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To examine retinal nerve fiber layer (RNFL) thickness, macular volumes (MV), and visual acuity in multiple sclerosis (MS) eyes, with and without history of acute optic neuritis (ON).

Optical coherence tomography (OCT) noninvasively quantifies retinal nerve fiber layer (RNFL) thickness. Studies show RNFL thinning in multiple sclerosis (MS), and we assessed its association with brain atrophy.

Bovine mastitis caused by Streptococcus agalactiaeis mainly subclinical and therefore can be diagnosed only in the laboratory. We developed a polymerase chain reaction (PCR)-based method for specific and sensitive detection of S. agalactiaein raw milk. The specificity of the PCR reaction is based on unique S. agalactiaeDNA sequences within the 16S subunit of the rRNA genes. Two pairs of sequences were used as positive controls; general streptococci primers, which anneal to conserved areas within the 16S rRNA subunit gene, and primers, which anneal to sequences within bovine mitochondrial DNA. The method of detection includes selective enrichment of S. agalactiaein the milk sample, followed by DNA extraction using a rapid and simple procedure developed for this purpose, and specific PCR reaction with appropriate controls. The method enables the detection of one bacterium in 1ml of raw milk. The method developed can be easily incorporated as part of routine screening of bulk milk collection tanks for early detection of infected cows in a herd.

The association of cell chimerism with the freemartin condition, and of the absence of cell chimerism with normal female development, was confirmed by PCR amplification of a Y-chromosome fragment in blood leukocytes and hair roots of cattle females co-twin to males.

Five proteins from human placenta capable of inhibiting pancreatic phospholipase A2 were purified. Two of these proteins were identified as lipocortins I and II. The other three proteins were immunologically distinct from lipocortins I and II and had apparent subunit molecular masses of 32, 33, and 73 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid sequence analysis of peptides produced by cyanogen bromide digestion indicated sequence homology of these proteins with lipocortin I and the heavy chain subunit of lipocortin II. Two of these proteins were identified as endonexin II and 67-kDa calelectrin. The third protein appears to be the human form of bovine endonexin I, also characterized as porcine protein II. Sedimentation equilibrium analysis of lipocortin I, endonexin I and II, and the 67-kDa calelectrin suggested monomer-dimer equilibria with dissociation constants in the range of 0.33-1.3 X 10(-3) M and monomer molecular masses of 38,050, 36,400, 36,850, and 73,610 Da, respectively. Self-association of lipocortin II was described by dimerization of a protomer (K12 = 5.3 x 10(-7) M), followed by an indefinite self-association of the dimer (isodesmic dissociation constant, Kiso = 3.6 x 10(-6) M). The protomer molecular mass was 48,800 Da, consistent with a heterodimeric structure composed of one heavy (38,600 Da) and one light (10,944 Da) chain as previously characterized for lipocortin II. Sedimentation equilibrium analysis of mixtures of individual protein inhibitors and purified pancreatic phospholipase A2 indicated weak association between enzyme and inhibitor (Kd greater than or equal to 3 x 10(-5) M), insufficient to account for the observed inhibition of enzyme activity.

The present study was undertaken to evaluate quantitatively the turnover of serum triacylglycerol (triglyceride) in the starved rat and to determine whether serum triacylglycerol recycled to liver contributes a significant fraction of the total hepatic triacylglycerol turnover. Serum was labelled in vitro with [3H]trioleoylglycerol (glycerol [3H]trioleate) to provide uniform labelling of all lipoprotein species. By using the curves describing disappearance of isotope from serum and its appearance in liver, rate constants for movement of triacylglycerol out of serum (0.29 min-1) and the uptake of serum triacylglycerol by liver (0.22 min-1) were calculated. The total rate of movement (flux) of triacylglycerol in these processes, the product of rate constant and serum pool size, was calculated to be 0.39 and 0.29 mg/min per 100 g body wt. respectively. A model is postulated for whole-body triacylglycerol metabolism consistent with the present data as well as most observations in the literature. From the model it can be predicted that: (1) the entire turnover of liver triacylglycerol in the starved rat can be accounted for on the basis of contributions from serum non-esterified fatty acid and serum triacylglycerol; (2) the entire turnover of the serum triacylglycerol pool can be accounted for quantitatively on the basis of contributions from intestine and liver; (3) the release rate for triacylglycerol from liver should be 0.34 to 0.35 mg/min per 100 g body wt.; (4) triacylglycerol synthesized by liver from non-esterified fatty acid of serum and by intestine can account quantitatively for the irreversible disposal rate of triacylglycerol from serum.

