Early mouse embryos produce and release factors with transforming growth factor activity

Summary Previous studies have shown that extracts from mouse embryos at mid and late stages of development contain factors that exhibit transforming growth factor activity. The work reported here demonstrates that cultured mouse embryos at significantly earlier stages of development produce and release factors that exhibit the characteristic property of transforming growth factors. Specifically, the data demonstrate that embryos cultured from the blastocyst stage in serum-containing medium or in serum-free medium release factors that promote the anchorage-independent growth of normal rat kidney fibroblasts. It is shown that these factors are produced and released by cells derived from the inner cell mass and by trophoblasts. The precise developmental stage when production of these factors first begins has not been determined but our findings suggest that these factors are produced by cell types associated with early postimplatation embryos. This work was supported by the Laboratory of Viral Carcinogenesis at the National Cancer Institute and by grants from the National Cancer Institute (CA-36727) and the University of Nebraska Medical Center (22-271-732). Editor's Statement This paper presents evidence that, in an in vitro assay system, early embryonic cells are capable of both synthesizing and secreting TGF-like growth factors, implicating the production of these factors in the events of early development. David W. Barnes     

Effect of domestic sewage on sand beach meiofauna at Goa,India

Meiofauna of a sewage-polluted sandy beach, where sand alone constituted > 90%, was surveyed. Nematodes dominated the fauna numerically at all stations, followed by harpacticoid copepods. Most of the animals were confined to the top 5 cm of the sediment. A seasonal pattern was observed in the distribution of the fauna. There were significant spatial and temporal variations in mean meiofauna density, attributed to organic discharge via sewage and prevailing environmental conditions in the study area.     

Direct analysis of growth factor requirements for isolated human fetal hepatocytes

Summary Hepatocytes were isolated from human fetal liver in order to analyze the direct effects of growth factors and hormones on human hepatocyte proliferation and function. Mechanical fragmentation and then dissociation of fetal liver tissue with a collagenase/dispase mixture resulted in high yield and viability of hepatocytes. Hepatocytes were selected in arginine-free, ornithine-supplemented medium and defined by morphology, albumin production and ornithine uptake into cellular protein. A screen of over twenty growth factors, hormones, mitogenic agents and crude organ and cell extracts for effect on the stimulation of hepatocyte growth revealed that EGF, insulin, dexamethasone, and factors concentrated in bovine neural extract and hepatoma cell-conditioned medium supported attachment, maintenance and growth of hepatocytes on a collagen-coated substratum. The population of cells selected and defined as differentiated hepatocytes had a proliferative potential of about 4 cumulative population doublings. EGF and insulin synergistically stimulated DNA synthesis in the absence of other hormones and growth factors. Although neural extracts enhanced hepatocyte number, no effect on DNA synthesis of neural extracts or purified heparin-binding growth factors from neural extracts could be demonstrated in the absence or presence of defined hormones, hepatoma-conditioned medium or serum. Hepatoma cell-conditioned medium had the largest impact on both hepatocyte cell number and DNA synthesis under all conditions. Dialyzed serum protein (1 mg/ml) at 10 times higher protein concentration had a similar effect to hepatoma cell-conditioned medium (100 μg/ml). The results suggest that hepatoma cell conditioned medium may be a concentrated and less complicated source than serum for purification and characterization of additional normal hepatocyte growth factors. This work was supported by NIH grant DK35310. Editor’s statement Many investigators have struggled with the special problems associated with culture of differentiated hepatocytes. In this paper attention is given to the specific growth factor requirements for fetal human hepatocytes. The observation that factors from hepatoma conditioned medium or neural extracts enhanced the growth of the cells may indicate that additional growth factors are to be identified that are important in the survival and proliferation of hepatocytes, and may also indicate that the malignant transformation of these cells may involve the production of autocrine growth stimulators.     

