Release of cell wall peptides into culture medium by exponentially growing Escherichia coli.

Escherichia coli W7 cells were found to release three different muropeptides into the culture medium: tetrapeptide (L-Ala-D-Glu-meso-diaminopimelic acid-D-Ala), tripeptide (L-Ala-D-Glu-meso-diaminopimelic acid), and a previously undescribed dipeptide (meso-diaminopimelic acid-D-Ala). From the rate of release of these three peptides, it was calculated that 6 to 8% of the murein in the sacculus was lost per generation.     

Release of antigens into the culture medium by LS fibroblasts in culture]

LS fibroblasts cultivated for 1 or more days in unsupplemented Eagle's MEM release antigens into the medium, without showing any evidence of lysis, but on the contrary continuing to proliferate. These antigens give up to three precipitation lines when tested by means of immunodiffusion and immunoelectrophoresis against specific rabbit antisera; one or two of them give identity reactions with antigens obtained by repeatedly washing the fibroblasts with balanced salt solutions. Evidence has been obtained that they are surface components, easily stripped or spontaneously shed by the cells.     

Intermediates in the limited proteolytic conversion of procollagen to collagen.

The conversion of chick bone procollagen to collagen proceeds in a stepwise fashion to produce a limited number of intermediates. Initial proteolytic cleavages remove NH2-terminal nonhelical extensions and yield an intermediate which remains disulfide-bonded via COOH-terminal extensions. Subsequent stepwise scission of one or two chains of the triple-stranded molecule in its COOH-terminal domain produces intermediates which can only be distinguished after dissociation of the noncovalently bonded alpha chains. A final cleavage in this region produces the collagen molecule and a disulfide-bonded triple-stranded fragment which represents the COOH-terminal domain. In all likelihood the endopeptidases which effect cleavage in the NH2- and COOH-terminal regions differ. More than two enzymes may be required for conversion of procollagen to collagen if the nonhelical domains are not released in an en bloc fashion.     

The in vivo effects of a culture medium. II. Influence of culture medium administered prior to irradiation on hemopoietic recovery of gamma-irradiated mice.

The culture medium administered to C57Bl/6 mice 18 h and 8 h before a single irradiation (9 Gy) had a radioprotective effect and clearly influenced postirradiation changes in haemopoiesis. Haemopoiesis recovery appeared to be faster in culture medium-pretreated animals than in those irradiated without such pretreatment. By 12-15 days after irradiation, the thymus cortex appeared to be repaired, on day 21 a multiple increase in extramedullary erythropoiesis, myelopoiesis and megakaryocytopoiesis in the red pulp was found and later, on day 28, the lymphopoiesis in the white pulp of spleen was restored. The rate of haemopoiesis proliferation of predominantly myeloid cells which reached a control level on day 28 following irradiation. Consequently, the regenerative processes in blood-forming organs were accompanied by considerable reticulocytosis and complete recovery of neutrophil and platelet counts in the peripheral blood as seen on day 21. Despite a slower rate complete recovery of the total leukocyte count was reached by day 180 after irradiation.     

Inhibiting effect of procollagen peptides on collagen biosynthesis in fibroblast cultures.

NH2-terminal extension peptides of type I and type III procollagens were isolated from dermatosparactic and normal fetal calfskin, respectively. Cell culture experiments showed that the globular domains of the tested procollagen peptides were biologically active but that peptides from the helical region of collagen had no effect. The peptides were added to the incubation medium of calf fibroblasts along with radioactive precursor amino acids, and the amount of newly synthesized collagen was determined. The experiments indicated that procollagen peptides exerted a feedback-like inhibitory effect specific for the synthesis of collagen. Neither degradation of collagen, hydroxylation of collagen alpha chains, nor synthesis of noncollagenous proteins were affected. Synthesis of type II collagen by calf chondrocytes was not reduced. In addition, it was shown that procollagen peptides from calf were equally effective when added to human fibroblast cultures, an observation that could be of considerable medical interest.     

Evaluation of a novel biphasic culture medium for recovery of mycobacteria: a multi-center study

Background

Mycobacterial culture and identification provide a definitive diagnosis of TB. Culture on Löwenstein-Jensen (L-J) medium is invariably delayed because of the slow growth of M. tuberculosis on L-J slants. Automated liquid culture systems are expensive. A low-cost culturing medium capable of rapidly indicating the presence of mycobacteria is needed. The aim of this study was to develop and evaluate a novel biphasic culture medium for the recovery of mycobacteria from clinical sputum specimens from suspected pulmonary tuberculosis patients.

