Associations among Farm, Breed, Lactation Stage and Parity, Gene Polymorphisms and the Fatty Acid Profile of Milk from Holstein, Simmental and Their Crosses.

This study aimed to analyze the factors affecting the fatty acid (FA) profile in cow's milk. The effects of a farm, lactation parity and stage, breed and polymorphisms in the AGPAT6, DGAT1, LEP, FASN and SCD1 genes were evaluated. A total of 196 Holstein cows, 226 Simmental cows and seven crosses were sampled 751 times. The cows were kept at five farms and were in the first up to the sixth lactation, and 49 individual FAs and 11 groups were analyzed. The farm significantly affected the proportion of all FAs except for C16:1n-7c and isoC14:0. Additionally, the lactation stage was significant for most FAs, and the opposite was true for lactation parity. The effect of the breed was negligible. For the gene polymorphisms, the SCD1 TT genotype exceeded the CC in C10:0, C12:0, C14:0, C16:1n-7c and C18:2, and the opposite was true for C10:1, C12:1, C14:1n-5c, isoC17:0, C16:1 and C18:1, i.e., the TT genotype was higher for saturated FAs, and the CT genotype was higher for monounsaturated FAs. The results hint at the intermediary heredity of the SCD1 gene. The FASN gene was strongly associated with four FAs and branched-chain FAs, and genotype AG was better than GG. LEP was significant for five individual FAs and branched-chain FAs. The differences in FA composition among genotypes were rather small, which could lead to overestimation of the effect and needs to be considered in the next research.

Comparison of Fatty Acid Proportions Determined by Mid-Infrared Spectroscopy and Gas Chromatography in Bulk and Individual Milk Samples.

Rapid analytical methods can contribute to the expansion of milk fatty acid determination for various important practical purposes. The reliability of data resulting from these routine methods plays a crucial role. Bulk and individual milk samples (60 and 345, respectively) were obtained from Czech Fleckvieh and Holstein dairy cows in the Czech Republic. The correlation between milk fatty acid (FA) proportions determined by the routine method (infrared spectroscopy in the mid-region in connection with Fourier transformation; FT-MIR) and the reference method (gas chromatography; GC) was evaluated. To validate the calibration of the FT-MIR method, a linear regression model was used. For bulk milk samples, the correlation coefficients between these methods were higher for the saturated (SFAs) and unsaturated FAs (UFAs) (r = 0.7169 and 0.9232; p < 0.001) than for the trans isomers of UFAs (TFAs) and polyunsaturated FAs (PUFAs) (r = 0.5706 and 0.6278; p < 0.001). Similar results were found for individual milk samples: r = 0.8592 and 0.8666 (p < 0.001) for SFAs and UFAs, 0.1690 (p < 0.01) for TFAs, and 0.3314 (p < 0.001) for PUFAs. The correlation coefficients for TFAs and PUFAs were statistically significant but too low for practical analytical application. The results indicate that the FT-MIR method can be used for routine determination mainly for SFAs and UFAs.

Identification of furan fatty acids in the lipids of common carp (Cyprinus carpio L.).

Fatty acid (FA) composition was analyzed in muscle and gonad tissues of marketed common carp (Cyprinus carpio). The extracted lipids were separated into four fractions: polar lipids (PL), diacylglycerols, free fatty acids and triacylglycerols (TAG) using thin layer chromatography. FA content within the lipid fractions was determined by gas chromatography with flame ionization detector (GC/FID). The muscle lipids consisted primarily of TAG (96.9% of total FA), while PL were the major component of both male (67.6%) and female gonad (58.6%) lipids. Polyunsaturated fatty acids predominated in PL of all tissues (52.2-55.8% of total FA); monounsaturated fatty acids were the most abundant FA group in TAG of muscle (51.8%) and female gonads (47.8%) whereas high proportion of furan fatty acids (F-acids) (38.2%) was detected in TAG of male gonads. Eight F-acids were identified by gas chromatography-mass spectrometry (GC/MS) in male gonad samples, including less common 12,15-epoxy-13,14-dimethylnonadeca-12,14-dienoic acid with even-numbered alkyl moiety.

Changes in the Content of Biogenic Amines and Fatty Acids in High Pressure-Processed Carp Flesh (Cyprinus carpio).

Biogenic amine and fatty acid contents were determined in vacuum-packed fillets of common carp (Cyprinus carpio). Samples were pressure treated at 300 and 500 MPa and were stored at 3.5 and 12°C for up to 28 days (control, 0 MPa) and 70 days (pressure-treated). The content of eight biogenic amines (putrescine, cadaverine, spermidine, spermine, histamine, tyramine, tryptamine, and phenylethylamine) were determined. Putrescine and cadaverine were influenced by all factors (temperature, pressurization level, and time of storage). Tyramine content was the most sensitive indicator of the improper status of sample; levels exceeding 10 mg/kg indicated both the loss of meat freshness and temperature abuse, in spite of persisting good sensory indices. Neither storage temperature nor pressurization level had a statistically important effect on the contents of fatty acids. Only polyunsaturated fatty acids decreased slightly if the storage time exceeded 42 days.

Interaction of late apoptotic and necrotic cells with vitronectin.