Different hormonal requirements for androgen-independent growth of normal and tumor epithelial cells from rat prostate

Summary The proliferation of isolated normal prostate epithelial cells from rat and man is androgen-independent and requires cholera toxin, insulin, dexamethasone, epidermal growth factor (EGF) and one or more polypeptide factors that are concentrated in bovine neural tissue. The active agents in the neural tissue extract are heparin-binding polypeptides (prostatropins), the predominant form of which has a molecular weight of 17400 and an acetylalanine at the aminoterminus. Prostatropins supported a half-maximal increase in normal prostate epithelial cell number at 50 picomolar. The proliferation of primary and serially-cultured epithelial cells from androgen-responsive Dunning R3327 rat prostate tumors was also androgen-independent, but exhibited dramatic alterations in response to hormones that stimulated normal cell proliferation. At low cell density, androgen-independent growth of isolated tumor-derived epithelial cells was independent on cholera toxin, was stimulated by dexamethasone, required insulin andeither EGFor prostatropin. The presence of either EGF or prostatropin masked the response to the other factor. In the absence of EGF, purified prostatropins supported a half-maximal increase in tumor cell number at 7 picomolar. Endogenous production of EGF-like and prostatropin-like factors or both was suggested by the reduced requirement for EGF and prostatropin at high prostate tumor cell density. These results suggest that anti-hormonal therapies against prostate tumor growth should be based on intervention with the activity of insulin (or insulin-like factors) or simultaneous intervention with both EGF and prostatropin (or their homologues). This work was supported by NIH grants CA 37589 and HL 33847, and grant 1718 from the Council for Tobacco Research. Editor’s Statement This paper is the first report of the comparison of the hormone requirements of primary cultures of normal and tumor prostate epithelial cells from the same system.     

High-cell-density fermentation studies of a recombinantE. coli that expresses atrial natriuretic factor

Summary Studies are presented on the fermentation of recombinantEscherichia coli that express rat atrial natriuretic factor (ANF) as a fusion protein. Our objective was to achieve high cell density while maintaining ANF expression at the same level as observed in shake flasks. Improved fermentation conditions included: maintaining glucose concentrations at 1 g/l, using an enriched medium, adding concentrates of medium throughout the fermentation, and blending oxygen for adequate aeration. Cell densities of 12 g/l (dry weight) were achieved, which represented a 10-fold increase over non-improved conditions, while maintaining ANF levels at 7 mg/g of dry cell mass. When galactose was used as an initial carbon source or as a feed supplement, there was a 2-3-fold increase in the expression of ANF from these high-cell-density fermentations. The recombinant ANF was biologically active.     

Environmental implications of recombinant DNA technology

Applications of recombinant DNA technology are discussed as a backdrop for evaluation of the environmental impacts of this technology. Some of applications include using traditional biological techniques for specific purposes, including nitrogen fixation, microbial pesticides, and waste treatment. In these instances the final product lies along a continuum, beginning with an organism marginally performing its function, and ending with one that is highly specialised and very efficient in what it does. One may move along this continuum toward the ‘perfect’ microorganism by using traditional methodologies of mutagenesis and selection, recombinant DNA technology, or a combination of the two.     

The postribosomal particle of rabbit liver contains protein-synthesis factors and serum albumin mRNA

As demonstrated by indirect immunoprecipitation and polyacrylamide gel electrophoresis, an 85S particle separated by sucrose density-gradient centrifugation from the postribosomal pellet of rabbit liver, is able to synthesize serum albumin if supplemented with both ribosomal subunits and sources of energy. It is retained on heparin bound to Sepharose 4B, contains translatable mRNA and apparently all protein factors required for translation. This particle may represent a highly organized protein synthesizing machinery, the combination of which with ribosomes results in formation of new protein molecules.     

