World Health Organization International Standards for highly purified human, porcine and bovine insulins

The 4th International Standard (IS) for Insulin, established in 1958, consists of a mixture of relatively impure bovine and porcine insulins and is not suitable as a standard for the assay of highly purified single-species insulins presently used in the treatment of diabetes. Preparations of human, bovine and porcine crystalline insulins, representative of current highly purified therapeutic insulins, have now been studied in an international collaborative study carried out by twenty-three laboratories in fifteen countries. In the collaborative study described here, each of the three preparations was found to be suitable for use as a standard for insulin for bioassay and each was established by WHO in 1986 as an international standard. The 4th IS of Insulin bovine/porcine (code numbered 58/6) has been discontinued. Insulin preparations should now be calibrated in terms of International Units defined by the standard for the appropriate species: the International Standard for Insulin, Human, the International Standard for Insulin, Bovine, or the International Standard for Insulin, Porcine.     

Drug-induced lysosomal storage of sulfated glycosaminoglycans in cultured bovine and human fibroblasts

Several di-cationic amphiphilic compounds are known to cause lysosomal accumulation of sulfated glycosaminoglycans (sGAG) in intact rats and in cultured rat fibroblasts. The purpose of the present investigation was to examine whether this drug side effect also occurs in bovine and human cells. Cultured fibroblasts from both species were exposed to tilorone (3 μM and 5 μM) for 72 h; lysosomal sGAG-storage was demonstrated by cytochemical staining with cuprolinic blue and by measuring the intracellular accumulation of [³⁵S]-GAG. The cytological alterations as well as the radiochemical results in both species were in good agreement with previous data from rat fibroblasts. The present findings indicate that the drug-induced lysosomal storage of sGAG is a species-independent phenomenon. Thus, cultured bovine and human fibroblasts are a suitable model for further studies concerning the as yet unknown molecular mechanisms underlying this adverse drug action.     

Drug-induced lysosomal storage of sulfated glycosaminoglycans in cultured bovine and human fibroblasts.

Several di-cationic amphiphilic compounds are known to cause lysosomal accumulation of sulfated glycosaminoglycans (sGAG) in intact rats and in cultured rat fibroblasts. The purpose of the present investigation was to examine whether this drug side effect also occurs in bovine and human cells. Cultured fibroblasts from both species were exposed to tilorone (3 microM and 5 microM) for 72 h; lysosomal sGAG-storage was demonstrated by cytochemical staining with cuprolinic blue and by measuring the intracellular accumulation of [35S]-GAG. The cytological alterations as well as the radiochemical results in both species were in good agreement with previous data from rat fibroblasts. The present findings indicate that the drug-induced lysosomal storage of sGAG is a species-independent phenomenon. Thus, cultured bovine and human fibroblasts are a suitable model for further studies concerning the as yet unknown molecular mechanisms underlying this adverse drug action.     

Analytical biotechnology of recombinant peptides and proteins. I. Analysis of the purity, composition and structure of human, porcine and bovine insulins

A method for analysis of the type, purity, and possible structural modifications of insulins of bovine, porcine, and human origin was proposed. It is based on a combination of narrow-bore reversed-phase HPLC and mass spectrometry. The hydrolysis of insulins with highly specific Glu-protease V8 from Staphylococcus aureus followed by peptide mapping of the hydrolysis products and mass spectrometry of the isolated fragments helps rapidly and reliably localize and identify substitutions of amino acid residues in insulin structure by using insulin samples of less than 1 nmol.     

Analytical biotechnology of recombinant peptides and proteins: I. determination of the purity, composition, and structure of human, porcine, and bovine insulins

A method for analysis of the type, purity, and possible structural modifications of insulins of bovine, porcine, and human origin was proposed. It is based on a combination of narrow-bore reversed-phase HPLC and mass spectrometry. The hydrolysis of insulins with highly specific Glu-protease V8 fromStaphylococcus aureus followed by peptide mapping of the hydrolysis products and mass spectrometry of the isolated fragments helps rapidly and reliably localize and identify substitutions of amino acid residues in insulin structure by using insulin samples of less than 1 nmol.     

