A spin label study of immobilized enzyme spectral subpopulations

Electron spin resonance (ESR) spin label studies have been carried out to examine the active site conformation of alpha-chymotrypsin before and after immobilization on two types of organic polymer supports: Amberlite XAD-8 and XAD-2. alpha-Chymotryspin was first chemically modified by reaction with methyl-4-phenylbutyrimidate and then inhibited by the active site spin label 4-(2,2,6,6-tetramethyl-piperdine-1-oxyl)-m-flurosulfonylbenzamide. In general, the ESR spectra of the active site lable revealed no significant changes in conformation for most of the enzyme before or after derivatization. On the other hand, two spectral subpopulations (A and B) of spin-labeled enzyme were characterized on the basis of their ESR spectra after immobilization on Amberlite XAD-8. Spectral subpopulation A (distinguished by a highly restrained spectrum) appeared to retain its active site structure and conformation and represented a large majority of the labeled chymotrypsin on the beads. Its presence correlated with the high activity and stability of phenylbutyramidinated chymotryspin on the Amberlite XAD-8 beads. Spectral subpopulation B (distinguished by a very weakly constrained spectrum) appeared to reflect loosely bound or denatured enzyme which was removable upon washing with 40% (v/v) ethylene glycol. Two methods for examining solvent accessibility to the active site lable of the kinetics of ascorbate reduction suggested that both spectral subpopulations had identical accessibilities to the bulk solvent. Paramagnetic broadening of the signal by K(3)Fe(CN)(6) revealed differences in the spin-spin broadening of the A and B components but is deemed and inappropriate indicator of solvent accessibility.     

Protection against acute paraquat toxicity by ambroxol

An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 g/ml/day using a 25 cm² tissue culture flask, even though the cell number was above 7×10⁵ cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter^TM (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 3 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1×10⁷ cells/ml), and the amount of G-CSF reached 41 g/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.Abbreviations ABTS 2,2-Azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid - BSA Bovine Serum Albumin - BSA-PBS Phosphate-buffered Saline without Ca²⁺ and Mg²⁺ containing Bovine Serum Albumin - dhfr Dihydrofolate Reductase - DO Dissolved Oxygen - G-CSF Granulocyte Colony-stimulating Factor - HEPES 4-(2-Hydroxyethyl)-1-piperazineethansulfonic Acid - IFN Interferon - MTX Methotrexate - PBS(-) Phosphate-buffered saline without Ca²⁺ and Mg²⁺ - Tween-PBS Phosphate-buffered saline without Ca²⁺ and Mg²⁺ containing 0.05% of Tween 20     

Influence of clay minerals on sorption of bacteriolytic enzymes

Myxobacteria presumably produce extracellular bacteriolytic enzymes when they are growing in soil. In order to study their ecological significance, adsorption experiments were performed with lytic enzymes produced byMyxococcus virescens in casitone media. Different soils as well as montmorillonite and kaolinite can rapidly adsorb the bacteriolytic but not the proteolytic enzymes. About 1 gm of montmorillonite per liter of cell-free culture solution is enough for the adsorption of 97% of the bacteriolytic enzymes. The adsorption per unit weight is about 100 times greater on montmorillonite than on kaolinite. About 40% of the adsorbed enzymes can be eluted with solutions of high pH or high ionic strength. The only desorbed bacteriolytic enzyme is the alanyl-∈-N-lysine endopeptidase.     

Distinct basolateral amygdala excitatory inputs mediate the somatosensory and aversive-affective components of pain

Pain is a multidimensional perception that includes unpleasant somatosensory and affective experiences; however, the underlying neural circuits that mediate different components of pain remain elusive. Although hyperactivity of basolateral amygdala glutamatergic (BLA^Glu) neurons is required for the somatosensory and emotional processing of pain, the precise excitatory inputs to BLA^Glu neurons and their roles in mediating different aspects of pain are unclear. Here, we identified two discrete glutamatergic neuronal circuits in male mice: a projection from the insular cortex glutamatergic (IC^Glu) to BLA^Glu neurons, which modulates both the somatosensory and affective components of pain, and a projection from the mediodorsal thalamic nucleus (MD^Glu) to BLA^Glu neurons, which modulates only the aversive-affective component of pain. Using whole-cell recording and fiber photometry, we found that neurons within the IC→BLA and MD→BLA pathways were activated in mice upon inflammatory pain induced by injection of complete Freund’s adjuvant (CFA) into their paws. Optical inhibition of the IC^Glu→BLA pathway increased the nociceptive threshold and induced behavioral place preference in CFA mice. In contrast, optical inhibition of the MD^Glu→BLA pathway did not affect the nociceptive threshold but still induced place preference in CFA mice. In normal mice, optical activation of the IC^Glu→BLA pathway decreased the nociceptive threshold and induced place aversion, while optical activation of the MD^Glu→BLA pathway only evoked aversion. Taken together, our results demonstrate that discrete IC^Glu→BLA and MD^Glu→BLA pathways are involved in modulating different components of pain, provide insights into its circuit basis, and better our understanding of pain perception.     

