An efficient method for routine epstein-barr virus immortalization of human B lymphocytes

Summary A variety of methods exist for the immortalization of B lymphocytes by Epstein-Barr virus due to the simplicity of such techniques to establish cell lines with stable genomic DNA. Two different methods for immortalizing lymphoblastoid cell lines were compared for differences in techniques and materials, time between initiation and immortalization, and success rate of immortalization. An incubation period in Epstein-Barr virus and the use of conditioned media improved immortalization efficiency from 86 to 98% and decreased the time (usually weeks) from culture initiation to cryopreservation. The resulting cell bank was used to produce DNA for genetic studies focusing on the genes involved in non-insulin-dependent diabetes mellitus.     

Use of Glycated Hemoglobin to Assess Glycemic Control in Wistar Diabetic Fatty Rats and Zucker Fatty Rats

The Wistar Diabetic Fatty rat (WDF fafa) is a con-genic strain of the Wistar Kyoto rat. Studies using blood glucose reveal that only fatty male (not female) WDF rats spontaneously develop hyperglycemia when fed a stock diet Blood glucose values have not provided consistent results for evaluation of glycemic status in fatty male WDF rats. Zucker fatty (fafa) rats, while sharing the fa gene and the development of hyperinsulinemia and hyperlipemia, do not spontaneously become hyperglycemic. In order to examine strain differences and the effects of age on long-term average glycemic status in WDF and Zucker rats, glycated hemoglobin (GHb) was analyzed. Glycated hemoglobin was measured in male lean and obese WDF and Zucker rats at 2,3,6, and 12 months of age. Nonfasted plasma glucose was measured in male lean and obese WDF rats at 2, 3, 6, and 12 months of age and in lean and obese Zucker rats at 3, 6, and 12 months of age. Plasma insulin was measured in lean and obese WDF and Zucker rats at 3, 6, and 12 months of age. Obese WDF rats had significantly elevated GHb compared to lean controls at 3, 6, and 12 months of age. Glycated hemoglobin was substantially above the normal range (3.8-6.5%) at 3 months of age (14.1%). Glycated hemoglobin significantly declined in the obese WDF rats between 6 and 12 months of age. Nonfasted plasma glucose was significantly elevated in the obese WDF rats at 3 months (14.1 ± 2.1 mM/L) and 6 months of age (16.2 ± 2.3 mM/L) compared to lean controls. At 12 months of age there was no difference in plasma glucose between obese and lean WDF rats. Obese and lean Zucker rats had similar levels of GHb and plasma glucose at all ages. In conclusion, GHb provides more integrated data for classifying disease status of WDF rats and evaluation of potential long-term complications associated with hyperglycemia.     

Characteristics of the Obesity Syndrome in Zucker-Brown Norway (ZBN) Hybrid Rats

The existence of a restriction fragment length polymorphism (RFLP) closely linked to the fatty locus between the Zucker (Z) and Brown Norway (BN) rat strains allows evaluation of early effects of the fatty (fa) gene using offspring of back-crosses (N₂) between F₁ females and Zucker obese males. We examined several metabolic characteristics of N₂ animals to determine if these hybrid animals exhibited similar characteristics of the obese syndrome to those of Zucker rats. Females from crosses of obese male Zucker (fd/fa) and lean female BN (+/+) rats were back-crossed to their sires, resulting in twelve N₂ litters. At 9 weeks of age, liver, spleen, interscapular brown fat (IBAT), and gonadal, retroperitoneal (RP), and inguinal fat depots were removed and weighed. Samples of the RP depot were analyzed for cell size and number. Obese N₂ rats were hyperphagic, with body weights in the range of those of obese Zucker rats. Obese N₂ rats were also hyperinsulinemic [mean f SEM, pU/ml: females, 7.9 ± 0.6 vs. 82.1 f 8.4 (lean vs. obese); males, 10.5 ± 1.6 vs. 128.5 ± 13.4 (lean vs. obese)] and mildly hyperglycemic [mean ± SEM, mg/dl: females, 104.1 ± 2.0 vs. 139.0 ± 14.7 (lean vs. obese); males, 100.9 ± 2.6 vs. 132.0 ± 2.8 (lean vs. obese) p ≤ 0.05]. White fat depots in obese tats were 3 to 7 times heavier than those in lean rats; adipocyte numbers in RP depots were 50% greater in obese than in lean rats; and cell size was more than 3 times larger. IBAT, liver, and spleen were also heavier in obese vs. lean rats, while tail lengths were shorter. Percent lean carcass mass and % carcass protein were about 30% greater in lean vs. obese rats, while % carcass fat in obese rats was 5 times greater than that of lean rats. Thus, phenotypic expression of the fa gene in ZBN hybrid animals, with approximately 25% of their genetic background coming from the BN strain, appears to be similar to that in Zucker rats. Given the similarity of phenotypic expression of the fa gene between the Zucker strain and ZBN hybrids, it is plausible to consider using ZBN hybrids for studies of early manifestations of fa gene action prior to onset of detectable obesity .     

