed the ovarian surface epithelium (OSE). Although extensive research has focused on established ovarian tumors, relatively little is known about the normal biology of the OSE that gives rise to ovarian cancer. The local expression and actions of growth factors are likely involved in both normal and tumorigenic OSE biology. The current study investigates the expression and action of keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and kit-ligand (KL) in normal ovarian surface epithelium (OSE). The actions of various growth factors on KGF, HGF, and KL expression are examined. Observations indicate that freshly isolated normal OSE express the genes for KGF, HGF, and KL and expression is maintained in vitro. KGF messenger RNA expression in OSE was found to be stimulated by KGF and HGF, but not KL. HGF expression in OSE was found to be stimulated by KGF, HGF, and KL. KL expression in OSE was also found to be stimulated by KGF, HGF, and KL. Therefore, the various growth factors can regulate the mRNA expression of each other in OSE. Effects of growth factors on OSE growth were examined. KGF, HGF, and KL stimulated OSE growth to similar levels as the positive control epidermal growth factor. Observations suggest that KGF, HGF, and KL interact to promote OSE growth and growth factor expression. The ability of these growth factors to interact in a positive autocrine feedback loop is postulated to be important for normal OSE biology. Paracrine interactions with the adjacent stromal cells will also be a factor in OSE biology. Abnormal interactions of these growth factors may be involved in the onset and progression of ovarian cancer.

Genes specific for the biosynthesis of clavam metabolites antipodal to clavulanic acid are clustered with the gene for clavaminate synthase 1 in Streptomyces clavuligerus.

Portions of the Streptomyces clavuligerus chromosome flanking cas1, which encodes the clavaminate synthase 1 isoenzyme (CAS1), have been cloned and sequenced. Mutants of S. clavuligerus disrupted in cvm1, the open reading frame located immediately upstream of cas1, were constructed by a gene replacement procedure. Similar techniques were used to generate S. clavuligerus mutants carrying a deletion that encompassed portions of the two open reading frames, cvm4 and cvm5, located directly downstream of cas1. Both classes of mutants still produced clavulanic acid and cephamycin C but lost the ability to synthesize the antipodal clavam metabolites clavam-2-carboxylate, 2-hydroxymethyl-clavam, and 2-alanylclavam. These results suggested that cas1 is clustered with genes essential and specific for clavam metabolite biosynthesis. When a cas1 mutant of S. clavuligerus was constructed by gene replacement, it produced lower levels of both clavulanic acid and most of the antipodal clavams except for 2-alanylclavam. However, a double mutant of S. clavuligerus disrupted in both cas1 and cas2 produced neither clavulanic acid nor any of the antipodal clavams, including 2-alanylclavam. This outcome was consistent with the contribution of both CAS1 and CAS2 to a common pool of clavaminic acid that is shunted toward clavulanic acid and clavam metabolite biosynthesis.