The lipid composition of muscle cells during development

Developmental changes in the phospholipid, cholesterol, and fatty acid composition of chicken pectoralis muscle cells were analyzed during development in vivo and in tissue culture. The phospholipid composition of muscle cells showed only minor changes during in vivo or in vitro development but there were significant alterations in fatty acid composition. During in vivo development between the 12th and 22nd days the concentration of palmitate and arachidonate decreased with increase in linoleate. In cultured muscle cells the fatty acid composition changes with surprising plasticity depending on the fatty acid supply. Compensatory changes were observed in the chain length and unsaturation of several fatty acids, aimed presumably at maintaining the physical properties of the lipid phase relatively constant.     

Purification and properties of glutamine synthetase from the plant cytosol fraction of lupin nodules

Glutamine synthetase from the plant cytosol fraction of lupin nodules was purified 89-fold to apparent homogeneity. The enzyme molecule is composed of eight subunits of M_r 44,700 ± 10%. Kinetic analysis indicates that the reaction mechanism is sequential and there is some evidence that Mg-ATP is the first substrate to bind to the enzyme. Michaelis constants for each substrate using the ammonium-dependent biosynthetic reaction are as follows: ATP, 0.24 mm; l-glutamate, 4.0–4.2 mm; ammonium, 0.16 mm. Using an hydroxamate-forming biosynthetic reaction the K_m ATP is 1.1 mm but the K_m for l-glutamate is not altered. The effect of pH on the K_m for ammonium indicates that NH₃ rather than NH₄⁺ may be the true substrate. At 10 mm Mg²⁺, the pH optimum of the enzyme is between 7.5 and 8, but increasing Mg²⁺ concentrations produce progressively more acidic optima while lower Mg²⁺ concentrations raise the pH optimum. The rate-response curve for Mg²⁺ is sigmoidal becoming bell-shaped in alkaline conditions. The enzyme is inhibited by l-Asp (K_i, 1.4 mm) and less markedly by l-Gln and l-Asn. Inhibition by ADP and AMP is strong, both nucleotides exhibiting K_i values around 0.3 mM. Investigations of the probable physiological conditions within the nodule plant cytosol indicate that in situ glutamine synthetase has an activity greater than that required to support the efflux of amino acid nitrogen from the nodule. A possible role for glutamine synthetase in the control of nodule ammonium assimilation is suggested.     

Enzymes of amide and ureide biogenesis in developing soybean nodules

Amide and ureide biogenic enzymes were measured in the plant fraction of soybean (Glycine max) nodules during the period 11 to 23 days after inoculation with Rhizobium japonicum (USDA 3I1b142). Enzymes involved in the initial assimilation of ammonia, i.e. glutamine synthetase, glutamate synthase, and aspartate aminotransferase, showed substantial increases in their specific activities over the time course. These increases paralleled the induction of nitrogenase activity in the bacteroid and leghemoglobin synthesis in the plant fraction. The specific activity of asparagine synthetase, however, showed a rapid decline after an initial increase in specific activity. Following the initial increases in the ammonia assimilatory enzymes, there was an increase in the activity of 5-phosphoribosylpyrophosphate amidotransferase, the enzyme which catalyzes the first committed step of de novo purine biosynthesis. This was followed by a dramatic increase in the purine oxidative enzymes, xanthine dehydrogenase and uricase. Smaller increases were observed in the activities of enzymes associated with the supply of metabolites to the purine biosynthetic pathway: phosphoglycerate dehydrogenase, serine hydroxymethylase, and methylene tetrahydrofolate dehydrogenase.     

