Point mutations in the abl SH2 domain coordinately impair phosphotyrosine binding in vitro and transforming activity in vivo.

We have constructed a series of point mutations in the highly conserved FLVRES motif of the src homology 2 (SH2) domain of the abl tyrosine kinase. Mutant SH2 domains were expressed in bacteria, and their ability to bind to tyrosine-phosphorylated proteins was examined in vitro. Three mutants were greatly reduced in their ability to bind both phosphotyrosine itself and tyrosine-phosphorylated cellular proteins. All of the mutants that retained activity bound to the same set of tyrosine-phosphorylated proteins as did the wild type, suggesting that binding specificity was unaffected. These results implicate the FLVRES motif in direct binding to phosphotyrosine. When the mutant SH2 domains were inserted into an activated abl kinase and expressed in murine fibroblasts, decreased in vitro phosphotyrosine binding correlated with decreased transforming ability. This finding implies that SH2-phosphotyrosine interactions are involved in transmission of positive growth signals by the nonreceptor tyrosine kinases, most likely via the assembly of multiprotein complexes with other tyrosine-phosphorylated proteins.     

Diurnal variation of milk somatic and differential leukocyte counts of Murrah buffaloes as influenced by different milk fractions,seasons and parities

Milk cell counts are good indicators of a mammary infection and milk quality. The present study was done to record diurnal rhythmicity in the milk somatic and differential cell counts during different seasons, milk strips and parity in Murrah buffaloes. Milk somatic cell counts (SCC) were measured by SCC counter and milk differential cell counts were measured microscopically after making a milk smear and staining it to identify neutrophils, lymphocytes and macrophages. Maximum milk SCC was observed in the summer season. Milk neutrophils were lowest during thermoneutral (TN), intermediate during the winter season and highest during the summer season. Milk lymphocytes were highest during the winter season, intermediate in the TN and lowest in the summer season. Diurnal rhythm in the milk SCC and neutrophil percentage is noticed in the summer season only. Maximum milk SCC values were observed in the late strip but neutrophils were highest in the early strip. Diurnal rhythm was observed in the late strip for neutrophils and in mid strip for the lymphocytes. Milk SCC and milk neutrophils were found to be the highest in the multiparous buffaloes and diurnal rhythm was observed only in the lymphocytes of primiparous buffaloes. Milk macrophages were higher in the morning samples of primiparous as compared to the multiparous buffaloes. In this pioneer study, diurnal rhythms in the milk cell counts of buffaloes have been studied extensively. This will help in maintaining low milk cell counts in buffalo and thus help in getting more milk per buffalo during stress periods.     

Bovine papillomavirus E5 protein induces the formation of signal transduction complexes containing dimeric activated platelet-derived growth factor beta receptor and associated signaling proteins

The bovine papillomavirus E5 protein binds to the cellular platelet-derived growth factor (PDGF) beta receptor, resulting in constitutive activation of the receptor and cell growth transformation. By subjecting extracts from E5-transformed or PDGF-treated cells to velocity sedimentation in sucrose gradients, activated PDGF beta receptor complexes were separated from monomeric, inactive receptor. Rapidly sedimenting activated complexes contained oligomeric (apparently dimeric), tyrosine-phosphorylated PDGF beta receptor, the E5 protein, and associated cellular signaling proteins including the p85 subunit of phosphoinositol 3'-kinase, phospholipase Cgamma, and Ras-GTPase activating protein. These signaling proteins made the major contribution to the increased sedimentation rate of the activated receptor complexes. Pairwise analysis of components of these complexes indicated that multiple signaling proteins and the E5 protein were simultaneously present in the activated complexes. Our results also showed that the E5 protein and PDGF activated only a small fraction of the total PDGF beta receptor, that not all receptor molecules associated with the E5 protein were tyrosine-phosphorylated, and that signaling proteins could bind to hemiphosphorylated receptor dimers. On the basis of these results, we propose a model for the assembly of multiprotein, activated PDGF beta receptor complexes in response to the E5 protein.     

