Broadband homonuclear chemical shift correlation at high MAS frequencies: a study of tanh/tan adiabatic RF pulse schemes without $${^{{\bf 1}}\hbox{{\bf H}}}$$ decoupling during mixing

At high magic angle spinning (MAS) frequencies the potential of tanh/tan adiabatic RF pulse schemes for ¹³C chemical shift correlation without ¹H decoupling during mixing has been evaluated. It is shown via numerical simulations that a continuous train of adiabatic ¹³C inversion pulses applied at high RF field strengths leads to efficient broadband heteronuclear decoupling. It is demonstrated that this can be exploited effectively for generating through-bond and through-space, including double-quantum, correlation spectra of biological systems at high magnetic fields and spinning speeds with no ¹H decoupling applied during the mixing period. Experiments carried out on a polycrystalline sample of histidine clearly suggest that an improved signal to noise ratio can be realised by eliminating ¹H decoupling during mixing.     

Evidence for mouse sulfhydryl oxidase‐assisted cross‐linking of major seminal vesicle proteins

Copulatory plug formation in animals is a general phenomenon by which competition is reduced among rival males. In mouse, the copulatory plug formation results from the coagulation of highly viscous seminal vesicle secretion (SVS) that is rich in proteins, such as dimers of SVS I, SVS I + II + III, and SVS II. These high‐molecular‐weight complexes (HMWCs) are also reported to be the bulk of proteins in the copulatory plug of the female mouse following copulation. In addition, mouse SVS contributes to the existence of sulfhydryl oxidase (Sox), which mediates the disulfide bond formation between cysteine residues. In this study, flavin adenine dinucleotide (FAD)‐dependent Sox was purified from mouse SVS using ion exchange and high‐performance liquid chromatography. The purified enzyme was identified to be Sox, based on western blot analysis with Sox antiserum and its capability of oxidizing dithiothreitol as substrate. The pH optima and thermal stability of the enzyme were determined. Among the metal ions tested, zinc showed an inhibitory effect on Sox activity. A prosthetic group of the enzyme was identified as FAD. The K_m and V_max of the enzyme was also determined. In addition to purification and biochemical characterization of seminal vesicle Sox, the major breakthrough of this study was proving its cross‐linking activity among SVS I–III monomers to form HMWCs in SVS.     

Rooting and shooting: dual function of thidiazuron in in vitro regeneration of soybean (<Emphasis Type="Italic">Glycine max.</Emphasis> L)

Thidiazuron (TDZ), primarily a cotton defoliant, has been later accepted as a plant growth regulator. In spite of extensive studies, the physiological function of TDZ is still uncertain. The aim of the present experiment was to study the activity of TDZ in in vitro regeneration of soybean. The seeds of soybean were cultured separately on MS and B5 medium supplemented with TDZ. The hypocotyls, cotyledons, cotyledonary nodes without axillary buds and cotyledonary nodes with axillary buds were used as explants and their capacity to direct regeneration was tested on both MS and B5 media containing TDZ (0.9–5.4 μM). Shoot formation was observed only on cotyledonary nodes with axillary buds cultured on MS and B5 basal media with TDZ (0.9–5.4 μM). All tested explants cultured on B5 medium with TDZ produced roots. Root formation was not observed on MS basal media supplemented with TDZ. Results show that TDZ functions as cytokinin (to produce the shoots) and auxin (to produce the roots) on various explants depending on the basal medium used.     

Highly-restricted, cell-specific expression of the simian CMV-IE promoter in transgenic zebrafish with age and after heat shock

Promoters with high levels of ubiquitous expression are of significant utility in the production of transgenic animals and cell lines. One such promoter is derived from the human cytomegalovirus immediate early (CMV-IE) gene. We sought to ascertain if the simian CMV-IE promoter (sCMV), used extensively in non-mammalian vertebrate research, also directs intense, widespread expression when stably introduced into zebrafish. Analysis of sCMV-driven expression revealed a temporal and spatial pattern not predicted by studies using the hCMV promoter in other transgenic animals or by observations of early F0 embryos expressing injected sCMV-reporter plasmids. Unexpectedly, in transgenic fish produced by both integration of linearized plasmid or Tol2-mediated transgenesis, sCMV promoter expression was generally observed in a small population of cells in telencephalon and spinal cord between days 2 and 7, and was thereafter confined to discrete regions of CNS that included the olfactory bulb, retina, cerebellum, spinal cord, and lateral line. In skeletal muscle, intense transgene expression was not observed until well into adulthood (>2-3 months post-fertilization). One final unexpected characteristic of the sCMV promoter in stable transgenic fish was tissue-specific responsiveness of the promoter to heat shock at both embryonic and adult stages. These data suggest that, in the context of stable transgenesis, the simian CMV-IE gene promoter responds differently to intracellular regulatory forces than other characterized CMV promoters.     

Variants in the CNR1 and the FAAH Genes and Adiposity Traits in the Community

Pharmacologic blockade of the endocannabinoid receptor 1 leads to weight loss and an improved metabolic risk profile in overweight and obese individuals. We hypothesize that common genetic variants in the CNR1 (encoding endocannabinoid receptor 1) and FAAH genes (encoding fatty acid amide hydrolase, a key enzyme hydrolyzing endocannabinoids) are associated with adiposity traits. We genotyped 18 single‐nucleotide polymorphisms (SNPs) in the CNR1 gene and 9 SNPs in the FAAH gene in 2,415 Framingham Offspring Study participants (mean age 61 ± 10 years; 52.6% women; mean BMI 28.2 ± 5.4 kg/m²; 30.3% obese) and studied them for association with cross‐sectional and longitudinal measures of adiposity (BMI, waist circumference, change over time in BMI and waist circumference, visceral and subcutaneous adipose tissue) using linear mixed‐effect models. The selected SNPs captured 85% (r² = 0.8) of the common variation (minor allele frequency >5%) at the CNR1 locus and 96% (r² = 0.8) of the common variation at the FAAH locus (defined as the genomic segment containing the gene +20 kb upstream and +10 kb downstream). After correction for multiple testing, none of the SNPs in the CNR1 gene or in the FAAH gene displayed statistical evidence for association with BMI, waist circumference, and visceral adipose tissue or subcutaneous adipose tissue (all P > 0.18). Despite comprehensive SNP mapping across the genes and their regulatory regions in a large unselected sample, we failed to find evidence for an association of common variants in the CNR1 and FAAH genes with measures of adiposity in our community‐based sample.     

Molecular cloning,overexpression and characterization of the raw-starch-digesting <Emphasis Type="Italic">α</Emphasis>-amylase of <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis>

Raw starch is the most abundant source of glucose in the world. Therefore, finding enzymes capable of digesting raw starch would find high industrial demand. The α-amylase gene of Bacillus amyloliquefaciens ATCC 23842 was amplified, cloned and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant enzyme was purified to apparent homogeneity using ion exchange and gel filtration chromatography. The raw-starch digestibility of the purified enzyme was characterized by studying the hydrolysis and adsorption rate on a variety of raw starches (potato, cassava, corn, wheat and rice). The raw-starch digestion was further confirmed by scanning electron microscopy studies, which revealed an effective rate of hydrolysis. The kinetic studies revealed a relatively low K _m of 2.76 mg/mL, exhibiting high affinity towards the soluble starch as the most preferred substrate and the inhibition kinetic studies revealed a high K _i value (350 mM).