Variation in protein metabolism biomarkers during the transition period and associations with health, colostrum quality, reproduction, and milk production traits in Holstein cows.

The aims of this study were to assess (1) the variation of protein metabolism biomarkers and factors affecting them during the transition period, (2) the association of each biomarker with skeletal muscle reserves and their changes, and (3) the association of these biomarkers with postpartum health, colostrum quality, reproduction, and milk production. For this purpose, 238 multiparous Holstein cows from 6 herds were used in a prospective cohort study. Plasma concentrations of 3-methylhistidine (3-MH) and 1-methylhistidine (1-MH) and serum concentrations of total protein (TP), albumin (ALB), urea nitrogen (BUN), and creatinine (SCR) were determined for each cow at -21, -7, 7, 21, and 28 d relative to calving. Clinical diseases were recorded during the first 28 d postcalving, and presence of subclinical ketosis (scKET) was investigated at 7 and 21 d. Colostrum quality was estimated by Brix refractometry. Reproduction data by 150 d in milk (DIM) and milk production records were also available. Linear mixed models including the fixed effects of time point, herd, parity, body condition score (-21 d), duration of dry period and postparturient diseases were fitted to assess the variation in each biomarker's concentration. The association between the biomarkers' concentration during the prepartum period with the odds for each postparturient disease and for a combined trait (CD_1-28), defined as the presence of at least one clinical condition during the first 28 d after calving, were assessed with separate binary logistic models for time points -21 d and -7 d. The relationship of each biomarker's concentration with longissimus dorsi thickness (LDT) and the changes in LDT (ΔLDT) was assessed with pairwise correlations. Separate general linear models were used to assess the association of each biomarker with colostrum Brix values and milk production traits. Finally, the associated hazard for first artificial insemination (AI) and for pregnancy by 150 DIM (PREG_150DIM) was assessed with Cox proportional hazard models, whereas odds for pregnancy to the first AI (PREG_1stAI) were assessed with binary logistic models. The level of 3-MH was affected mainly by herd, time points, and their interaction. Higher 3-MH was associated with increased odds for metritis and CD_1-28, increased hazard for PREG_150 DIM and with increased milk production. 1-Methylhistidine was affected mainly by herd, scKET and occurrence of displaced abomasum. Higher 1-MH was associated with better colostrum quality, increased odds for scKET, increased hazard for first AI by 150 DIM and with decreased milk production. Both 3-MH and 1-MH were weakly to moderately negatively correlated with LDT and moderately to strongly negatively correlated with ΔLDT at the corresponding time periods. Additionally, higher TP was associated with increased odds for metritis and CD_1-28 and increased milk production, while higher ALB was associated with increased odds for scKET and increased milk production. Moreover, higher BUN was associated with decreased odds for scKET, increased odds for PREG_1stAI and increased milk production. Higher SCR was associated with decreased odds for retained fetal membranes, metritis, and CD_1-28. Periparturient protein metabolism is significantly associated with postpartum health, colostrum quality, reproduction, and milk production; mechanisms involved require further investigation.

Substrate specificity and subsite mobility in T. aurantiacus xylanase 10A.

The substrate specificity of Thermoascus aurantiacus xylanase 10A (TAX) has been investigated both biochemically and structurally. High resolution crystallographic analyses at 291 K and 100 K of TAX complexes with xylobiose show that the ligand is in its alpha anomeric conformation and provide a rationale for specificity on p-nitrophenyl glycosides at the -1 and -2 subsites. Trp 275, which is disordered in uncomplexed structures, is stabilised by its interaction with xylobiose. Two structural subsets in family 10 are identified, which differ by the presence or absence of a short helical stretch in the eighth betaalpha-loop of the TIM barrel, the loop bearing Trp 275. This structural difference is discussed in the context of Trp 275 mobility and xylanase function.

High resolution structure and sequence of T. aurantiacus xylanase I: implications for the evolution of thermostability in family 10 xylanases and enzymes with (beta)alpha-barrel architecture.

Xylanase I is a thermostable xylanase from the fungus Thermoascus aurantiacus, which belongs to family 10 in the current classification of glycosyl hydrolases. We have determined the three-dimensional X-ray structure of this enzyme to near atomic resolution (1.14 A) by molecular replacement, and thereby corrected the chemically determined sequence previously published. Among the five members of family 10 enzymes for which the structure has been determined, Xylanase I from T. aurantiacus and Xylanase Z from C. thermocellum are from thermophilic organisms. A comparison with the three other available structures of the family 10 xylanases from mesophilic organisms suggests that thermostability is effected mainly by improvement of the hydrophobic packing, favorable interactions of charged side chains with the helix dipoles and introduction of prolines at the N-terminus of helices. In contrast to other classes of proteins, there is very little evidence for a contribution of salt bridges to thermostability in the family 10 xylanases from thermophiles. Further analysis of the structures of other proteins from thermophiles with eight-fold (beta)alpha-barrel architecture suggests that favorable interactions of charged side chains with the helix dipoles may be a common way in which thermophilic proteins with this fold are stabilized. As this is the most common type of protein architecture, this finding may provide a useful guide for site-directed mutagenesis aimed to improve the thermostability of (beta)alpha-barrel proteins. Proteins 1999;36:295-306.