The kinetics of insulin binding to isolated rat epididymal fat cells was investigated at 15 degrees C, at which temperature the system was simplified by the absence of lysosomal insulin degradation. The data were fit by maximum likelihood criteria with differential equations describing a number of models for the interaction of insulin and cells. Among those models that yielded a fit, the selection criteria were minimization of the Akaike information criterion and compatibility of the overall equilibrium constant for the system calculated from rate constants with the previously obtained experimental value. The results of the analysis indicated that insulin, I, first reversibly bound to cell surface receptors, R, whereupon this initial insulin-receptor complex, RI, reversibly altered its state or cellular location to R'I, according to the following equation. (Formula: see text) No evidence was found that insulin could either associate or dissociate from R'I directly. The association rate constant was kappa 12 = 1.6 x/divided by 1.4 X 10(5) liter mol-1 s-1, a value shown to be incompatible with diffusion control. The other rate constants were: kappa 21 = 3.4 x/divided by 1.6 X 10(-3) s-1, kappa 23 = 3.2 x/divided by 1.5 X 10(-4) s-1, and kappa 32 = 2.0 x/divided by 1.5 X 10(-4) s-1. From these rate constants, an equilibrium constant of 8.4 x/divided by 1.5 nM was calculated, in excellent agreement with the previously measured value of 8.8 x/divided by 1.3 nM (Lipkin, E. W., Teller, D. C., and de Haën, C. (1986) J. Biol. Chem. 260, 1694-1701). The kinetic analysis also yielded receptor numbers similar to those obtained by equilibrium binding studies. The nature of the R'I state is discussed in terms of an internalized state, in terms of insulin receptor complex in caveolae, in terms of receptor aggregates, and in terms of being a Michaelis complex between insulin bound to the receptor and cell surface-bound insulin protease.

Equilibrium binding of insulin to isolated rat epididymal fat cells was investigated. A temperature of 15 degrees C was chosen for the study to minimize lysosomal degradation of insulin. Indeed, medium insulin lost only 1% of its precipitability in trichloroacetic acid during the 4-h incubation required to approach equilibrium. Binding was measured by a method that did not perturb the equilibrium of the system. A new formalism for analyzing binding data in general was introduced. A correction for trapping of insulin in the interstitial space of cell pellets was both necessary and sufficient to derive specific binding data from raw observations. Thus, so-called “nonspecific binding” was unmasked as a misnomer, and the expression “correction for trapping” was proposed as a substitute. Equations for one and two independent classes of binding sites were fit to the data by the method of maximum likelihood, and the best fit was selected based on Akaike's information criterion, as adapted for a constant fractional error. More than 99.7% of the binding sites were found to be describable by a simple binding isotherm with Kd,app = 8.8 multiplied by over divided by 1.3 nM. Less than 0.3% sites had a higher affinity (Kd approximately equal to 8 multiplied by over divided by 3 pM). There were 99,000 x/divided by 1.6 binding sites/cell. These equilibrium parameters are in agreement with values derived from a kinetic analysis, presented in the subsequent paper (Lipkin, E. W., Teller, D. C., and de Haën, C. (1986) J. Biol. Chem. 260, 1702-1711).

We compared a fluorescein-conjugated monoclonal antibody (FA) direct specimen test (MicroTrak; Syva Co., Palo Alto, Calif.) with culture (TC) in McCoy cells (vials, with blind passage and iodine staining of inclusions) for diagnosis of Chlamydia trachomatis infection in the cervix. Duplicate specimens were collected from 1,230 women, but for 262 of these subjects, both results were unavailable (150 FA smears were inadequate, indicating a need for clinical training in specimen collection), leaving 968 comparisons. Prevalence of chlamydiae by culture was 13% (126/968). Compared with TC results, the sensitivity of FA was 70% (88/126) and the specificity was 94% (795/842). There was a 91% agreement (883/968). The predictive value of a positive FA test was 65% (88/135), and that of a negative FA was 95% (795/833). We reexamined 38 smears for which paired results were discrepant, and the reread would have changed the result in only 5 of these. TC is less than 100% sensitive and some FA-positive, TC-negative specimens represent positive specimens not detected by TC. Unfortunately, it is not possible to identify which results in this group are truly false-positive. Clearly, the FA procedure has a performance profile which would make it a useful tool in screening high-risk populations (particularly when TC is not available) but it is less suited to screening low-risk populations, for which false-positive results are more important. The greater utility of the FA procedure in a venereal disease clinic was confirmed by testing 172 evaluable specimen pairs, of which 34 (20%) were Chlamydia isolate positive. The FA sensitivity was 76% (26/34) and specificity was 96% (133/138), giving a predictive value of 84% (26/31) for a positive test.

