Effects of Confluent, Roscovitine Treatment and Serum Starvation on the Cell-cycle Synchronization of Bovine Foetal Fibroblasts

The present study was designed to examine the effects of cell-cycle synchronization protocols, such as confluent, roscovitine treatment and serum starvation, in bovine foetal fibroblasts on synchronization accuracy at G0/G1, viability, apoptosis, necrosis and ploidy for use as a nuclei donor. The cells in 5-10 passages were randomly allocated into three treated groups. Cells were cultured either in Dulbecco's modified Eagle's medium (DMEM) + 10% foetal bovine serum (FBS) until 90% confluent (group 1, confluent), in DMEM + 10% FBS + 30 microM roscovitine for 12 h (group 2, roscovitine), or in DMEM + 0.5% FBS for 5 days (group 3, serum starvation). Most of the cells (>80%) in all groups were arrested at the G0/G1 stage. Although the rates did not differ, cells in group 1 showed an increased cell population arrested at the G0/G1 phase. Significantly (p < 0.05) higher rates of apoptosis occurred in group 3 than in group 1 and 2 (10% vs 6% and 6%, respectively). No differences in chromosomal abnormality were observed among groups. However, by increasing the number of cell culture passages up to 15, significantly (p < 0.05) higher chromosomal abnormality was observed than in 5 and 10 passages (39% vs 28% and 23%, respectively) in group 1. The results clearly indicated that bovine foetal fibroblasts could be effectively synchronized at G0/G1 stages by all the three different treatments, confluent, roscovitine and serum starvation. However, cells in confluent showed reduced apoptosis and necrosis when they underwent 5-10 passages, exhibiting increased percentage of cells with stable chromosome diversity. Hence, cells in confluent merit further studies before they could be used as nuclear donors.     

Alteration of stimulus-specific guard cell calcium oscillations and stomatal closing in Arabidopsis det3 mutant

Cytosolic calcium oscillations control signaling in animal cells, whereas in plants their importance remains largely unknown. In wild-type Arabidopsis guard cells abscisic acid, oxidative stress, cold, and external calcium elicited cytosolic calcium oscillations of differing amplitudes and frequencies and induced stomatal closure. In guard cells of the V-ATPase mutant det3, external calcium and oxidative stress elicited prolonged calcium increases, which did not oscillate, and stomatal closure was abolished. Conversely, cold and abscisic acid elicited calcium oscillations in det3, and stomatal closure occurred normally. Moreover, in det3 guard cells, experimentally imposing external calcium-induced oscillations rescued stomatal closure. These data provide genetic evidence that stimulus-specific calcium oscillations are necessary for stomatal closure.     

Occurrence and diversity of coccidia in indigenous, Saanen and crossbred goats in South Africa

Occurrence and diversity of Eimeria species in two groups of indigenous South African goats kept under traditional management systems, as well as in a mixed herd of Saanen, indigenous and crossbred goats kept under an intensive management system were examined. Infection rates ranged from 88.7 to 100% in the various groups. Mean OPG of immature goats (< 1 year old) exceeded that of adult goats at all three sites. There was no consistent difference between adult OPG counts at the three sites. Under the intensive system, adult crossbred goats had significantly higher OPG counts than adult Saanen or indigenous goats. Overall, OPG counts of immature goats were significantly higher during the dry season (winter) than during the wet season (summer). Ten Eimeria species were identified, Eimeria arloingi being the most prevalent species at all three sites, followed by Eimeria hirci. Up to seven Eimeria species were recovered from individual specimens.     

Characterization of a wild-type strain of Francisella tularensis isolated from a cat.

