Purification and characterization of the high molecular weight microtubule associated proteins from neonatal rat brain

The changes in the levels of microtubule-associated proteins (MAPs) during advanced embryonic stages, neonatal and adult organisms reflect the importance of these cytoskeletal proteins in relation to the morphogenesis of the central nervous system. MAP-1B is found in prenatal brains and it appears to have the highests levels in neonatal rat brains, being a developmentally-regulated protein. In this research, a fast procedure to isolate MAP-1B, as well as MAP-2 and MAP-3 from neonatal rat brains was designed, based on the differential capacity of poly L-aspartic acid to release MAPs during temperature-dependent cycles of microtubule assembly in the absence of taxol. The high molecular weight MAP-1B was recovered in the warm supernatants after microtubular protein polymerization in the presence of low concentrations of polyaspartic acid. Instead, MAP-2 and a 180 kDa protein with characteristics of MAP-3 remained associated to the polymer after the assembly. Further purification of MAP-1B was attained after phosphocellulose chromatography. Isolation of MAP-2 isoforms together with MAP-3 was achieved on the basis of their selective interactions with calmodulin-agarose affinity columns. In addition, MAP-2 and MAP-3 were also purified on the basis of their capacities to interact with the tubulin peptide -II (422–434) derivatized on an Affigel matrix. However, MAP-1B did not interact with the -II tubulin fragment, but it showed interaction with the Affigel-conjugated -I (431–444) tubulin peptide. The different MAPs componentes were characterized by western blots using specific monoclonal antibodies. A salient feature of neonatal rat brain MAP-3 was its interactions with site-directed antibodies that recognize binding epitopes on the repetitive sequences of tau and MAP-2. However, these site-specific antibodies did not interact with MAP-1B from the neonatal rat brain tissue.Abbreviations PAA poly (L-aspartic acid) - HMW-MAPs high molecular weight microtubule associated proteins     

Nonlinear effect of dynamic self-organization in macromolecular systems caused by photocontrolled electron flux

We use the electron-conformational interaction approach to develop a physical model which self-consistently describes the photomobilized electron transfer kinetics and structure conformational transitions in reaction centers (RCs) of purple bacteria. We consider the kinetics of electron transition from pigment onto primary acceptor and the subsequent charge recombination accounting for the change of distance between the above-mentioned cofactors. It is shown that, given natural values of RC parameters, the kinetic constant's dependence on the acting light intensity is monotone. As opposed to the previous case, similar dependencies for the chain of electron transfer between primary and secondary quinone acceptors revealed anS-like relationship. This can lead to bistability of the RC optical transmission coefficient and a fundamental dependence of charge recombination kinetics upon the prehistory of the RC's interaction with exciting radiation.     

QUANTITATIVE GENETICS OF RESPONSE TO COMPETITORS IN NEMOPHILA MENZIESII: A GREENHOUSE STUDY

To determine the potential for adaptation to a local biotic environment, we examined the magnitude and nature of genetic variation in response to neighboring plants within a natural population of the native California annual, Nemophila menziesii. A total of 22 plants from a natural population were crossed in three reciprocal factorials. The progeny were grown in a greenhouse in nine treatments that varied in conspecific density and in the density of a naturally co-occurring grass species, Bromus diandrus. Increasing the density of each species significantly reduced individual survival, fruit number, and dry weight. Among survivors, we found small to moderate heritability of dry weight within treatments. Additive genetic correlations (r_A) of dry weight between competitive regimes were generally large and positive. In no case were they significantly different from 1, as expected under the null hypothesis that the relative performance of the genotypes under consideration is the same in all environments. On the basis of these results, we cannot conclude that the structure of genetic covariation within this population would promote genetic differentiation in response to locally varying conditions of density of these two species. Aspects of the experiment that may have compromised our ability to detect r_A differing from 1 are discussed.     

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.     

