Reduction of lactic acid secreted from SV3T3 cells by a serum factor and biotin and its relationship to cell growth

Supplementation of media containing a low concentration (0.15–0.30% $\text{v}\text{>v}$) of calf serum with biotin or a low molecular weight serum growth factor (Peak III) reduces the amount of lactic acid secreted by simian virus 40-transformed 3T3 cells. While biotin and Peak III (which has been tentatively identified as biotin) can stimulate “stationary phase” cells to resume viable cell division, this growth promotion is not due to an alleviation of lactic acid toxicity per se. This conclusion is based on the finding that, although higher concentrations of lactic acid are cytotoxic, lactic acid added at concentrations found during “stationary phase” to cells plated in fresh medium is not growth inhibitory. These results suggest, instead, a possible major role for biotin and Peak III in energy production.     

The second large simian virus 40 T-antigen exon contains the information for maximal cell transformation

Microinjection of early simian virus 40 (SV40) DNA fragments has shown that maximal transformation of rat cells (Ref 52) is a property of the second SV40 T-antigen exon. Expression of this particular T-antigen region was obtained by coinjection of the $\text{Taq}\text{Bam}$ DNA fragment with the early promoter/enhancer $\text{Hpa}\text{II}\text{Bgl}\text{I}$ fragment. Microinjection of the DNA fragment mixture induced two categories of transformants; namely, maximally and minimally transformed cells. The maximally transformed cells synthesize two $\text{Taq}\text{Bam}$-specific polypeptides, and the minimally transformed cells only the lower molecular weight form. Both types of transformants contain the cellular p52 protein at high concentrations. Furthermore, maximal transformation of Ref 52 cells requires the carboxy terminus of the T-antigen. Cells transformed by microinjection of the SV40 Pst A-fragment display different parameters of maximally transformed cells but not anchorage-independent growth.     

Mutations in the right operator of bacteriophage lambda: evidence for operator-promoter interpenetration

A set of virulent mutants of bacteriophage lambda have been selected from λv2 v3. The sites of mutation form two microclusters, both close to v3. Some of the mutants, selected for their ability to grow on a λ-lysogen, can also grow on a λdv carrier strain. They are called “supervirulent” and a mutation conferring super-virulence is called vs. The sites of mutation to vs lie between the presumed promoter mutants (x3, x7) and x13, implying that the operator and promoter interpenetrate each other. The relative affinities of λ repressor for binding, in vitro, to λv⁺, λv3, λvs326, and λv3 vs327 DNA were 1, $\text{1}\text{4}$, $\text{1}\text{20}$, and $\text{1}\text{4000}$, respectively. We suggest that two separate mutations in the right operator are needed to confer virulence because promoter sites lie within the operator.     

The non-covalent binding of benzo[a]pyrene and its hydroxylated metabolites to intracellular proteins and lipid bilayers

The non-covalent interactions of benzo[a]pyrene (BP) and several of its hydroxylated metabolites with ligandin, aminoazodye-binding protein A (Z-protein, fatty acid binding protein) and lecithin bilayers have been studied by equilibrium dialysis, an adsorption technique and fluorescence spectroscopy. Binding affinities expressed as $\text{v}\text{/c}$ (where $\text{v}$ = moles of BP or BP metabolite bound per mole of protein or lipid and c = unbound concentration), were measured at concentrations sufficiently low that there was no self-association of the unbound compounds as judged by their fluorescence characteristics. 3-Hydroxybenzo[a]pyrene (BP-3-phenol), 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene (BP-4,5-dihydrodiol) and 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (BP-7,8-dihydrodiol) bind more strongly ($\text{v}\text{/c = 10}{}^5\text{?5 · 10}{}^5\text{l}\text{·}\text{mol}{}^{\mathrm{?1}}$) to all three binders than does BP itself ($\text{v}\text{/c = 10}{}^4\text{?7 · 10}{}^4\text{l}\text{·}\text{mol}{}^{\mathrm{?1}}$). 9,10-Dihydro-9,10-dihydroxybenzo[a]pyrene (BP-9,10-dihydrodiol) binds to ligandin with an affinity similar to those of the other BP metabolites studied here, but binds much less strongly to both protein A and lecithin ($\text{v}\text{/c = 10}{}^4$ and 3 · 10⁴ l · mol^?1, respectively). The low affinity of BP-9,10-dihydrodiol for lecithin would account for earlier findings that on incubation of BP with isolated rat hepatocytes, this metabolite egressed from the cells to the extracellular medium much more readily than either BP-4,5-dihydrodiol or BP-7,8-dihydrodiol.Calculations based on these results suggest that within hepatocytes BP and its metabolites, including BP-9,10-dihydrodiol, will be found almost exclusively associated (>98%) with lipid membranes.     