Methods and Findings

The biphasic medium consisted of 7 ml units of L-J slant medium, 3 ml units of liquid culture medium, growth indicator and a mixture of antimicrobial agents. The decontamination sediments of sputum specimens were incubated in the biphasic culture medium at 37°C. Mycobacterial growth was determined based on the appearance of red granule sediments and the examination using acid-fast bacilli (AFB). The clinical sputum specimens were cultured in the biphasic medium, on L-J slants and in the Bactec MGIT 960 culture system. Among smear-positive specimens, the mycobacteria recovery rate of the biphasic medium was higher than that of the L-J slants (P<0.001) and similar to that of MGIT 960 (P>0.05). Among smear-negative specimens, the mycobacterial recovery rate of the biphasic medium was higher than that of L-J slants (P<0.001) and lower than that of MGIT 960 (P<0.05). The median times to detection of mycobacteria were 14 days, 20 days and 30 days for cultures grown in MGIT, in biphasic medium, on L-J slants for smear negative specimens, respectively (P<0.001).

Conclusions

The biphasic culture medium developed in this study is low-cost and suitable for mycobacterial recovery. It does not require any expensive detection instrumentation, decreases the time required for detection of M. tuberculosis complex, and increases the detection rate of M. tuberculosis complex.     

Release into culture medium of membrane-associated, tumor-specific antigen by B-16 melanoma cells.

Serum-free supernatant fluids from monolayer cultures of B-16 mouse melanoma cells were found to contain a soluble membrane associated tumor-specific antigen. The 100,000 g supernatant of the culture fluid induced an antibody response to the B-16 cells both in rabbits and in the mouse strain of origin (C57Bl/6J). Similar supernatant fluids derived from an unrelated cell line (L-929) or from normal C57Bl/6 fibroblasts did not contain the B-16 specific material. Preliminary results indicate that the B-16 specific material is a protein of low molecular weight which is released into the culture fluid chiefly by living cells and, to a lesser extent, by autolysing cells.     

A serum-free medium for the growth of muscle cells in culture.

Rates of cell proliferation essentially equal to those in 10% serum were obtained when Yaffe's L6 myoblasts were incubated in Ham's F-12 medium containing 10(-5) M fetuin, 10(-6) M insulin, and 10(-7) M dexamethasone; we have designated this mixture muscle medium-1 (MM-1). Addition of other growth factors and hormones in various combinations did not increase the proliferation of myoblasts above the rate in MM-1, and neither fetuin nor insulin could be replaced by other growth factors. All glucocorticoids tested (but no other steroid hormones) were active. Fetuins prepared by the rather different procedures of Pedersen, Deutsch, and Spiro were all active, and the active material was heat labile and nondialyzable; this is the first cell culture system in which highly purified Spiro fetuin has been found active. Primary rat myoblasts proliferated more rapidly that fibroblasts in parallel cultures when incubated in MM-1. This simple medium, composed of relatively inexpensive and readily available components, should be useful for the study of muscle cell growth and differentiation.     

Release of peptides from sustained delivery systems (microcapsules and microparticles) in vivo. A histological and immunohistochemical study

Sustained delivery systems (microcapsules, microparticles, or implants) developed for once a month administration of peptides are efficacious and convenient. Long acting formulations of several bioactive peptides are based on microcapasules of a biodegradable polymer poly(DL-lactide-co-glycolide) (PLG), but a better understanding is required of the mechanism of the peptide release from the microcapsules, which is assumed to be primarily by diffusion through pores. In order to clarify this mechanism, microcapsules and microparticles of the agonist [D-Trp6]-LHRH and microcapsules of the LHRH antagonist SB-75 were given i.m. to rats 2 h and 1, 2, 4, 7, 14 and 21 days before histological and immunohistochemical investigation. Signs of biodegradation of the PLG matrix could be seen the first day after the injection, in a form of vacuole development in the interior of the particles and connected with the presence of macrophages within the matrix. The microcapsules showed excellent tissue-compatibility, and no significant foreign body reaction was detected. Immunohistochemical study on the microcapsules revealed no visible decrease in peptide concentration in the remnants of the matrix even 2 weeks after the injection. Evaluation of serum [D-Trp6]-LHRH showed that after an initial burst, both microcapsules and microparticles maintained elevated serum [D-Trp6]-LHRH levels for more than 3 weeks. Our results suggest that the previously proposed mechanisms do not reflect the experimental findings, particularly for the insoluble peptides. The peptide release from the PLG microcapsules or microparticles appears to be controlled mostly by the speed of the biodegradation of the polymer matrix and the diffusion of the peptides from the PGL is negligible.     