A stimulatory subunit in the polypeptide elongation factor-1 of the chick brain

Abstract: The higher-molecular-weight elongation factor-1 (EF-1_H) of the chick brain was observed to contain three subunits (denominated α, β, and γ), contrary to a previous report that the brain EF-1_H consisted of aggregates of low-molecular-weight elongation factor- 1 (EF-1_L). Crude EF-1_H, obtained from 20-day embryonic brain, was treated with 0.4 M ammonium chloride and 0.1 mM GTP, and EF-1_βγ, was obtained using a DEAE-Sephadex column equilibrated in 0.025 mM GTP. Both EF-1_β, and EF-1_γ, were isolated by means of a DE-52 column equilibrated in 6 M urea and were found to have molecular weights of 2.8 and 4.8 × 10⁴, respectively. EF-1_β and EF-1_γ were also obtained from young rat and calf brains by the same procedures. The molecular weight of the isolated EF-1_α was 5 × 10⁴. It was found that EF-1_β stimulated the two EF-1_α-dependent reactions, i.e., phenylalanyl-tRNA binding (reaction 1) and polyphenylalanine synthesis (reaction 2), and also stimulated the nucleotide exchange reaction in the EF- 1_α-guanine nucleotide binary complex (reaction 3). The degrees of stimulation of reactions 1, 2, and 3 by the addition of EF-1_β were 2 to 3 times, about 18 times, and 2 to 3 times as much as with EF-1_α alone, respectively. The amino acid compositions of EF-1_α -1_β, and -1_γ and EF-2 were very similar to those of other eukaryotic tissues. Thus the constituents and properties of EFs of the brain were found to be basically similar to those of other tissues of eukaryotes, although EF-1_β, and EF-1, had not been reported in the brain. A possible physiological significance of EF-1_β during brain development is also discussed.     

Multiple Molecular Forms of Enkephalins in the Guinea Pig Hippocampus

Abstract: The combined techniques of HPLC and radioimmunoassay were used to identify and quantitate enkephalin-related peptides in the guinea pig hippocampus. Both met- and leu-enkephalin were identified, in approximately a 2:1 ratio, as well as a third enkephalin-like molecule that is neither met- nor leu-enkephalin. The third enkephalin elutes earlier than met- or leu-enkephalin from a reversed-phase column, has a molecular weight similar to the other enkephalins, and is as active as these enkephalins are in inhibiting binding of labeled opiates to rat brain membranes. All regions of the hippocampus (dentate gyrus, CA1–2, CA3–4, and subiculum) contain all three immunoreactive peptides. Immunocytochemical techniques, using antisera raised against met-enkephalin, show with one antiserum immunoreactivity in the granule cell-mossy fiber system, and with the other scattered immunoreactive cells mostly in the CA2 region. Enkephalins are not confined to the mossy fiber system, as previously suggested, but may be a component of another hippocampal innervation.     

Requirements for antiribosomal activity of pokeweed antiviral protein

It has been known for some time that pokeweed antiviral protein acts by enzymatically inhibiting protein synthesis on eucaryotic ribosome systems. The site of this action is known to be the ribosome itself. In this paper we show that the pokeweed antiviral protein reaction against ribosomes is a strong function of salt concentrations, where 160 mM K⁺ and 3 mM Mg²⁺ retards the reaction, while 20 mM K⁺ and 2 mM Mg²⁺ allows maximum reaction rate. It is also shown, however, that an unidentified protein in the postribosomal supernatant solution, together with ATP, allows the ribosome to be attacked even in the presence of high salt. Kinetic analysis of the antiviral protein reaction has been carried out under both sets of conditions, and reveals that the turnover number for the enzyme is about 300–400 mol/mol per min. in each case. The K_m for ribosomes is 1 μM in the presence of low salt and 0.2 μM at higher salt in the presence of postribosomal supernatant factors plus ATP. The antiviral protein reaction is also shown to be pH dependent and is controlled by a residue with pK_a value of approx. 7.0, apparently a histidine. Stoichiometric reaction of the enzyme with iodoacetamide results in a significant loss of antiribosomal activity.