Specificity of synexin-induced chromaffin granule aggregation

Preparations of synexin (1) exhibit a self-interaction in the absence of chromaffin granules as evidenced by an increase in absorbance at the wave-length used for observing granule aggregation (2). We incorporated this observation into a new formula for calculating the synexin-induced chromaffin granule aggregation. According to this amended analysis, synexin-induced aggregation is specific for chromaffin granules or their membranes. Treatment of intact chromaffin granuleswith trypsin or pronase renders the granules unresponsive to synexin.     

Isolation and characterization of chromaffin granules from a pheochromocytoma (PC 12) cell line

Incubation of rat kangaroo PtK2 cells with increasing concentrations of dimethylsulfoxide (DMSO) in the growth medium results in striking rearrangements of actin containing structures. After 1 h at concentrations of DMSO between 7.5 and 15%, immunofluorescence microscopy reveals actin containing inclusions within the nucleus of a large proportion of interphase cells. These paracrystals, which seem identical to those described by Fukui by electron microscopy [1], appear not to contain the microfilament-associated proteins tropomyosin, α-actinin or myosin and disappear within 1 h when the cells are shifted to normal medium. Electron microscopy confirms the intranuclear location. At concentrations above 20% DMSO the cells do not recover upon incubation in DMSO-free medium. When DMSO is present at a concentration of 50% the cells appear fixed, no paracrystals are formed and the actin profile resembles that seen in normal cells. Nuclear actin inclusions which appear similar to those induced by DMSO are also found upon incubation of PtK2 cells with the ionophore A23187 in the presence of high levels of magnesium ions. These conditions also result in striking morphological changes of the PtK2 cells. The data suggest that A23187 and DMSO may affect cellular morphology by changing the permeability of the cell to divalent cations, and that at least some of the actin found in the nuclear inclusions is of cytoplasmic origin.     

Purification, characterization, and partial amino acid sequence of nerve growth factor from cobra venom.

The nerve growth factor (NGF) from Naja naja (cobra) venom has been purified and its structure compared to the NGF from mouse submaxillary gland. A two-step purification procedure has been devised, consisting of a gel filtration step in 1 M acetic acid followed by chromatography of the active pool on carboxymethylcellulose at pH 5. The molecular weight of the native protein was found to be 28000, and this value was reduced by approximately one-half under denaturing conditions. These values are comparable to those obtained for mouse 2.5S or betaNGF. Tryptic peptide maps of S-[14C]carboxymethyl NGF gave the number of labeled peptides expected for a structure composed of two identical or very similar subunits. Thus, the quaternary structures of mouse and cobra NGF are the same. Cyanogen bromide (CNBr) treatment of Naja naja NGF produced three fragments, of which two were purified to homogeneity. These fragments and the whole protein were analyzed in the automated protein Sequencer. The amino-terminal CNBr fragment of the protein was also subjected to digestion by thermolysin and the resultant peptides were purified and characterized. These data plus those from the characterization of the tryptic peptides provided the basis of the construction of a tentative primary structure of Naja naja NGF which is approximately 60% identical with mouse NGF.     

Stimulation of rat adrenal medulla can induce differential changes in the peptide and mRNA levels of chromogranins, neuropeptides and other constituents of chromaffin granules

The levels of various components of chromaffin granules were determined in rat adrenals after treatment with several stimulants. After reserpine the levels of calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and chromogranin B but not those of chromogranin A and secretogranin II were elevated. On the other hand, the mRNA of chromogranins A, B and secretogranin II were significantly increased. Treatment with oxotremorine or nicotine (multiple injections for 2 or 3 days) induced analogous changes for peptide and mRNA levels, however, the increases were smaller and for the mRNA less consistent. A single injection of oxotremorine or nicotine raised only the levels of CGRP and NPY and of the NPY mRNA whereas those of the chromogranins and their respective mRNAs remained unaltered. Amongst the membrane proteins only the levels of dopamine beta-hydroxylase are increased after prolonged stimulation, whereas those of cytochrome b-561, carboxypeptidase H and synaptin/synaptophysin (SYN) remain unaltered. Thus, the biosynthesis of chromaffin granules can be regulated in quite sophisticated patterns.     

Chromaffin granule and PC12 cell chondroitin sulfate proteoglycans and their relation to chromogranin A

Two major proteoglycans, which appear to be structurally closely related, were isolated from bovine chromaffin granule matrix proteins by ion-exchange chromatography. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis they have apparent average molecular sizes of 35-40 kDa (range of 23-75 kDa) and generate a 14-kDa core glycoprotein after chondroitinase treatment. Previous studies demonstrated that these two major chromaffin granule proteoglycans are very similar in terms of their peptide mapping patterns and carbohydrate composition (having a high proportion of tri- and tetraantennary N-glycosidic oligosaccharides, and O-glycosidic oligosaccharides consisting predominantly of disialyl derivatives of galactosyl(beta 1-3)N-acetylgalactosamine), and that they differed in these respects from the chromogranins. By using antisera to five synthetic peptide fragments of chromogranin A to stain immunoblots of purified chromaffin granule proteoglycans before and after chondroitinase treatment, we have now shown that these major proteoglycans are not immunochemically related to chromogranin A. However, it has recently been reported that some chromogranin A-immunoreactive material disappears after chondroitinase treatment, and our studies demonstrate that approximately 1-2% of the chromogranin A occurs in the form of a 110-kDa proteoglycan, which is converted to a 95-kDa core glycoprotein after chondroitinase treatment. Similar chromogranin A proteoglycans could be detected in rat PC12 pheochromocytoma cells, where they have a molecular size of 115-145 kDa and yield a 105-kDa core protein after chondroitinase treatment. Studies using antibodies to synthetic peptide fragments of chromogranin B (secretogranin I) did not provide any evidence that this related protein occurs in a proteoglycan form.     

Preparative separation and amino acid composition of neurofilament triplet proteins

Abstract: Intact neurofilaments were isolated from bovine spinal cord white matter, washed by sedimentation in 0.1 m -NaCl, and extracted with 8 m -urea. Solubilized neurofilament triplet proteins of molecular weights approximately 68,000 (P68), 150,000 (P150), and 200,000 (P200) were purified by preparative electrophoresis, using an LKB 7900 Uniphor apparatus. The method provides for an enhanced yield of purified protein and has markedly reduced admixture of electrophoresed protein with acrylamide and associated protein contaminants. Amino acid compositions of the purified neurofilament triplet proteins are reported and compared.     

<i>Trichoderma</i> spp. fostering growth on <i>Capsicum chinense</i> Jacq. seedlings and antagonistic against <i>Meloidogyne incognita</i>

Fourteen native strains of Trichoderma spp. from wildand agricultural pathosystems in the state of Yucatan, Mexico, with growth-promoting ability of Capsicum chinense Jacq. seedlings were evaluated and antagonistic effect of their filtrate against second-stage juveniles (J2) of Meloidogyne incognita. The strains Th05-02 and Th27-08 showed the best significant effects on plant hight variable increments 55.57 and 47.62%, theTh07-04 with 29.48% more root length, theTh02-01 and Th07-04 isolates increased from 48.71 to 84.61% in volume radical and 53.40% of total dry biomass. Statistical analysis (p≤0.001) of Th43 and Th43-13-14 filtrates caused 100% mortality at 24 and 48h. In the test of reversibility to 24 h after replacing the filtrates Th43-13, Th43-14, TH09-06 and TH20-07 by sterile distilled water, the J2 did not recover their viability, so they were considered as the best potential strains of Trichoderma spp. with antagonistic capacity in J2 of M.incognita.