Germ cell development in Meishan and White Composite gilts

This study compared dynamics of the germ cell population in two swine breeds that differ in prolifacy, White Composite (WC) and Meishan (MS), during fetal and neonatal life and in mature sows. Germ cell populations developed in a similar pattern in these two diverse breeds during fetal life. Maximal germ cell number was observed at 90 days postcoitum (dpc) in both WC and MS gilts, and substantial oogonial apoptosis was evident thereafter with approximately 30% of maximal numbers present at 25 days postpartum (dpp). Neither gilt nor sow germ cell number was correlated with maternal ovulation rate. Postnatal MS gilts had larger pools of primordial follicles and consistently greater proportions and numbers of primary and secondary follicles compared to postnatal WC gilts, indicative of enhanced follicular recruitment and primordial follicle activation. Occasional antral follicles were present in MS ovaries by 25 dpp and numerous surface follicles were observed at 56 dpp in MS but not WC ovaries, indicative of more rapid ovarian maturation and early onset of puberty. Total germ cell number is unlikely to influence or to predict subsequent ovulation rate. These observations highlight important developmental events during late fetal and early postnatal life that prepare the ovarian environment for early onset of puberty and subsequent ovulation in MS gilts.     

Determination of thiopurine S-methyltransferase activity in erythrocytes using 6-thioguanine as substrate and a non-extraction liquid chromatographic technique

A non-extraction high-performance liquid chromatographic (HPLC) method has been developed for the determination of 6-methylthioguanine (6-MTG), as part of the determination of thiopurine S-methyltransferase activity (TPMT) in erythrocytes. Erythrocyte lysate is added to a glass vial containing substrates and incubation buffer, which is then sealed for the rest of the analysis. Enzyme incubation, sample preparation, and analysis are then undertaken without further sample-handling steps. The need for a solvent extraction step has been overcome by heating the incubate to 85 degrees C to stop the enzyme reaction. The heat inactivation step precipitates protein which upon centrifugation forms a thin film in the bottom of the glass vial enabling the supernatant to be injected directly onto the HPLC system. The assay shows excellent precision and recovery with a within-batch imprecision giving a co-efficient of variation of 2.9% (mean=41.5 nmol 6-MTG/gHb/h, n=10) and 5.1% (mean=12.6 nmol 6-MTG/g Hb/h, n=10). The between-batch imprecision gives a co-efficient of variation of 8.2% (mean=11.1 nmol 6-MTG/gHb/h, n=11) and 7.3% (mean=41.0 nmol 6-MTG/gHb/h, n=16). Determination of the TPMT activity in 120 people shows a range of enzyme activity of 11.3-63.8 nmol 6-MTG/gHb/h with a mean and median activity of 34.8 and 34.2 nmol 6-MTG/gHb/h, respectively. TPMT is increasingly used in clinical practice to ensure optimisation of treatment with thioguanine drugs. This direct HPLC method minimises sample-handling, reduces inherent imprecision, the possibility of laboratory error and with the potential for further automation, makes it ideal for use in a regional referral laboratory.     

Starch-binding domain shuffling in Aspergillus niger glucoamylase

Aspergillus niger glucoamylase (GA) consists mainly of two forms, GAI [from the N-terminus, catalytic domain + linker + starch-binding domain (SBD)] and GAII (catalytic domain + linker). These domains were shuffled to make RGAI (SBD + linker + catalytic domain), RGAIDeltaL (SBD + catalytic domain) and RGAII (linker + catalytic domain), with domains defined by function rather than by tertiary structure. In addition, Paenibacillus macerans cyclomaltodextrin glucanotransferase SBD replaced the closely related A.niger GA SBD to give GAE. Soluble starch hydrolysis rates decreased as RGAII approximately GAII approximately GAI > RGAIDeltaL approximately RGAI approximately GAE. Insoluble starch hydrolysis rates were GAI > RGAIDeltaL > RGAI > GAE approximately RGAII > GAII, while insoluble starch-binding capacities were GAI > RGAI > RGAIDeltaL > RGAII > GAII > GAE. These results indicate that: (i) moving the SBD to the N-terminus or replacing the native SBD somewhat affects soluble starch hydrolysis; (ii) SBD location significantly affects insoluble starch binding and hydrolysis; (iii) insoluble starch hydrolysis is imperfectly correlated with its binding by the SBD; and (iv) placing the P.macerans cyclomaltodextrin glucanotransferase SBD at the end of a linker, instead of closely associated with the rest of the enzyme, severely reduces its ability to bind and hydrolyze insoluble starch.     

Analysis of a conserved hydrophobic pocket important for the thermostability of Bacillus pumilus chloramphenicol acetyltransferase (CAT-86)

Site-directed mutagenesis was carried out on Bacillus pumilus chloramphenicol acetyltransferase (CAT-86) to determine the effects of substitution at a conserved hydrophobic pocket identified earlier as important for thermostability. Mutations were introduced that would substitute residues at consensus positions 33, 191 and 203 in the enzyme, both individually and in combination. Two mutants, SDM1 (CAT-86 Y33F, A203V) and SDM5 (CAT-86 A203I), were more thermostable than wild-type and two mutants, SDM4 (CAT-86 I191V) and SDM7 (CAT-86 A203G), were less stable. Reconstruction of the residues of this hydrophobic pocket to that of a more thermostable CAT-R387 enzyme pocket (as a Y33F, I191V, A203V triple mutant) increased the thermostability of the enzyme above the wild-type, but its stability was less than that of SDM1 and SDM5. The K(m) values of the mutant enzymes for chloramphenicol and acetyl-CoA were essentially unaltered (in the ranges 15-30 and 26-35 microM respectively) and the specific activity of purified enzyme was in the range 270-710 units/mg protein. The possible effects of the amino acid substitutions on the CAT-86 structure were determined by homology modelling. A reduction in conformational strain and optimized hydrophobic interactions are predicted to be responsible for the increased thermostability of the SDM1 and SDM5 mutants.     

Proapoptotic p53-interacting protein 53BP2 is induced by UV irradiation but suppressed by p53

p53 is an important mediator of the cellular stress response with roles in cell cycle control, DNA repair, and apoptosis. 53BP2, a p53-interacting protein, enhances p53 transactivation, impedes cell cycle progression, and promotes apoptosis through unknown mechanisms. We now demonstrate that endogenous 53BP2 levels increase following UV irradiation induced DNA damage in a p53-independent manner. In contrast, we found that the presence of a wild-type (but not mutant) p53 gene suppressed 53BP2 steady-state levels in cell lines with defined p53 genotypes. Likewise, expression of a tetracycline-regulated wild-type p53 cDNA in p53-null fibroblasts caused a reduction in 53BP2 protein levels. However, 53BP2 levels were not reduced if the tetracycline-regulated p53 cDNA was expressed after UV damage in these cells. This suggests that UV damage activates cellular factors that can relieve the p53-mediated suppression of 53BP2 protein. To address the physiologic significance of 53BP2 induction, we utilized stable cell lines with a ponasterone A-regulated 53BP2 cDNA. Conditional expression of 53BP2 cDNA lowered the apoptotic threshold and decreased clonogenic survival following UV irradiation. Conversely, attenuation of endogenous 53BP2 induction with an antisense oligonucleotide resulted in enhanced clonogenic survival following UV irradiation. These results demonstrate that 53BP2 is a DNA damage-inducible protein that promotes DNA damage-induced apoptosis. Furthermore, 53BP2 expression is highly regulated and involves both p53-dependent and p53-independent mechanisms. Our data provide new insight into 53BP2 function and open new avenues for investigation into the cellular response to genotoxic stress.