Hypothalamic Monoaminergic Activity in 11-Week-Old Cold-Exposed Female Lean (Fa/Fa) and Obese (fa/fa) Zucker Rats

We previously reported that serotonergic activity was reduced in the ventromedial hypothalamic nucleus (VMN) of obese vs. lean male Zucker rats. To verify that this reduction was associated with genotype rather than gender, we measured monoamines and their major metabolites in hypothalamic nuclei of ll-week-old female lean (Fa/Fb) and obese (fa/fb) Zucker rats. In addition, since the thermic response to cold is reported to differ between lean and obese rats, some rats were also exposed to 9° or 22° C for 2h to determine if cold exposure altered hypothalamic monoaminergic activity. As in males, levels of 5-hydroxyindoleacetic acid [5-HIAA; major metabolite of serotonin (5-HT)] and the ratio of 5-HIANS-HT were lower in the VMN of obese vs. lean females (P = 0.008, 0.001, respectively). S-HIANS-HT was also reduced in the paraventricular (PVN) and suprachiasmatic nuclei (SCN) of the obese compared to the lean females. Cold exposure significantly stimulated brown fat mitochondria1 GDP binding in lean but not obese rats. Similarly, levels of norepinephrine, dopamine (DA), 5-HIAA, and 5-HT in the PVN, and 5-HIAA in the SCN increased in cold-exposed lean but not obese rats. In contrast, VMN and preoptic 3,4-dihydroxyphenylacetic acid (DOPAC; major metabolite of DA) increased in the cold-exposed obese but not lean animals. We conclude that: (1) the blunted peripheral response to cold in obese vs. lean Zucker rats is accompanied by altered hypothalamic monoaminergic activity, the physiological role of which needs further evaluation; and 2) depressed VMN serotonergic activity is associated with the obese genotype (fa/fa) rather than gender and as such may contribute to the reduced sympathetic and enhanced parasympathetic outflow from the VMN .     

A Commentary on Multiple Screening for Aminoacidopathies in the Newborn Infant

Multiple screening for aminoacidopathies has been attempted with a simple micromethod (Lancet, 2: 230, 1964) requiring only 10 μl. of plasma collected in a capillary tube and processed by a chromatographic technique. In a survey of 1250 infants, two newborn infants with phenylketonuria, two older infants with hypermethioninemia-tyrosinemia and hepatic cirrhosis were found, and approximately 10% of the premature infants had marked hypertyrosinemia. Multiple screening therefore has the advantage of multiple diagnosis over other techniques which screen specifically for a single disease. Pilot screening projects with this and other techniques could improve our knowledge of heritable metabolic disease so that mass screening for such problems may eventually be justifiable.     

With no gap to mind: a shallow genealogy within the world's most widespread small pelagic fish

Species‐delimitation studies across wide geographic ranges often reveal insights that ultimately improve our understanding of biogeographic and evolutionary processes. Here we investigated species delimitation and the global coastal pelagic population structures of the marine sardine species from the economically important subgenus Sardinella (Clupeidae). The main purpose of this study was to relate morphological and genetic discontinuities to biogeography, in a taxonomic and systematic context. Morphological examinations have first reduced the currently recognized five species of the subgenus to two distinct morphospecies with parapatric relationship. Genetic analyses further showed a remarkable shallow genealogy across a global scale, yet to be encountered among small pelagic fishes. Additional three species‐delimitation analyses have failed to delimit the five putative species, indicating the possible existence of only a single cosmopolitan species with two ecophenotypic variations, thus entitling Sardinella aurita as the world's most widespread small pelagic fish. Subsequent population‐structure investigations revealed distinct geographical intraspecific sub‐divisions, flagging the West Pacific Ocean through gene‐flow computations as the probable source of future speciation for the subgenus. Considering its utmost importance to fisheries, this finding of a remarkable global genetic homogeneity should attract future attention among population geneticists and fishery researchers.     

Arbuscular mycorrhizal fungi associated with <Emphasis Type="Italic">Leptospermum scoparium</Emphasis> (mānuka): effects on plant growth and essential oil content

Leptospermum scoparium or mānuka is a New Zealand native medicinal plant that produces essential oils with antimicrobial properties. This study investigated the arbuscular mycorrhizal fungi (AMF) community in mānuka by culture dependent (trap culture) and independent (denaturing gradient gel electrophoresis) approaches. Furthermore, to assess whether mycorrhizal inoculation could alter growth and essential oil composition of mānuka, plants of a single regional chemotype were grown in unsterilized soil and inoculated with five AMF isolates. Leaf essential oil compositions and yields were determined by microscale solvent extraction and gas chromatography-mass spectrometry (GC-MS) analysis. AMF inoculation significantly increased growth compared to uninoculated plants. Qualitative i.e. different relative proportions of compounds, which are distinctive in chemotypes and quantitative (i.e. absolute concentrations of compounds, expressed as mg/g of dry leaf or equivalent) effects of AMF inoculation on mānuka essential oil composition depended on the isolate. AMF inoculation modified the Gammaproteobacterial community on roots and this may have contributed to changes in essential oil composition. Overall, these results demonstrated that AMF can improve the growth of mānuka and affect plant secondary metabolites in leaves, which would be valuable in commercial essential oil production from plantation-grown mānuka.