Identification and partial characterisation of the non-collagenous amino- and carboxyl-terminal extension peptides of cartilage procollagen.

Procollagen secreted by embryonic chick cartilage cells has been partially purified by (NH₄)₂SO₄ precipitation and DEAE-cellulose chromatography. Analyses of the products of digestion by human rheumatoid synovial collagenase and bacterial collagenase indicated the presence of non-collagenous peptide sequences at the N- and C-termini. Both regions were found to incorporate [³⁵S]cystine but inter-chain disulphide bonds were restricted to a C-terminal location. Electrophoretic analysis gave apparent molecular weights of 13000 and 36000 daltons for the respective N- and C-terminal extensions.     

A simple medium for the study of hepatocyte growth in culture under defined conditions

Summary The combination (1∶1) of Dulbecco's modified Eagle's medium and Waymouth's medium MAB 87/3 was found to provide favorable conditions for serum-free culture and growth of adult rat hepatocytes. In this simple medium, a majority of hepatocytes stimulated by epidermal growth factor plus insulin entered S phase and divided, with a normal (13 h) interval between DNA synthesis and cell division. The proliferative response did not require extra substratum or the presence of serum, even during cell isolation and plating. This work was supported by the Norwegian Cancer Society.     

Shape and assembly of type IV procollagen obtained from cell culture.

Type IV procollagen was isolated from the culture medium of the teratocarcinoma cell line PYS-2 by affinity chromatography on heparin-Sepharose. Immunological studies showed that type IV procollagen is composed of pro-alpha 1(IV) and pro-alpha 2(IV) chains and contains two potential cross-linking sites which are located in the short triple-helical 7S domain and the globular domain NC1 . The 7S domain was also identified as the heparin binding site. Rotary shadowing visualized type IV procollagen as a single triple-helical rod (length 388 nm) with a globule at one end. Some of the procollagen in the medium, however, had formed aggregates by alignment of 2-4 molecules along their 7S domains. After deposition in the cell matrix, non-reducible cross-links between the 7S domains are formed while the globules of two procollagen molecules connect to each other. The latter may require a slight proteolytic processing of the globular domains NC1 . The shape of type IV procollagen and the initial steps in its assembly are compatible with a recently proposed network of type IV collagen molecules in basement membranes. Since both type IV collagen and laminin bind to heparin, the formation of higher ordered structures by interaction of both proteins with heparan-sulfate proteoglycan may occur in situ.     

The disulphide-bonded nature of procollagen and the role of the extension peptides in the assembly of the molecule.

1. The molecular weights of chick tendon and cartilage procollagens, and their constituent polypeptides, were determined by gel filtration and gel electrophoresis. The values obtained are in good agreement and indicate that the mol.wts. of the secreted procollagens (types I and II) and their individual pro-alpha-chains are of the order of 405 000-445 000 and 137 000-145 000 respectively.2. Digestion of tendon procollagen with human rheumatoid synovial collagenase gave products consistent with the presence of large non-helical peptide extensions at both N-and C-termini. Electrophoretic analysis gave apparent mol.wts. of 17 500 and 36 000 for the respective N- and C-terminal extensions of pro-alpha1(I)-and pro-alpha2-chains, and inter-chain disulphide bonds were restricted to the C-terminal location. 3. During the biosynthesis of procollagen by tendon and cartilage cells a close correlation was observed between the extent of inter-chain disulphide bonding and the proportion of procollagen polypeptides having a triple-helical conformation. These processes appeared to commence in the rough endoplasmic reticulum and be completed in the smooth endoplasmic reticulum, but the rate at which they occur in cartilage cells is markedly slower than that found in tendon cells. 4. When the intracellular [14C]procollagen polypeptides present in the rough-endoplasmic-reticulum fractions of tendon and cartilage cells were analysed under non-reducing conditions on agarose/polyacrylamide composite gels, no significant pools of dimeric intermediates were detected. 5. In both cell types, inter-chain disulphide-bond formation occurred even when hydroxylation, and hence triple-helix formation, was inhibited. The presence of pro-alpha1- and pro-alpha2-components in a ratio of 2:1 in the disulphide-linked unhydroxylated procollagen isolated from tendon cells demonstrated that correct chain association occurs in the absence of hydroxylation. This observation is consistent with a model for the assembly of pro-gamma112-chains in which the recognition and selection of pro-alpha1-and pro-alpha2-chains in a 2:1 ratio are directed by the non-helical C-terminal extension peptides of tendon procollagen.     