Sphingomyelinases in human, bovine and porcine seminal plasma

The seminal plasma of man, boar and bull was found to have a sphingomyelinase (SMase) activity hydrolysing [N-methyl-14C]sphingomyelin. The human and porcine enzymes had an acid pH optimum and were not influenced by divalent metal ions or chelating agents. They were closely similar with the lysosomal enzyme in many tissues. The bovine seminal plasma SMase was partially purified. The enzyme was a glycoprotein with pH optimum at 6.5, a broad pI 4.2-4.8 and molecular mass of 160 and 60 kDa, respectively, in native and SDS-PAGE. The enzyme was activated by Co greater than Mn greater than Cd greater than Ni and inhibited by chelating agents, Cu, Fe, Pb and Zn. The enzyme was clearly distinct from the acid lysosomal SMase and the previously described neutral Mg2+-dependent and independent activities. It had a wide distribution in the bull reproductive tissues.     

Ultrastructure of cultured human orbital fibroblasts

The fine structure of cultured human orbital fibroblasts was investigated by transmission electron microscopy. One culture was derived from a patient with severe Graves' ophthalmopathy, the other from a donor without inflammatory orbital disease. Despite their known differences in metabolism, orbital fibroblasts from either source revealed no ultrastructural differences. The cells had extensive thin cytoplasmic processes. The perinuclear areas contained multiple assemblies of Golgi membranes, modest amounts of rough endoplasmic reticulum, intermediate filaments, and lysosome-like structures. Glycogen deposits were noted both in the perinuclear cytoplasm and in the thin processes. These ultrastructural features of orbital fibroblasts are the same as those of fibroblasts from other anatomic regions.     

Intracytoplasmic injection of porcine, bovine, mouse, or human spermatozoon into porcine oocytes

We determined the incidence of activation, male pronuclear formation, and apposition of pronuclei in porcine oocytes following intracytoplasmic injection of various porcine sperm components and foreign species spermatozoa, such as that of cattle, mouse or human. The porcine oocytes were activated by injection of a spermatozoon or an isolated sperm head. In contrast, injection of either sperm tail or a trypsin- or NaOH-treated sperm head failed to induce oocyte activation. Because injection of mouse, bovine, or human spermatozoon activated porcine oocytes, the sperm-borne activation factor(s) is not strictly species-specific. Male pronuclear formation and pronuclear apposition were observed in porcine oocytes following injection of porcine, bovine, mouse or human spermatozoa. Electrical stimulation following sperm cell injection did not enhance the incidence of male pronuclear formation or pronuclear apposition compared with sperm cell injection alone (P > 0.1). Following porcine sperm injection, the microtubular aster was organized from the neck of the spermatozoon, and filled the whole cytoplasm. In contrast, following injection of bovine, mouse, or human spermatozoon, the maternal-derived microtubules were organized from the cortex to the center of the oocytes, which seems to move both pronuclei to the center of oocytes. Cleavage to the two-cell stage was observed at 19-21 hr after injection of porcine spermatozoon. However, none of the oocytes following injection of mouse, bovine, or human spermatozoa developed to the mitotic metaphase or the two-cell stage. These results suggested that the oocyte activating factor(s) is present in the perinuclear material and that it is not species-specific for the porcine oocyte. Self-organized microtubules seemed to move the pronuclei into center of oocytes when foreign species spermatozoa were injected into porcine oocytes.     

The glycopeptides from the human,bovine and porcine stomachs

• 1.1. Glycopeptides were isolated from tryptic digests of human, bovine and porcine gastric mucosae.
• 2.2. Fractionation of the glycopeptide by preparative zone electrophoresis on a Pevikon C-870 block, yielded one glycopeptide fraction from the mucosa of the human stomach, whereas from both mucosae of the bovine fourth stomach and swine stomach it yielded three glycopeptide fractions.
• 3.3. They were characterized by an increase in sialic acid and sulfate as the mobility of Pevikon block electrophoresis increased.

     

L-iduronidase in cultured human fibroblasts and liver

Extracts of normal human livers and skin fibroblasts cultured from normal individuals and patients with the Hurler syndrome released L-iduronic acid when incubated with desulfated dermatan sulfate. Enzyme extracts of normal fibroblasts liberated larger amounts of L-induronic acid, as judged by paper chromatography, than did homogenates from Hurler fibroblasts. Preliminary studies with desulfated heparan sulfate using the same enzyme systems have also shown material with the R_f of iduronolactone on paper chromatography.     

Glycine transport by cultured human fibroblasts

The transport of glycine was studied in cultured human fibroblasts. The amino acid entered the cell by Na+-dependent and Na+-independent mechanisms. Na+-independent glycine (0.1 mM) transport was less than 10% of total uptake and occurred by a mechanism formally indistinguishable from diffusion. Two distinct routes contributed to Na+-dependent glycine transport. The first route was identified with system A because it was inhibited by MeAIB and underwent adaptive regulation. The second route was identified with system ASC as it was inhibited by L-alanine, but not by MeAIB. Kinetic analysis revealed that the two systems operated glycine transport with the same Km of 1.6 mM, a value unusually high for system ASC.     

