A simple enzymeimmunoassay of milk progesterone for monitoring fertility in dairy buffaloes

A simple, sensitive, direct (without extraction) enzymeimmunoassay (EIA) was usec to determine progesterone levels in whole milk samples of 400 Nili-Ravi dairy buffaloes. The mean progesterone values 22 d after A.I. were significantly higher in pregnant (16.6 +/- 9.2 ng/ml) than nonpregnant (below 5 ng/ml) animals. The mean progesterone values were below 0.34 +/- 0.12 (the detection limit) both at estrus and in cases of clinically diagnosed inactive ovaries, 3.18 +/- 1.9 at proestrus, 2.25 +/- 1.2 postestrus and 13.22 +/- 6.74 at Day 10 of the estrous cycle. Twenty buffaloes confirmed pregnant for 2 to 3 mo, had a mean value of 20.3 +/- 4.5 ng/ml. The EIA test is very reliable in the selection of nonpregnant buffaloes (100 %) and the confirmation of inactive ovaries and of estrus. Differential diagnosis of inactive or active ovaries can be made by analyzing two milk samples at a 7-d interval.     

Freeze Thaw: A Simple Approach for Prediction of Optimal Cryoprotectant for Freeze Drying

The present study evaluates freeze thaw as a simple approach for screening the most appropriate cryoprotectant. Freeze–thaw study is based on the principle that an excipient, which protects nanoparticles during the first step of freezing, is likely to be an effective cryoprotectant. Nanoparticles of rifampicin with high entrapment efficiency were prepared by the emulsion-solvent diffusion method using dioctyl sodium sulfosuccinate (AOT) as complexing agent and Gantrez AN-119 as polymer. Freeze–thaw study was carried out using trehalose and fructose as cryoprotectants. The concentration of cryoprotectant, concentration of nanoparticles in the dispersion, and the freezing temperature were varied during the freeze–thaw study. Cryoprotection increased with increase in cryoprotectant concentration. Further, trehalose was superior to fructose at equivalent concentrations and moreover permitted use of more concentrated nanosuspensions for freeze drying. Freezing temperature did not influence the freeze–thaw study. Freeze-dried nanoparticles revealed good redispersibility with a size increase that correlated well with the freeze–thaw study at 20% w/v trehalose and fructose. Transmission electron microscopy revealed round particles with a size ∼400 nm, which correlated with photon correlation spectroscopic measurements. Differential scanning calorimetry and X-ray diffraction suggested amorphization of rifampicin. Fourier transfer infrared spectroscopy could not confirm interaction of drug with AOT. Nanoparticles exhibited sustained release of rifampicin, which followed diffusion kinetics. Nanoparticles of rifampicin were found to be stable for 12 months. The good correlation between freeze thaw and freeze drying suggests freeze–thaw study as a simple and quick approach for screening optimal cryoprotectant for freeze drying.     

Analysis and identification of beta-turn types using multinomial logistic regression and artificial neural network

MOTIVATION: So far various statistical and machine learning techniques applied for prediction of beta-turns. The majority of these techniques have been only focused on the prediction of beta-turn location in proteins. We developed a hybrid approach for analysis and prediction of different types of beta-turn. RESULTS: A two-stage hybrid model developed to predict the beta-turn Types I, II, IV and VIII. Multinomial logistic regression was initially used for the first time to select significant parameters in prediction of beta-turn types using a self-consistency test procedure. The extracted parameters were consisted of 80 amino acid positional occurrences and 20 amino acid percentages in beta-turn sequence. The most significant parameters were then selected using multinomial logistic regression model. Among these, the occurrences of glutamine, histidine, glutamic acid and arginine, respectively, in positions i, i + 1, i + 2 and i + 3 of beta-turn sequence had an overall relationship with five beta-turn types. A neural network model was then constructed and fed by the parameters selected by multinomial logistic regression to build a hybrid predictor. The networks have been trained and tested on a non-homologous dataset of 565 protein chains by 9-fold cross-validation. It has been observed that the hybrid model gives a Matthews correlation coefficient (MCC) of 0.235, 0.473, 0.103 and 0.124, respectively, for beta-turn Types I, II, IV and VIII. Our model also distinguished the different types of beta-turn in the embedded binary logit comparisons which have not carried out so far. AVAILABILITY: Available on request from the authors.     