Insulin binding to isolated rat white adipocytes at 15 degrees C, a temperature at which cellular degradation of insulin is negligible, has been found to be described by the Two-step Binding Model: R + I in equilibrium RI in equilibrium R′I (Lipkin, E. W., Teller, D. C., and de Haën, C. (1986) J. Biol. Chem. 261, 1702-1711). RI is the initially formed complex between the receptor, R, and insulin, I, and R′I is the complex in an altered state or cellular location. Here the possibility was examined that R′I results from disulfide exchange between the receptor and insulin, an exchange proposed by Clark and Harrison (Clarke, S., and Harrison, L. C. (1986) J. Biol. Chem. 257, 12239-12244) to occur at 37 degrees C. A number of sulfhydryl reagents representing various chemical reactivities did not affect insulin binding. The exception was 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), which enhanced the number of insulin-binding sites up to 2-fold with no effect on the equilibrium constant. The data suggested that this enhancement was due to activation of cryptic binding sites pre-existing on the cell surface, possibly by increasing the valency of the receptor from 1 to 2. Insulin binding was also enhanced by structural congeners of DTNB devoid of sulfhydryl reactivity, the simplest one being benzoic acid. It was concluded that the effects were not related to modification of sulfhydryl groups, that modification of sulfhydryl groups on the receptor either did not take place or was without effect on binding, and finally, that disulfide exchange between insulin and the receptor was an unlikely explanation for the formation of R′I. Also, since it is possible to show insulin action at 15 degrees C, contrary to the proposal by Clark and Harrison (Clark, S., and Harrison, L. C. (1983) J. Biol. Chem. 258, 11434-11437), disulfide exchange does not appear to be necessary for signal transmission by the occupied receptor.

AbstractA selective DNA pooling approach using milk samples was employed to map QTL affecting milk somatic cells count (MSCC) in the Italian Brown Swiss dairy cattle population. The mapping population consisted of five half sib daughter families of Brown Swiss bulls, sires of 1000 to 3600 daughters. Two hundred highest and 200 lowest daughters, ranked by dam-corrected EBV, were selected from the high and the low tail. Four independent replicate pools, each made of 50 randomly chosen daughters, were prepared for each sire-tail combination. Dinucleotide microsatellite markers were used to scan the genome. Sire marker allele frequencies were estimated by densitometry and shadow correction analysis. Significance threshold of 10% aFDR was used at the marker level, and resulted in a critical CWER P-value of 0.054. A threshold of 20% aFDR within the significant markers was used at the sire-marker level and resulted in a critical P-values of 0.058. Out of 145 markers, 41 were significant. Out of 122 sire-marker tests, at the significant markers, 58 resulted significant. QTL regions will be selected for further intensive study. This is the first complete genome scan for MSCC in the Brown Swiss breed.

AbstractBovine chromosome 20 (BTA20) was studied to identify QTL for milk yield and protein percentage in the Italian Holstein-Friesian breed using a selective milk DNA pooling strategy in a daughter design with sire haplotype analysis. Several QTL were identified. The effect of the known GHR F279Yand PRLR S18Nmutations were in for the most part confirmed. However, it was also shown that these markers cannot explain all significant effects observed on BTA20 for the investigated traits.

The Baladi is a native Bos taurusbreed of the Mediterranean basin known for its hardiness, disease resistance, tolerance of poor care, meager diet and adverse climate conditions. In Israel, and probably in other Mediterranean countries, the Baladi breed is in danger of extinction due to the introduction of larger more productive European breeds. Domestication of cattle occurred independently in two or three sites: Southwest Asia (Bos taurus), Southeast Asia (Bos indicus), and possibly North Africa (Bos taurus). In this study, global admixture analysis of pure Baladi and 15 other cattle breeds around the world were used to investigate the ancestral components of the Baladi genome. The Baladi was revealed as a unique combination of the three main postulated ancestral cattle domestications, having each component in roughly equal proportions, and thus represents an enormous store of cattle genetic diversity. Local admixture analysis exposed regions characterized by positive deviation for the Indicine and African Taurine genome components of the Baladi genome and mostly negative selection against the Southwest Taurine component. Thus, these regions may harbor genomic elements increasing the Baladi local adaptation. The Baladi share similar global and local admixture profiles with three Turkish breeds. Thus, all four breeds may be considered in large part as independent replicates taken from the same Middle East ancestral mix. This brings enormous potential for high-resolution validated mapping of chromosomal regions contributing to adaptation. Thus, from both a practical and preserving biodiversity views, the Baladi is a prime candidate for conservation.

