Mixed-Valence Cytoplasmic Iron Granules Are Linked to Anaerobic Respiration

Intracellular granules containing ferric and ferrous iron formed in Shewanella putrefaciens CN32 during dissimilatory reduction of solid-phase ferric iron. It is the first in situ detection at high resolution (150 nm) of a mixed-valence metal particle residing within a prokaryotic cell. The relationship of the internal particles to Fe(III) reduction may indicate a respiratory role.     

Isolation of Rat Pituitary Prolactin Isohormones Differing in Charge,Size, and Specific Immunological Activity

A method has been described for the isolation of three differently charged isohormones of rat prolactin from a discard fraction obtained after extraction of gonadotropins, thyrotropin and growth hormone from homogenized frozen pituitaries. The procedure involved extraction at pH 9.8, ammonium sulphate fractionation, molecular sieve chromatography on Sephadex G-100, and column electrophoresis in agarose suspension. The purification was monitored by radioimnunoassays and the recovered components were all found to possess a specific immunoactivity exceeding that of the standard preparation (RP-1) supplied by the NIAMDD, Bethesda, U.S.A. Increased acidity among these isohormones was found to be paralleled by significantly decreased immunopotency. Each component showed biological activity in radioreceptor assay.

A high degree of purity of the isolated components was shown by analytical electrophoresis in polyacrylamide gel. Sodium dodecyl sulphate electrophoresis in the same medium showed no size heterogeneity and yielded a value of approximately 25 000 for the molecular weight of the isohormones.

In addition a large form of prolactin, suggested to represent a dimer, was isolated by a further extraction step (pH 10.5) followed by molecular sieve chromatography on Sephadex G-100 and electrophoresis in agarose suspension. The large form was associated with both biopotency and immuno-potency. The electrophoresis resolved the prolactin activity into three or four immunoactive components. This pleomorphism of the large prolactin was confirmed by analytical polyacrylamide gel electrophoresis.

Amino acid analyses revealed a close similarity between the three monomers and the major dimeric form of the hormone.     

Isolation and Characterization of Cytoplasmic Cofilin-Actin Rods

Cofilin-actin bundles (rods), which form in axons and dendrites of stressed neurons, lead to synaptic dysfunction and may mediate cognitive deficits in dementias. Rods form abundantly in the cytoplasm of non-neuronal cells in response to many treatments that induce rods in neurons. Rods in cell lysates are not stable in detergents or with added calcium. Rods induced by ATP-depletion and released from cells by mechanical lysis were first isolated from two cell lines expressing chimeric actin-depolymerizing factor (ADF)/cofilin fluorescent proteins by differential and equilibrium sedimentation on OptiPrep gradients and then from neuronal and non-neuronal cells expressing only endogenous proteins. Rods contain ADF/cofilin and actin in a 1:1 ratio. Isolated rods are stable in dithiothreitol, EGTA, Ca²⁺, and ATP. Cofilin-GFP-containing rods are stable in 500 mm NaCl, whereas rods formed from endogenous proteins are significantly less stable in high salt. Proteomic analysis of rods formed from endogenous proteins identified other potential components whose presence in rods was examined by immunofluorescence staining of cells. Only actin and ADF/cofilin are in rods during all phases of their formation; furthermore, the rapid assembly of rods in vitro from these purified proteins at physiological concentration shows that they are the only proteins necessary for rod formation. Cytoplasmic rod formation is inhibited by cytochalasin D and jasplakinolide. Time lapse imaging of rod formation shows abundant small needle-shaped rods that coalesce over time. Rod filament lengths measured by ultrastructural tomography ranged from 22 to 1480 nm. These results suggest rods form by assembly of cofilin-actin subunits, followed by self-association of ADF/cofilin-saturated F-actin.     

