Molecular characterization, polymorphism and association study of lysozyme gene with milk production and somatic cell trait in Bos indicus × Bos taurus cattle

Lysozyme, an anti-bacterial enzyme, is mostly found in the body fluids, various tissues and secretions of animals and humans, and confers immunity against a wide range of bacterial species. The present study was carried out to elucidate the gene sequence of this enzyme in Indian Sahiwal × Holstein Friesian crossbred cattle and to explore the polymorphism of the gene as well as their association with milk production and somatic cell traits. The total length of lysozyme cDNA was found to be of 447 bp. The similarity with Bos taurus, human, pig, monkey, gorilla, mice, rat, chicken, dog and sheep was estimated as 99.1%, 85%, 81.0%, 85.2%, 84.3%, 77.9%, 77.9%, 41.4%, 40.5% and 24.6%, respectively. Polymorphism study of two fragments, extended exon1 including promoter, exon1 and partial intron1 (268 bp), and extended exon2 including partial intron1 and 2, and exon2 region (287 bp) of milk lysozyme gene was carried out by employing single-stranded conformation polymorphism (SSCP). In the extended exon1 fragment, three alleles namely A, B and C with frequencies of 0.59, 0.28 and 0.12 were observed while in the exon extended fragment, three different alleles - P, Q and R with respective frequencies of 0.61, 0.38 and 0.01 - were determined in Indian Sahiwal × Holstein Friesian cross. A total of eight haplotypes were found in this population where the most predominant one was h1 (0.52). Genotypes of exon1 extended fragment showed significant association with total milk yield, daily milk yield, peak yield and somatic cell score at P < 0.05 while that of exon2 extended fragment had significant correlation with only total lactational milk yield. Haplotype combinations also revealed significant association with total milk production where h1h1 homozygous showed highest yield during first lactation.     

Isolation and characteristics of the melanocortin 1 receptor gene (MC1R) in the Chinese yakow (Bos grunniens×Bos taurus)

The Chinese yakow is the offspring of yak (Bos grunniens) and Yellow cattle (Bos taurus). The melanocortin 1receptor gene (MC1R) plays a crucial role in determining coat colour of mammals. To investigate the relationship of polymorphism of the MC1R with coat colour in the Chinese yakow, the coding sequence (CDS) and the flanking region of MC1R were sequenced from 84 Chinese yakow samples and compared with the sequences of the MC1R from other bovid species. A fragment of 1134 base pair (bp) sequences including the full CDS (954bp) and parts of the 5'- and 3'-untranslated regions (162 and 18bp, respectively) of the Chineseyakow MC1R were obtained. A total of 13 single nucleotide polymorphisms (SNPs) including 4 SNPs (T-129C, A-127C, C-106T, G-1A) in the 5'-untranslated region and 9 SNPs (C201T, T206C, C340A, C375T, T663C, G714C, C870T, G871A and T890C) in the CDS were identified, revealing high genetic variability. Four novel SNPs including T206C, G714C, C870T and T890C, which have not been reported previously in bovid species, were retrieved. Within 9 coding SNPs, C201T, C375T, T663C and C870T were silent mutations, while T206C, C340A, G714C, G871A and T890C were mis-sense mutations, corresponding to amino acid changes p.L69P, p.Q114K, p.K238N, p.A291N and p.I297T, respectively. Amino acid sequences alignment showed a more than 96% similarity with other ruminates. However, three classical bovine MC1R loci the E(D), E(+) and e were not retrieved in the Chinese yakow, indicating other genes or factors could be involved in affecting coat colour in this species.     

Farm-level emission intensities of smallholder cattle (Bos indicus; B. indicus–B. taurus crosses) production systems in highlands and semi-arid regions

