K252a Modulates the Expression of Nerve Growth Factor-Dependent Capsaicin Sensitivity and Substance P Levels in Cultured Adult Rat Dorsal Root Ganglion Neurones

Abstract: K252a, an inhibitor of trk phosphorylation and nerve growth factor signal transduction in PC12 cells, blocked nerve growth factor-induced responses in cultured adult rat dorsal root ganglion sensory neurones. The nerve growth factor-dependent appearance of capsaicin sensitivity and accumulation of the neuropeptide substance P were inhibited when dorsal root ganglion neurones were grown in the presence of low concentrations (100 n M ) of K252a. At higher concentrations (3 µ M ), however, K252a stimulated the development of capsaicin sensitivity and the accumulation of substance P even in the absence of nerve growth factor. By using a wide dose range, therefore, we showed that K252a could either inhibit or mimic nerve growth factor's actions on sensory neurones. These results may explain the apparent paradox in the literature that some groups show a blocking effect of K252a on nerve growth factor-dependent survival of dorsal root ganglion sensory neurones, whereas others report that K252a can substitute for nerve growth factor or other trophic factors and promote neuronal survival.     

Purification and PCR-based cDNA cloning of a plastidial n-6 desaturase

A plastidial membrane-bound n-6 desaturase from spinach (Spinacia oleracea) was purified from chloroplast envelope membranes by anion exchange, cation exchange and ferredoxin-affinity chromatography. The molecular mass of the protein was estimated by SDS-PAGE to be 40 kDa. The highest specific activity of the desaturase in the final preparation was 196 nmol/min per mg protein with free oleic acid as the substrate. The N-terminal amino acid sequence of the blotted protein was determined and used for the construction of a degenerated and inosine-containing oligonucleotide primer for PCR experiments with cDNA transcribed from leaf mRNA. A 3-RACE experiment with this primer amplified a single band of 1500 bp that after sequencing showed an open reading frame of 382 amino acids corresponding to a protein of 43 kDa. The 5 end of the cDNA was amplified by a 5-RACE experiment and isolated as a 500 bp fragment. Sequencing of this DNA revealed an additional 65 amino acids at the N-terminus of the native protein that are attributed to a plastidial leader peptide. With appropriate primers derived from these sequences a full-length clone was amplified by PCR and sequenced. Comparison of the plastidial oleate desaturase with the homologous enzyme from cyanobacteria showed about 50% amino acid homology. Comparison with other desaturases revealed three histidine boxes with the general sequence HXXXH that are highly conserved in all membrane-bound desaturases. These boxes might be involved in metal ion complexation required for reduction of oxygen.     

Monoclonal antibodies Ki-S3 and Ki-S5 yield new data on the 'Ki-67'proteins

Abstract. The monoclonal antibody (mab) Ki-67 has been used for about 10 years, mainly in tissue sections, to monitor proliferating cells, but so far only very little is known about the proteins it recognizes. The new mabs Ki-S3 and Ki-S5 detect proliferating cells in frozen and paraffin-embedded tissues. They recognize proteins with the same molecular mass as Ki-67 in Western blot and for the first time also in immunoprecipitation experiments. With these mabs we were able to enrich and purify the Ki-67 proteins. Protein sequencing of four peptides of the digested proteins corresponded to the cDNA-deduced amino acid sequence already published for the Ki-67 proteins.
Since we were able to immunoprecipitate the Ki-67 proteins, we performed various immunoprecipitation experiments to obtain more information about the nature of these proteins. After radiolabelling L428 cells with [³⁵S]-methionine we were able to immunoprecipitate the Ki-67 proteins after only 5 min of labelling time. In turnover experiments the Ki-67 proteins could not be detected 3 h after the end of labelling. These data indicate a halt-life of the Ki-67 proteins of about 90 min.
Labelling experiments with [³²P]-orthophosphate revealed that the Ki-67 proteins are phosphorylated. After dephosphorylation was blocked with okadaic acid or cell growth was arrested by means of Colcemid, the phosphorylation of the Ki-67 proteins was greatly increased, indicating that the Ki-67 proteins are phosphorylated via serine and threonine, and that the phosphorylation of the Ki-67 proteins increases in cycling cells. Labelling experiments with [³H]-mannose and [³H]-glucose revealed that the protein is weakly N -glycosylated.     