Staining of Some Specific Regions of Human Chromosomes,particularly the Secondary Constriction of No. 9

SEVERAL procedures have been described recently which produce specific patterns of differential staining in human chromosomes^1–9. Techniques which involve DNA denaturation and reannealing reveal deeply stained areas on centromere and secondary constriction regions which have been equated with constitutive heterochromatin⁹.     

The mode of action of thidiazuron: auxins,indoleamines, and ion channels in the regeneration of <Emphasis Type="Italic">Echinacea purpurea</Emphasis> L.

The biochemical mechanisms underlying thidiazuron (TDZ)-induced regeneration in plant cells have not been clearly elucidated. Exposure of leaf explants of Echinacea purpurea to a medium containing TDZ results in undifferentiated cell proliferation and differentiated growth as mixed shoot organogenesis and somatic embryogenesis. The current studies were undertaken to determine the potential roles of auxin, indoleamines, and ion signaling in the dedifferentiation and redifferentiation of plant cells. E. purpurea leaf explants were found to contain auxin and the related indoleamine neurotransmitters, melatonin, and serotonin. The levels of these endogenous indoleamines were increased by exposure to TDZ associated with the induction of regeneration. The auxin-transport inhibitor 2,3,5-triiodobenzoic acid and auxin action inhibitor, p-chlorophenoxyisobutyric acid decreased the TDZ-induced regeneration but increased concentrations of endogenous serotonin and melatonin. As well, inhibitors of calcium and sodium transport significantly reduced TDZ-induced morphogenesis while increasing endogenous indoleamine content. These data indicate that TDZ-induced regeneration is the manifestation of a metabolic cascade that includes an initial signaling event, accumulation, and transport of endogenous plant signals such as auxin and melatonin, a system of secondary messengers, and a concurrent stress response.     

Evolution of resident bird breeding phenology in a landscape with heterogeneous resource phenology and carryover effects

It is generally expected that, in environments with pronounced seasonal resource peaks, birds’ reproductive success will be maximised when nestlings’ peak food demand coincides with the timing of high food availability. However in certain birds that stay resident over winter, earlier breeding leads juveniles to join the winter flock earlier, which by the prior residence effect increases their success in breeding territory competition. This trade-off between reproduction and competition may explain why, in certain species, breeding phenology is earlier and asynchronous with the resource. This study extends a previous model of the evolution of breeding phenology in a single habitat type to a landscape with two habitat types: ‘early’ and ‘late’ resource phenology. The offspring’s natal habitat type has a carryover effect upon their competitive ability regardless of which habitat type they settle in to potentially breed. We find that, when the difference in resource phenology between habitats is small (weak carryover effect), breeding phenology in the late habitat evolves to occur earlier and more asynchronously than in the early habitat, to compensate for the competitive disadvantage to juveniles raised there. However if the difference is large (strong carryover effect), then the reproductive cost of earlier breeding outweighs the benefit of the compensation, so instead breeding phenology in the late habitat evolves to become more synchronous with the resource. Recruitment is generally asymmetric, from early to late habitat type. However if the early habitat is less frequent in the landscape or produces fewer offspring, then the asymmetry is reduced, and if there is some natal habitat-type fidelity, then recruitment can have an insular pattern, i.e. most recruits to each habitat type come from that same habitat type. We detail the different scenarios in which the different recruitment patterns are predicted, and we propose that they have implications for local adaptation.     

Auxin-like activity of extract from hypertrophied tissue of <Emphasis Type="Italic">Acacia eburnea</Emphasis> infected with <Emphasis Type="Italic">Ravenelia esculenta</Emphasis>

Ravenelia esculenta Naras. and Thirum. is a rust, pathogenic to Acacia eburnea Willd. The infection leads to hypertrophy changing the morphology with bizarre shapes of plant organs. Healthy and infected tissues were subjected to extraction of IAA and indole derivatives and were estimated by spectrophotometric methods. The hypertrophy produced was presumed to be due to increase in the indole-3-acetic acid (IAA) content in the infected tissue, however, the amount of IAA in infected tissues decreased with the progression of disease. Concomitantly, the infected tissue showed the presence of a novel, slow migrating, indole derivative on TLC. Cultured shoot tips of Withania somnifera were dosed with the methanolic extract of the infected hypertrophied tissue (MEHT) (0.25, 0.5, 0.75, 1.00 and 1.25 mg/l). The stimulation in shoot growth along with profuse rooting was observed in a dose dependent manner with maximum at 1.00 and 1.25 mg/l concentration.