Relationship of plasma estradiol-17beta, total estrogen, and progesterone to estrus behavior in mithun (Bos frontalis) cows

The objectives of this study were (1) to establish the characteristics of estrus behavior in mithun cows (n = 12) and (2) to determine the relationships between this behavior and the plasma concentrations of estradiol-17beta (E2), total estrogen, and progesterone. Estrus was detected by visual observations of estrus signs, per recta examination of genitalia and bull parading thrice a day for three consecutive cycles. Among the behavioral signs of estrus, the cow to be mounted by bull (100%) was the best indicator of estrus followed by standing to be mounted (92%). Per rectum examination of genital organs revealed relaxed and open os externa of cervix, turgid uterus, and ovaries having palpable follicles in all animals. The mean (+/-SEM) length of estrus cycle and duration of estrus were recorded to be 21.8 +/- 0.69 days and 12.6 +/- 1.34 h, respectively. Endocrine profiles during the peri-estrus period showed that the mean highest peak concentrations of E2 (27.29 +/- 0.79 pg/ml) and total estrogen (45.69 +/- 2.32 pg/ml) occurred at -3.90 +/- 2.27 and -3.89 +/- 2.26 h prior to the onset of estrus, respectively. Plasma progesterone concentration was basal (0.14 +/- 0.001 ng/ml) during the peri-estrus period. Plasma E2 and total estrogen were found to increase from 6 days before estrus to reach a peak level on the day of estrus and decline thereafter to basal level on day 3 of the cycle. The plasma progesterone concentration was the lowest on the day of estrus showing gradual increase to register a peak level on day 15 of the cycle. Estrus behavior was found to be positively correlated with the maximum peak concentration of E2 (r = 0.89; P < 0.0001) and total estrogen (r = 0.66; P = 0.019) during the peri-estrus period. The mean total estrogen concentration during the peri-estrus period was significantly correlated with estrus behavior (r = 0.60; P = 0.04). The correlations between the estrus behavior and E2:progesterone ratios at 6 days before the onset of estrus (r = 0.92) and on the day of estrus (r = 0.95) was significant. The total estrogen:progesterone ratios at 6 days before the onset of estrus and on the day of estrus were also positively correlated with the estrus behavior (r = 0.86 and 0.88). In conclusion, our results suggest that the maximum peak concentration of E2 and total estrogen and mean level of total estrogen during the peri-estrus period and the E2:progesterone and total estrogen:progesterone ratios on 6 days before the onset of estrus and on the day of estrus are the important factors contributing the behavioral manifestation of estrus in mithun cows.     

Effect of plantain banana on gastric ulceration in NIDDM rats: role of gastric mucosal glycoproteins, cell proliferation, antioxidants and free radicals

Methanolic extract of Musa sapientum var. Paradisiaca (MSE, 100 mg/kg) was studied for its antiulcer and mucosal defensive factors in normal and non-insulin dependent diabetes mellitus (NIDDM) rats. NIDDM was induced by administering streptozotocin (STZ, 70 mg/kg, ip) to 5 days old rat pups. The animals showing blood glucose level >140mg/dL after 12 weeks of STZ administration were considered as NIDDM positive. Effects of MSE were compared with known ulcer protective drug, sucralfate (SFT, 500 mg/kg) and anti-diabetic drug glibenclamide (GLC, 0.6 mg/kg) when administered orally, once daily for 6 days against gastric ulcers (GU) induced by cold-restraint stress (CRS) and ethanol and subsequent changes in gastric mucosal glycoproteins, cell proliferation, free radicals (lipid peroxidation and nitric oxide) and anti-oxidants enzymes (super oxide dismutase and catalase) and glutathione (GSH) levels. MSE showed better ulcer protective effect in NIDDM rats compared with SFT and GLC in CRS-induced GU. NIDDM caused a significant decrease in gastric mucosal glycoprotein level without having any effect on cell proliferation. However, all the test drugs reversed the decrease in glycoprotein level in NIDDM rats, but cell proliferation was enhanced in case of MSE alone. Both CRS or NIDDM as such enhanced gastric mucosal LPO, NO and SOD, but decreased CAT levels while CRS plus NIDDM rats caused further increase in LPO and NO level without causing any further changes in SOD and CAT level. MSE pretreatment showed reversal in the levels of all the above parameters better than GLC. Ethanol caused a decrease in glutathione level which was further reduced in NIDDM-ethanol rats. MSE reversed the above changes significantly in both normal as well as in NIDDM rats, while GLC reversed it only in NIDDM rats. However, SFT was ineffective in reversing the changes induced by CRS or ethanol or when given in NIDDM-CRS or NIDDM-ethanol rats. The results indicated that the ulcer protective effect of MSE could be due to its predominant effect on mucosal glycoprotein, cell proliferation, free radicals and antioxidant systems.     