Relationships between fertility, peak milk yields and lactational persistency in dairy cows

Peak milk yield, lactational persistency and conception rates were studied using 5928 lactation records of high milk-producing cows at three California dairies. Log-linear analysis was used to study relationships between peak milk yield, lactational persistency, dairy of origin, lactation number and conception rates in 3850 completed lactations. Cows with peak milk yields greater than the median (38.2 kg milk per day) were less likely to have conceived in one or two breedings than cows with peak milk yields lower than or equal to the median. Cows with a higher than median (0.755) lactational persistency were less likely to have conceived in one or two breedings than cows with a lactational persistency lower than or equal to the median. Dairy of origin had a significant effect on the probability of conceiving in one or two breedings. Cows in the first lactation were more likely than those in subsequent lactations to conceive in one or two breedings. This retrospective study demonstrated that subfertility is associated with high peak lactational yields in high milk-producing California cows.     

Effective human defense against E. histolytica: high amoebicidal activity of lymphocytes and monocytes in amoebic liver abscess patients until 3 months follow-up

Adherence of pathogenic Entamoeba histolytica trophozoites mediated by Gal/GalNAc lectin is a prerequisite for killing na?ve T cells and monocytes but the activated T cells and monocyte derived macrophages (MDMs) not only resist the attack but can kill the parasite. In the present study, we have analysed the adherence and cytotoxicity of the immunecompetent cells from patients of amoebic liver abscess at the time of their diagnosis and after 3 months to elucidate the development of cell mediated cytotoxicity, a major mechanism of resistance to amoebic infection. The results show that CD3+ cells from amoebic liver abscess cases, when stimulated, in vitro, bound E. histolytica trophozoites with increased intensity and their viability was also increased. The activated lymphocytes (taken at 3 months post treatment) were also able to kill amoebae. MDMs bound amoebae with greater intensity than lymphocytes, until 3 months post infection. These MDMs were effective in killing approximately 40% amoebae which was significantly less than at the time of diagnosis but was very significant as compared to the controls. The data suggest that cell mediated cytotoxic responses are maximum until 1 month post treatment and are significantly reduced thereafter.     

Relationship of body condition score and blood urea and ammonia to pregnancy in Italian Mediterranean buffaloes

The relationship of body condition score (BCS) and blood urea and ammonia to pregnancy outcome was examined in Italian Mediterranean Buffalo cows mated by AI. The study was conducted on 150 buffaloes at 145 +/- 83 days in milk that were fed a diet comprising 14.8% crude protein, 0.9 milk forage units.kg-1 dry matter and a non-structural carbohydrate/crude protein ratio of 2.14. The stage of the oestrous cycle was synchronised by the Ovsynch-TAI programme and blood urea and ammonia levels were assessed on the day of AI. Energy corrected milk (ECM) production and BCS were recorded bi-weekly. The pregnancy risk was 46.7% and was slightly lower in buffaloes with BCS < 6.0 and BCS > 7.5. There were no significant differences in ECM, urea and ammonia between pregnant and non-pregnant buffaloes. However, pregnancy outcome was higher (P = 0.02) in buffaloes with blood urea < 6.83 mmol.L-1. The likelihood of pregnancy for buffaloes with low urea blood level was 2.6 greater than for high urea level and exposure to a high urea level lowered the probability of pregnancy by about 0.25. The findings indicate that buffaloes are similar to cattle and increased blood levels of urea are associated with reduced fertility when animals are mated by AI.     

Stable association of activated pp60src with two tyrosine-phosphorylated cellular proteins.

We have identified two phosphotyrosine-containing cellular proteins with relative molecular masses of 130,000 (pp130) and 110,000 (pp110) daltons in chicken embryo cells that coimmunoprecipitated with pp60v-src and activated forms of chicken pp60c-src (pp60(527)F). Most if not all of the tyrosine-phosphorylated forms of pp130 and pp110 could be immunoprecipitated from lysates with any of several src protein-specific monoclonal antibodies directed against at least three spatially distinct epitopes. Consequently, of the more than 15 prominent phosphoproteins detected on immunoblots with phosphotyrosine-specific antibodies, pp130 and pp110 were selectively removed by src protein-specific immunoprecipitation, and their presence in the immunoprecipitates appears to have been due to a direct interaction with activated src proteins. src protein variants that induce different morphological phenotypes were altered in their ability to form detergent-stable complexes with pp130 and pp110 or with pp110 alone. Mutant src proteins, defective for myristylation, showed increased tyrosine phosphorylation of and association with pp110. Expression of src variants with mutations in the A box (pp60dl92/527F) or B box (pp60dl155/527F) of the src homology region induced differences in phosphorylation of pp130 and pp110, as well as changes in their association with variant src proteins. Sequences within the B-box region appeared to be necessary for stable complex formation with pp130 and pp110 and may be involved in the interaction of activated src proteins with cellular substrates.     