Cryopreservation of Plasmodium chabaudi I: Protection by glycerol and dimethyl sulfoxide during cooling and by glucose following thawing

Two additives, glycerol and dimethyl sulfoxide (Me₂SO), were investigated for toxic and protective effects for the intraerythrocytic stages of Plasmodium chabaudi. After incubation for 15 min, at 0 °C in Me₂SO and at 37 °C in glycerol, with various concentrations of these additives, half the blood from each treatment was cryopreserved in glass capillary tubes cooled at approximately 3600 °C min^?1 by plunging into liquid nitrogen. Warming was rapid, approximately 12000 °C min^?1, produced by agitation in a water bath at 40 °C for 1 min. The effect of dilution in phosphate-buffered saline (PBS) supplemented with various concentrations (5 to 25% $\text{v}\text{v}$) of glucose was also investigated in conjunction with the two cryoprotectants. Survival of both the frozen and the unfrozen control parasites was assayed by the mean time taken for the parasitemia in groups of five mice to reach a level of 2% following intraperitoneal injection of 10⁶ parasitized erythrocytes into each mouse. Glycerol was toxic at concentrations above 10% $\text{v}\text{v}$ and Me₂SO above approximately 15%. The use of glucose in the recovery medium resulted in a substantial improvement in the survival of frozen and unfrozen parasites previously incubated in either cryoprotectant. The amount of glucose required varied with the concentration of additive used, and optimum survival of cryopreserved parasites was obtaind with 10% $\text{v}\text{v}$ glycerol or 15% $\text{v}\text{v}$ Me₂SO and with 15% $\text{w}\text{v}$ glucose in the diluent medium.     

The effects of lead upon collagen synthesis and proline hydroxylation in the Swiss mouse 3T6 fibroblast.

The effects of lead upon collagen synthesis and proline hydroxylation were examined in the Swiss mouse 3T6 fibroblast. The results indicate that lead reduces proline hydroxylation in stationary phase cultures of 3T6 cells, resulting in increased cellular retention of unhydroxylated procollagen. Inhibition of proline hydroxylation by lead was prevented by increasing the extracellular $\text{Fe}{}^{\mathrm{2+}}\text{Pb}{}^{\mathrm{2+}}$ molar ratio. Interference by lead in the hydroxylation of proline in logarithmic phase cultures of 3T6 cells resulted in increases in the 0.5 n HClO₄ soluble/insoluble hydroxyproline ratio. This was attributed to an increase in the rate of breakdown of lead-induced unhydroxylated procollagen. Kinetic analysis of the lead-iron interaction with proline hydroxylase suggests that the mechanism is competitive.     

Human complex-forming glycoprotein, heterogeneous in charge: the primary structure around the cysteine residues and characterization of a disulfide bridge

L-929 cell surface membranes have been assayed in vitro and found to contain significant protein kinase activity. A steady-state kinetic analysis indicated that at least two distinct protein kinases were present. Plots of reaction velocity (v) against substrate (ATP) concentration were distinctly biphasic, as were Lineweaver-Burk plots of $\text{1}\text{v}$ versus $\text{1}\text{ATP}$. Michaelis constants of the two enzymes were calculated to be 22 and 173 μm, respectively. Sodium dodecyl sulfate polyacrylamide gel analysis of the phosphorylated membrane proteins provided additional support for the existence of more than one protein kinase. Different endogenous proteins were phosphorylated at 1 μm ATP compared to 1 μm ATP. Further studies of the low K_m (22 μm) enzyme suggested that it is a typical cyclic 3′,5′-AMP-independent protein kinase. Its activity was dependent on the presence of Mg²⁺, but it was not affected by cyclic 3′,5′-AMP, cyclic 3′,5′-GMP, or the heat-stable inhibitor of cyclic 3′,5′-AMP-dependent protein kinases. ATP and GTP, but not other nucleoside triphosphates, could serve as phosphoryl donor and maximum kinase activity was expressed at pH 7.0. Phosvitin and casein were superior to histones as exogenous substrates for the low K_m enzyme.     