Interactions of Catecholestrogens with Cytoplasmic and Nuclear Estrogen Receptors in Rat Pituitary Gland and Hypothalamus

Abstract: The affinity of a series of catecholestrogens for 7S cytoplasmic receptor proteins from hypothalamus and pituitary gland of ovariectomised rats was assessed in vitro by a competitive charcoal binding assay at 4°C. The equilibrium dissociation constants ( K _i) of catecholestrogens 4-hydroxyestradiol, 4-hydroxyethynylestradiol, 2-hydroxyestradiol, 2-hydroxyethynylestradiol, and 4-hydroxyestrone were of the same order ( K _i 0.3–0.6 n m ) as those of estradiol and ethynylestradiol ( K _i: 0.1 n m ). Methylation of 2-hydroxyestradiol led to a substantial loss of binding affinity. Tritium-labelled receptor complexes were demonstrated in KCl extracts of purified nuclei from pituitary and hypothalamic tissue 1 h after intravenous injection of 0.1 mCi tritiated 2- or 4-hydroxyestradiol. These macromolecular complexes sedimented in the 5-6S region of 5–20% (w/v) sucrose gradients containing 0.4 m -KCl. Further evidence for the translocation of estrogen receptors by catecholestrogens into the nuclei of rat pituitary and hypothalamus was the increase in nuclear receptor concentrations, measured by exchange assay, 1 h after the intraperitoneal injection of 0.1 mg unlabelled catecholestrogen. Administration of 4-hydroxyestradiol and 4-hydroxyethynylestradiol increased nuclear receptor concentrations to the same maximal levels as those following application of the same dose of estradiol or ethynylestradiol, whereas the respective 2-hydroxylated compounds exhibited only 60–70% of the maximal translocating capacity. The in vivo translocating capacities of the various catecholestrogens tested at this dose correlated well with their binding affinities for cytosol receptors determined in vitro.     

Protein Kinases Are Associated with Multiple,Distinct Cytoplasmic Granules in Quiescent Yeast Cells

The cytoplasm of the eukaryotic cell is subdivided into distinct functional domains by the presence of a variety of membrane-bound organelles. The remaining aqueous space may be further partitioned by the regulated assembly of discrete ribonucleoprotein (RNP) complexes that contain particular proteins and messenger RNAs. These RNP granules are conserved structures whose importance is highlighted by studies linking them to human disorders like amyotrophic lateral sclerosis. However, relatively little is known about the diversity, composition, and physiological roles of these cytoplasmic structures. To begin to address these issues, we examined the cytoplasmic granules formed by a key set of signaling molecules, the protein kinases of the budding yeast Saccharomyces cerevisiae. Interestingly, a significant fraction of these proteins, almost 20%, was recruited to cytoplasmic foci specifically as cells entered into the G₀-like quiescent state, stationary phase. Colocalization studies demonstrated that these foci corresponded to eight different granules, including four that had not been reported previously. All of these granules were found to rapidly disassemble upon the resumption of growth, and the presence of each was correlated with cell viability in the quiescent cultures. Finally, this work also identified new constituents of known RNP granules, including the well-characterized processing body and stress granule. The composition of these latter structures is therefore more varied than previously thought and could be an indicator of additional biological activities being associated with these complexes. Altogether, these observations indicate that quiescent yeast cells contain multiple distinct cytoplasmic granules that may make important contributions to their long-term survival.     

DNA-Dependent RNA Polymerase Activity Associated with Subviral Particles of Polyhedral Cytoplasmic Dexoyribovirus

Polyhedral cytoplasmic deoxyribovirus virions contain a DNA-dependent RNA polymerase which catalyzes the incorporation of ribonucleotides into an acid-precipitable product. Treatment of virions with sodium deoxycholate and dithiothreitol resulted in the formation of subviral particles which could be separated from virions by rate zonal centrifugation in sucrose gradients. Subviral particles were RNA polymerase-positive and more active per unit mass of protein than virions. In vitro enzyme activity associated with subviral particles required addition of ribonucleotides, Mg(2+), and exogenous denatured DNA template. Optimal enzyme activity occurred over a broad pH (7.2 to 8.8) and Mg(2+) concentration (2 to 10 mumol) range. The specific activity of the RNA polymerase was maximal at 37 C. Addition of DNase or actinomycin D to the reaction mixture reduced the incorporation of [(3)H]UMP into an acid-precipitable product. The product of the reaction was sensitive to degradation by RNase but not to DNase or Pronase. These data suggest that the enzyme copies DNA into RNA.     

