Mineral salt requirements of Bacillus globisporus subsp. marinus strains

The mineral salt requirements of four isolates of Bacillus globisporus subsp. marinus and of two terrestrial strains of B. globisporus were investigated. In contrast to the terrestrial reference strains the marine isolates showed an obligate requirement for sodium and potassium. The sodium ion could not be replaced by potassium or by osmotically equivalent concentrations of mannitol. None of the strains proved to be dependent upon Mg²⁺ or Ca²⁺. The B. globisporus subsp. marinus strains are considered true marine bacteria. The criteria used to distinguish between marine and terrestrial Gram-negative bacteria can also be applied for Gram-positive bacteria.     

Growth factor requirement of Thiobacillus novellus

Thiobacillus novellus cannot be grown in mineral salts media unless supplied with yeast extract. The requirement is only for miniscule amounts of yeast extract and is not fully expressed unless cells grown in a complex medium are allowed to multiply in a mineral salts medium for four to five generations. Individual sulfur-containing organic compounds, namely biotin, coenzyme A, and lipoic acid, but not reduced inorganic sulfur compounds, can substitute for the yeast extract requirement. Biotin can fully satisfy this requirement at a concentration insufficient to fulfill the biosynthetic sulfur needs; further, the organisms continue to incorporate ³⁵SO₄ into cellular protein in the presence of yeast extract or biotin. It is concluded that biotin is required as a growth factor and not owing to an inability to obtain sulfur from sulfate; the reasons why coenzyme A and thiamine pyrophosphate can substitute for biotin are discussed.Non-standard Abbreviations MS Mineral Salts Base     

Observations on the life history of the belted sandfish,Serranus subligarius (Serranidae)

Synopsis The biology of Serranus subligarius, a typical inhabitant of low limestone reef outcroppings and artificial reefs from North Carolina to Florida and in the northern Gulf of Mexico, was studied on inshore jetties in the northeastern Gulf of Mexico from February 1974 to April 1975. The length-weight relationship was expressed by the equation W = 0.000006L^3.431. Growth was greatest in the first year (43.5 mm SL) and declined in the remaining three years of life (17.3, 10.5, and 7.1, respectively). In individuals larger than 40 mm SL, gammaridean and caprellid amphipods were the most numerous food items but crabs, shrimp, and fishes were the most important food items by weight. In individuals less than 40 mm SL, copepods were the most numerous food item, but gammaridean amphipods and shrimp were the most important items by weight. Two incidents of cannibalism were recorded. The ovotestis of the belted sandfish is composed of two lobes which are broadly united posteriorly, with the testicular tissue restricted to a broad band encircling the posterior region of the gonad. Age-group 1 fish are functional synchronous hermaphrodites. Testicular tissue matures in the fall or winter of a fish's first year (all specimens examined histologically, including those from the nonspawning season, contained flagellated spermatozoa) while ovarian tissue matures in the following spring. Spawning season in 1974 extended from late April or early May to September. Crossfertilization is the usual reproductive mode, although individuals are capable of selffertilization.     

Properties of receptors for epidermal growth factor in detergent solution: Evidence for heterogeneous aggregated states

Between 60% and 100% of epidermal growth factor (EGF) binding activity was recovered from membranes of the A431 human epidermoid carcinoma cell line treated with solutions containing the nonionic detergent Triton X-100. Approximately half of the recovered binding activity was sedimented at low centrifugal forece and hence was operationally insoluble in nonionic detergent solution. Receptors in both the detergent-soluble and -insoluble fractions displayed similar affinities for ¹²⁵I-EGF, and the values were in good agreement with those obtained for receptors in untreated membranes. The receptors in both fractions also formed identical direct linkage complexes with ¹²⁵I-EGF in similar yield, providing no evidence for partitioning of different molecular species of EGF receptors in the detergent-soluble and -insoluble fractions. Gel chromatography of the detergent-soluble membrane fraction on Sepharose 6-B revealed heterogeneity of ¹²⁵I-EGF binding activity; the smallest and most monodisperse peak of activity resolved by this technique was eluted at a Stokes radius of 95 Å. Operationally soluble ¹²⁵I-EGF binding activity also behaved heterogeneously during velocity sedimentation; more than half the activity sedimented more rapidly than the apparently monidisperse, 7S form. An average of less than half the nonionic detergent-solubilized activity recovered from 10 independent membrane preparations behaved as an apparently monodisperse entity. Since a maximum of 60% of ¹²⁵I-EGF binding activity was operationally soluble, less than 25% of the total EGF binding activity was recovered in an apparently monodisperse form. The remaining 75% of the EGF receptors displayed a marked tendency to exist as aggregates in nonionic detergent solutions.     

