Enzymatic and chemical properties of an endopeptidase from the larva of the hornet Vespa crabro.

An endopeptidase from the larvae of the hornet Vespa crabro has been purified to homogeneity. The enzyme has been characterized with respect to molecular weight, amino acid compositon, and amino- and carboxyl-terminal sequences. The catalytic properties of the hornet protease are similar to those of bovine chymotrypsin with respect to inactivation by phenylmethanesulfonyl fluoride and carbobenzoxyphenylalanine chloro ketone and preferential peptide bond cleavage at aromatic amino acid residues. In contrast to bovine chymotrypsin, the hornet protease is not inhibited by the basic pancreatic Kunitz inhibitor, soybean inhibitor, or chicken ovomucoid. The molecular weight, as determined by several independent methods, was found to be 14 500. The protease is a single-chain protein containing two disulfide bonds. The terminal sequences are: NH2-Ile-Val-Gly-Gly-Ile-Asp.....Gly-Lys-Tyr-Pro-Tyr-Gln-Val-Ser-Leu-Arg-COOH.     

Intracellular pH of Thermoplasma acidophila

Thermoplasma acidophila, a mycoplasma-like organism, was grown at 56 °C and pH 2. The intracellular pH of this organism is close to neutral as measured by the distribution of a radioactive weak organic acid, 5,5-dimethyl-2,4-oxazolidinedione, across the plasma membrane. The cell can maintain the pH gradient when subjected to heat or to metabolic inhibitors. Our experiments seem to indicate that a major portion of the pH gradient is not maintained by active processes, but rather by a Donnan potential across the cell membrane.     

不同胁迫预处理提高水稻幼苗抗寒性期间蛋白质的变化

水稻(Oryza sativa L.)幼苗经盐、热激和冷三种不同胁迫预处理均提高了幼苗的抗寒性。与未预处理苗相比,在处理后、低温伤害后和常温下恢复2d的三个时期,不同胁迫预处理苗的可溶性和热不稳定蛋白含量变化趋势甚为相似,但热稳定蛋白含量变化则各有异同。SDS-PAGE图谱分析显示,不同胁迫预处理提高水稻幼苗抗寒性时,其可溶性蛋白、热稳定和热不稳定蛋白组成变化亦各有异同。除诱导出共有的新多肽外,还各自诱导出特有的新多肽。结果表明,植物对不同胁迫的交叉适应存在一定的共同机理,但亦可看出植物对同一种环境胁迫似乎不是以同一的机理去适应。     

Nanomoulding of Functional Materials,a Versatile Complementary Pattern Replication Method to Nanoimprinting

We describe a nanomoulding technique which allows low-cost nanoscale patterning of functional materials, materials stacks and full devices. Nanomoulding combined with layer transfer enables the replication of arbitrary surface patterns from a master structure onto the functional material. Nanomoulding can be performed on any nanoimprinting setup and can be applied to a wide range of materials and deposition processes. In particular we demonstrate the fabrication of patterned transparent zinc oxide electrodes for light trapping applications in solar cells.     

Evolution of insect wings and development – new details from Palaeozoic nymphs

The nymphal stages of Palaeozoic insects differ significantly in morphology from those of their modern counterparts. Morphological details for some previously reported species have recently been called into question. Palaeozoic insect nymphs are important, however – their study could provide key insights into the evolution of wings, and complete metamorphosis. Here we review past work on these topics and juvenile insects in the fossil record, and then present both novel and previously described nymphs, documented using new imaging methods. Our results demonstrate that some Carboniferous nymphs – those of Palaeodictyopteroidea – possessed movable wing pads and appear to have been able to perform simple flapping flight. It remains unclear whether this feature is ancestral for Pterygota or an autapomorphy of Palaeodictyopteroidea. Further characters of nymphal development which were probably in the ground pattern of Pterygota can be reconstructed. Wing development was very gradual (archimetaboly). Wing pads did not protrude from the tergum postero‐laterally as in most modern nymphs, but laterally, and had well‐developed venation. The modern orientation of wing pads and the delay of wing development into later developmental stages (condensation) appears to have evolved several times independently within Pterygota: in Ephemeroptera, Odonatoptera, Eumetabola, and probably several times within Polyneoptera. Selective pressure appears to have favoured a more pronounced metamorphosis between the last nymphal and adult stage, ultimately reducing exploitation competition between the two. We caution, however, that the results presented herein remain preliminary, and the reconstructed evolutionary scenario contains gaps and uncertainties. Additional comparative data need to be collected. The present study is thus seen as a starting point for this enterprise.     