A Zonal Density Gradient Centrifuge for Separating Cytoplasmic Granules from Trypanosoma brucei

SYNOPSIS. A small zonal density gradient centrifuge has been designed and constructed for the separation of cell granules from Trypanosoma brucei. Two distinct bands were obtained on a simple sucrose density gradient of the following granules which were found in corresponding fractions of the effluent by electron microscopy: Type I granules (lysosomes) which formed a clear-cut band toward the periphery of the rotor, and Type II (endoplasmic reticular) granules which formed a more diffuse band and showed great diversity of size, shape and state of aggregation. This aggregation is related to the structure of these granules which are formed in the endoplasmic reticular tubules and are therefore not easily dispersed during rupture of the cell body.     

The Isolation of Bacterial Polysaccharides with Anti-inflammatory Activity

It was found in the previous paper that wheat gluten polypeptides gave higher molecular weights in SDS-PAGE than in sedimentation equilibrium. To clear the cause of the abnormality of gluten polypeptides in SDS-PAGE, behaviors of the SDS complex of gliadin IV were investigated in comparison with those of the standard proteins. The amount of bound SDS for reduced and alkylated gliadin IV was not different from the value obtained with usual proteins. In accordance with the results of Reynolds et al., the plot of log [η] against log M gave a slope of 1.1, supporting a rodlike structure of the complex. The intrinsic viscosity of the SDS complex of gliadin IV gave a higher value of 0.28 dl/g in comparison with the corresponding value of 0.15 dl/g for the standard proteins. The sedimentation constant was lower in gliadin IV (1.61 S) than in the standard (1.77 S). These facts indicate that the gliadin IV complex has a higher frictional coefficient for its molecular weight of protein, suggesting a more elongated structure than usual. The helix content of the complex of gliadin IV was extremely low (12%). It was suggested that the high proline content of gliadin gives an elongated structure to the SDS complex and this structure causes a low electrophoretic migration mobility and overestimation of molecular weight in SDS-PAGE.     

Isolation of Three Electrophoretic Variants of Rat Pituitary Growth Hormone

A procedure has been developed for the isolation of rat pituitary growth hormone and for the subsequent resolution of the preparation into three variants by preparative electrophoresis. The starting material was whole frozen glands and the process involved homogenization and extraction at pH 6.2, ammonium sulfate fractionation and molecular-sieve chromatography on Sephadex G-100. The separation into charge variants was achieved by zone electrophoresis in agarose suspension at alkaline pH.

The purfication was monitored by radioimmunoassay and the specific activities were expressed in terms of the rat growth hormone reference preparation (RP-1) supplied by the NIADDK, Bethesda, U. S. A.

The three-component preparation and its constituents all had activities in the same range, exceeding the activity of the reference by a factor up to 20 times. Bioassay of the three-component preparation, based on measurement of longitudinal bone growth in hypophysectomized rats gave a potency of 4–5 IU/mg. The reference was the 1st International Standard (bovine) for growth hormone. The yield of the three-component preparation was 3.3 mg per gram pituitary tissue.

Different electrophoretic analyses revealed the efficiency of the preparative procedure in separating the variants. The results of the analyses also support the view that difference in electrophoretic behaviour is due to a difference of a single net charge between adjacent variants.

In addition, growth hormone was prepared from two side extracts (at pH 7.0 and pH 9.8, respectively), provided by a procedure developed earlier for rat prolactin. The three preparations gave electrophoretic patterns of equal appearance although the relative proportions of the activity peaks differed.     