Dual X-ray absorptiometry model for characterizing water in the human forearm using multiple frequency bioimpedance analysis

The purpose of this study was to develop a method for measuring intracellular (ICW) and extracellular water (ECW) in the human forearm using multiple frequency bioimpedance analysis (MFBIA). The approach was (i) to measure whole-body and forearm fat-free mass using dual X-ray absorptiometry (DXA); (ii) to use these measurements to estimate the fat-free mass (FFM) resistivity in both the forearm and in the whole body; and (iii) to use the ratio of these FFM resistivities to estimate the resistivity in the ICW and ECW compartments of the forearm. To first demonstrate the accuracy of the DXA software in differentiating lean body mass from fat and bone within a volume of tissue, ex-vivo bovine muscle tissue samples (n = 3) were used to approximate the physical properties of the human forearm. It was found that although the human whole-body software overestimates FFM, it was slightly underestimated by the small animal software. Using this technique, DXA measures of FFM were obtained from human volunteers (n = 11; age = 20 +/- 5 years; height = 170 +/- 12 cm; mass = 64 +/- 16 kg). These measures were used in conjunction with MFBIA measures of impedance of the whole body and of the forearm to determine the resistivities of the ICW and ECW compartments of the forearm, namely 375.8 +/- 25.2 ohms cm and 55.6 +/- 3.7 ohms cm, respectively. These were used in MFBIA equations to calculate the ICW, ECW, and total arm water (TAW) volumes of the human forearm. The calculated TAW and the ECW (+/- SD) volume fraction (667.29 +/- 200.15 mL and 0.169 +/- 0.039 mL, respectively) were in agreement with literature values. MFBIA results were compared with those obtained using nuclear magnetic resonance relaxometry (NMRR). MFBIA was performed on 15 subjects before and after an intense maximal handgrip exercise to estimate changes in water volume in muscle. Following exercise, the total and intracellular water of the forearm increased on average by 8% +/- 3% and 10% +/- 4% (mean +/- SD), respectively. In 5 healthy volunteers, MFBIA and NMRR were performed before and after a similar exercise of the forearm muscle. The changes with exercise of intracellular and total arm water volumes as measured by MFBIA were estimated. The percent increases in total water were found to be 9.4% +/- 4.2% and 9.4% +/- 2.6% and in intracellular water were found to be 10.6% +/- 4.6% and 12.0% +/- 2.8% (mean +/- SD) for NMRR and MFBIA, respectively. The results show that the exercise-induced changes in ICW and TAW determined with the MFBIA model are consistent with those observed with NMRR and radiotracer literature.     