The preservation of Mycoplasma capricolum cell intactness after phospholipase A2 treatment

(1) By treating Mycoplasma capricolum cells with phospholipase A₂ about 80% of membrane phospholipids were rapidly hydrolyzed. The rate and extent of hydrolysis (at 37°C) were the same in intact cells and in isolated unsealed membranes. (2) Due to the low endogenous lysophospholipase activity detected in M. capricolum, phospholipase A₂ treatment resulted in the accumulation of lysophospholipids and free fatty acids. The free fatty acids were efficiently extracted from the cells by 1% bovine serum albumin whereas the lysophospholipids were almost fully retained within the cell membrane. (3) Following phospholipase A₂ treatment in the presence of 1% bovine serum albumin, cell intactness was preserved as indicated by the constant absorbance of the cell suspension and the retention of nucleic acids and NADH dehydrogenase activity within the cells. The treated cells showed, however, a slight decrease in K⁺ content and a decrease in cell viability. Viability was fully preserved after phospholipase A₂ treatment of cells grown with exogenous sphingomyelin. (4) Adapting M. capricolum to a cholesterol-poor medium resulted in a marked decrease in the cholesterol to phospholipid molar ratio (from about 1.1 to 0.3). Phospholipase A₂ treatment of the cholesterol-poor cells resuted in cell lysis. Cell lysis was induced in the cholesterol-rich cells by hydrolysing the lysophospholipids accumulated following phospholipase A₂ treatment. (5) It is suggested that after phospholipase A₂ treatment of M. capricolum cells, a relatively stable cell membrane is maintained and cell intactness is preseved due to the interaction of cholesterol, present in high amount in this membrane, with the lysophospholipids formed.     

500 MHz H-NMR studies of bile salt-phosphatidylcholine mixed micelles and vesicles Evidence for differential motional restraint on bile salt and phosphatidylcholine resonances

¹H nuclear magnetic resonance (NMR) spectra at 500 MHz have been obtained for taurocholate/egg phosphatidylcholine mixtures of varying composition. The excellent chemical shift dispersion permits identification of most resonances for each component. This high-resolution character of the NMR spectra is retained until the phosphatidylcholine (PC) mole fraction exceeds 60–70% (the exact limit depends on ionic strength). ¹H linewidths have been monitored as a function of solute composition in order to evaluate trends in local molecular mobility of each component as the distribution of aggregate particles is varied, and to examine the effects of added NaCl in altering micellar size and shape. Although prior light scattering studies (Mazer, N.A., Benedek, G.B. and Carey, M.C. (1980) Biochemistry 19, 601–615) and our own work indicate a 6-fold increase in particle hydrodynamic radius from pure taurocholate micelles to 1 : 1 taurocholate/PC mixtures containing 150 mM NaCl, both lipid components retain substantial motional freedom and exhibit narrow NMR signals in this compositional region. As the solubilization limit for PC is approached (approx. 2:1 PC:taurocholate), differential behavior is observed for the two components: the motion of taurocholate becomes preferentially restricted, while polar portions of the PC remain mobile until large multilayers predominate.     

Interactions between immature porcine Leydig and Sertoli cells in vitro

Summary Interactions between Leydig and Sertoli cells, as well as a stimulatory effect of FSH on Leydig cell activity, have been reported in many studies. In order to investigate these interactions, the ultrastructure of immature pig Leydig cells under different culture conditions has been studied. When cultured alone in a chemically defined medium, there is a marked regression of the Leydig cell smooth endoplasmic reticulum and a swelling of the mitochondria. Addition of FSH or hCG does not prevent these phenomena. Co-culturing of Leydig cells with Sertoli cells from the same animal maintains the smooth endoplasmic reticulum at the level seen in vivo and in freshly isolated Leydig cells. The addition of FSH to the co-culture stimulates its development and increases Leydig cell activity, as assessed by an increase in hCG binding sites and an increased steroidogenic response to hCG. These results suggest that Sertoli cells exert a trophic effect on Leydig cells, and that the stimulatory effect of FSH on Leydig cell function is mediated via the Sertoli cells. These results reinforce the concept of a local regulatory control of Leydig cell steroidogenesis.Post-Doctoral fellow supported by CIRIT, Generalitat de Catalunya, Spain     