Simian virus 40 rapidly lowers cAMP levels in mouse cells

The addition of SV40 to contact inhibited $\text{Balb}\text{3T3}$ cells causes a 2-fold decrease in intracellular cAMP levels. The levels reach a minimum 3 hours after virus addition, and after a few hours begin to rise toward normal. No significant changes in cAMP levels are observed after cells are exposed to UV-inactivated virus or are mock-infected. This is the earliest known effect of SV40 infection. We propose that SV40 induces host DNA synthesis by lowering cAMP levels.     

Conformation of 1- and 3-deazaadenosines in solution as studied by 1H nuclear magnetic resonance spectroscopy.

¹H nuclear magnetic resonance spectra of 1 - (II) and 3-deazaadenosines (III) together with adenosine (I) in dimethylsulfoxide have been examined. Features of coupling constants indicate that the furanose rings of I, II, and III have similar conformational preferences and that conformations about the 4′-C–5′-C bond are preferentially $\text{gauche-gauche}$. Nuclear Overhauser effect and spin-lattice relaxation-time measurements demonstrate that II predominantly adopts the $\text{syn}$-conformation similar to that of I, whereas that of III has a greater $\text{anti}$ (freely rotating) component. The results suggest that the $\text{syn}$-conformation in II as well as I is stabilized presumably through a hydrogen bond between the 3-N and 5′-hydroxyl group.     

Effects of cytochalasin B on division and calcium carbonate extrusion in a calcareous alga.

Cytochalasin B (CB) (100 μg/ml) reversibly blocked cell division and cuased the formation of abnormal cytoplasmic bodies in the alga Cricosphaera carterae. Concentrations of 20 μg/ml and 40 μg/ml CB were without effects. In the presence of CB, calcified bodies (coccoliths) which form in Golgi vesicles and are normally extruded through the plasma membrane were not extruded and accumulated within the cell. CB appeared to alter the membranes of Golgi vesicles containing coccoliths. DMSO (10% $\text{v}\text{v}$), the solvent for CB, was without effect on cell division and coccolith extrusion. A concentration of 20% $\text{v}\text{v}$ DMSO inhibited cell division irreversibly.     

Synthesis of simian virus 40 DNA in isolated nuclei

The presence or absence of calcium ions during the isolation of nuclei from SV40-infected African green monkey kidney cells significantly affects the size of SV40 DNA synthesized $\text{in vitro}$. When Ca⁺⁺ is present during the nuclear isolation procedure, the 3–7S fragments of SV40 DNA synthesized $\text{in vitro}$ mature into long chains; in the absence of Ca⁺⁺ they do not.     

Rat lymphocyte subsets: Cellular requirements for the generation of T-cell growth factor

In this study, the cells producing T-cell growth factor (TCGF) in the rat MLR were characterised with respect to the antigens defined by $\text{W}\text{3}\text{13}\text{,}\text{W}\text{3}\text{25}$, and OX8 monoclonal antibodies. Unfractionated lymphocytes and cells depleted of OX8 positive cells were found to be fully capable of producing TCGF, whereas lymphocytes depleted of $\text{W}\text{3}\text{13}\text{and}\text{W}\text{3}\text{25}$ positive cells had lost this ability. Parallel experiments demonstrated that cells selected by the fluorescence-activated cell sorter for the expression of $\text{W}\text{3}\text{13}\text{and}\text{W}\text{3}\text{25}$ defined antigens were potent producers of TCGF. Further studies suggested a functional role for the antigen defined by $\text{W}\text{3}\text{25}$ antibody because the addition of this antibody to a MLR abrogates TCGF production. These findings suggest that the important immunomodulatory functions of $\text{W}\text{3}\text{25}$ positive lymphocytes could be exercised via the synthesis of essential lymphokines.     