Ruminants are central to the economic and nutritional life of much of sub-Saharan Africa, but cattle are now blamed for having a disproportionately large negative environmental impact through emissions of greenhouse gas (GHG). However, the mechanism underlying excessive emissions occurring only on some farms is imperfectly understood. Reliable estimates of emissions themselves are frequently lacking due to a paucity of reliable data. Employing individual animal records obtained at regular farm visits, this study quantified farm-level emission intensities (EIs) of greenhouse gases of smallholder farms in three counties in Western Kenya. CP was chosen as the functional unit to capture the outputs of both milk and meat. The results showed that milk is responsible for 80–85% of total CP output. Farm EI ranged widely from 20 to >1 000 kg CO₂-eq/kg CP. Median EIs were 60 (Nandi), 71 (Bomet), and 90 (Nyando) kg CO₂-eq/kg. Although median EIs referenced to milk alone (2.3 kg CO₂-eq/kg milk) were almost twice that reported for Europe, up to 50% of farms had EIs comparable to the mean Pan-European EIs. Enteric methane (CH₄) contributed >95% of emissions and manure ～4%, with negligible emissions attributed to inputs to the production system. Collecting data from individual animals on smallholder farms enabled the demonstration of extremely heterogeneous EI status among similar geographical spaces and provides clear indicators on how low EI status may be achieved in these environments. Contrary to common belief, our data show that industrial-style intensification is not required to achieve low EI. Enteric CH₄ production overwhelmingly drives farm emissions in these systems and as this is strongly collinear with nutrition and intake, an effort will be required to achieve an “efficient frontier” between feed intake, productivity, and GHG emissions.     

Amino acid differences in interferon-tau (IFN-τ) of Bos taurus Coreanae and Holstein

Interferons (IFNs) are commonly grouped into type I and type II IFN. Type I IFNs are known as antiviral IFNs including IFN-α, IFN-β, and IFN-ω whereas type II IFN is referred to immune IFN and IFN-γ is only member of the type II IFN. Type I IFNs are induced by virus invading however type II IFN is produced by mitogenic or antigenic stimuli. IFN-τ was first identified in ruminant ungulates as a pregnancy recognition hormone, trophoblastin. IFN-τ constitutes a new class of type I IFN, which possesses the common features of type I IFN, such as the ability to prevent viral infection and to limit cell proliferation. In addition, IFN-τ is unique in that it is induced by pregnancy unlike other type I IFNs. We cloned Bos taurus (B. T.) Coreanae IFN-τ from peripheral blood mononuclear cells. The amino acid sequence of B. T. Coreanae IFN-τ shares only 90.3% identity with that of Holstein dairy cow. Recombinant B. T. Coreanae and Holstein IFN-τ proteins were expressed in Escherichia coli and the antiviral activity of IFN-τ proteins were examined. Both recombinant proteins were active and protected human WISH and bovine MDBK cells from the cytopathic effect of vesicular stomatitis virus. The recombinant IFN-τ protein of B. T. Coreanae and Holstein properly induced the expression of antiviral genes including 2',5'-oligoadenylate synthetase (OAS) and Mx GTPase 1 (Mx-1).     

TGF-��1 and BRCA2 Expression are Associated with Clinical Factors in Breast Cancer

The objective of this study was to investigate the possible association between the expression of transforming growth factor beta-1 (TGF-β1) and breast cancer type2 susceptibility protein (BRCA2) with clinical factors in breast cancer. TGF-β1, BRCA2, human epidermal growth factor receptor2 (HER2), estrogen receptor, and progesterone receptor protein levels were measured in 67 samples from breast cancer patients by immunohistochemistry. The expression of these proteins was correlated with various clinical factors including age, pathohistological grade and status of axillary lymph node implication. TGF-β1 and BRCA2 were expressed in breast cancer tissues and expression of HER2 and TGF-β1 was significantly correlated with BRCA2. The authors conclude that elevated expression of BRCA2 correlates with TGF-β1 and HER2 in breast cancer and these three factors act in synergy to promote cancer. Thus, detection of both TGF-β1 and BRCA2 may therefore assist in the prognosis and treatment of breast cancer.     

Novel Polymorphisms in Porcine 3′UTR of the Leptin Gene,Including a Rare Variant within Target Sequence for MIR-9 Gene in Duroc Breed,not Associated with Production Traits

The leptin gene (LEP) is considered as a functional candidate for production traits in livestock due to its crucial role in energy homeostasis. Because polymorphisms in regulatory sequences may affect gene expression, we searched for them in the 3′UTR of LEP and analyzed their association with production traits. Four breeds and a composite line were studied. In the Polish Landrace and Polish Large White breeds, 8 SNPs and 1 indel were observed; whereas, in the Duroc breed, 9 specific SNPs were found. Pietrain and Line 990 were monomorphic. One SNP (g.+168C>T), observed in the Duroc breed only, was located within a target site for microRNA (miR-9). Association studies showed a weak association between one SNP (c.+846C>T) and abdominal fat weight in the Polish Landrace only. Thus, we concluded that contribution of polymorphisms in the 3′UTR to phenotypic variability of pig production traits is marginal. Moreover, we presented an overview of known polymorphisms (128) in the pig leptin gene.     