Bovine respiratory disease (BRD), caused by multiple pathogens that become more virulent in response to stress, is the leading cause of morbidity and mortality in feedlot cattle. As various preventive strategies failed, marker or gene assisted selection for resistance becomes attractive. In the present study, selective DNA pooling was applied in a genome-wide association study to map BRD QTLs in Israeli Holstein male calves. Kosher scoring at the abattoir was used to allocate 122 and 62 animals to High and Low BRD resistant groups, respectively. Kosher scoring involves examination of the lungs for adhesions, a consequence of secondary bacterial infection in BRD, and hence a marker for individual history of BRD infections. Animals are graded Glatt (adhesions absent; assigned to High group), Kosher (moderate adhesions) and Treif (severe adhesions; assigned to Low group). All pools were genotyped by Illumina BovineHD BeadChip. Moving average of-logP was used to map QTLs and 1 Log drop was used to define QTL boundaries (QTLRs). The combined procedure was efficient for high resolution mapping. Nineteen QTLRs distributed over 13 autosomes were found, some overlapping previous studies. The QTLRs contain polymorphic functional and expression candidate genes, with putative immunological and wound healing activities that might affect kosher grade. Kosher grading was shown to be a low cost, easily collected phenotype for mapping QTLs affecting BRD morbidity.

Ecological and economic concerns drive the need to improve feed utilization by domestic animals. Residual Feed Intake (RFI) is one of the most acceptable measures for feed efficiency (FE). However, phenotyping RFI related traits is complex, expensive, and requires special equipment. Advances in marker technology allow the development of various DNA-based selection tools. To assimilate these technologies for the benefit of RFI-based selection, reliable phenotypic measures are prerequisite. In the current study, we used genomic DNA of individuals presenting RFI phenotypic consistency across different ages and diets (stages 1–3), for targeted sequencing of chromosomal regions associated with FE and RFI related traits. Forty-eight top single nucleotide polymorphisms (SNPs), significantly associated with at least one of three stages were identified. Eleven of these SNPs were harbored by the fatty acid binding protein 4 (FABP4). While ten significant SNPs found in FABP4, were common for stage 1 and stage 3, one SNP (FABP4_5; A < G substitution), in the promoter region of the gene, was significantly associated with all three stages. As the three stages reflect changing diets and ages with concomitant RFI phenotypic consistency, the above polymorphisms and in particular FABP4_5, might be considered possible markers for RFI-based selection for FE in the Holstein breed, following a larger scale validation.

AbstractMilk contains a number of micro-components (“functional foods”) having specific health promoting activities. Important among these compounds are the conjugated linoleic acids (CLAs) consistingof a mixture of isomers of C18:2 fatty acid with conjugated doublebonds. The predominant form in milk fat is the cis-9,trans-11isomer, accounting for 75 to 90% of the total CLA content. Biomedical studies with animal models have demonstrated a variety of preventive health effects from CLA including anticarcinogenic, antiatherogenic, antiobesity, immune system enhancement and antidiabetic benefits. Linolenic acid does not form CLA as an intermediate in rumen biohydrogenation, but it does form vaccenic acid (C18:1,t11) that enters the bloodstream from the rumen, through the abomasum and intestine, and is transported to the mammary gland. Several investigations using direct and indirect approaches established that endogenous synthesis of CLA via ∆9-desaturase action on vaccenic acid (VA, C18:1,t11) is the predominant source of the CLA found in milk fat across a range of diets. Thus, rumen VA production and mammary tissue ∆9-desaturase (D9D) are of key importance in determining the CLA content of milk fat. Mammary tissue D9D activity is measured indirectly as the milk desaturase index (DI), calculated as the ratio of milk CLA to the sum of milk CLA and VA. The study is centered on the Italian Brown Swiss and Israeli Holstein dairy cattle populations. Objectives of the project are: i) to identify environmental and physiological factors (e.g., herd, season, parity and stage of lactation) affecting milk CLA content and DI, and determine appropriate correction factors, if needed; ii) to estimate heritability of milk CLA content and DI in the study populations; iii) to implement a total genome scan for QTL affecting milk CLA VA content and DI in the two study populations. using a selective DNA pooling approach based on milk samples for the initial scan, followed by individual genotyping to confirm suggestive QTL regions. For each of the studied populations,, the research includes determination of CLA, VA and DI for more than 2500 individuals of five large sire half-sib families (for the genome scan) and an additional 400 individuals of 20 small sire half-sib families for the heritability analysis. Milk fat is extracted and transmethylated according to Chouinard et al.(1999). Fatty acid methyl esters are analyzed by GC-FID with a highly polar 100 m SP-2560 column, using GLC-60 supplemented with CLA and VA as a standard. Based on the results obtained to date for the Italian Brown Swiss population, the mean (fatty acid composition over the total fatty acid) ±SD( minimum and maximum) are respectively: VA, 1.97±0.77 (0.63 - 5.40); CLA, 0.63±.0.47 (0.35 - 5.31); DI, 0.25±0.097 (0.063 - 0.64). The means for VA and CLA content found are similar to those reported by other studies (control treatment) and reviewed by Collomb et al(2006). The slightly larger variability here reported, anyhow within the limit of values found in literature, is possibly affected by the wide farming conditions of the individuals sampled.