Tyrosine phosphoproteomics of fibroblast growth factor signaling: a role for insulin receptor substrate-4

Signal transduction by receptor tyrosine kinases is initiated by recruitment of a variety of signaling proteins to tyrosine-phosphorylated motifs in the activated receptors. Several signaling pathways are thus activated in parallel, the combination of which decides the cellular response. Here, we present a dual strategy for extensive mapping of tyrosine-phosphorylated proteins and probing of signal-dependent protein interactions of a signaling cascade. The approach relies on labeling of cells with "heavy" and "light" isotopic forms of Arg to distinguish two cell populations. First, tyrosine-phosphorylated proteins from stimulated ("heavy"-labeled) and control samples ("normal"-labeled) are isolated and subjected to high sensitivity Fourier transform ion cyclotron resonance mass spectrometry analysis. Next, phosphopeptides corresponding to tyrosine phosphorylation sites identified during the tyrosine phosphoproteomic analysis are used as baits to isolate phosphospecific protein binding partners, which are subsequently identified by mass spectrometry. We used this approach to identify 28 components of the signaling cascade induced by stimulation with the basic fibroblast growth factor. Insulin receptor substrate-4 was identified as a novel candidate in fibroblast growth factor receptor signaling, and we defined phosphorylation-dependent interactions with other components, such as adaptor protein Grb2, of the signaling cascade. Finally, we present evidence for a complex containing insulin receptor substrate-4 and ShcA in signaling by the fibroblast growth factor receptor.     

Tyrosine phosphorylation of the alpha subunit of transducin and its association with Src in photoreceptor rod outer segments

Recent evidence indicates that tyrosine phosphorylation may play important roles in retinal photoreceptor rod outer segments (ROS). We investigated the tyrosine phosphorylation of endogenous proteins in isolated bovine ROS. Several proteins with apparent molecular masses of 31, 39, 60, 83, 90, 97, 120, 140, and 180 kDa were tyrosine-phosphorylated in ROS incubated with Mg(2+), ATP, and orthovanadate. Several tyrosine kinase inhibitors significantly inhibited tyrosine phosphorylation of these proteins in ROS. The 39- and 60-kDa tyrosine-phosphorylated proteins were identified as the alpha subunit of the G protein transducin (Talpha) and the tyrosine kinase Src, respectively. The presence of Src and tyrosine kinase activity in bovine ROS was confirmed by their cofractionation with rhodopsin and Talpha on continuous sucrose gradients. Several tyrosine-phosphorylated proteins, including Src, coimmunoprecipitated with Talpha. The association of Src with Talpha was detected in the absence of tyrosine phosphorylation, but was enhanced with increased tyrosine phosphorylation of ROS. Moreover, tyrosine kinase activity also associated with Talpha was sevenfold higher under tyrosine-phosphorylating conditions. The recovery of transducin by hypotonic GTP extraction from tyrosine-phosphorylated ROS was significantly less than that from nonphosphorylated ROS. We localized the site on Talpha phosphorylated by Src to the amino-terminal half by limited tryptic digests, and further mapped it by ion trap mass spectrometry to Tyr(142) in the helical domain of Talpha. Talpha was also tyrosine-phosphorylated in vivo in rat retina, but this phosphorylation was not affected by light.     