Experimental studies on a mutant gene (e) preventing the differentiation of eye and normal hypothalamus primordia in the axolotl

The phenotype of axolotls (Ambystoma mexicanum) homozygous for the mutant gene e (“eyeless”) is different from normal in that (1) no optic vesicles develop in $\text{e}\text{e}$ embryos, (2) $\text{e}\text{e}$ larvae from posthatching onward are darker than normal white larvae, and (3) fully grown $\text{e}\text{e}$ animals are sterile.Experiments reported here show that eyelessness in $\text{e}\text{e}$ embryos results from a direct effect of the gene on presumptive forebrain ectoderm; not on the mesoderm that induces the ectoderm to form eyes. Homotopic grafts of normal presumptive ectoderm on $\text{e}\text{e}$ blastula hosts differentiated complete eyes, but reciprocally grafted embryos were always eyeless. Similarly, grafts of either $\text{e}\text{e}$ or normal presumptive prechordal mesoderm into normal hosts gave normal eyes, but in the mutant hosts no eyes developed. Thus the e gene affects only the ectodermal component of the inductive system for eye formation.Genetically eyeless (pigmented) cells, when interspersed prior to gastrulation among genetically eyed (albino) cells in the eye preprimordium, are induced to form clones of pigmented retinal epithelium in the albino host eye.The sterility of $\text{e}\text{e}$ larvae appears also to be due to a direct effect of the e gene on the ectodermal (neural plate) primordium of the hypothalamus. Grafts of normal cells which included the hypothalamic, but not the optic or anterior pituitary primordia, always restored fertility to $\text{e}\text{e}$ recipients.The mutant pigmentation phenotype was demonstrated to be a consequence of eyelessness and, therefore, an indirect effect of the gene. The pigment pattern of normal embryos from which both optic vesicles were removed resembles that of the mutants. In addition, implantation of a single full-sized, functional eye was able to restore the normal pigmentation, but not fertility, to $\text{e}\text{e}$ recipients.     

Liquid scintillation counting of aqueous samples using Triton-containing scintillants

Markedly unstable count rates were observed using a toluene-Triton (2:1, $\text{v}\text{v}$) scintillant during counting of water-soluble radioactive compounds when < 5% ($\text{v}\text{v}$) water was present, because of the separation of phases. Efficiency correction in these instances could not be made by using ³H₂O as internal standard, because under the same conditions count rates with tritiated water were stable. Increasing water to ≥6% stabilized the count rates. With toluene-Triton (2:1, $\text{v}\text{v}$) scintillant, the water level should preferably be maintained between either 6 and 12 or 18 and 24% for ¹⁴C- and ³H-labeled compounds for counting at 6°C or at ambient temperature (but only between 6 and 12% for ³H counting at room temperature). With a “Tritosol” (Anal. Biochem.63, 555 (1975) modified to contain 35 ml of ethylene glycol, 140 ml of ethanol, 250 ml of Triton X-100, 575 ml of xylene, 3 g of PPO, and ±200 mg of POPOP, water levels of up to 23% were acceptable for ¹⁴C and ³H for counting at room temperature or at 6°C. Within these limitations, with the toluene-Triton or with the modified Tritosol as scintillant, both polar and apolar radioactive compounds exhibited similar efficiencies and gave quench-correction curves, based on the external standard ratio, that were linear for both ¹⁴C and ³H-labeled compounds.     