Extracellular matrix and capillary ingrowth in interspecies chimeric kidneys

The migration of capillaries into mouse embryonic kidneys grafted on quail chorioallantoic membrane (CAM) was analyzed by two monoclonal antibodies against quail endothelial and haematopoietic cells. As shown by immunohistochemistry, the quail chorioallantoic vessels invaded the kidney explant. Initially, the capillaries were detected in the interstitial stroma and, soon thereafter, tightly adjacent to the branches of the ureteric bud. The induced mesenchymal cell condensates, the prospective nephric vesicles, were avascular, but when the early S-shaped body was formed, the capillaries invaded its lower crevice. Finally chimeric glomeruli consisting of mouse podocytes and quail endothelial cells, were formed and, contemporarily, the capillaries ceased to migrate. Within the endothelial-mesangial area of the chimeric glomeruli, all cells expressed the quail-type nuclear structure and were stained by the quail endothelial-specific antibodies. The pattern of migrating capillaries was compared to the distribution of the extracellular matrix (ECM) molecules by double staining with polyclonal antibodies against laminin or fibronectin, and monoclonal quail endothelial-specific antibodies. Initially, the capillaries migrated in a fibronectin-rich matrix, devoid of laminin, but when the epithelial kidney tubules formed, some capillaries attached to the newly formed epithelial basement membrane. At no stage were the capillaries seen to penetrate the epithelial basement membrane. The orderly branching of the ureteric bud, followed by the formation of nephrons and the shift in the ECM, might create pathways for an oriented capillary migration. The fibronectin-rich areas could be a scaffold for the capillary migration, and the attachment to the basement membranes a means for their cessation.     

A region in domain 1 of CD4 distinct from the primary gp120 binding site is involved in HIV infection and virus-mediated fusion.

The high affinity binding site for human immunodeficiency virus (HIV) envelope glycoprotein gp120 resides within the amino-terminal domain (D1) of CD4. Mutational and antibody epitope analyses have implicated the region encompassing residues 40-60 in D1 as the primary binding site for gp120. Outside of this region, a single residue substitution at position 87 abrogates syncytium formation without affecting gp120 binding. We describe two groups of CD4 monoclonal antibodies (mAbs) which recognize distinct epitopes associated with these regions in D1. These mAbs distinguish between the gp120 binding event and virus infection and virus-induced cell fusion. One cluster of mAbs, which bind at or near the high affinity gp120 binding site, blocked gp120 binding to CD4 and, as expected, also blocked HIV infection of CD4+ cells and virus-induced syncytium formation. A second cluster of mAbs, which recognize the CDR-3 like loop, did not block gp120 binding as demonstrated by their ability to form ternary complexes with CD4 and gp120. Yet, these mAbs strongly inhibited HIV infection of CD4+ cells and HIV-envelope/CD4-mediated syncytium formation. The structure of D1 has recently been solved at atomic resolution and in its general features resembles IgVk regions as predicted from sequence homology and mAb epitopes. In the D1 structure, the regions recognized by these two groups of antibodies correspond to the C'C" (Ig CDR2) and FG (Ig CDR3) hairpin loops, respectively, which are solvent-exposed beta turns protruding in two different directions on a face of D1 distal to the D2 domain. This face is straddled by the longer BC (Ig CDR1) loop which bisects the plain formed by C'C' and FG. This structure is consistent with C'C' and FG forming two distinct epitope clusters within D1. We conclude that the initial interaction between gp120 and CD4 is not sufficient for HIV infection and syncytium formation and that CD4 plays a critical role in the subsequent virus-cell and cell-cell membrane fusion events. We propose that the initial binding of CD4 to gp120 induces conformational changes in gp120 leading to subsequent interactions of the FG loop with other regions in gp120 or with the fusogenic gp41 potion of the envelope gp160 glycoprotein.     

Atrial natriuretic peptide (ANP) is a high-affinity substrate for rat insulin-degrading enzyme.

A cytosolic protein specifically binding to and degrading atrial natriuretic peptide (ANP) was purified from rat brain homogenate. Based on partial amino acid sequences and enzymatic properties, this protein with an apparent molecular mass of 112 kDa has been identified as the rat insulin-degrading enzyme (IDE). In addition to the known substrates, insulin and transforming-growth-factor alpha IDE binds also with high affinity (apparent Kd 60 nM) to ANP. Competition studies with structural variants of ANP demonstrate that both the C terminus and the disulfide loop of the molecule are essential for high-affinity binding. The data suggest that IDE might be involved in the cellular processing and/or metabolic clearance of ANP.     

R15 alpha 1 and R 15 alpha 2 peptides from Aplysia: comparison of bioactivity, distribution, and function of two peptides generated by alternative splicing

The mRNA precursor encoded by the R15 gene is alternatively spliced in different neurons to form two related variants, R15-1 and R15-2 mRNA. One of the peptides encoded by the R15-2 mRNA, the R15 alpha 1 peptide, is expressed in the endogenously bursting neuron R15 and mediates some of its central and peripheral synaptic actions. In this study we found that the R15 alpha 2 peptide, which is encoded by the R15-1 mRNA, is synthesized in other neurons in the abdominal ganglion and is also bioactive. The R15 alpha 1 and R15 alpha 2 peptides were found to exert many similar actions on the cardiovascular, digestive, respiratory, and reproductive systems. However, the differences between many of the pharmacological effects of the R15 alpha 1 and R15 alpha 2 peptides indicate that alternative splicing in this system results in two functionally different peptides. Widespread immunoreactivity was found for an antibody directed against the R15 alpha 2 peptide, both in the central nervous system and the periphery. But because of the shared sequence with the R15 alpha 1 peptide, the antibody cross-reacts with the R15 alpha 1 peptide. To distinguish immunocytochemically between the two peptides, we also raised a second antibody that recognizes only the R15 alpha 1 peptide. This antibody labeled the cell body of only one neuron in the central nervous system, R15, although widespread immunoreactivity was found in axons and varicosities in the periphery.