Hansjürgen Ristow

Properties of two growth-stimulating proteins isolated from fetal calf serum.

Two proteins (S1, S2) which stimulate cell growth in cultures of embryonic rat fibroblasts, have been isolated from fetal calf serum. S1 is a protein complex consisting of an α2-macroglobulin and insulin, asc ould be shown by immunological methods. For growth-activating activity both components are essential; when added separately to the cultures, they are ineffective. S2 has a molecular weight of about 26 000 D and does not cross-react with S1 in immunological tests. For optimal stimulation of the cultures both factors must be present together in the medium. As demonstrated by autoradiographic studies, there probably exist two different cell populations in the rat fibroblast cultures which either respond to S1 or S2. S1 and S2 strongly influence the metabolism of isolated fat cells. S1 behaves like insulin; S2 stimulates glucose oxidation and lipogenesis, but, in contrast to insulin and non-suppressible insulin-like activity, also activates the adenylate cyclase in these cells.

A system in vitro for the synthesis of RNA and protein by isolated salivary glands and by nuclei from Chironomus larvae

Abstract Salivary glands from the front ends of the Dipteran larvae Chironomus can be isolated by sucrose gradient centrifugation. The isolated glands incorporate [3H]uridine and [14C]leucine in vitro. Extraction and gradient centrifugation of the labelled RNA reveals peaks, at about 16 S and 21 S, which do not coincide with the absorbance peaks of the ribosomal RNA. In addition, intact nuclei can be obtained from the salivary glands by the use of detergents. These isolated nuclei also incorporate [3H]uridine and [14C]leucine. The sedimentation behaviour of the RNA synthesized in vitro is the same as that found in total glands.

Induction of sporulation in Bacillus brevis by peptide antibiotics.

THE regulatory mechanisms of the process of bacterial sporulation are unknown, although some authors have speculated that sporulation and the synthesis of peptide antibiotics are somehow connected1–4. Paulus and coworkers proposed that tyrocidine and gramicidin D, both synthesised by Bacillus brevis, may interact with the transcriptional process during sporogenesis5. They have isolated a mutant of B. brevis which is unable to synthesise gramicidin D and which produces defective spores. The defects could be cured by exogenous gramicidin D, suggesting that this peptide is involved in sporogenesis6. In this report we demonstrate that sporulation of early vegetative growing B. brevis cells can be induced by exogenous tyrothricin, a mixture of tyrocidine and gramicidin D, provided that the cells are exposed to a culture medium lacking the nitrogen source. The induction of sporulation occurs concomitantly with a significant increase in RNA synthesis. The results suggest that sporulation and the action of these two peptides are causally connected. Both peptides may play a positive regulatory role in cell differentiation.

The peptide antibiotic gramicidin D A specific reactivator of tyrocidine-inhibited transcription

The sporulating bacterial strain Bacillus brevis (ATCC 8185) produces two types of peptide antibiotics, the linear gramicidins and the cyclic tyrocidines. Their effects on transcription in vitro were studied: 1. RNA synthesis catalized by RNA polymerase and DNA as template, both from B. brevis cells, is inhibited by the addition of gramicidin maximally to 50% as compared to controls without gramicidin. This inhibition is dependent on the concentration of gramicidin as well as on that of RNA polymerase. 2. Transcription of DNA is also inhibited by tyrocidine. This inhibition is partially compensated by the addition of gramicidin. Here, the action of gramicidin is again dependent on its concentration and on that of RNA polymerase. 3. This counteraction of gramicidin occurs only with RNA polymerase from B. brevis. Enzyme preparations of other origin are additively inhibited by gramicidin when previously inhibited by tyrocidine. The specific action pattern of gramicidin and tyrocidine in connection with the B. brevis RNA polymerase supports the notion that the two peptide antibiotics may be involved in a gene regulatory mechanism during sporogenesis.

Fibroblastoid and epithelioid cells in tissue culture: differences in sensitivity to ouabain and to phospholipid composition.

Abstract Investigations on nine different mammalian cell lines revealed that permanently growing cells of one morphological class have numerous membrane properties in common which are different or even lacking in the other cell class. With electrophysiological methods it is shown that the sensitivity of the Na+-K+ pump to ouabain is three orders of magnitude higher in the ionically non-coupled epithelioid cells than in the ionically coupled fibroblastoid cells which respond like primary cultures. This is accompanied by considerably higher binding constants for ouabain of the epithelioid cells as was shown by [3 H]ouabain binding and membrane potential measurements. The epithelioid cells also revealed a 50% lower relative amount of phosphatidylethanolamine and a 60-fold less net synthesis of phospatidylinositol. Finally, although primary cultures cannot proliferate without serum, permanent fibroblastoid cells have a reduced serum requirement and permanent epithelioid cells can proliferate without any serum.

Polysomal precursors in KB cells. I.

