Regeneration of the active zone at the frog neuromuscular junction

The active zone is a unique specialization of the presynaptic membrane and is believed to be the site of transmitter release. The formation of the active zone and the relationship of this process to transmitter release were studied at reinnervated neuromuscular junctions in the frog. At different times after a nerve crush, the cutaneous pectoris muscles were examined with intracellular recording recording and freeze-fracture electron microscopy. The P face of a normal active zone typically consists of two double rows of particles lined up in a continuous segment located opposite a junctional fold. In the initial stage of reinnervation, clusters of large intramembrane particles surrounding membrane elevations appeared on the P face of nerve terminals. Like normal active zones, these clusters were aligned with junctional folds. Vesicle openings, which indicate transmitter release, were seen at these primitive active zones, even though intramembrane particles were not yet organized into the normal pattern of two double rows. The length of active zones at this stage was only approximately 15% of normal. During the secondary stage, every junction was reinnervated and most active zones had begun to organize into the normal pattern with normal orientation. Unlike normal, there were often two or more discontinuous short segments of active zone aligned with the same junctional fold. The total length of active zone per junctional fold increased to one-third of normal, mainly because of the greater number of segments. In the third stage, the number of active zone segments per junctional fold showed almost no change when compared with the secondary stage. However, individual segments elongated and increased the total length of all active zone segments per junctional fold to about two-thirds of the normal length. The dynamic process culminated in the final stage, during which elongating active zones appeared to join together and the number of active zone segments per junctional fold decreased to normal. Thus, in most regions, regeneration of the active zones was complete. These results suggest that the normal organization of two double rows is not necessary for the active zone to be functional. Furthermore, localization of regenerating active zones is related to junctional folds and/or their associated structures.     

Platelet derived growth factor BB is a ligand for dermatan sulfate chain(s) of small matrix proteoglycans from normal and fibrosis affected fascia

Structural requirements of the short isoform of platelet derived growth factor BB (PDGF-BB) to bind dermatan sulfate (DS)/chondroitin sulfate (CS) are unknown. Meanwhile the interaction may be important for tissue repair and fibrosis which involve both high activity of PDGF-BB and matrix accumulation of DS. We examined by the solid phase assay the growth factor binding to DS chains of small proteoglycans from various fasciae as well as to standard CSs. Before the assay a structural analysis of DSs and CSs was accomplished involving the evaluation of their epimerization and/or sulfation patterns. In addition, in vivo acceptors for PDGF-BB in fibrosis affected fascia were detected. PDGF-BB binding sites on DSs/CSs are located in long chain sections with the same type of hexuronate isomer however without any apparent preference to glucuronate or iduronate residues. Alternatively, the interaction seems to involve two shorter DS chain sections assembling disaccharides with the same type of hexuronate isomer which are separated by disaccharide(s) with another hexuronate one. Moreover, DS/CS affinity to the growth factor most probably depends on an accumulation of di-2,4-O-sulfated disaccharides in binding site while the presence of 6-O-sulfated N-acetyl-galactosamine residues rather attenuates the binding. All examined fascia DSs and standard CSs showed significant PDGF-BB binding capability with the highest affinity found for normal palmar fascia decorin DS. In fibrosis affected palmar fascia DS/CS proteoglycans are able to form with PDGF-BB supramolecular complexes also including other matrix components such as type III collagen and fibronectin which bind the growth factor covalently. Our results suggest that DS chains of fascia matrix small PGs may regulate PDGF-BB availability leading to restriction of fibrosis associated with Dupuytren's disease or to control of normal fascia repair.     

Pyrazolone-fused combretastatins and their precursors: synthesis,cytotoxicity, antitubulin activity and molecular modeling studies

A series of pyrazolone-fused combretastatins and precursors were synthesized and their cytotoxicity as well as antitubulin potential was evaluated. The hydrazide 9f and the pyrazolone-fused combretastatins 12a, 12b and 12c were highly cytotoxic against various tumor cell lines including cisplatin resistant cells. The same compounds were also the best inhibitors of tubulin polymerization. Molecular modeling results showed that they bind the colchicine binding site at the tubulin heterodimer. The hydrazide 9f arrested HeLa cells in the G2/M phase of the cell cycle and strongly affected cell shape and microtubule network.     

Fine needle aspiration cytology in lupus lymphadenopathy. A case report.

This case report describes the aspiration cytologic characteristics of histologically proven acute lupus lymphadenitis. The aspirate contained numerous lymphoid cells and many amorphous, basophilic, hematoxylin-stained bodies dispersed in a granular, necrotic background that lacked polymorphonuclear leukocytes. The lymphadenopathy was diagnosed cytologically as necrotizing lymphadenitis and histologically as acute lupus lymphadenitis.     

Expression and characterization of recombinant human ciliary neurotrophic factor from Escherichia coli

The gene for ciliary neurotrophic factor (CNTF) was cloned from a human genomic DNA library by screening with a DNA fragment amplified from human genomic DNA using the polymerase chain reaction. A DNA sequence coding for human CNTF was placed under control of an regulatable promoter in the expression vector pJU1003 and transformed into Escherichia coli strain BL21(DE3). Induction of expression in cultures of this transformant led to the accumulation of approx. 25 mg/l per A600 unit of human CNTF. CNTF was purified to homogeneity from cell lysates via anion-exchange, cation-exchange and Zn(2+)-affinity chromatography. Purified CNTF contained less than 0.1% contaminating E. coli proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analysis and reversed-phase high-pressure liquid chromatography (HPLC). The protein exhibited an ultraviolet absorption maximum at 279 nm with a calculated extinction coefficient of A1%(279) = 9.0. Peptide map and amino acid sequence analyses confirmed that the expressed protein has the amino acid sequence expected for human CNTF, except for the absence of the amino-terminal methionine. High-purified recombinant human CNTF supported the survival of chick embryo parasympathetic, sympathetic and sensory neurons in culture at low picomolar concentrations. These results indicate that the biological activities previously ascribed to impure CNTF preparations indeed reside in one molecule.     

Obituary for Roland Schauer

Glycoconjugate Journal -     

Binding of antigen in the absence of histocompatibility proteins by arsonate-reactive T-cell clones

Inducer T-cell clones reactive to the p-azobenzenearsonate (arsonate) hapten possess binding sites for radioactive arsanylated proteins, which are not present on clones with other antigen specificities. Binding occurred in the absence of histocompatibility proteins. Binding was specific for the p-azobenzenearsonate hapten, since unconjugated proteins and proteins conjugated to the nonactivating o-azobenzenearsonate hapten neither bound to the clones nor competed binding of radioactive antigen. One of the clones was studied in more detail, using a panel of structural analogs of arsonate conjugated to the carrier protein ovalbumin. All conjugates that activated the clone in the presence of antigen-presenting cells also competed binding of radioactive antigen in the absence of antigen-presenting cells. Nonactivating conjugates did not compete binding. Based on evidence in this and the succeeding paper (Rao et al., accompanying paper), we suggest that these arsonate-binding sites may include the physiological antigen receptors of arsonate-reactive T-cell clones.     

Primary tuberculosis of the parotid gland

A very unusual presentation of Mycobacterium tuberculosis in the parotid gland substance is described to suggest reexamination of the place of tuberculosis in the differential diagnosis of a parotid mass. In this patient, diagnosis was made postoperatively only by histologic examination of the excised specimen. When M. tuberculosis etiology is suspected, either clinically or at operation, culture confirmation should be tried, and a Mantoux test should be performed to complete the investigation.