Samuel J. DiMari

Use of a ternary gradient for the separation of phenylthiohydantoin-amino acids, including the methyl esters of aspartic and glutamic acids, by high-performance liquid chromatography

Abstract The phenylthiohydantoin (PTH) derivatives of the common amino acids can be resolved in a single high-performance liquid chromatographic analysis by elution from a cyanopropylsilane column with a ternary gradient of methanol, acetonitrile, and ammonium acetate. The system is compatible with an automated sequencer-automated converter combination that produces the methyl esters of PTH-aspartic and PTH-glutamic acids.

Characterization of tetanus toxins and toxin components by amino terminal analyses

Abstract Extract tetanus toxin, filtrate tetanus toxin, and the heavy and light chains of filtrate toxin were analyzed for their amino termini with 4- N,N -dimethylaminoazobenzene-4′isothiocyanate and phenylisothiocyanate. Extract toxin (intracellular toxin) is a single-chain polypeptide with proline as the amino terminus. Filtrate toxin (extracellular toxin) is a mixture of species produced by endogenous proteases, and showed three major amino terminal residues, proline, asparagine, and serine. Cleavage points in the filtrate toxin molecule appear to be on either side of a disulfide bond. Reductive and nonreductive preparative electrophoresis of filtrate toxin produce different species of light and heavy chains. The light chains have a single amino terminus of proline, indicating that the light chain is the amino terminal portion of the toxin molecule. The heavy chains showed no proline but rather asparagine and serine as the major amino termini. Small amounts of other amino terminal residues were present, indicating microheterogenity at the cleavage sites in the toxin. The results permit the construction of a model of tetanus toxin which is consistent with the fragments obtained from either reductive or nonreductive preparative electrophoresis of filtrate toxin.

The cytotoxins of cobra venoms: Isolation and partial characterization

Eight basic proteins which lyse virus-transformed mouse fibroblasts in culture have been isolated from the venoms of six Asian Naja naja subspecies. These cytotoxins appear to represent an homologous series of proteins, all within the molecular weight range of 7000-8000. They have been divided into three arbitrary types on the basis of amino acid composition, electrophoretic mobilities and elution order upon ion-exchange chromatography. The rate at which the toxins effect cell lysis: (1) appears to be a function of the basicity of each toxin; (2) is dependent upon toxin concentration; (3) is temperature dependent; and (4) is inhibited by heparin sulfate. In view of the physical changes, which the cell undergoes during lysis and of the various factors which affect the action of these proteins, it is proposed that interaction of membrane receptors with the toxin, leading to alteration of cell membrane structure, is the principal event which ultimately leads to the disruption of the cell.

Characterization of the Collagen Phenotype of Rabbit Proximal Tubule Cells in Culture

Studies were performed to characterize the collagen phenotype of cultured rabbit proximal tubule (RPT) epithelial cells grown on plastic and on the reconstituted basement membrane preparation, Matrigel. When grown on a plastic substratum, RPT cells display a cobblestone appearance characteristic of glomerular epithelial cells. While initially forming an interlocking network of cells after subculture on Matrigel, this pattern of culture morphology rapidly develops into one characterized by isolated, organized groups of cells. Notwithstanding the effects of Matrigel on culture morphology, total cellular proliferation was reduced only 25% when RPT cells were grown on this substrate. Greater than 90% of the collagen synthesized by RPT cells grown on plastic was secreted into the culture medium. Qualitative analysis by SDS-PAGE revealed components exhibiting electrophoretic mobilities corresponding to the chains present in type IV and type I collagens. Quantitative analysis by CM-Trisacryl chromatography established that approximately 2/3 of the total collagen synthesized by RPT cells grown on plastic was type IV and approximately 1/3 type I. Quantitative analysis of the collagens produced by RPT cells grown on Matrigel again indicated the synthesis of only type IV and type I molecules but in a slightly more equal ratio of both collagen types and in the ratio of secreted to cell-associated molecules. However, the total amount of collagen synthesized by RPT cells grown on Matrigel was reduced to approximately 1% of the level synthesized by the cells grown on plastic. On plastic, approximately 3/4 of the type I collagen produced was recovered as the type I homotrimer, but on Matrigel type I homotrimers represented only approximately 55% of the total type I collagen synthesized. On Matrigel, the majority of the type IV collagen was recovered as heterotrimers containing alpha1(IV) and alpha2(IV) chains. In contrast, RTP cells grown on plastic predominantly produced type IV homotrimers containing only the alpha1(IV) chain. These data represent the initial report describing the collagens produced by nonimmortalized cultured proximal tubule cells. The finding that a significant amount of the total collagen synthesized was type IV (basement membrane) collagen, regardless of culture substrate, suggests that the RPT cells have maintained a significant degree of differentiation in culture, and thus establishes RPT cells as an appropriate model for investigating ECM changes in proximal tubule cells that occur in kidney disease. Finally, the observation that culture of RPT cells on a reconstituted basement membrane preparation results in a significant reduction in total collagen production and alterations in the molecular forms of type IV and type I molecules synthesized indicates that integrity of the tubular basement membrane may represent an important component in preventing the development of tubulointerstitial fibrosis.