Development of a biotin-streptavidin amplified enzyme immunoassay for oxytocin and its application during milk ejection and the reproductive cycle in the mithun (Bos frontalis)

Oxytocin is a key hormone involved in milk ejection. It plays a key role in regulation of reproductive cyclicity in female mammals by taking part in the process of luteolysis. Determination of oxytocin is, therefore, important for studying the control of its secretion and its role in reproduction of the mithun. A simple and sufficiently sensitive enzyme immunoassay (EIA) for oxytocin determination in mithun plasma using the biotin-streptavidin amplification system and second antibody coating technique was therefore developed. Biotin was coupled to oxytocin and used to bridge between streptavidin-peroxidase and the immobilized oxytocin antiserum in a competitive assay. The EIA was conducted directly in 200 microl of unknown mithun plasma. Standards prepared in hormone-free plasma were used. The lowest detection limit was 0.5 pg/ml plasma. Plasma volumes for the EIA (50, 100, and 200 microl) did not influence the shape of standard curve, even though a drop in OD450 was seen with higher plasma volumes. A parallelism test was carried out to compare endogenous mithun oxytocin with a bovine oxytocin standard. The former showed good parallelism with the bovine standard curve. For biological validation of the assay, plasma oxytocin was measured in the blood samples collected before, during, and after milking in three mithun cows and in six non-lactating cyclic mithuns during the entire estrous cycle. A sharp release of oxytocin shortly after udder stimulation was observed. A high level of oxytocin was maintained during milking, falling sharply thereafter. The mean plasma oxytocin concentration was different on different days of the estrous cycle (P < 0.001). Two peaks of oxytocin were recorded, one at day 6 and another at day 18 of the estrous cycle. In conclusion, a simple, sufficiently sensitive and direct EIA procedure has been developed for the first time to determine plasma oxytocin levels in mithuns. Apart from being non-radioactive, the EIA procedure described here also utilizes a highly stable biotinalyted hormone which has a shelf life of several years, unlike the short shelf life of iodinated tracer used in RIA procedures.     

CYP17, SRD5A2, CYP1B1, and CYP2D6 gene polymorphisms with prostate cancer risk in North Indian population

To investigate the involvement of the CYP17, SRD5A2, CYP1B1, and CYP2D6 variants with prostate cancer, a case-control study of 100 patients and an equal number of age-matched control men was conducted. There appears to be a nonsignificant increase with risk of prostate cancer for individuals carrying one copy of the CYP17 A2 allele (OR, 1.80; 95% CI, 0.99-3.29, P=0.05). The risk was increased in individuals having two A2 alleles (OR; 2.81, 95% CI, 1.06-7.40, P=0.03). Compared with men having the VV genotype of SRD5A2 gene, there was no significant association between the VL genotype and the risk of prostate cancer (OR; 0.54, 95% CI; 0.29-1.03, P=0.06). There was no difference in the occurrence of the genotype LL between controls and prostate cancer patients (OR; 0.90, 95% CI; 0.43-1.89, P=0.79). There was a nonsignificant increased risk of prostate cancer for individuals carrying the CYP1B1Leu/Val genotype (OR, 1.70, 95% CI, 0.91-3.17, P =0.09), which was increased in those having the Val/Val allele (OR, 3.38; 95% CI, 1.13-10.07, P=0.02). Relative to men homozygous for the wild-type allele in CYP2D6 gene, those heterozygous for the B allele had an odds ratio of 1.78 (95% CI, 0.76-4.17, P=0.18) for patients, and for homozygous individuals, it was 1.95 (0.55-6.93, P=0.30). These observations have suggested that the CYP17 A2/A2, CYP1B1 Val/Val, and CYP2D6 genotypes may be associated with an altered risk of prostate cancer, while the CYP2D6 and SRD5A2 V89L polymorphism have no association with its risk in the North Indian population.     

Three-dimensional culture regulates Raf-1 expression to modulate fibronectin matrix assembly