Ovulation rate in ewes after single oral glucogenic dosage during a ram-induced follicular phase

A 2-factor factorial array with three replicates (N = 280) was used to simultaneously assess the effects on ovulation rate of two alternative doses of medroxy-progesterone acetate (MPA) (10 or 60 mg), applied during a 6-day priming period, and the effect of a single dosage of a glucogenic formulation, administered immediately before ram exposure to groups of adult seasonally anovular Corriedale ewes. The glucogenic formulation contained 1,2,3-propanetriol (glycerol; 70% $\text{v}\text{v}$), 1,2-propanediol (propylene glycol; 20% $\text{v}\text{v}$) and distilled water (10% $\text{v}\text{v}$). At sponge withdrawal, a single oral dose of 100 ml of this formulation or the same volume of distilled water was administered to treated and control groups, respectively, and ewes were immediately exposed to rams and hormonally-induced oestrous ewes. Data from an ancillary experiment (n = 10) showed significantly (P < 0.005) above normal plasma glucose levels in treated animals at 3 and 6 h after dosage. A significant interaction (P = 0.0006) between MPA priming doses and glucogenic supplementation was detected. Supplemented ewes, among those exposed to the lower dose of MPA, exhibited a higher (P = 0.0098) mean ovulation rate (1.56 ± 0.076) than ewes that did not receive glucogenic treatment (1.31 ± 0.060). In contrast, ovulation rate was significantly decreased (P = 0.021) from 1.30 ± 0.058 to 1.13 ± 0.042 after glucogenic treatment in ewes that were primed with sponges containing 60 mg of MPA. Ewes exposed to 60 mg of MPA were marked by the rams at a significantly later (P < 0.00001) mean time (54.8 ± 1.44 h) than ewes receiving 10 mg sponges (43.6 ± 1.08 h). These results reveal the potential for modifying ovulation rate through short-term glucogenic manipulations, at least during the compressed follicular phase typical of ram-induced ovulations.     

Two tissue culture media for production of lepidopteran cells and nuclear polyhedrosis viruses

Two media supporting the growth of several established lepidopteran cell lines in monolayer and suspension culture are described. The medium designated $\text{BML-TC}\text{10}$ was developed specifically as an inexpensive medium for production of cells of Spodoptera frugiperda and the homologous nuclear polyhedrosis virus (NPV) of this species. Simultaneously, a second medium was formulated in which the amino acid requirements were provided by enzymatic protein hydrolysates, one of which was termed $\text{BML-TC}\text{7A}$. Several cell lines could be adapted easily to this medium. $\text{BML-TC}\text{10}$ supported growth of S. frugiperda cells and production of the NPV's of S. frugiperda and Autographa californica. $\text{BML-TC}\text{7A}$ supported the growth of cells of S. frugiperda. Carpocapsa pomonella, Heliothis zea, and Trichoplusia ni. Cells of the latter produced the polyhedra of T. ni and A. californica NPV's in this medium.     

Cyclic AMP-dependent protein kinase of Neurospora crassa

$\text{Neurospora}\text{crassa}$ was surveyed for cyclic AMP-dependent protein kinase activity. Two peaks (I and II) of protein kinase activity were demonstrated by DEAE-cellulose chromatography of wild type $\text{Neurospora}$ extracts. Peak I was stimulated by cyclic AMP, eluted below 60 mM NaCl and had high activity using histone H2B as substrate. Peak II eluted at 200–250 mM NaCl; its activity was not cyclic AMP stimulated and was highest with dephosphorylated casein as a substrate. Cyclic AMP binding to a protein associated with the protein kinase is specifically inhibited by certain cyclic AMP analogs.     

An hydroxylapatite batch assay for the quantitation of 1alpha,25-dihydroxyvitamin D3-receptor complexes.

A versatile hydroxylapatite batch assay for 1α,25-dihydroxyvitamin D₃-receptor complex from chick intestinal mucosa has been developed. The assay has been characterized with respect to time and temperature of incubations, protein concentration, amount of hydroxylapatite required to bind receptor-steroid complexes, pH, and effects of KCl and phosphate. Triton X-100 (0,5%, $\text{v}\text{v}$) was found to be essential for the removal of nonspecifically bound ligand. The hydroxylapatite was shown to bind the 1α,25-dihydroxy-vitamin D₃ receptor as demonstrated by the specificity and high affinity for 1α,25-dihydroxy-vitamin D₃ and the sedimentation properties of the phosphate-extracted hydroxylapatite-bound complex on sucrose density gradients. Binding appears to be nearly quantitative. The efficient separation of bound from free ligand utilizing this assay makes it possible to examine a number of aspects of the binding of this steroid hormone to its cytoplasmic receptor that has not previously been possible.