Interaction of plasma apolipoproteins with lipid monolayers

The monolayer technique has been used to study the interaction of lipids with plasma apolipoproteins. Apolipoprotein C-II and C-III from human very low density lipoproteins, apolipoprotein A-I from human high density lipoproteins and arginine-rich protein from swine very low density lipoproteins were studied. The injection of each apoprotein underneath a monolayer of egg phosphatidyl[¹⁴C]choline at 20 mN/m caused an increase in surface pressure to approximately 30 mN/m. With apolipoprotein C-II and apolipoprotein C-III there was a decrease in surface radioactivity indicating that the apoproteins were removing phospholipid from the interface; the removal of phospholipid was specific for apolipoprotein C-II and apolipoprotein C-III. Although there was a removal of phospholipid from the monolayer, the surface pressure remained constant and was due to the accumulation of apoprotein at the interface. The rate of surface radioactivity decrease was a function of protein concentration, required lipid in a fluid state and, of the lipids tested, was specific for phosphatidylcholine. Cholesterol and phosphatidylinositol were not removed from the interface. The addition of 33 mol% cholesterol to the phosphatidylcholine monolayer did not affect the removal of phospholipid by apolipoprotein C-III.The addition of phospholipid liposomes to the subphase greatly facilitated the apolipoprotein C-II-mediated removal of phospholipid from the interface.     

Isolation of a high molecular weight glycoconjugate derived from the surface of S purpuratus eggs that is implicated in sperm adhesion

Sea urchin sperm–egg adhesion is mediated by bindin, a sperm surface protein that has lectin-like activity. Bindin agglutinates eggs, and this interaction has been shown to be inhibited by glycopeptides released from the egg surface by protease treatment. In this study, we report the purification and properties of such an egg surface glycoconjugate that may be involved in sperm adhesion. The glycoconjugate was partially purified by gel filtration and affinity chromatography on bindin particles. Upon gel filtration on Sepharose CL 4-B, the glycoconjugate elutes near the void volume, suggesting that it has a molecular weight in excess of one million. In addition, we have found that the egg surface glycoconjugate agglutinates bindin particles, indicating that it is multivalent. Carbohydrate analysis indicates that the glycoconjugate is composed primarily of fucosc, xylose, galactose, and glucose. This purified egg surface component is the most potent inhibitor of bindin-mediated egg agglutination yet described.     

Foraging behavior of specialist and generalist caterpillars on plantain (Plantago lanceolata) altered by predatory stinkbugs

Summary To examine the effects of predators and plant genotype on the behavior, patterns of herbivory, growth and survivorship of caterpillars, we used an experimental garden in which we contrasted two hostplant genotypes of plantain (Plantago lanceolata), two kinds of herbivores (specialist Junonia coenia vs. generalist Pyrrharctia isabella) and two levels of caterpillar predation (with and without Podisus maculiventris stinkbugs). Each of the replicate plots per treatment contained two plants of the same genotype. The stinkbugs reduced the survivorship of the specialist caterpillars but not that of the generalists, which reflects the differences in predatoravoidance behaviors of these species. Nonetheless, the stinkbugs influenced the behavior of both caterpillar species. When stinkbugs were present, both specialist and generalist caterpillars were less likely to be found on the plant upon which they were initially placed (=initial plant), and they were more likely to be off both plants within the plot than larvae in the absence of predators. Consequently in the presence of the stinkbug predators, the proportion of the initial plants consumed was less than in the absence of the predators. Plant genotype influenced plant size and the proportion of individual plants eaten, but it did not affect larval location on the plots. Neither presence of predators nor plant genotype had an effect on relative growth rate of the caterpillars.