Effect of feeding protected fat and proteins on milk production,composition and nutrient utilization in Murrah buffaloes (Bubalus bubalis)

Objective of this study was to investigate the effect of feeding protected fat and proteins on milk production, composition and nutrient utilization in Murrah buffaloes (Bubalus bubalis). Eighteen buffaloes were divided into two groups (9 each) on the basis of most probable production ability. Buffaloes in control group (C group; most probable production ability 2204 kg) were fed chaffed wheat straw, chopped maize fodder and concentrate mixture as per requirements. Buffaloes in supplemented group (S group; most probable production ability 2211 kg) were fed same ration as C group plus 2.5% rumen protected fat (on dry matter intake basis) and formaldehyde treated mustard and groundnut oil cake (1.2 g formaldehyde/100 g crude protein) in place of unprotected cakes. Group S buffaloes were supplemented rumen protected fat and protein 60 days pre-partum to 90 days postpartum and persistence of milk production was monitored up to 210 days of lactation. Milk yield during supplementation period (90 days) in S group was 13.11 kg/d and was 19% higher (P<0.01) than the C group (11.01 kg/d), whereas after supplement withdrawal (120 days), it was 11.04 kg/d and was 15% higher (P<0.01) than the C group (9.61 kg/d). There was no effect on total solid, protein, solid-not fat (SNF) and lactose contents in the two groups, whereas milk fat yield was increased (P<0.05) and level of milk urea nitrogen was decreased (P<0.01) in S group. Moreover, the supplement produced noticeable changes in the fatty acid profile of the milk fat, i.e., reduction in the concentration of saturated fatty acids (SFA) by 19% and an increase in that of unsaturated fatty acids (USFA) by 36%. Besides, digestibility of dry matter, crude protein, acid detergent fiber and neutral detergent fiber were not affected, whereas ether extract digestibility was higher (P<0.05) in S group. There was no effect on plasma glucose, non-esterified fatty acids, triglycerides and cholesterol concentrations between two groups, whereas blood urea nitrogen concentration was lower (P<0.01) in S group. Supplementation of protected nutrients to buffaloes increased milk production and unsaturated fatty acids content in milk fat and persistence of lactation after supplements were withdrawn.     

Analysis of culling probability in dairy buffalo using survival models

In order to contribute to the genetic breeding programs of buffaloes, this study aimed to determine the influence of environmental effects on the stayability (ST) of dairy female Murrah buffalo in the herd. Data from 1016 buffaloes were used. ST was defined as the ability of the female to remain in the herd for 1, 2, 3, 4, 5 or 6 years after the first calving. Environmental effects were studied by survival analysis, adjusted to the fixed effects of farm, year and season of birth, class of first-lactation milk yield and age at first calving. The data were analyzed using the LIFEREG procedure of the SAS program that fits parametric models to failure time data (culling or ST = 0), and estimates parameters by maximum likelihood estimation. Breeding farm, year of birth and first-lactation milk yield significantly influenced (P < 0.0001) the ST to the specific ages (1 to 6 years after the first calving). Buffaloes that were older at first calving presented higher probabilities of being culled 1 year after the first calving, without any effect on culling at older ages. Buffaloes with a higher milk yield at first calving presented a lower culling probability and remained for a longer period of time in the herd. The effects of breeding farm, year of birth and first-lactation milk yield should be included in models used for the analysis of ST in buffaloes.     

Milk progesterone levels in crossbred buffaloes (swamp x murrah) and a comparison to the original breeds

Progesterone levels in milk fat and whole milk were determined in eight crossbred buffaloes (swamp x murrah). Progesterone peaks in milk fat and whole milk during the luteal phase ranged from 44.23 to 224.50 ng/ml and 8.58 to 20.22 ng/ml, respectively. Progesterone levels were measured in two murrah cows and seven swamp cows for comparison with the levels in the crossbred buffaloes. Variation in milk progesterone levels between breeds was indicated and discussed. Radioimmunoassay for progesterone in milk fat and whole milk of buffaloes was validated.     

