Peptide recognition by PTB and PDZ domains

Protein tyrosine binding (PTB) and ‘post synaptic density disc-large zo-1’ (PDZ) domains bind to short peptidic ligands by augmentation of one of the domain's β sheets and other recognition mechanisms. The two domain classes have a superficial resemblance to each other, even though no sequential homology exists. The structural bases of the interactions are well understood for the domains now experimentally determined, and ligand—target pairs can probably be identified in favorable cases by analogy with the known domains. For both PTB and PDZ classes, functional activities are still not fully defined: it is possible that these domain classes, along with pleckstrin homology domains, have multiple roles.     

Calpain: A Cytosolic Proteinase Active at the Membranes

Conclusion  Membrane association is essential for GRK function and because of this the GRKs have evolved complex regulatory mechanisms for associating with the membrane. Although the GRKs are highly homologous, each kinase utilizes a distinct mechanism for associating with the membrane, which makes it unique within the family. Initially, the carboxyl terminus of the GRKs was identified as the “membrane association domain” but recent evidence suggests that the amino terminus may also play a critical role in localizing the kinases to the membrane (Murga et al., 1996; Pitcher et al, 1996). It is within these two domains that the GRKs are most variable at the amino acid level. The GRKS exhibit an absolute requirement for phospholipids not only for association with the membrane but also for activity. There are differences in preference and binding sites for the phospholipids within the GRK family, which may reflect differential targeting of the GRKs to G protein-coupled receptors situated in different lipid environments. There are hundreds of G protein-coupled receptors and only six known GRKs. All the GRKs appear to phosphorylate the same receptor substrates in vitro (Sterne-Marr & Benovic, 1995; Premont et al., 1995). Receptor specificity, in a cellular     

BCL-2-Related Protein Expression in Apoptosis: Oxidative Stress Versus Serum Deprivation in PC12 Cells

Abstract: Expression of the BCL-2 protein family members, BAX, BAK, BAD, BCL-xL, BCL-xS, and BCL-2, was measured (by western blotting using specific antibodies) in PC12 cells before and during apoptosis induced by either H₂O₂ treatment or by serum deprivation and during rescue from apoptosis by nerve growth factor (NGF). H₂O₂-induced apoptosis, as measured by DNA fragmentation, caused: (a) a dose-dependent increase in BAX, (b) a dose-independent increase in BAK, and (c) a dose-dependent inhibition of BAD expression. By comparison, apoptosis induced by serum deprivation resulted in a time-dependent decrease in both BAX and BAK, along with a dramatic and sudden decrease in BAD expression. However, when PC12 cells were incubated in an apoptosis-sparing medium (i.e., NGF-supplemented serum-free medium), both BAX and BAK were increased significantly, whereas BAD expression remained inhibited. BCL-xL expression was increased by H₂O₂ but unaffected by serum deprivation or long-term NGF treatment. Neither BCL-2 nor BCL-xS expression could be detected in PC12 cells under the experimental conditions tested. Our results show that the expression of BAX, BAK, BAD, and BCL-xL is altered in a stimulus-dependent manner but cannot be used to define whether a cell will undergo or survive apoptosis. The similarity between changes in expression of BCL-2-related proteins induced by H₂O₂ exposure and NGF rescue could reflect activation in part of a common antioxidant pathway.     

A novel approach to the analysis of the initiation of embryo development in gramineae

An in vivo model system to study the initiation of embryo development is presented. From the so-called Salmon system of wheat (alloplasmic lines with a 1BL-1RS chromosome translocation), three completely isogenic and homozygous lines were produced by selection for uniformity in about 20 selfing/backcross generations as well as between sublines of doubled haploids. The line (aestivum)-Salmon is male fertile and sexual. The lines (caudata)-Salmon and (kotschyi)-Salmon are male sterile and have a parthenogenetic capacity of about 90%. The expression of nuclear-cytoplasmic male sterility is different for the two parthenogenetic lines. The initiation of autonomous embryo development at defined developmental stages of the ovaries and the maximum degree of parthenogenesis are identical in both parthenogenetic lines as proved by the auxin test and progeny analyses. The protein patterns from ovary extracts of the three isogenic lines were identical for more than 200 spots of 2-D polyacrylamide gels, confirming their homogeneity. However, one protein (P 115.1) was found 3 days before and during anthesis only in ovaries of the parthenogenetic lines. It seems to be involved in the initiation of parthenogenesis.     