Murine mesenchymal stem cells isolated by low density primary culture system

Murine mesenchymal stem cells (mMSC) and the difficult task of isolation and purification of them have been the subject of rather extensive investigation. The present study sought to isolate these cells from two different mouse strains, one outbred and the other inbred, primarily through a relatively simple but novel approach, the most important feature of which was the low density primary culture of bone marrow cells. For this purpose, mononuclear cells from either NMRI or BALB/c bone marrow were plated at about 500 cells per well of 24-well plates and incubated for 7 days. At this point, the fibroblastic clones that had emerged were pooled together and expanded through several subcultures. To investigate the mesenchymal nature, we differentiated the cells into the osteoblastic, chondrocytic and adipocytic lineages in different subcultures up to passage 10. According to the results, 1 week after culture initiation, several clones each comprising several fibroblastic cells appeared in each plate. The cells from different passages were capable of differentiating into corresponding skeletal tissues. In the present investigation, the best culture condition for maximum proliferation and also the expression of certain surface marker on isolated cells were examined. In this term the two murine strains showed some differences.     

Novel rearranged abietane diterpenoids from the roots of Salvia sahendica

Two naphthoquinone diterpenoids, 1 and 2, one tricyclic, and one tetracyclic rearranged abietane ('4,5-seco-10,5-friedo-abietane') diterpenoids, 3 and 4, respectively, together with horminone (5) have been isolated from the roots of Salvia sahendica. Compounds 2 and 3 are new, and the 13C-NMR assignment for compound 4 was modified using ' Heteronuclear Multiple-Bond Correlation' (HMBC) spectroscopic data. The structures of the compounds have been established by using different spectral data including 1D- and 2D-NMR, IR, UV, and MS. The elemental composition for the major peaks of 3 and 4 were determined by ' High-Resolution Electron Impact Mass Spectrometry' (HR-EI-MS). The relative configurations of the new compounds were determined by 1H-NMR and 'Rotating-Frame NOES' (ROESY) spectroscopy. Compounds 1, 2, and 5 showed antifungal activities when tested on Blakeslea trispora. Lapachol, a prelynated naphthoquinone, was used as a positive control. The biological activities of the related naphthoquinones and abietane diterpenoids were discussed.     

Optical PCR: Genomic analysis by long-range PCR and optical mapping

Optical mapping is an approach for the rapid, automated, non-electrophoretic construction of ordered restriction maps of DNA from ensembles of single molecules. Previously, we used optical mapping to make high-resolution maps of large insert clones such as bacterial artificial chromosomes (BAC) and large genomic DNA molecules. Here, we describe a combination of optical mapping and long-range polymerase chain reaction (PCR), in a process we term optical PCR, which enables automated construction of ordered restriction maps of long-range PCR products spanning human genomic loci. Specifically, we amplified three long PCR products, each averaging 14.6 kb in length, which span the 37-kb human tissue plasminogen activator (TPA) gene. PCR products were surface mounted in gridded arrays, and samples were mapped in parallel with either ScaI, XmnI, HpaI, ClaI, or BglII. A contig of overlapping high-resolution maps was generated, which agreed closely with maps predicted from sequence data. The data demonstrate an approach to construct physical maps of genomic loci where very little prior sequence information exists, since the only sequence needed is that required to anchor PCR primers. Large segments of genomic DNA (within the practical limits imposed by long-range PCR) can be mapped quickly and to high resolution without the use of cloning vectors. Received: 9 February 1999 / Accepted: 26 May 1999     

Repulsive guidance molecule (RGMa), a DRAGON homologue, is a bone morphogenetic protein co-receptor