Circadian Rhythm in Mammary Cytoplasmic Estrogen Receptor Content of Balb/C Female Mice with and Without Pituitary Isografts

Cytoplasmic estrogen receptors were determined by the dextran-coated charcoal method in inguinal breast tissue of three groups of Balb/C female mice 6-8 weeks following subcutaneous implantation into the intact animals of three pituitary glands and three pieces of skeletal muscle (group I), three pituitary glands and three segments of hypothalamic tissue (group II), or three pieces of skeletal muscle (group III) obtained from animals of the same inbred strain as control. A circadian rhythm in estrogen receptor content was statistically quantified by cosinor analysis in the muscle implanted control and the pituitary and hypothalamic implant groups. In the pituitary and muscle implant group the circadian rhythm is of borderline significance with a P-value between 0.05 and 0.10. The timing (acrophase) and extent of change (amplitude) are similar in all three treatment groups. The average receptor content (MESOR) in the two pituitary-implanted groups, which in previous studies were shown to have an increased breast cancer incidence is about twice that of the control group. The reduction in the pituitary induced breast cancer rate by hypothalamic tissue addition to a cancer incidence between the animals with pituitary and muscle isograft and the mice carrying no pituitary at all has also been shown previously in this strain of mice and is not reflected in receptor content.     

Circadian Rhythm in Mammary Cytoplasmic Estrogen Receptor Content of Balb/C Female Mice with and Without Pituitary Isografts

Cytoplasmic estrogen receptors were determined by the dextran-coated charcoal method in inguinal breast tissue of three groups of Balb/C female mice 6–8 weeks following subcutaneous implantation into the intact animals of three pituitary glands and three pieces of skeletal muscle (group I), three pituitary glands and three segments of hypothalamic tissue (group II), or three pieces of skeletal muscle (group III) obtained from animals of the same inbred strain as control. A circadian rhythm in estrogen receptor content was statistically quantified by cosinor analysis in the muscle implanted control and the pituitary and hypothalamic implant groups. In the pituitary and muscle implant group the circadian rhythm is of borderline significance with a P-value between 0.05 and 0.10. The timing (acrophase) and extent of change (amplitude) are similar in all three treatment groups. The average receptor content (MESOR) in the two pituitary-implanted groups, which in previous studies were shown to have an increased breast cancer incidence is about twice that of the control group. The reduction in the pituitary induced breast cancer rate by hypothalamic tissue addition to a cancer incidence between the animals with pituitary and muscle isograft and the mice carrying no pituitary at all has also been shown previously in this strain of mice and is not reflected in receptor content.     

Elongation of the Cytoplasmic Tail Interferes with the Fusion Activity of Influenza Virus Hemagglutinin

The hemagglutinin (HA) of fowl plague virus was lengthened and shortened by site-specific mutagenesis at the cytoplasmic tail, and the effects of these modifications on HA functions were analyzed after expression from a simian virus 40 vector. Elongation of the tail by the addition of one to six histidine (His) residues did not interfere with intracellular transport, glycosylation, proteolytic cleavage, acylation, cell surface expression, and hemadsorption. However, the ability to induce syncytia at a low pH decreased dramatically depending on the number of His residues added. Partial fusion (hemifusion), assayed by fluorescence transfer from octadecylrhodamine-labeled erythrocyte membranes, was also reduced, but even with the mutant carrying six His residues, significant transfer was observed. However, when the formation of fusion pores was examined with hydrophilic fluorescent calcein, transfer from erythrocytes to HA-expressing cells was not observed with the mutant carrying six histidine residues. The addition of different amino acids to the cytoplasmic tail of HA caused an inhibitory effect similar to that caused by the addition of His. On the other hand, a mutant lacking the cytoplasmic tail was still able to fuse at a reduced level. These results demonstrate that elongation of the cytoplasmic tail interferes with the formation and enlargement of fusion pores. Thus, the length of the cytoplasmic tail plays a critical role in the fusion process.     