Preliminary studies on bovine embryo survival following short-term storage at 4 degrees C

A total of 113 non-surgically collected bovine embryos, 5-8 days of age, were stored for 48 hours at 4 degrees C in a modified phosphate-buffered saline solution (PBS). Following storage, embryos were cultured for 8-12 hours at 37 degrees C, and those which were morphologically normal were transferred to synchronized recipients by several methods designed to achieve twin pregnancies. Embryos which were collected and transferred on the same day served as controls. Of 113 embryos stored, 47 (42%) appeared to be transferable after the brief culture period. There was a marked breed effect on viability after refrigeration, with Hereford embryos surviving significantly better than Angus embryos (71% vs. 12%, respectively, p < .001). Post-transfer embryo survival of stored and control embryos, based on actual calvings, was 34 and 48 percent, respectively, a difference which was not significant (p=0.3). A marked difference in pregnancy rate following non-surgical transfer by 2 different technicians was noted (50% vs. 21.7%, respectively).     

Induction of parturition in the pig using a new prostaglandin analogue (K11941)

Eighty seven sows, taken from a herd whose mean gestation length was 116 days, were allotted at random on day 114 of gestation to one of five treatment groups as follows: (1) 175 mug prostaglandin analogue (closprostenol I.C.I. Ltd) (2) 1 mg K11941 (alfaprostol VETEM Ltd), (3) 2 mg K11941, (4) 3 mg K11941 (5) 2 ml saline (control). All prostaglandin analogues induced parturition significantly earlier than controls (P < 0.05), but there were no differences between any of the prostaglandin treatments in the interval to birth of the first piglet (P > 0.05). The mean intervals to parturition were 24.6, 22.3, 24.9, 30.7 and 53.0 hours for treatments 1, 2, 3, 4 and 5 respectively. Almost two-thirds of treated sows farrowed during the working period of the day following injection compared with a small proportion of control animals. Induction of parturition had no effect on birth weight, piglet survival to 3 weeks or on subsequent weaning to service interval in treated sows. Results show that this new analogue of prostaglandin (K11941) is effective in inducing farrowing in the sow.     

Preparation of sarcoplasmic reticulum with high calcium-sensitive ATPase and stable calcium transport function from rat skeletal muscle

A method was developed for the isolation from rat skeletal muscle of sarcoplasmic reticulum vesicles in which the calcium transport function does not decay during storage. High initial and maximum uptake of calcium and calcium-dependent ATPase activity were obtained for membranes isolated from mixed muscles or pure red fibers. Unstable vesicles resulted when 2 mM EDTA was included in the isolation medium. The calcium uptake activity was lost upon ageing at 0 degrees C, probably due to conversion of the calcium-dependent ATPase to a calcium-independent form. Addition of Ca2+ counteracted the affects of EDTA, suggesting their involvement in maintaining the structure of the calcium transport system. This is supported by the fact that different structural states of the ATPase in stable and unstable vesicles were detected by DEAE-cellulose column chromatography.     

Frequency of chromosomal abnormalities in embryos from superovulated merino ewes

Chromosomal analysis was carried out on 48 Day 2-7 embryos collected from superovulated Merino ewes. Three embryos had abnormal chromosome complements (1 X 1N, 1 X 1N/2N, 1 X 3N), yielding an incidence of 6.25% abnormal embryos. It is concluded that superovulation does not cause an increase in the incidence of chromosomal abnormalities in embryos of Merino sheep.     

The new sequencer on the block: comparison of Life Technology’s Proton sequencer to an Illumina HiSeq for whole-exome sequencing

We assessed the performance of the new Life Technologies Proton sequencer by comparing whole-exome sequence data in a Centre d’Etude du Polymorphisme Humain trio (family 1463) to the Illumina HiSeq instrument. To simulate a typical user’s results, we utilized the standard capture, alignment and variant calling methods specific to each platform. We restricted data analysis to include the capture region common to both methods. The Proton produced high quality data at a comparable average depth and read length, and the Ion Reporter variant caller identified 96 % of single nucleotide polymorphisms (SNPs) detected by the HiSeq and GATK pipeline. However, only 40 % of small insertion and deletion variants (indels) were identified by both methods. Usage of the trio structure and segregation of platform-specific alleles supported this result. Further comparison of the trio data with Complete Genomics sequence data and Illumina SNP microarray genotypes documented high concordance and accurate SNP genotyping of both Proton and Illumina platforms. However, our study underscored the problem of accurate detection of indels for both the Proton and HiSeq platforms.