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor beta (TGF-beta) superfamily of ligands, which regulate many mammalian physiologic and pathophysiologic processes. BMPs exert their effects through type I and type II serine/threonine kinase receptors and the Smad intracellular signaling pathway. Recently, the glycosylphosphatidylinositol (GPI)-anchored protein DRAGON was identified as a co-receptor for BMP signaling. Here, we investigate whether a homologue of DRAGON, repulsive guidance molecule (RGMa), is similarly involved in the BMP signaling pathway. We show that RGMa enhances BMP, but not TGF-beta, signals in a ligand-dependent manner in cell culture. The soluble extracellular domain of RGMa fused to human Fc (RGMa.Fc) forms a complex with BMP type I receptors and binds directly and selectively to radiolabeled BMP-2 and BMP-4. RGMa mediates BMP signaling through the classical BMP signaling pathway involving Smad1, 5, and 8, and it up-regulates endogenous inhibitor of differentiation (Id1) protein, an important downstream target of BMP signals. Finally, we demonstrate that BMP signaling occurs in neurons that express RGMa in vivo. These data are consistent with a role for RGMa as a BMP co-receptor.     

Milk progesterone profiles in various reproductive states in dairy buffaloes under field conditions

Fifty-one dairy buffaloes in the last two months of gestation were selected at seven private peri-urban farms in the Peshawar district. Observations were recorded in buffaloes during normal (NBS, August to January) and low breeding seasons (LBS, February to July). After parturition, rectal examination of reproductive organs was carried out. Estrus detection was made through visual observation and the use of intact bull. Postpartum ovulation was confirmed by ovarian palpation per rectum and milk progesterone levels (MPL), determined through radio-immunoassay. MPL was higher (p < 0.01) at various intervals in NBS calves (1.97 +/- 0.30 ng/ml) as compared to LBS calves (0.68 +/- 0.08 ng/ml). During LBS, MPL remained < 0.30 ng/ml up to the third fortnight and started rising later, reaching a peak of 1.27 ng/ml during the sixth fortnight. During NBS, there was a sharp rise in MPL during the second fortnight, reaching 3.64 ng/ml during the sixth fortnight. MPL was significantly different on different experimental farms (p < 0.01). MPL reached the lowest levels on the day of estrus (0.10 ng/ml), reached it's peak on day 7 and started declining on day 17 of estrus. MPL showed two postpartum elevations. In true anestrus buffaloes, MPL remained consistently low. However, in the anestrus period, silent ovulations were also noted, as reflected by increasing MPL without estrus signs. In pregnant buffaloes, MPL remained > 1 ng/ml. Results of the study showed that the low postpartum reproductive performance in dairy buffaloes during LBS was primarily due to inadequate functioning of the corpus luteum in secreting optimum concentrations of progesterone. The higher incidence of silent estrus during LBS indicated improved management for the detection of estrus.     

DRAGON, a bone morphogenetic protein co-receptor

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF)beta superfamily of ligands that regulate many crucial aspects of embryonic development and organogenesis. Unlike other TGFbeta ligands, co-receptors for BMP ligands have not been described. Here we show that DRAGON, a glycosylphosphatidylinositol-anchored member of the repulsive guidance molecule family, which is expressed early in the developing nervous system, enhances BMP but not TGFbeta signaling. DRAGON binds directly to BMP2 and BMP4 but not to BMP7 or other TGFbeta ligands. The enhancing action of DRAGON on BMP signaling is also reduced by administration of Noggin, a soluble BMP antagonist, indicating that the action of DRAGON is ligand-dependent. DRAGON associates directly with BMP type I (ALK2, ALK3, and ALK6) and type II (ActRII and ActRIIB) receptors, and its signaling is reduced by dominant negative Smad1 and ALK3 or -6 receptors. In the Xenopus embryo, DRAGON both reduces the threshold of the ability of Smad1 to induce mesodermal and endodermal markers and alters neuronal and neural crest patterning. The direct interaction of DRAGON with BMP ligands and receptors indicates that it is a BMP co-receptor that potentiates BMP signaling.