BACKGROUND: Vitronectin is an abundant plasma glycoprotein identified also as a part of extracellular matrix. Vitronectin is substantially enriched at sites of injured, fibrosing, inflamed, and tumor tissues where it is believed to be involved in wound healing and tissue remodeling. Little is known about the mechanism of vitronectin localization into the damaged tissues. METHODOLOGY/PRINCIPAL FINDINGS: 2E12 antibody has been described to bind a subset of late apoptotic cells. Using immunoisolation followed by mass spectrometry, we identified the antigen recognized by 2E12 antibody as vitronectin. Based on flow cytometry, we described that vitronectin binds to the late apoptotic and necrotic cells in cell cultures in vitro as well as in murine thymus and spleen in vivo. Confocal microscopy revealed that vitronectin binds to an intracellular cytoplasmic structure after the membrane rupture. CONCLUSIONS/SIGNIFICANCE: We propose that vitronectin could serve as a marker of membrane disruption in necrosis and apoptosis for flow cytometry analysis. Moreover, we suggest that vitronectin binding to dead cells may represent one of the mechanisms of vitronectin incorporation into the injured tissues.

Kit- and Fc epsilonRI-induced differential phosphorylation of the transmembrane adaptor molecule NTAL/LAB/LAT2 allows flexibility in its scaffolding function in mast cells.

The transmembrane adaptor protein (TRAP), NTAL, is phosphorylated in mast cells following FcvarepsilonRI aggregation whereby it cooperates with LAT to induce degranulation. The Kit ligand, stem cell factor (SCF), enhances antigen-induced degranulation and this also appears to be NTAL-dependent. However, Kit and FcvarepsilonRI appear to utilize different mechanisms to induce NTAL phosphorylation. Thus, we examined whether the responsible kinases selectively phosphorylated distinct tyrosines in NTAL and explored the implications for downstream signaling. Whereas FcvarepsilonRI required Lyn and Syk for NTAL phosphorylation, Kit appeared to directly phosphorylate NTAL. Furthermore, co-transfection studies with NTAL constructs revealed that Lyn, Syk, and Kit phosphorylate different tyrosines in NTAL. The tyrosines principally phosphorylated by Syk were recognized as Grb2-binding sites, whereas Lyn and Kit phosphorylated other tyrosines, both inside and outside of these motifs. Pull down studies revealed that PLCgamma1 associated with the two terminal Syk-phosphorylated Grb2-binding sites, which would help to explain the observed decrease in antigen-induced calcium signal and degranulation in NTAL-knock down-human mast cells. The observations reported herein support the conclusion that NTAL may be differentially utilized by specific receptors for relaying alternative signals and this suggests a flexibility in the function of TRAPs not previously appreciated.

Single and combined deletions of the NTAL/LAB and LAT adaptors minimally affect B-cell development and function.

NTAL (non-T-cell activation linker, also called LAB) and LAT (linker for activation of T cells) are evolutionarily related transmembrane adaptor proteins that are phosphorylated upon immunoreceptor engagement. Using quantitative reverse transcription-PCR, both NTAL and LAT were found to be expressed in B cells. However, LAT expression was limited to early B cells, whereas NTAL expression typified mature B cells. To delineate their roles in B-cell development and function, Ntal-deficient mice were generated and crossed with Lat-deficient mice. B cells developed in Lat(-/-) Ntal(-/-) double-deficient mice and in mice lacking either of the two adaptors with the same efficiency as in wild-type mice. Upon B-cell antigen receptor cross-linking, Ntal(-/-) B cells exhibited slightly increased Ca(2+) mobilization and proliferation. In addition, Ntal-deficient mice had increased levels of natural antibodies and slightly increased humoral response to a T-dependent antigen. Normal titers of serum-specific immunoglobulins were produced in response to a T-cell-independent antigen. Although NTAL is also expressed in plasma cells, its absence did not affect the hypergammaglobulinemia E and G1 that developed in mice with a mutation in tyrosine 136 of LAT. Therefore, NTAL does not play a role in B cells symmetric to the role played by LAT in T cells.

LIME: a new membrane Raft-associated adaptor protein involved in CD4 and CD8 coreceptor signaling.

Lymphocyte membrane rafts contain molecules critical for immunoreceptor signaling. Here, we report identification of a new raft-associated adaptor protein LIME (Lck-interacting molecule) expressed predominantly in T lymphocytes. LIME becomes tyrosine phosphorylated after cross-linking of the CD4 or CD8 coreceptors. Phospho-LIME associates with the Src family kinase Lck and its negative regulator, Csk. Ectopic expression of LIME in Jurkat T cells results in an increase of Csk in lipid rafts, increased phosphorylation of Lck and higher Ca2+ response to CD3 stimulation. Thus, LIME appears to be involved in regulation of T cell activation by coreceptors.

Non-T cell activation linker (NTAL): a transmembrane adaptor protein involved in immunoreceptor signaling.

A key molecule necessary for activation of T lymphocytes through their antigen-specific T cell receptor (TCR) is the transmembrane adaptor protein LAT (linker for activation of T cells). Upon TCR engagement, LAT becomes rapidly tyrosine phosphorylated and then serves as a scaffold organizing a multicomponent complex that is indispensable for induction of further downstream steps of the signaling cascade. Here we describe the identification and preliminary characterization of a novel transmembrane adaptor protein that is structurally and evolutionarily related to LAT and is expressed in B lymphocytes, natural killer (NK) cells, monocytes, and mast cells but not in resting T lymphocytes. This novel transmembrane adaptor protein, termed NTAL (non-T cell activation linker) is the product of a previously identified WBSCR5 gene of so far unknown function. NTAL becomes rapidly tyrosine-phosphorylated upon cross-linking of the B cell receptor (BCR) or of high-affinity Fcgamma- and Fc epsilon -receptors of myeloid cells and then associates with the cytoplasmic signaling molecules Grb2, Sos1, Gab1, and c-Cbl. NTAL expressed in the LAT-deficient T cell line J.CaM2.5 becomes tyrosine phosphorylated and rescues activation of Erk1/2 and minimal transient elevation of cytoplasmic calcium level upon TCR/CD3 cross-linking. Thus, NTAL appears to be a structural and possibly also functional homologue of LAT in non-T cells.