Francisella tularensis type A is the primary cause of tularemia in animals and humans in North America. The majority of research on F. tularensis has been done with the attenuated live vaccine strain (LVS), which is a type B, but very few wild-type F. tularensis strains have been characterized. A gram-negative coccobacillus that was isolated in pure culture from the lungs of a cat that died after being lost for 5 days was received for identification at the Virginia-Maryland Regional College of Veterinary Medicine Teaching hospital. The isolate (strain TI0902) was not identified (or was misidentified) by commercial identification systems; however, it was identified as F. tularensis subspecies tularensis (type A) by sequencing a portion of the 16S ribosomal RNA gene. Furthermore, repetitive extragenic palindromic sequences-polymerase chain reaction amplified a 4-kb DNA fragment from TI0902 that was characteristic of F. tularensis type A but not type B. The electrophoretic profile of the lipopolysaccharide of strain TI0902 was identical to that of the LVS by Western blotting with antiserum to LVS. The protein-enriched outer membrane of strain TI0902 contained 6-8 proteins, which were similar in molecular size to those from the LVS. Electron microscopy of negatively stained and alcian blue-stained LVS and TI0902 cells showed that both strains were coccobacillary in shape and may be encapsulated. However, after mouse challenge, the TI0902 strain was clearly more virulent than the LVS strain. Results of this study indicate that the genotype and phenotype of wild-type F. tularensis type A strain TI0902 is similar, but not identical, to that of the LVS strain. Further studies will help determine whether pathogenesis and host-pathogen interactions are also similar between the 2 strains.     

Metribuzin degradation in soil: II—effects of tillage

A 140-day laboratory incubation, using surface soil from a long-term soybean tillage study, evaluated tillage influence on [¹⁴C]metribuzin degradation. Higher plant residue conditions in no-tillage (NT) soil inhibited metribuzin mineralization to [¹⁴C]carbon dioxide as compared to metribuzin degradation patterns observed in conventional tillage (CT) soil. At 140 days, relative abundance of extractable ¹⁴C components in NT included polar metabolites > metribuzin = deaminated metribuzin (DA) = deaminated diketometribuzin (DADK), while in CT, components included metribuzin > polar metabolites > DADK?DA. Conditions in NT apparently inhibited polar ¹⁴C degradation, and resulted in its accumulation, while in CT polar ¹⁴C degradation proceeded relatively rapidly. For both NT and CT, more ¹⁴ C was measured in an unextractable fraction than in any other fraction. A greater portion of the unextractable fraction in NT was associated with decomposed plant residue than in CT. Surface accumulation of crop residue, such as occurs under NT, provided a soil environment which altered metribuzin degradation patterns.     

Age-related variations in hematologic and plasma biochemical test results in Beagles and Labrador Retrievers

OBJECTIVE: To investigate age-related variations in results of hematologic and plasma biochemical tests performed on dogs of 2 common breeds. DESIGN: Prospective cohort study. ANIMALS: 34 Beagles and 44 Labrador Retrievers. PROCEDURE: Blood samples were collected throughout the dogs' lives; 589 samples were collected from the Beagles and 964 samples were collected from the Labrador Retrievers (age at the time of sample collection ranged from 22 days to 15 years). White blood cell and RBC counts; hemoglobin concentration; Hct; mean cell volume; mean cell hemoglobin concentration; alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase activities; and calcium, phosphorus, cholesterol, urea, protein, and albumin concentrations were measured. RESULTS: For all tests, there were significant effects of age on test results. There was a significant interaction between age and breed for all tests except hemoglobin, albumin, and phosphorus concentrations. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that there were age-related changes in hematologic and plasma biochemical test results in these 2 breeds of dogs. Changes were most evident during the first year of life, reflecting growth and maturation of the puppies. In some instances, values for puppies diverged markedly from those for adults, necessitating the use of age-specific reference ranges for the interpretation of clinical data.     

The effects of beta agonists on muscle cells in culture

Increases in protein synthesis of 12% were found with two myogenic cell lines (L6 and G8-1) on treatment for 6 hr with the beta-adrenergic agonist cimaterol. In L6 cells, propranolol blocked the effect. Protein breakdown measured over 18–24 hr was unchanged. The K_d for cimaterol binding to the L6 beta-receptor was 26 nM which was compatible with its EC₅₀ for the stimulation of protein synthesis (approx 5 nM). Evidence provided with muscle cell lines indicatesa direct effect of cimaterol on protein synthesis, which may contribute to muscle accretion in cimaterol-fed animals.