Identification of a candidate integrin-fraction associated with the activated form of the PDGF-receptor

The activated PDGF-receptor has been shown to coimmunoprecipitate together with alpha v beta 3 integrin out of the 15,000g soluble supernatant of non-ionic detergent cell lysates. I have now further characterized this complex by ultracentrifugation analysis. The ultracentrifugation-conditions were chosen so that the phosphorylated form of the PDGF-receptor was pelleted out of the 15,000g soluble supernatant. Together with the tyrosine-phosphorylated PDGF-receptor small amounts of integrins, cytoskeletal- and extracellular matrix proteins were recovered in the pelleted material. The results show that (i) the candidate-fraction of integrins interacting with the activated PDGF-receptor is small compared to the overall integrin population in the cell lysate, (ii) several proteins known to be present in focal adhesions such as FAK, talin, and vinculin are absent from the integrin-growth factor receptor complexes, while on the other hand (iii) a tyrosine-phosphorylated protein migrating at 120 kDa was highly enriched in the ultracentrifugation-pellet, and finally (iv) non-ionic detergent cell lysates appear to contain quantitatively small fractions of complexed proteins that are qualitatively distinct from their total cellular population. Thus, the separation of protein-complexes from the total cellular proteom may be instrumental for the investigation of cellular protein complexes in general.     

Comparison of non-linear models to describe the lactation curves for milk yield and composition in buffaloes (Bubalus bubalis)

In order to describe the lactation curves of milk yield (MY) and composition in buffaloes, seven non-linear mathematical equations (Wood, Dhanoa, Sikka, Nelder, Brody, Dijkstra and Rook) were used. Data were 116 117 test-day records for MY, fat (FP) and protein (PP) percentages of milk from the first three lactations of buffaloes which were collected from 893 herds in the period from 1992 to 2012 by the Animal Breeding Center of Iran. Each model was fitted to monthly production records of dairy buffaloes using the NLIN and MODEL procedures in SAS and the parameters were estimated. The models were tested for goodness of fit using adjusted coefficient of determination root means square error (RMSE), Durbin–Watson statistic and Akaike’s information criterion (AIC). The Dijkstra model provided the best fit of MY and PP of milk for the first three parities of buffaloes due to the lower values of RMSE and AIC than other models. For the first-parity buffaloes, Sikka and Brody models provided the best fit of FP, but for the second- and third-parity buffaloes, Sikka model and Brody equation provided the best fit of lactation curve for FP, respectively. The results of this study showed that the Wood and Dhanoa equations were able to estimate the time to the peak MY more accurately than the other equations. In addition, Nelder and Dijkstra equations were able to estimate the peak time at second and third parities more accurately than other equations, respectively. Brody function provided more accurate predictions of peak MY over the first three parities of buffaloes. There was generally a positive relationship between 305-day MY and persistency measures and also between peak yield and 305-day MY, calculated by different models, within each lactation in the current study. Overall, evaluation of the different equations used in the current study indicated the potential of the non-linear models for fitting monthly productive records of buffaloes.     

Differential expression of immune response genes associated with subclinical mastitis in dairy buffaloes

Buffalo milk production has become of significant importance on the world scale, however, there are few studies involving biotechnological tools specifically for buffalo. To verify the effects caused by subclinical mastitis on the components of milk and to study the innate immune system in the udder of dairy buffaloes with subclinical mastitis, we evaluated the levels of expression of the lactoferrin (LTF), tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-8 (IL-8), and toll-like receptors 2 (TLR-2) and 4 (TLR-4) genes in buffaloes with and without subclinical mastitis. Milk samples were collected for the determination of milk components: somatic cell score (SCS), fat, protein, lactose, total solids and solids-not-fat (SNF), as well as for RNA extraction of milk cells, complementary DNA synthesis, and expression profile quantification by quantitative real-time PCR. For gene expression, the ΔΔCt was estimated using contrasts of the target genes expression adjusted for the expression of the housekeeping genes between both groups. Linear regression analysis was performed to determine the relationship between the genes studied and the milk components. Subclinical mastitis induced changes in the fat, lactose and SNF in milk of buffaloes, and the messenger RNA abundance was upregulated for TLR-2, TLR-4, TNF-α, IL-1β and IL-8 genes in milk cells of buffaloes with subclinical mastitis, whereas the LTF gene was not differentially expressed. Results of linear regression analysis showed that TLR-2 gene expression most explains the variation in SCS, and the change in a unit of ΔCt of the TNF-α gene would result in a higher increase in SCS. The study of these immune function genes that are active in the mammary gland is important to characterize the action mechanism of the innate immunity that occurs in subclinical mastitis in dairy buffaloes and may aid the development of strategies to preserve the health of the udder.     