Abstract Small particles are found in the cytoplasm of KB cells which contain ribosomal RNA. These so-called 40-S and 60-S particles are precursors for polysomes. This conclusion is drawn from the following experiments: 1. 1. In the cytoplasm, labeled uridine is incorporated first into 40-S particles and can be chased later into the polysomes. The turnover of the 60-S particles and the single ribosomes remains low during the course of such experiments. 2. 2. Polyuridylic acid forms aggregates with these particles which have the size of polysomes. 3. 3. Since uridine is incorporated simultaneously with labeled leucine, the 40-S and 60-S fractions are ribonucleoprotein particles; about 70 % of their RNA content is not digested by ribonuclease.

Increased phosphatidylinositol synthesis in rat embryo fibroblasts after growth stimulation and its inhibition by δ-hexachlorocyclohexane

When resting rat embryo fibroblasts are stimulated to grow, a substantial increase in phosphatidylinositol synthesis can be observed. This increase cannot be explained by increased glucose uptake or glycolysis. delta-Hexachlorocyclohexane having the same configuration as myo-inositol, inhibits phosphatidyl inositol synthesis as well as DNA synthesis and mitosis, but has no effect on phosphatidyl choline synthesis. When delta-hexachlorocyclohexane is added to fibroblast cultures during the first hours after stimulation, a delay of DNA synthesis and mitosis compared to uninhibited cultures can be observed. Since delta-hexachlorocyclohexane also inhibits the uptake of nucleotides, hexoses and amino acids, it is suggested that phosphatidylinositol is necessary for the proper functioning of those receptors and carriers which are an essential part of the early cellular processes after growth stimulation, and this role of phosphatidyl-inositol may explain its increased turnover in growing cells. The increased phosphatidylinositol synthesis could not be associated to one of the subcellular fractions. When cells were labeled with [32P]orthophosphate during the first 10 min after growth stimulation and were subsequently separated into cellular fractions such as nuclei, mitochondria, plasma membranes and microsomes, no significant differences in radioactivity of phosphatidylinositol among those fractions could be observed.

Messenger RNA associated with polysomes, ribosomes and subribosomal particles in KB cells.

Abstract Ribosomes and subribosomal particles in the cytoplasm of KB cells contain a class of RNA which is different from ribosomal 18-S and 28-S RNA. It is the first RNA fraction in the cytoplasmic particles which is labelled. It has a heterogeneous sedimentation profile. It is easily digested by ribonuclease while on the particles, and it stimulates protein synthesis in vitro . It has properties identical to those of the messenger RNA (mRNA) associated with the polysomes, and we therefore propose that mRNA is a constituent of ribosomes and subribosomal particles in KB cells.

Peptide antibiotics and sporulation: induction of sporulation in asporogenous and peptide-negative mutants of Bacillus brevis

Mutants of Bacillus brevis ATCC 8185 were isolated which were unable to produce detectable amounts of either tyrocidine or linear gramicidin, or both peptide antibiotics. Tyrocidine-negative mutants (BM5, BM21, BM44) sporulated normally. Gramicidin-negative mutants (BM2, BM24) were oligosporogenous, and mutants unable to produce both peptides (S18, S19) were asporogenous. Addition of tyrocidine and/or gramicidin to asporogenous mutants in rich medium did not stimulate sporulation. However, these mutants formed normal spores after being transferred to nitrogen-free medium and upon the addition of tyrocidine. It was demonstrated that nutrient broth has a suppressive effect on tyrocidine-induced sporulation of S18. The tyrocidine-negative mutant BM44, sporogenous in rich medium, could sporulate under nitrogen deprivation only if supplemented with tyrocidine. The significance of the peptide antibiotics for a regulatory role in sporogenesis of B. brevis is discussed.

Induction of Sporulation in Bacillus brevis. 1. Biochemical Events and Modulation of RNA Synthesis during Induction by Tyrocidine

Under conditions of severe nitrogen starvation, brought about by nutritional shift-down, Bacillus brevis ATCC 8185 was unable to sporulate unless supplemented with the peptide antibiotic tyrocidine. The induction of sporulation was highly specific for tyrocidine and required only very low concentrations of the peptide (5 microM). Tyrocidine-induced sporulation was accompanied by the typical sporulation-specific events (e.g. extracellular protease production and dipicolinate synthesis) as well as the formation of linear gramicidin. The addition of tyrocidine produced acute inhibition of RNA synthesis that was followed by a limited activation of transcription near the time of onset of linear gramicidin synthesis, when the first sporulation-specific changes were observed. These results provide direct evidence for a role of tyrocidine in sporulation of B. brevis and suggest that the action of the peptide antibiotic may involve the control of transcription. Such a notion is supported by earlier studies on the effects of tyrocidine and linear gramicidin on purified RNA polymerase.