Suicidal Behavior and Haplotypes of the Dopamine Receptor Gene (DRD2) and ANKK1 Gene Polymorphisms in Patients with Alcohol Dependence – Preliminary Report

Suicide is a significant public health issue and a major cause of death throughout the world. According to WHO it accounts for almost 2% of deaths worldwide. The etiology of suicidal behavior is complex but the results of many studies suggest that genetic determinants are of significant importance. In our study,- we have analyzed selected SNPs polymorphisms in the DRD2 and ANKK1 genes in patients with alcohol dependence syndrome (169 Caucasian subjects) including a subgroup of individuals (n = 61) who have experienced at least one suicide attempt. The aim of the study was to verify if various haplotypes of selected genes, comprising Taq1A, Taq1B, and Taq1D single nucleotide polymorphisms (SNP), play any role in the development of alcohol dependence and suicidal behavior. The control group comprised 157 unrelated individuals matched for ethnicity, gender,- and age and included no individuals with mental disorders. All subjects were recruited in the North West region of Poland. The study showed that alcohol dependent subjects with a history of at least one suicidal attempt were characterized by a significantly higher frequency of the T-G-A2 haplotype when compared to individuals in whom alcohol dependence was not associated with suicidal behavior (p = 0.006). It appears that studies based on identifying correlation between SNPs is the future for research on genetic risk factors that contribute to the development of alcohol addiction and other associated disorders. To sum up, there is a necessity to perform further research to explain dependencies between the dopaminergic system, alcohol use disorders and suicidal behavior.     

Expression of Cyclin D1 Is Associated with ��-Catenin Expression and Correlates with Good Prognosis in Colorectal Adenocarcinoma

Background

The aim of this study is to investigate the prevalence and prognostic impact of β-catenin and cyclin D1 expression in colorectal carcinoma (CRC) patients.

Method

We evaluated immunohistochemial expression of β-catenin and cyclin D1 using 2-mm cores from 220 CRC patients for tissue microarray, and its significance was statistically evaluated.

Result

Positive expression of β-catenin and cyclin D1 was found in 72.5% (158 of 218 cases) and 59.4% (129 of 217 cases) of CRC patients, respectively. Expression of β-catenin was significantly correlated with tumor location (P = .017), differentiation (P = .010), lymph node metastasis (P = .032), preoperative carcinoembryonic antigen level (P = .032), and cyclin D1 expression (P = .005). Expression of cyclin D1 was significantly correlated with recurrence and/or metastasis (P = .004). In univariate analysis, β-catenin expression predicted more favorable overall survival (P = .022) and cyclin D1 expression predicted both more favorable overall survival and relapse-free survival (P = .004 and P = .006, respectively). Multivariate analysis showed that tumor stage and expression of cyclin D1 were independent prognostic factors significantly associated with overall survival and relapse-free survival.

Conclusion

This study shows that expression of β-catenin and cyclin D1 is associated with favorable clinicopathologic variables and it is a clinically significant prognostic indicator for CRC patients.     

Ovarian responses in Bos indicus heifers treated to synchronise ovulation with intravaginal progesterone releasing devices, oestradiol benzoate, prostaglandin F(2α) and equine chorionic gonadotrophin