Interaction of calf suckling, use of oxytocin and milk yield with reproductive performance of dairy buffaloes

Calf suckling and oxytocin injections are commonly used for pre-milking stimulus in dairy buffaloes under field conditions. A study was conducted to investigate effect of these treatments on reproductive performance. Fifty one Nili-Ravi buffaloes were monitored from parturition up to 150 days postpartum through rectal examination. Data on milk yield, body condition score (BCS) and reproductive parameters were recorded weekly. Postpartum ovulation interval (POI) was determined by presence of an ovulation depression or a very soft corpus luteum haemorrhagicum and was confirmed through milk progesterone levels (MPL). Suckling was used to stimulate milk let down, and where the calf had died, injection of oxytocin was resorted to. Milk samples were analyzed for MPL using radioimmunoassay (RIA) and fat; and milk yield was converted to 4% fat corrected milk (FCM). The mean postpartum uterine involution length (PUI) was 34.30 ± 1.33 days. Mean POI was 59.37 ± 4.76 days and mean postpartum estrus interval (PEI) was 69.03 ± 6.03 days. Suckling period averaged 26.40 ± 5.57 days and correlated with POI (r = 0.19, P < 0.01) and PEI (r = 0.23, P < 0.01). POI was shortest in buffaloes suckled for one month (P < 0.05). Oxytocin was used with a mean dosage of 7.50 IU, delaying placental expulsion time (PET) and POI but shortening PEI. BCS shortened PET, POI and PEI (P < 0.01). Mean FCM was 14.50 ± 0.20, ranging from 2 to 35 kg/d; and was higher in estrus group; correlating positively with POI (r = 0.31, P < 0.01). MPL were 1.37 ± 0.17 ng/ml and increased after ovulation, remaining greater than 1.5 ng/ml from Day 4 to 14 of the estrus cycle, followed by a rapid decline up to next estrus. BCS in buffaloes resuming oestrus was constantly higher than those failing to resume ovarian cyclicity. Live weight, prepartum was 510.0 ± 5.9 kg with a loss of 3.7 ± 2.12 kg, 30 days postpartum. The present study suggests a lower reproductive efficiency of dairy buffaloes under the peri-urban farming system reflected by ovarian cyclicity in 68.63% buffaloes within 150 days postpartum and silent estrus in 51.5% of the cases. Increasing suckling duration and use of oxytocin delayed POI, however, POI was shortest in buffaloes suckled for one month. The high yielding buffaloes also manifested better reproductive cyclicity; while moderate yielder showed shorter ovulation intervals and higher conception rate.     

Differential protein phosphorylation in human gastric adenocarcinomas.

A qualitative analysis of endogenous protein phosphorylation in microsomal fractions from surgical specimens of human gastric cancer, benign gastric ulcers and normal gastric tissues is presented. Fractions were incubated in the presence of (gamma-32P)ATP to measure the transfer of (gamma-32P) to natural substrates mediated by endogenous protein kinases. Phosphoproteins were characterized through PAGE-SDS and detected by autoradiography. KOH at high temperature was used to select for tyrosine-phosphorylated polypeptides on dried gels. We report a notorious enhancement in overall protein phosphorylation in gastric cancer samples over benign ulcers and normal controls as well. Moreover, a highly basic-low molecular weight phosphoprotein is found through 2-D protein gel analysis and a 50 kDa protein is detected only in the presence of Mg2+ after KOH treatment. These two proteins might become putative molecular markers to detect this type of neoplasia.     

Evaluation of type traits in relation to production,and their importance in early selection for milk performance in dairy buffaloes