Visualization of thrombin receptors on mouse embryo fibroblasts using fluorescein-amine conjugated human α-thrombin

The localization of thrombin receptors on mouse embryo (ME) cells has been examined by direct fluorescence microscopy using a fluorescein aminelabeled thrombin. Two fluorescein amines, 4-(N-6-aminoethyl thioureal)-fluorescein and 4-(N-6-aminohexyl thioureal)-fluorescein, were synthesized and attached to the carbohydrate moiety of highly purified human α-thrombin by periodate oxidation of the carbohydrate and selective reduction of the Schiff's base using sodium cyanoborohydride. Preparations of fluorescent thrombin with from 1 to 4 fluoresceins per molecule of thrombin retained their ability to proteolytically cleave fibrinogin to form fibrin clots, to bind to thrombin receptors on ME cells, and to initiate cell division. After incubating mitogenic concentrations of the fluorescein amine labeled thrombin with ME cells at 4°C, a diffuse fluorescent pattern was observed over the surface of the ME cells. This diffuse pattern was specific: it was not observed on cells from parallel cultures incubated with fluorescent thrombin plus a 20-fold excess of unlabeled thrombin. Thus, thrombin receptors appear to be distributed randomly over the surface of ME cells prior to interaction with thrombin. Increasing the temperature to 37°C following binding at 4° C resulted in a rapid dissociation of the fluorescent pattern from the cells leaving only the autofluorescent vesicles. This result may reflect the unique ability of thrombin to proteolytically cleave its own receptor.     

Correlation between cell density,membrane fluidity,and the availability of transferrin receptors in friend erythroleukemic cells

Undifferentiated Friend erythroleukemic cells (FL cells) acquire membrane microviscosity ( ), in accord with the culture cell density. At low cell density poise, whereas at confluency it increases to poise. Concomitantly, the total number of available transferrin receptors per cell decreases by about 80% upon increase in cell density. Modulation of membrane microviscosity, by artificial alteration of the membrane cholesterol level, mediates similar modulations of the availability of the transferrin receptors. The correlation between the availability of the transferrin receptors and the membrane lipid fluidity may take part in the overt decrease in iron uptake by erythroid cells along the erythropoiesis pathway.     

The effect of cessation of growth on protein synthesis in a mutant of Chinese hamster ovary cells with a temperature-sensitive leucyl-tRNA synthetase

A temperature-sensitive mutant of Chinese hamster ovary cells with an altered leucyl-tRNA synthetase fails to grow and to incorporate amino acids into protein properly at or near the non-permissive temperature. This mutant was used to determine whether cessation of growth at the elevated temperature affected elongation factor EF-1, since the activity of EF-1 is markedly lower in non-growing cells in stationary phase than in rapidly-growing cells in exponential phase. Cell-free extracts prepared from cells maintained at 39°C for 24 h showed a marked decrease in the ability to translate natural mRNAs, compared to cells incubated at 34°C. However, the ability to translate poly(U), which requires elongation factor EF-1 (and EF-2), was not affected. Analyses of activities involved in the initiation of protein synthesis and in the activation of amino acids revealed that, with the exception of leucyl-tRNA synthetase, the rest of the components required for translation also appeared to be relatively stable even after 24 h at the elevated temperature. The effects of elevated temperature on cell-free extracts were also investigated. The results were similar to those obtained with intact cells; that is, except for leucyl-tRNA synthetase which was rapidly inactivated in vitro at 39°C, other aminoacyl-tRNA synthetases and translational components involved in chain initiation and elongation were relatively stable. Thus, no change in EF-1 activity was detected as a result of arrested cell growth, an inherent lability of the elongation factor, or metabolic degradation as a consequence of a rapid turnover rate in the absence of protein synthesis.     