Calcium fluxes at plasmalemma and tonoplast

Abstract. An account is given of the characterization of calcium fluxes across plasmalemma and tonoplast membranes of root cortical cells, using compartmental analysis. Some of the assumptions associated with the method are discussed. Recent evidence regarding the concentration of free Ca²⁺ in plant cells, and the mechanisms driving active calcium transport across cell membranes, is reviewed. It is proposed that the evidence from whole cell studies and work at the molecular level is mutually supportive, and some speculation is ventured about the general pattern of calcium transport in higher plant cells.     

Evidence for intracellular formation of angiotensins: coexistence of renin and angiotensin-converting enzyme in Leydig cells of rat testis

Leydig cells were purified from rat testes by discontinuous metrizamide density gradient and were shown to contain renin (EC 3.4.99.1), angiotensin-converting enzyme (dipeptidyl carboxypeptidase, (EC 3.4.15.1), and the peptide hormone angiotensins I, II and III as determined by the combined HPLC and radioimmunoassay. In germinal cells only angiotensin II (AII) was found at a significant level. These findings provide evidence for intracellular formation of AII in testicular cells and demonstrate that an intracellular renin-angiotensin system exists in normal non-transformed cells.     

Brain CCK receptors are structurally distinct from pancreas CCK receptors

Brain and pancreas cholecystokinin (CCK) receptors differ markedly in their selectivity for CCK analogs. To determine the size and subunit structure of the brain CCK receptor and compare it to that of the pancreas, 125I-CCK33 was covalently cross-linked with ultraviolet light to its receptor on mouse brain particles and purified pancreatic plasma membranes. When CCK was crosslinked to brain membranes, a single consistent major labeled protein band of Mr = 55,000 was observed in both the presence and the absence of DTT. These data with brain receptors contrast to results with pancreatic receptors where two bands of Mr = 120,000 and 80,000 are labeled in the absence and presence of DTT, respectively. These studies indicate, therefore, that the brain and pancreas CCK receptors are structurally and functionally distinct.     

Resolution of multiple fluorescence lifetimes in heterogeneous systems by phase-modulation fluorometry

Procedures are described for the treatment of phase and modulation lifetime data in fluorescent systems having multiexponential decay. All computer procedures (called FIT programs) arise from the lifetime resolution theory for phase-modulation measurements (Weber, G (1981) J. Phys. Chem. 85, 949–953). The programs most successful in resolving heterogeneous lifetimes use a Monte Carlo approach in which phase and modulation lifetime data at three modulation frequencies are simultaneously utilized. These programs are shown to have more utility than the final closed form procedure presented by Weber (1981). The FIT routines are simple and require little computer time while yielding excellent results. To illustrate the applicability of these programs, defined binary (carbazole and pyrene) and ternary systems (carbazole, pyrene and POPOD) were examined. In most cases, the resolved lifetimes were within 5% of the independently measured value and the fractional fluorescence contributions were within 10% of that expected. These results demonstrate that phase-modulation measurements analyzed by appropriate computer programs are capable of solving for lifetimes in both binary and, in selected cases, ternary systems. An example is given from the recent literature (Dalbey, R., Weiel, J. and Yount, R.G. (1983) Biochemistry 22, 4696–4706) in which the above programs allowed the resolution of both binary and ternary lifetimes of a dansyl label on myosin, where Förster energy transfer was occuring. These lifetimes] were used to quantify changes in distances between two activity-related thiols on myosin upon the addition of Mg-ATP or its analogs.     

Field evaluation of symbiotic nitrogen fixation by rhizobial strains using15N methodology

Summary Small differences in N₂ fixation by nodulated soybeans (Glycine max. (L.) Merr.), inoculated with various strains ofRhizobium japonicum, were assessed in field experiments using¹⁵N methodology, and compared with yields of plant dry matter and total N. Percentage of plant-N derived from atmospheric N₂ and from fertilizer, and values of %¹⁵N atom excess had lower coefficients of variation than did total N and dry matter yield. Nevertheless the precision of estimates of kg N/ha fixed were sufficient to differentiate only the extremes of the range of strains tested, and there were discrepancies between ranking of strains based on % N derived from fertilizer and on total N yield.