The Effects of Tissue Pretreatment and Pepsin Levels on the Isolation of Collagens from Human Placenta

A comparative study has been undertaken to ascertain the effects of different tissue pretreatment procedures on the recovery of the major genetic types of collagen from human placenta. Essentially the same recovery of types I, III, IV and V collagen was obtained from placenta which was directly processed, from placenta which was stored at -70 degrees C after washing, and from dried acetone extracts of this tissue. Each collagen type isolated from the treated tissue preparations displayed properties consistent with those exhibited by its counterpart obtained from fresh tissue which was directly processed. Furthermore, it was observed that while the amount of types I and III collagen recovered was directly proportional to the level of pepsin employed, the recovery of types IV and V collagen was inversely related to this isolation parameter. These results establish that human placenta can be either stored frozen or as a dry acetone extract without affecting either the yield or the profile of the different genetic types of collagen recovered and that the amounts of different genetic types of collagen recovered can be modulated by the amount of pepsin employed in the isolation.

Purification of tetanus toxin and its peptide components by preparative polyacrylamide gel electrophoresis

Abstract A discontinuous preparative gel electrophoresis system has been devised and used successfully to separate the different tetanus toxin forms and fragments into highly purified preparations. A major feature of the system is the interaction of toxin, a suitable reducing agent and a critical concentration of denaturant during electrophoresis. With this procedure, filtrate (nicked) toxin has been separated into two distinct, but closely related molecular species. They appear to be nicked close to but on either side of an interchain disulfide bond, yielding heavy and light chains. The heavy- and light-chain components of each form of nicked toxin have been prepared and characterized. The system was also used to prepare extract (unnicked) toxin to a degree of purity not previously achieved in this laboratory. Nicked and unnicked toxin as well as the two forms of both heavy and light chain can consistently be prepared in sufficient purity and quantity to allow extensive biological, chemical, and physical characterization of each.

Effects of epidermal growth factor on collagen expression by rat kidney mesangial cells in culture

Increased collagen production by mesangial cells plays a key role in the development and progression of glomerular sclerosis. These changes reflect in part the impact of growth factors on mesangial cells. Since mesangial cells possess receptors for epidermal growth factor (EGF) and since previous studies have documented that EGF affects collagen synthesis in other cell types, we have examined the effects of EGF on collagen biosynthesis by rat kidney mesangial (RKM) cells in culture. Exposure for 24 h to EGF did not substantially affect the growth rate of RKM cells. While the types of collagen produced by RKM cells (types I, III, IV and V) were unaltered by exposure to EGF, total collagen production was reduced ( approximately 50%). This decrease in collagen expression was not uniform for each collagen type. Type I collagen production was inhibited by approximately 50%, both type III and type IV expression were each reduced by approximately 30%, but type V collagen production was suppressed by only approximately 15%. The reduction in type I collagen synthesis was accounted for mainly by a decrease in type I homotrimer production. Since type I molecules represent approximately 95% of the total collagen produced, the decrease in overall collagen expression reflects a specific suppression by EGF on type I homotrimer production in mesangial cells. As EGF exposure resulted in a decrease in collagen production, these results suggest that the increases in synthesis and deposition of collagen observed in several glomerular diseases likely do not reflect the short-term effects of EGF on mesangial cells. Rather, these findings suggest the possibility that EGF or EGF-like growth factors may ameliorate the effects of other soluble factors that cause enhanced matrix production and deposition in renal diseases.