Perfusion system for studying peptide secretion from endocrine cells with high time resolution

To study the secretion from endocrine cells in culture, we have developed a cell column perfusion system with a time resolution of 4 s. The core of the system is a perfusion chamber with a cell-supporting matrix of monosized polystyrene beads. The particles are solid and can withstand a high pressure gradient without deformation. The minimum amount of cell material required to obtain detectable levels of secretory products is a function of the assay sensitivity, perfusion flow, fraction volume and time resolution. The volume of the perfusion chamber is therefore adjustable to satisfy varying demands of minimum cell number. The general flow characteristics of the system were characterized using radiolabeled substances of various molecular sizes. Using the system in secretory studies of rat pituitary tumor (GH4C1) cells, we have identified differences in secretion profiles that may be related to the kinetics of the different transmembrane and intracellular mechanisms involved.     

Degradation of the type I inositol 1,4,5-trisphosphate receptor by caspase-3 in SH-SY5Y neuroblastoma cells undergoing apoptosis

The type I inositol 1,4,5-trisphosphate (IP(3)) receptor is selectively down-regulated in several neurodegenerative diseases, including Alzheimer's disease, Huntington's chorea, and ischemia, all conditions in which apoptotic neuronal loss occurs. In the present study, we used a neuronal cell line, human neuroblastoma SH-SY5Y cells, to investigate whether the levels of IP(3) receptor are changed during apoptosis in these cells. Following induction of apoptosis by staurosporine, the immunoreactivity of the type I IP(3) receptor in microsome preparations from SH-SY5Y cells was reduced within 2 h, with a further reduction during subsequent hours. Immunoblot analyses, using antibodies to poly(ADP-ribose) polymerase and spectrin breakdown products, revealed proteolysis of these caspase-3 substrates within 3 h, confirming that IP(3) receptor cleavage is an early consequence of apoptosis. In vitro incubation of SH-SY5Y microsomes or immunopurified IP(3) receptor from rat cerebellum with recombinant caspase-3 led to generation of immunoreactive breakdown products similar to those observed in intact cells, suggesting that the type I IP(3) receptor is a potential substrate for caspase-3. Preincubation of the neuroblastoma cells with the caspase-3 inhibitor Z-Asp-Glu-Val-Asp-fluoromethyl ketone prevented IP(3) receptor degradation. These results show that the type I IP(3) receptor is a substrate for caspase-3 in neuronal cells and indicate that apoptotic down-regulation of IP(3) receptor levels may contribute to the pathology of neurodegenerative conditions.     

Molecular determinants of KATP channel inhibition by ATP.

ATP-sensitive K+ (KATP) channels are both inhibited and activated by intracellular nucleotides, such as ATP and ADP. The inhibitory effects of nucleotides are mediated via the pore-forming subunit, Kir6.2, whereas the potentiatory effects are conferred by the sulfonylurea receptor subunit, SUR. The stimulatory action of Mg-nucleotides complicates analysis of nucleotide inhibition of Kir6. 2/SUR1 channels. We therefore used a truncated isoform of Kir6.2, that expresses ATP-sensitive channels in the absence of SUR1, to explore the mechanism of nucleotide inhibition. We found that Kir6.2 is highly selective for ATP, and that both the adenine moiety and the beta-phosphate contribute to specificity. We also identified several mutations that significantly reduce ATP inhibition. These are located in two distinct regions of Kir6.2: the N-terminus preceding, and the C-terminus immediately following, the transmembrane domains. Some mutations in the C-terminus also markedly increased the channel open probability, which may account for the decrease in apparent ATP sensitivity. Other mutations did not affect the single-channel kinetics, and may reduce ATP inhibition by interfering with ATP binding and/or the link between ATP binding and pore closure. Our results also implicate the proximal C-terminus in KATP channel gating.