The objectives were: (i) improve understanding of the ovarian responses of Bos indicus heifers treated with different ovulation synchronisation protocols, (ii) compare ovarian responses of B. indicus heifers treated with intravaginal progesterone releasing device (IPRD)+oestradiol benzoate (ODB) versus a conventional prostaglandin F(2α) (PGF(2α)) protocol and (iii) investigate whether reducing the amount of progesterone (P(4)) in the IPRD, and treatment with equine chorionic gonadotrophin (eCG) would increase the proportion of heifers with normal ovarian function during the synchronised and return cycles. Two-year-old Brahman (n=30) and Brahman-cross (n=34) heifers were randomly allocated to three IPRD-treatment groups: (i) standard-dose IPRD (Cue-Mate(?) 1.56g P(4); n=17); (ii) half-dose IPRD (Cue-Mate(?) 0.78g P(4); n=15); (iii) half-dose IPRD+300IU eCG at IPRD removal (n=14), and a non-IPRD control group (iv) 2×PGF(2α) (500μg cloprostenol) on Days -16 and -2 (n=18). IPRD-treated heifers received 250μg cloprostenol at IPRD insertion (Day -10) and IPRD removal (Day -2) and 1mg ODB on Days -10 and -1. Ovarian function was evaluated by ultrasonography and plasma P(4) throughout the synchronised and return cycles. The mean diameter of the dominant follicle observed at 54-56h after IPRD removal, was greater for heifers which ovulated than heifers which did not ovulate (P<0.001; 14.5±1.1 vs. 9.3±0.6mm, respectively). The prevalence of IPRD-treated heifers with ovarian dysfunction (persistent CL, failure to re-ovulate, shortened luteal phase) was 39%. This relatively high prevalence of ovarian dysfunction may explain the commonly reported, lower than expected pregnancy rates to FTAI in B. indicus heifers treated to synchronise ovulation.     

Production of N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) fused with secretory signal Igκ in insect cells

The human UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) is one of the key enzymes that initiate synthesis of hinge-region O-linked glycans of human immunoglobulin A1 (IgA1). We designed secreted soluble form of human GalNAc-T2 as a fusion protein containing mouse immunoglobulin light chain kappa secretory signal and expressed it using baculovirus and mammalian expression vectors. The recombinant protein was secreted by insect cells Sf9 and human HEK 293T cells in the culture medium. The protein was purified from the media using affinity Ni-NTA chromatography followed by stabilization of purified protein in 50mM Tris-HCl buffer at pH 7.4. Although the purity of recombinant GalNAc-T2 was comparable in both expression systems, the yield was higher in Sf9 insect expression system (2.5mg of GalNAc-T2 protein per 1L culture medium). The purified soluble recombinant GalNAc-T2 had an estimated molecular mass of 65.8kDa and its amino-acid sequence was confirmed by mass-spectrometric analysis. The enzymatic activity of Sf9-produced recombinant GalNAc-T2 was determined by the quantification of enzyme-mediated attachment of GalNAc to synthetic IgA1 hinge-region peptide as the acceptor and UDP-GalNAc as the donor. In conclusion, murine immunoglobulin kappa secretory signal was used for production of secreted enzymatically active GalNAc-T2 in insect baculovirus expression system.     

Back fat thickness and meat tenderness are associated with a 526 T→A mutation in the exon 1 promoter region of the MyF-5 gene in Chinese Bos taurus

Qualitative trait loci (QTL) for growth and meat quality traits in cattle (Bos taurus) have been previously mapped to three chromosome regions, 0 to 30, 55 to 70, and 70 to 80 cM on chromosome 5. We evaluated the allele frequencies and gene-specific single nucleotide polymorphisms (SNPs) of bovine myogenic factor 5 (MyF-5) in the QTL regions and their associations with live weight and meat characteristics in indigenous Chinese cattle breeds. PCR-SSCP methodology showed a T>A mutation at 526 bp. Least square analysis revealed a significant association of this SNP with backfat thickness and meat tenderness (P < 0.05), while no significant association was found with live weight, loin eye height, loin eye area, rib area, or water holding capacity. Allele frequencies of MyF-5-A/B in the five breeds were 0.760/0.239, 0.752/0.247, 0.629/0.370, 0.715/0.284, and 0.750/0.250, for JiaXian red, Luxi, Nanyang, Qinchuan, and XiaNan crossbreed, respectively. The genotype distributions for these alleles in two of the Chinese cattle breeds (Luxi and Qinchuan) were not in Hardy-Weinberg equilibrium (P < 0.05); while those for the other three breeds (JiaXian red, Nanyang, and XiaNan) were in agreement with Hardy-Weinberg equilibrium (P > 0.05). The genotypic frequencies among all five cattle breeds showed moderate diversity (0.25 < polymorphism information content < 0.5). Based on our findings, we suggest that the MyF-5 gene influences back fat thickness and meat tenderness in Chinese Bos taurus. This SNP could be useful for marker-assisted selection for meat quality traits in these cattle.     