Oncogenic transformation has been associated with decreased fibronectin (FN) matrix assembly. For example, both the HT-1080 fibrosarcoma and MAT-LyLu cell lines fail to assemble a FN matrix when grown in monolayer culture (2-dimensional [2D] system). In this study, we show that these cells regain the ability to assemble a FN matrix when they are grown as aggregates (3-dimensional [3D] system). FN matrix assembly in 3D correlates with decreased Raf-1 protein expression compared with cells grown in monolayer culture. This effect is associated with reduced Raf-1 mRNA levels as determined by quantitative RT-PCR and not proteasome-mediated degradation of endogenous Raf-1. Interestingly, transient expression of a Raf-1 promoter-reporter construct demonstrates increased Raf-1 promoter activity in 3D, suggesting that the transition to 3D culture may modulate Raf-1 mRNA stability. Finally, to confirm that decreased Raf-1 expression results in increased FN matrix assembly, we used both pharmacological and small interfering RNA knockdown of Raf-1. This restored the ability of cells in 2D culture to assemble a FN matrix. Moreover, overexpression of Raf-1 prevented FN matrix assembly by cells cultured in 3D, resulting in decreased aggregate compaction. This work provides new insight into how the cell microenvironment may influence Raf-1 expression to modulate cell-FN interactions in 3D.     

Cell cycle-dependent roles for the FCH-domain protein Cdc15p in formation of the actomyosin ring in Schizosaccharomyces pombe

Cell division in the fission yeast Schizosaccharomyces pombe requires the formation and constriction of an actomyosin ring at the division site. The actomyosin ring is assembled in metaphase and anaphase A, is maintained throughout mitosis, and constricts after completion of anaphase. Maintenance of the actomyosin ring during late stages of mitosis depends on the septation initiation network (SIN), a signaling cascade that also regulates the deposition of the division septum. However, SIN is not active in metaphase and is not required for the initial assembly of the actomyosin ring early in mitosis. The FER/CIP4-homology (FCH) domain protein Cdc15p is a component of the actomyosin ring. Mutations in cdc15 lead to failure in cytokinesis and result in the formation of elongated, multinucleate cells without a division septum. Here we present evidence that the requirement of Cdc15p for actomyosin ring formation is dependent on the stage of mitosis. Although cdc15 mutants are competent to assemble actomyosin rings in metaphase, they are unable to maintain actomyosin rings late in mitosis when SIN is active. In the absence of functional Cdc15p, ring formation upon metaphase arrest depends on the anillin-like Mid1p. Interestingly, when cytokinesis is delayed due to perturbations to the division machinery, Cdc15p is maintained in a hypophosphorylated form. The dephosphorylation of Cdc15p, which occurs transiently in unperturbed cytokinesis, is partially dependent on the phosphatase Clp1p/Flp1p. This suggests a mechanism where both SIN and Clp1p/Flp1p contribute to maintenance of the actomyosin ring in late mitosis through Cdc15p, possibly by regulating its phosphorylation status.     

Highly sensitive second antibody format enzymeimmunoassay for determination of 13,14-dihydro-15-keto-PGF2alpha in blood plasma of mithun (Bos frontalis)

As an alternative to radioimmunoassays, a simple, highly sensitive and quick enzymeimmunoassay (EIA) for determination of 13,14-dihydro-15-keto-PGF(2alpha) (PGFM) in blood plasma of mithun (Bos frontalis; bovine) on microtitreplates using second antibody coating technique and PGFM-horseradish peroxidase as a label has been developed. The wells of the microtitreplate were coated with affinity-purified goat IgG (antirabbit IgG) that binds the hormone specific antibody. The EIA was carried out directly in 20microl plasma. The PGFM standard curve, with doses ranging from 0.1 to 50pg/well was linear. The sensitivity of the assay was 5pg/ml. PGFM standard curve in buffer showed parallelism with serially diluted mithun plasma containing high endogenous PGFM. Plasma PGFM concentrations estimated by using the developed EIA and commercially available PGFM EIA kit in the same samples were significantly correlated (r=0.98) and showed linearity. Intra- and inter-assay coefficients of variation were below 7%. Recovery of known concentrations of added PGFM in charcoal stripped plasma was linear (r=0.99). The developed EIA was further validated biologically by estimating PGFM in cyclic cows for the entire estrous cycle and in peri-parturient cows beginning day 7 prior to calving till day 30 post-calving; the concentrations were along with the expected lines as reported in bovine. In conclusion, the EIA developed in this study is simple, highly sensitive, valid and sufficiently reliable method for estimation of PGFM directly in bovine plasma.