Interaction of filipin with junctional membrane at different stages of the junction's life history

The relationship of filipin-sterol complexes to tight and gap junctions during their formation, maturation, internalization, and degradation was studied in separate cell lines. Filipin-sterol complexes tended to be excluded from mature junctions in tight junction forming COLO 316 cells and gap junction forming SW-13 cells. Once internalized, unlabeled junctional membrane appeared to fuse with heavily labeled vesicles, presumably lysosomes. Although the absence of filipin-sterol complexes from junctional membrane does not necessarily reflect the absolute sterol content of this membrane, the fact that filipin-sterol complexes are largely excluded from these areas indicates that this membrane is different from surrounding membrane. The absence of filipin-sterol complexes also permits the visualization of 'mixing' of this specialized unlabeled membrane domain with other filipin labeled membrane systems.     

Comparison of AMP and NADH binding to glycogen phosphorylase b

The binding sites for the allosteric activator, AMP, to glycogen phosphorylase b are described in detail utilizing the more precise knowledge of the native structure obtained from crystallographic restrained least-squares refinement than has hitherto been available. Localized conformational changes are seen at the allosteric effector site that include shifts of between 1 and 2 A for residues Tyr75 and Arg309 and very small shifts for the region of residues 42 to 44 from the symmetry-related subunit. Kinetic studies demonstrate that NADH inhibits the AMP activation of glycogen phosphorylase b. Crystallographic binding studies at 3.5 A resolution show that NADH binds to the same sites on the enzyme as AMP, i.e. the allosteric effector site N, which is close to the subunit-subunit interface, and the nucleoside inhibitor site I, which is some 12 A from the catalytic site. The conformations of NADH at the two sites are different but both conformations are "folded" so that the nicotinamide ring is close (approx. 6 A) to the adenine ring. These conformations are compared with those suggested from solution studies and with the extended conformations observed in the single crystal structure of NAD+ and for NAD bound to dehydrogenases. Possible mechanisms for NADH inhibition of phosphorylase activation are discussed.     

Characterization by human autoantibody of a nuclear antigen related to the cell cycle

Using a serum from a patient with an autoimmune disease, we have recently described a novel 55 000-dalton antigen (p55) in the nucleus of several animal cells including human ones. This antigen, designated PSL, was not related to the previously defined antigens recognized by sera from patients with systemic rheumatic diseases (Sm, n-RNP, SS-B, Scl-70). We have now found that p55 is associated with chromatin structures as it is released from the nucleus of mink cell fibroblasts by saline + DNase treatments. Analysis by sucrose gradient centrifugation of the nuclear material released in these conditions indicated that p55 co-migrated with core histones. Meanwhile, p55 was absent from the residual nuclear matrices (achromatinic nuclei). Localization of p55 in synchronized cells was performed by indirect immunofluorescence and immunoprecipitation. P55 appeared to accumulate in the nucleus during the S phase. Finally, it was not recognized by an anti-SV40 tumor serum that specifically precipitated the protein p53, which has been recently related to cell proliferation. Thus, PSL an p53, although apparently not antigenically related, appear to be implicated in the same step of the cell cycle.     

Spontaneous action potentials and resting potential shifts in fertilized eggs of the tunicate Clavelina

Electrical activity in the fertilized egg of the tunicate Clavelina was studied with microelectrode recording and voltage clamp techniques. The resting potential could assume either of two stable values (approximately ?70 or ?30 mV) and could be shifted between these values by direct current stimulation. Spontaneous shifts between two stable resting potentials were also seen. Egg cells produced action potentials spontaneously and in response to depolarizing stimuli. Inward currents were carried by both Na and Ca ions and a prominent outward potassium current was seen with depolarization to voltages above ?15 mV. The steady-state current-voltage relationship (I–V curve) of the membrane showed two voltages where the net membrane current equaled zero: approximately ?35 and ?70 mV. Between these two voltages, membrane current was inward and carried by noninactivating Na and Ca currents. Inward rectification, which was blocked by external Rb, occurred at voltages below ?70 mV. The voltage dependence of inward rectification is thought by the authors to be important for establishing the more negative resting potential; it is also thought the presence of inward current which does not inactivate completely at voltages more negative than about ?20 mV is an important determinant of the more depolarized resting potential.     

The use of thioesterase II as a rat mammary epithelial cell-specific marker

The avidin-biotin-peroxidase complex immunoperoxidase technique was employed to determine the intercellular distribution of thioesterase II in rat mammary glands. This enzyme is responsible for shifting the product specificity of the fatty-acid synthetase enzyme complex from long to medium chain fatty acids. Thioesterase II was found exclusively in the cells lining the lumen of the ductal and alveolar structures in glands from mature virgin (150 days old) and pregnant rats. The ductal cell staining intensity was considerably less than that of the alveolar cells in the mature virgin rat glands. No immunoreactive thioesterase II was found in the stromal, adipose, vascular, or myoepithelial components of the gland in the developmental stages examined. In the glands from immature virgin rats (40-45 days old) thioesterase II was again found only in the epithelial cells lining the lumen of the ductal and end-bud structures although this layer was usually more than one cell thick. Quantitative determination of thioesterase II activity in cytosol preparations revealed similar levels in mammary fragments from enzymatically-dissociated glands obtained from mature virgins and in end buds derived from immature virgins, but somewhat higher levels in mammary structures derived from late-pregnant animals. These immunohistological and biochemical results demonstrate thioesterase II's usefulness as a mammary epithelial cell-specific marker.     