Biolistic Transfer of the Gene uidA and Its Expression in Haploid Embryo-like Structures of Triticale (×Triticosecale Wittmack)

Haploid embryo-like structures (ELS) of triticale were obtained by in vitro androgenesis. These structures were bombarded with gold microparticles 1 µm in diameter coated with the plasmid pAHGUS, using the Dupont PDS He/1000 apparatus. Analysis was made of the influence of genotype, duration of ELS pre-culture, helium pressure and shooting distance on the transfer and expression of the gene uidA. No significant differences were seen between genotypes or duration of pre-culture; differences were found, however, with respect to helium pressure and shooting distance. The combination of 1100 psi helium and a 6 cm shooting distance led to the greatest mean number of uidA expression foci in the ELS (1.63), and the greatest mean number of ELS showing at least one focus (0.38). With a pressure of 1800 psi and a 9 cm distance, only 0.35 foci were obtained per ELS, and only 0.18 ELS had one or more foci. These results are being used as references in a program for obtaining double haploid and transgenic triticale plants.     

Incidence of and Risk Factors Associated with Pulmonary and Extra-Pulmonary Tuberculosis in Saudi Arabia (2010–2011)

Setting

National Tuberculosis Program, Department of Public Health, Ministry of Health, Kingdom of Saudi Arabia (KSA).

Objective

To summarize data on the incidence of tuberculosis and associated risk factors for cases reported during 2010–2011.

Design

Retrospective analysis of routinely collected data through an established national disease notification system of the Ministry of Health in KSA.

Results

The estimated incidence of all forms of tuberculosis fell from 15.8/100000 (95% CI: 15.3/100,000–16.3/100,000) in 2010 to 13.8/100,000 (95% CI: 13.4/100,000–14.2/100,000) in 2011. Saudis experienced a decrease from 11.8/100,000 (95% CI: 11.3/100,000 to 12.3/100,000) in 2010 to 9.9/100,000 (95% CI: 9.5/100,000–10.4/100,000) in 2011 while the incidence in non-Saudis declined from 24.7/100,000 (95% CI: 23.6/100,000 to 25.7/100,000) in 2010 to 22.5/100,000 (95% CI: 21.5/100,000 to 23.4/100,000) in 2011. The proportion of Extra Pulmonary TB (EPTB) which increased minimally from 30% in 2010 to 32% in 2011 was higher than global figures and strongly associated with age, sex, nationality and occupation.

Conclusion

The current estimated incidence of about 14/100,000 in 2011 is less than half its estimated value of 44/100000 in 1990. Without prejudice to any under-reporting, the KSA appeared to be on the course for TB elimination by 2050 having reached the first milestone set by WHO. The proportion of EPTB remains higher than global figure and age, sex, nationality and occupation were significant independent predictors of EPTB.     

Photodynamic Action of Proflavine on Coliphage T3 III. Damages to the Deoxyribonucleic Acid Associated with R×1 and R×2

Ultracentrifugational studies with deoxyribonucleic acid (DNA) extracted from phage exposed to light in the presence of either 0.25 or 8.5 mug of proflavine per ml reveal that the lethal damage of Rx1 renders DNA alkali-labile, with lethality resulting from damage that occurs singly on either strand or simultaneously on both strands. Apparently nonlethal damages temporally associated with Rx1 consist of (i) heat and alkali-labile cross-links (which produce undenatured DNA that migrates at 45S) and (ii) heat-labile bonds. The formation of 45S material is a linear function of light dose, and the production of this material ceases when Rx2 appears at the higher dye concentration. No tendency to plateau is seen at 0.25 mug of dye per ml. The nature of the lethal damage of Rx2 could not be determined. Damages that were temporally associated with Rx2 at 8.5 mug of dye per ml were heat-labile, alkali-stable cross-links (undenatured DNA, 38 to 41S; alkali-denatured-reneutralized DNA, 85 to 89S) and some double-stranded breaks. No such changes were seen at 0.25 mug of dye per ml.