Anthramycin inhibition of restriction endonuclease cleavage and its use as a reversible blocking agent in DNA constructions.

Anthramycin can form a stable complex with DNA which does not dissociate upon repeated ethanol precipitations. The complex forms in less than one hour at pH 5.5. Bound anthramycin seems to be located in the minor groove of the DNA helix in the anthramycin DNA complex, since methylation of adenosine residues at N-3 by dimethylsulfate is reduced. The anthramycin-DNA complex is resistant to digestion by an excess of a number of restriction enzymes. Anthramycin can be removed from DNA by incubation at acid pH. The released DNA can then be cleaved by restriction enzymes. Anthramycin-DNA complexes can be acted upon by T4 polynucleotide ligase to form longer DNA molecules. The ability of anthramycin to form a stable but reversible complex which is not cleaved by restriction enzymes but can engage in joining reactions may allow a wider variety of DNA fragments to be more readily constructed in vitro.     

The effects of short-term isolation on systolic blood pressure and heart rate in rats

The effect of short-term isolation on the systolic blood pressure and heart rate of rats has been studied. Five days continuous isolation in glass metabolic cages caused systolic arterial hypertension in all animals. Isolation in standard cages for this time period caused hypertension, but only in 55% of the animals. Both forms of isolation caused an initial tachycardia. Handling and contact with other animals for 1 hr daily prevented the development of hypertension in some animals but did not alter the blood pressure once the hypertension had developed. Group-housing of animals after a 3 week period of isolation restored blood pressure to control levels within 24 hr. It is possible that stress imposed by isolation activated the sympatho-adrenal system and thereby caused these changes.     

Additional observations on the blood parasites of Ugandan birds.

One thousand and seventy-six birds of 26 families and 127 species were examined for hemoprotozoa; 404 birds (37%) of 41 species representing 17 families harbored one or more blood parasites. Most parasites were species of Haemoproteus which represented 95% of all parasitic infections detected. Prevalence of blood parasites in birds collected from four areas over a period of six years was relatively stable.     

Evidence against the release of prostaglandin-like material from isolated intestinal tissue by pure cholera toxin.

Prostaglandin-like material was released from finely cut guinea-pig ileum or human intestinal mucosa during incubation with Krebs solution. The tissue inactivated some significant change in release of prostaglandin-like material when pure cholera toxin was incubated with guinea-pig ileum or human intestinal mucosa. The work is discussed in relation to the action of cholera toxin in vivo.     

Characterization of glycerol nonutilizing and protoperithecial mutants ofNeurospora

Summary Mutants defective in polyol metabolism and/or in protoperithecial development were selected inNeurospora tetrasperma, a species in which protoperithecial development occurs at nonpermissively high temperature if certain polyols are used in lieu of sucrose as carbon source. Mutants selected for nonutilization of one of the four polyols tested, glycerol, mannitol, sorbitol, or xylitol, were usually found to be nonutilizers of the other three polyols as well. Mutants blocked at various stages of protoperithecial development complemented pairwise to produce more advanced developmental stages, usually mature protoperithecia and, when of opposite mating type, mature perithecia. About one-third of the mutants manifested both polyol auxotrophy and defective protoperithecial development upon initial isolation, but protoperithecial defectiveness in such mutants usually showed erratic segregation in crosses and/or instability to repeated vegetative transfer, whereas polyol auxotrophy usually did not and was, therefore, studied further. Two glycerol nonutilizing strains were introgressed intoN. crassa to facilitate genetic analysis. One,glp-4, lacked both inducible and constitutive glycerol kinase and mapped to linkage group VI, betweenad-1 andrib-1; the other,glp-5, lacked glyceraldehyde kinase and mapped to linkage group I, proximal toad-9. Another mutant,gly-u(234), has been reported by other investigators to lack inducible glycerol kinase but to map to linkage group I, distal toad-9.     

Effect of hydrocortisone on cell morphology in C6 cells

Hydrocortisone has been found to induce cell spreading in rat glial C6 cells by 24 hours after its addition. This spreading phenomenon is correlated with an increase in the fraction of the peripheral cytoplasm occupied by microfilaments. Cytochalasin B causes disorganization of microfilaments in the peripheral cytoplasm of the cells. Additionally, it also prevents cell spreading in response to hormonal stimulation. High levels of calcium prevent recovery of normal microfilament organization and cell spreading following removal of cytochalasin B, but have no effect on normal microfilament organization alone. Additionally both the hydrocortisone induced spreading of C6 cells and increases in peripheral microfilaments are shown to be dependent on RNA ans protein synthesis. The levels of protein co-electrophoresing with actin are not effected by hydrocortisone.     

Relationship of F9 antigen and genes of theT/t complex

Serological studies relating F9 antigen of embryonal carcinoma cells to at the murineT/t complex have been extended and confirm that only the lethal haplotype t¹²- and none of the other five lethal haplotypes-affects the quantitative expression of F9 antigen on sperm. Cytotoxicity tests on preimplantation embryos show that t¹² homozygotes are less susceptible to antiF9 serum than t^w5 homozygotes, and that using specific antimutant haplotype antisera prepared against sperm, t¹² antigen is detectable on morulae, whereas t^w5 antigen is not.     

Association of spectrin with its membrane attachment site restricts lateral mobility of human erythrocyte integral membrane proteins

Interactions between spectrin and the inner surface of the human erythrocyte membrane have been implicated in the control of lateral mobility of the integral membrane proteins. We report here that incubation of “leaky” erythrocytes with a water-soluble proteolytic fragment containing the membrane attachment site for spectrin achieves a selective and controlled dissociation of spectrin from the membrane, and increases the rate of lateral mobility of fluorescein isothiocyanate-labeled integral membrane proteins (> 70% of label in band 3 and PAS-1). Mobility of membrane proteins is measured as an increase in the percentage of uniformly fluorescent cells with time after fusion of fluorescent with nonfluorescent erythrocytes by Sendai virus. The cells are permeable to macromolecules since virus-fused erythrocytes lose most of their hemoglobin. The membrane attachment site for spectrin has been solubilized by limited proteolysis of inside-out erythrocyte vesicles and has been purified (V). Bennett, J Biol Chem 253:2292 (1978). This 72,000-dalton fragment binds to spectrin in solution, competitively inhibits association of ³²P-spectrin with inside-out vesicles with a K_i of 10^?7M, and causes rapid dissociation of ³²P-spectrin from vesicles. Both acid-treated 72,000-dalton fragment and the 45,000 dalton-cytoplasmic portion of band 3, which also was isolated from the proteolytic digest, have no effect on spectrin binding, release, or membrane protein mobility. The enhancement of membrane protein lateral mobility by the same polypeptide that inhibits binding of spectrin to inverted vesicles and displaces spectrin from these vesicles provides direct evidence that the interaction of spectrin with protein components in the membrane restricts the lateral mobility of integral membrane proteins in the erythrocyte.