Probabilistic and multiple regression modeling of the biologist’s decision processes and its implications for medical diagnosis

Current psychological research into the inference (diagnostic) process is briefly reviewed, using as a vehicle an investigation of the prediction of the probability of success of hypothetical applicants to a graduate program in biology. Brunswik’s lens model and multiple regression analysis are used, as is a Bayesian approach. Four judges’ (biologists’) predictions are analyzed. Some general conclusions about inference, drawn from the current data in psychology, are presented. This investigation was supported by PHS Research Grant No. MH-17487-01 from the National Institute of Mental Health.     

R Factor Proteins Synthesized in Escherichia coli Minicells: Incorporation Studies with Different R Factors and Detection of Deoxyribonucleic Acid-Binding Proteins

Analysis of the protein synthesized by Escherichia coli minicells containing R factors demonstrated a variety of low- and high-molecular-weight polypeptides in sodium dodecyl sulfate (SDS)-polyacrylamide gels. Only half of this protein was released into a soluble fraction on lysis of these minicells. The other half remained associated with the minicell envelope. The efficiency of precursor incorporation into protein and the kinds of proteins synthesized changed with the age of the minicells at the time of harvest. About 1 to 2% of the soluble R factor-coded protein bound to calf thymus, E. coli, or R factor DNA-cellulose. Although most of these proteins were excluded from Sephadex G-100 columns, they migrated chiefly as low-molecular-weight-polypeptides (13,000 to 15,000) in SDS-polyacrylamide gels. Additional DNA-binding proteins that appeared to be higher-molecular-weight peptides were noted in extracts from younger minicells. At least one protein, identified as an SDS band, appeared to bind selectively to R factor DNA-cellulose. Minicells with R factors also contained DNA-binding proteins of cell origin, including the core RNA polymerase. No such binding proteins were found in R(-) minicells. These studies suggest that: (i) R factors code for proteins that may be involved in their own DNA metabolism; (ii) R factor DNA-binding proteins may be associated with larger host cell DNA-binding proteins or subunits of larger R factor proteins; and (iii) the age of the minicell influences the extent of protein synthesis and the kinds of proteins synthesized by R factors in minicells.     

Identity of the two 8S RNA components of the mouse sarcoma virus (Moloney)

The two 8S A and B RNAs of the Mouse Sarcoma Virus (Moloney) can be converted by heating into a homogeneous population. After digestion with T₁ RNAse, they give identical fingerprints. It is concluded that these two molecules represent conformational isomers of the same molecular species.     

Light-dependent hydrogen evolution by Scenedesmus

Summary The effect of glucose and the uncoupler Cl-CCP upon hydrogen production was studied in adapted cells of Scenedesmus obliquus D₃. Cl-CCP at 10^-5M concentration completely inhibited the evolution of H₂ in the dark and increased the apparent rate of H₂ evolution in the light. At 10^-5M Cl-CCP, photosynthesis and photoreduction by anaerobically adapted algae were only temporarily inhibited; O₂ evolution reappeared after approximately 1 hr of illumination if CO₂ was present. Increasing the Cl-CCP concentration to 5 x 10^-5M led to a maximum rate of photohydrogen production and fully inhibited H₂ evolution, photoreduction and dark H₂ evolution. H₂ evolution was accompanied by a release of varying amounts of CO₂ in the light, as well as in the dark. Dark CO₂ production was stimulated by Cl-CCP. H₂ evolution in the light was stimulated by adding glucose to autotrophically grown cells or by growing the cells heterotrophically with glucose; starvation had an opposite effect. Adapted cells released ¹⁴CO₂ from the 3 and/or 4 position of specifically labeled glucose, indicating that degradation occurred via the Embden-Meyerhof pathway. The amount of H₂ released by autotrophically grown cells was the same either with continuous illumination or with short periods of light, followed by darkness. Scenedesmus mutant No. 11, which is unable to evolve O₂ was not inhibited in its capacity to evolve H₂ in the light. These data indicate that the evolution of H₂ in the light by adapted Scenedesmus depends upon the degradation of organic material and does not require the production of free O₂ by photosystem II.The following abbreviations are used: Cl-CCP = carbonyl cyanide m-chlorophenylhydrazone; DCMU = 3-(3,4-dichlorophenyl)-1,1-dimethylurea, DNP = 2,4-dinitrophenol.This work was supported by contract AT-(40-1)-2687 from the U.S. Atomic Energy Commission.