The transport of alanine, serine, and cysteine in cultured human fibroblasts

The transport of L-alanine, L-serine, and L-cysteine has been studied in skin-derived diploid human fibroblasts in culture. Competition analysis, mathematical discrimination by nonlinear regression, and conditions varying the relative contribution of the various mediations have been used to characterize the systems engaged in the inward transport of these amino acids. All the adopted criteria yielded results showing that L-alanine, L-serine, and L-cysteine enter the cell by two Na+-dependent systems, System A and System ASC, and by a Na+-independent route, whose major component has been identified as System L. The apparent affinity of L-alanine, L-serine, and L-cysteine for the putative carrier was higher for System ASC than for System A. The transport Vmax for System A increased in response to cell starvation; after 12 h, its values were similar or higher than those exhibited by System ASC. At amino acid concentrations approaching those present in human plasma, System ASC appeared to be the primary mediation for the inward transport of L-alanine, L-serine, and L-cysteine in human fibroblasts. The contribution of System A was negligible in nonstarved cells and became appreciable under conditions of cell starvation. The Na+-independent System L made no substantial contribution to the uptake of L-alanine and L-serine and accounted for approximately one-fourth of the total uptake of L-cysteine.     

Metabolism of ganglioside-amides in cultured human fibroblasts

Metabolism of [3H]ganglioside derivatives GM3-amide and GM2-amide has been investigated in normal human skin fibroblasts. In a cell-free system the ganglioside analogues have been shown to enter biosynthetic pathways, their degradation, however, was curtailed at an early stage, as GM3-amide could not be hydrolysed by sialidase action. GM2-amide was susceptible to beta-hexosaminidase degradation yielding GM3-amide. When incorporated into fibroblasts [3H]GM2-amide was degraded to [3H]GM3-amide presumably in the lysosomes, and at the same time glycosylation to [3H]GD1a-monoamide took place most likely in the Golgi apparatus. [3H]GM3-amide, however, did not seem to reach the glycosylation sites in the Golgi apparatus. It could be detected in the lysosomes, where it was not degraded due to its sialidase resistance. From these results we conclude that in cells exogenously administered [3H]GM3-amide and [3H]GM2-amide both are directed to the lysosomes and that [3H]GM2-amide also reaches the Golgi apparatus. The synthesis of higher [3H]ganglioside-amides from incorporated [3H]GM2-amide can occur by direct glycosylation. [3H]GM3-amide, however, even if it reaches the Golgi compartment, does not enter the biosynthetic pathway.     

Immobilized glycolipids from human diploid fibroblasts inhibit DNA synthesis of cultured human fibroblasts

Several previous studies have shown that glycolipids isolated from plasma membranes of cultured cells and added to cells in culture inhibit the growth rate in a concentration-dependent fashion. In order to investigate the possible involvement of glycolipids in the growth regulation of normal cells by cell-cell contacts, we tested the effect of immobilized glycolipids, isolated from human fibroblasts, on the DNA synthesis of freshly seeded fibroblasts. Gangliosides inhibited DNA synthesis to a great extent, whereas neutral glycolipids had only a minor effect. The degree of inhibition of DNA synthesis by immobilized gangliosides depended both on the cell density of the cultures from which the gangliosides were isolated and on the pretreatment of the immobilized gangliosides: Preincubation with DMEM without FCS of immobilized gangliosides, isolated from confluent cultures, resulted in a 75% inhibition of growth rate of embryonal human lung fibroblasts (FH109) cultured on immobilized gangliosides. Under the same conditions, gangliosides from sparse cultures reduced the growth rate by about 30%. On the other hand, the degree of inhibition exerted by immobilized gangliosides isolated from confluent cultures was found to be greatly reduced by preincubation with DMEM with FCS, whereas the slight inhibition of growth rate, exerted by gangliosides from sparse cultures, was found to be reversed into a slight stimulation of growth rate after preincubation with complete medium. Concomitantly with the reduction of the inhibition of DNA synthesis, it was found that the complete medium, used for preincubation of the gangliosides, was no longer able to support DNA synthesis to the same extent as untreated complete medium. The data suggest that gangliosides bind growth-supporting factors of the serum, gangliosides isolated from sparse cultures being more potent in the binding of these molecules than gangliosides isolated from dense cultures.