Type traits (TTs) can contribute to breeding animals with good economic traits such as production, longevity, fertility, and profitability. Dairy buffaloes are the second largest source of milk supply in the world, and their TTs should be taken into consideration in future dairy buffalo breeding programmes. However, the relationship between TTs and milk production traits in buffalo remains largely unknown. The study aimed to establish an early selection method for buffaloes with desirable milk performance by TTs. Using 1 908 records from 678 buffaloes, the relationship between TTs and milk production traits was analysed and the optimal growth curves of TTs related to milk production traits were constructed. We examined the correlations between 45 TTs (33 body structural, 12 udder and teat morphological traits) and three milk production traits (milk yield (MY), milk fat percentage (MF), and milk protein percentage (MP)). The results showed that the highest correlation was found between MY and udder circumference (r = 0.438), teat length (r = ?0.380) or heart girth (r = ?0.341). The teat distance and teat circumference exhibited a significant negative correlation with MF and MP. Rump length was the only trait that had a significant positive correlation with milk production traits, suggesting that milk performance could be comprehensively improved by including rump length in the selection procedure. Notably, we found that high milk production traits was obtained from the buffaloes with short teats (<6 cm), small heart girth (<200 cm), large udder circumference (>104 cm), long rump (>39 cm), and small distance between teats. Moreover, an early selection method for buffaloes with excellent milk performance was developed based on the non-linear models. Brody model exhibited the best fitting effect for heart girth and rump length, while the Logistic model displayed the best fitting effect for teat length. Our findings provide theoretical basis for the early selection of buffaloes with desirable milk performance.     

Separation and Characterization of the Activated Pool of Colony-Stimulating Factor 1 Receptor Forming Distinct Multimeric Complexes with Signalling Molecules in Macrophages

Colony-stimulating factor 1 (CSF-1) triggers the activation of intracellular proteins in macrophages through selective assembly of signalling complexes. The separation of multimeric complexes of the CSF-1 receptor (CSF-1R) by anion-exchange chromatography enabled the enrichment of low-stoichiometry complexes. A significant proportion of the receptor in CSF-1-stimulated cells that neither possessed detectable tyrosine kinase activity nor formed complexes was separated from the receptor pool displaying autokinase activity that formed chromatographically distinct multimeric complexes. A small pool of CSF-1R formed a multimeric complex with phosphatidylinositol-3 kinase (PI-3 kinase), SHP-1, Grb2, Shc, c-Src, Cbl, and a significant number of tyrosine-phosphorylated proteins in CSF-1-stimulated cells. The complex showed a considerable amount of CSF-1R complex-associated kinase activity. A detectable level of the complex was also present in untreated cells. PI-3 kinase in the multimeric complex displayed low lipid kinase activity despite the association with several proteins. The major pool of activated CSF-1R formed transient multimeric complexes with distinctly different tyrosine-phosphorylated proteins, which included STAT3 but also PI-3 kinase, Shc, SHP-1, and Grb2. A significant level of lipid kinase activity was detected in PI-3 kinase in the latter complexes. The different specific enzyme activities of PI-3 kinase in these complexes support the notion that the activity of PI-3 kinase is modulated by its association with CSF-1R and other associated cellular proteins. Specific structural proteins associated with the separate CSF-1R multimeric complexes upon CSF-1 stimulation and the presence of the distinct pools of the CSF-1R were dependent on the integrity of the microtubular network.     

Identification of a tyrosine-phosphorylated CCCTC-binding nuclear factor in capacitated mouse spermatozoa

The molecular basis of mammalian sperm capacitation, either in vivo in the female reproductive tract, or in vitro, is poorly understood. It is well known that sperm capacitation is associated with an increase in tyrosine phosphorylation of a subset of proteins. We resolved the phosphoproteins in the cell lysate of mouse sperm after capacitation by 2-DE. One tyrosine-phosphorylated 130-kDa spot was trypsin-digested, and six oligopeptide sequences were established from the MS data. These were confirmed in a CCCTC-binding nuclear factor (CTCF), a widely expressed and highly conserved protein. Further, both an anti-phosphotyrosine antibody and an anti-CTCF antibody showed immunoreactivity to a 130-kDa component in the immunoprecipitates obtained after incubation of the cell lysate from the capacitated sperm using another anti-CTCF antibody. The data support the presence of a tyrosine-phosphorylated CTCF in the capacitated sperm. Immunolocalization of the CTCF revealed fluorescent staining in the acrosome region in both capacitated and incapacitated sperm. The electrophoretic mobility shift assay, using a CTCF target sequence 5'-GGCGGCGCCGCTAGGGGTCTCTCT-3' found in the promoter of the amyloid beta-protein precursor, manifested that, relative to CTCF in the incapacitated sperm, the tyrosine-phosphorylated protein in the capacitated sperm had stronger affinity to the CTCF target sequence.