The preferred conformations of protected homodito homoheptamethionine peptides. A1 H n.m.r. study in deuterochloroform medium

Detailed analyses of the conformations of the homo-oligopeptide series, Boc-(L-Met)n-OME n = 2--7, in deuterochloroform have been carried out with proton n.m.r. and IR spectroscopy. Well-resolved high field n.m.r. spectra with assignments for the NH and alpha-CH resonances of these homo-methionine peptides are presented. Extensive n.m.r. concentration-dependent chemical shift studies are combined with IR results to delineate the involvement of the various methionine NH protons in intra- and/or intermolecular hydrogen bonding. N.m.r. chemical shift dependencies with temperature and solvent, DMSO-d6, are used to explore the strength of the hydrogen bonds for the various oligopeptides. At low concentrations, where peptide aggregation is absent, the dipeptide is found to be disordered. The tetra- to heptapeptides possess intramolecular hydrogen bonded seven-membered rings at internal residues. The number of internal rings and the oligopeptide self-association increase with increasing peptide chainlength. At intermediate concentrations associations of peptide molecules with folded structures occur with initial association at the C-terminal region. At high concentrations, "in-register" associated extended beta structures are formed.     

Hyperlipidemic rabbit serum reduces recovery of acidic glycosaminoglycans from tissue culture medium

Hyperlipidemic rabbit serum and its lipid extract were found to impair the precipitation of glycosaminoglycans by cetylpyridinium chloride. The inhibition was 60–80% in the case of sulfated glycosaminoglycans and 75–85% in the case of hyaluronic acid. The interfering compounds could be removed by extracting the samples twice with a six-fold volume of ethanol-ether (2:1) for 1 h.     

Intermediates in the conversion of procollagen to collagen. Evidence for stepwise limited proteolysis of the COOH-terminal peptide extensions.

Intermediates in the conversion of procollagen to collagen were isolated from radioactively labeled chick cranial bones by ion-exchange chromatography. Cleavage of these proteins with vertebrate collagenase revealed that each of the several forms of these intermediates lacked NH2-terminal but retained COOH-terminal extensions. The chain composition of each intermediate was resolved by two-dimensional slab gel electrophoresis. The intermediates differed from each other in having sustained cleavages in zero, one or two pcalpha chains. The relative proportions of intermediates with different intact pcalpha chains, observed in conversion of procollagen, have enabled us to construct a detailed model of the stepwise limited proteolysis of procollagen.     

The role of glycosylation in the enzymatic conversion of procollagen to collagen: studies using tunicamycin and concanavalin A

The enzymatic conversion of chick embryo cranial bone procollagen was studied in vitro using procollagen proteases isolated from the culture medium of chick tendon fibroblasts. During the normal conversion process, chains intermediate in length between proα and α chains, as well as the COOH-terminal extension peptides, can be identified. Underglycosylated procollagen, synthesized by bones treated with an inhibitor of protein glycosylation (tunicamycin), was processed by these proteases in a manner similar to that of intact procollagen. However, medium from cells cultured with tunicamycin lacked the COOH-terminal procollagen protease activity; this did not result from a direct inhibition of the protease by the drug. Concanavalin A also inhibited the conversion of procollagen to collagen by fibroblasts in culture. In an in vitro system, Concanavalin A inhibited the COOH-terminal procollagen protease, and this inhibition was reversed by methyl-α-d-glucopyranoside. These data suggest that the COOH-terminal procollagen protease contains oligosaccharide side chains that are recognized by concanavalin A and that tunicamycin affects the secretion, activity, or activation of the enzyme.     

Studies on the nutrition of rice cell culture I. A simple, defined medium for rapid growth in suspension culture

We investigated the nutrient requirements of rice in liquidculture and developed a revised medium of mineral salts, sucrose,thiamine and 2,4-dichlorophenoxyacetic acid. The following majornutrients at the indicated concentrations were beneficial: NO₃-N(40 mM), NH₄-N (5.0 mM), P (2.0 mM) and K (40 mM). Cobalt, iodine,pyridoxine, nicotinic acid and m-inositol were not essentialand were excluded from the revised R-2 medium. Growth was betterwith ammonium and nitrate together than with nitrate as thesole nitrogen source. Cell growth in the R-2 medium was superiorto that obtained in B5, Heller, Murashige-Skoog and White media. (Received March 31, 1973; )