A hepatic membrane-associated factor stimulates nuclear DNA synthesis in cultured fibroblastic cells

Nuclear DNA replication in cultured mouse fibroblasts is stimulated by isolated hepatic plasma membranes in a time- and concentration-dependent manner. The plasmalemmal activity is susceptible to trypsin treatment, and to treatment with protein modifying agents, N-ethylmaleimide, N-bromosuccinimide, and 2-hydroxy-5-nitro-benzylbromide.     

Discrimination between aminoacyl groups on su+ 7 tRNA by elongation factor Tu

The su⁺7 nonsense suppressor of Escherichia coli is a mutant tRNA^Trp that can be aminoacylated with either tryptophan or glutamine. We have compared the ternary complexes of glutaminyl and tryptophanyl-su⁺7 tRNA with elongation factor Tu and GTP. Glutaminyl-su⁺7 tRNA binds more strongly than tryptophanyl-su⁺7 tRNA to EF Tu · GTP. The greatest distinction between the two species of the tRNA is seen in their dissociation rates from the complex, which differ by as much as fivefold. The distinction is affected by pH values around neutrality. These results show that EF Tu can distinguish between two aminoacyl-tRNAs which differ only in the aminoacyl group. The implications for the unusual amino acid specificity of su⁺7 tRNA are discussed.     

Kinetics of drug-DNA interaction. Dependence of the binding mechanism on structure of the ligand

Kinetic and equilibrium studies of the binding of several phenanthridines and acridines to DNA have been performed to investigate the physical processes underlying the direct ligand transfer mechanism of drug-DNA interaction· Substitution of the 6-phenyl ring of dimidium with a p-carboxyl residue, or complete removal of either the 6-substituent or the 3-amino group, does not prevent the phenanthridine chromophore from transferring directly between binding sites. Loss of the aromatic ring increases association rate constants three- to ninefold and enhances dissociation rates by factors of up to 12; the rates of direct transfer and dissociation from site 1 are the most perturbed. The presence of a phenyl ring stabilizes the site 1 complex and lowers the binding constant to site 2. Introduction of the p-carboxyl group does not affect the equilibrium distribution of bound forms but produces equivalent increases (2·5-fold) in forward and reverse rate constants for binding to site 1 and for the direct transfer step. The 3-amino group greatly stabilizes the site 1 complex. Its removal accelerates all kinetic processes except for the reverse transfer step; the transfer rate is enhanced 25-fold and binding to site 2 is increased 12-fold. The dissociation rate from site 1 rises by a factor of 45 and that from site 2 by a factor of 5·8.10-Methyl-9-aminoacridine binds via the direct transfer pathway with rate and equilibrium constants similar to those of the 3-desamino derivative of ethidium. This compound provides the first fully characterized example of an acridine that utilizes bimolecular transfer. By contrast, rivanol (6,9-diamino-2-ethoxyacridine) interacts with DNA via a two-step sequential mechanism analogous to that seen with proflavine, yet its intrinsic association constant is three times higher. This results from tighter ‘external’ attachment to the helix, together with a decrease in equilibrium constant for the insertion step, which is markedly slower than that of proflavine. There appears to be a simple relation between the apparent enthalpy of binding and the number of extracyclic amino substituents on the intercalating chromophore.We propose that the two bound forms that participate in direct ligand transfer represent molecules intercalated via one or other of the grooves of DNA, and that the transfer pathway corresponds to exchange of drug between the wide groove of one helix and the narrow groove of another. The ability to form strongly bound complexes at the surface of the helix appears to play a major role in determining the mechanism of ligand binding.