Isolation of Melanin Granules from Dark Hair of Human

A new method for the isolation of melanin granules from human dark hair is described. S-melanin prepared from dark hair of human³⁾ was treated with 6 M urea solution of pH 8 at 30°C in order to remove the accompanying keratin moiety. Melanin granules thus obtained seemed to be pure by electron microscopy, and were composed of melanin-pigment, protein and minerals. Visible absorption as well as infrared spectra of the granules were examined.     

具有抑菌活性的海洋细菌的分离与鉴定

分离并筛选具有抑菌活性的海洋细菌对于开发和利用海洋微生物具有重要意义,该研究从6份海泥样品中共分离到78株海洋细菌,以6种细菌作为敏感指示菌,采用覆盖技术对分离菌株进行拮抗试验,17株海洋细菌具有抑菌活性,对其中2株具有较强抑菌活性的海洋细菌进行革兰氏染色,耐盐试验,运动性观察,过氧化氢酶测定,明胶液化试验,硫化氢产生试验,石蕊牛奶试验,糖类发酵,硝酸盐还原等特性分析,依据《伯杰氏细菌鉴定手册》进行分类鉴定,它们分别应归属为气单孢菌属（Aeromonas sp.）和假单孢菌属（Pseudomonas sp.）。     

Isolation and Characterization of Microorganisms with Alternan Hydrolytic Activity

Alternan is an unusual α-D-glucan containing alternating (1 → 3), (1 → 6) linkages that exhibits remarkable resistance to enzymatic hydrolysis. The commercial potential of the polysaccharide may be enhanced by the ability to economically modify the native form into fractions of varying molecular weight. By employing isolation procedures with covalently dyed alternan as the substrate, several bacterial isolates that produced endohydrolytic activity were obtained in pure culture. The activity was confirmed by decreases in viscosity and by direct examination of the hydrolysis products with thin layer chromatography. Analysis of the hydrolysis products established that all isolates produced enzymes with identical alternan depolymerizing activity, producing a cyclic tetrasaccharide as a major product. All alternanase activity was shown to be extracellularly located. A single strain exhibited constitutive production of alternanase, while all other isolates required the presence of alternan in the growth media for enzyme production. All isolates were phenotypically similar, produced heat-resistant spores, and were tentatively identified as members of the genus Bacillus.     

Isolation and Characterization of Bacterial Strains with Inulinase Activity

Thirty-two bacterial strains growing on inulin as the sole carbon and energy source were isolated from soil samples by enrichment culture on a mineral medium. Twenty of the strains were identified as Flavobacterium multivorum. All the bacteria contained a β-fructosidase that was active on both inulin and sucrose. The enzyme activity was cell bound and was produced at the end of the growth phase. These enzymes have potential uses in the preparation of fructose syrups from inulin and invert sugar from sucrose.     

Mitogenic Activity of Cytoplasmic Membranes Isolated from L-Forms of Staphylococcus aureus

Cytoplasmic membranes of L-forms of Staphylococcus aureus exerted a strong mitogenic effect on splenocytes of athymic nude mice as well as normal mice, while a cytoplasmic fraction of the same bacteria did not show definite mitogenicity. The mitogenic principle(s) of the membrane fraction was resistant to treatment with trypsin and was heat stable (at 100 C for 10 min). The active principle(s) in the insoluble residue of the membrane fraction digested with trypsin was not extracted with cold acetone, but could be solubilized by extraction with a cold chloroform-methanol mixture (2:1, v/v). The mitogenic principle(s) in the extract was fractionated by silicic acid column chromatography. Among five fractions separated by chromatography, fractions eluted with chloroform-methanol mixtures (1:1 and 1:20, v/v) were found to be strongly mitogenic. The cytoplasmic membranes of the L-forms also exerted a definite mitogenic effect on guinea pig splenocytes, but not on the thymocytes.