One-step purification of metallothionein extracted from two different sources

We describe a one-step purification of hepatic metallothionein from the Amazon fish Colossoma macropomum injected with cadmium and from the copper-loaded metallothionein from the yeast Saccharomyces cerevisiae, performed by affinity chromatography through metal-chelating columns. Yeast metallothionein was purified from Cu2+-loaded resin and eluted by a continuous EDTA gradient whereas hepatic metallothionein extracted from fishes was purified by Ni2+-loaded resin and eluted by a continuous imidazol gradient. Purified metallothioneins were evaluated by SDS-PAGE and characterized by UV spectra of the apo- and Cd2+-loaded protein. This method allowed high purity and yield as well as rapid one-step extraction of both metal-loaded and apoprotein.     

Effect of glycerol on gluconeogenesis in isolated rabbit kidney cortex tubules

In renal tubules isolated from fed rabbits glycerol is not utilized as a glucose precursor, probably due to the rate-limiting transfer of reducing equivalents from cytosol to mitochondria. Pyruvate and glutamate stimulated an incorporation of [14C]glycerol to glucose by 50- and 10-fold, respectively, indicating that glycerol is utilized as a gluconeogenic substrate under these conditions. Glycerol at concentration of 1.5 mM resulted in an acceleration of both glucose formation and incorporation of [14C]pyruvate and [14C]glutamate into glucose by 2- and 9-fold, respectively, while it decreased the rates of these processes from lactate as a substrate. In the presence of fructose, glycerol decreased the ATP level, limiting the rate of fructose phosphorylation and glucose synthesis. As concluded from the 'cross-over' plots, the ratios of both 3-hydroxybutyrate/acetoacetate and glycerol 3-phosphate/dihydroxyacetone phosphate, as well as from experiments performed with methylene blue and acetoacetate, the stimulatory effect of glycerol on glucose formation from pyruvate and glutamate may result from an acceleration of fluxes through the first steps of gluconeogenesis as well as glyceraldehyde-3-phosphate dehydrogenase. As inhibition by glycerol of gluconeogenesis from lactate is probably due to a marked elevation of the cytosolic NADH/NAD+ ratio resulting in a decline of flux through lactate dehydrogenase.     

The transfer of bovine J blood group activity to erythrocytes: chemical nature of transferable and of non-transferable J1

The bovine J blood group substance exists as a glycosphingolipid (ceramide deca-hexoside as well as ceramide dodecahexoside) and as a glycoprotein. The lipidic form occurs in erythrocyte membranes, both forms are found in serum. The lipidic J substances were isolated from erythrocytes and from serum, and identified by thin-layer chromatography with lipidic J substances isolated from spleen. The glycoprotein nature of the non-lipidic J of serum was evident by pronase-catalysed hydrolysis yielding J-active glycopeptides of lower molecular weights. The lipidic J was completely extracted from lyophilized stroma with chloroform/methanol. From lyophilized serum, however. it was completely extracted only in the presence of water, indicating different binding partners in serum and in erythrocyte membranes. The J lipid was incorporated as intact molecule into the erythrocyte membrane by a simple incubation technique. The incorporation was inhibited by various glyc-erophospholipids (called blockers). The J glycoprotein could not be transferred to the erythrocyte membrane. Three methods are descrjbed which are suitable for the preparation of a blocker-free fraction enriched with J lipids from J-positive serum.     

Ferulic acid degradation encoded by a catabolic plasmid

Abstract A strain of Pseudomonas fluorescens was isolated from soil with ferulic acid as sole source of carbon and energy. The ability to degrade this compound iis plasmid-encoded, as shown by the transfer of such degradative capacity from the wild-type into a recipient strain by conjugation and isolation of a plasmid from the exconjugants, identical to that from the wild-type and the exconjugants.     

Plasma membrane glycoproteins of tumour cells in enzootic bovine leukosis compared with normal bovine lymphoid cells

Plasma membranes from tumor cells in enzootic bovine leukosis and from normal bovine lymphoid cells of different sources have been isolated and characterized. The glycoprotein composition of the different membranes has been studied by SDS-PAGE and by analysis of the carbohydrate composition as well as by lectin binding of glycoprotein fractions obtained by phenol treatment of the membranes. No differences in the electrophoretical glycoprotein pattern could be detected comparing (nonmalignant) cells from persistent lymphocytosis and tumour cells, suggesting that malignancy is not associated with the gain or loss of a major glycoprotein. A 151.5 kD glycoprotein, present in membranes from normal lymph node lymphocytes, seems to be absent in the membranes of peripheral blood lymphocytes as well as of tumour cells. The loss of this glycoprotein might thus be associated with a loss of sessility of bovine lymphoid cells. The carbohydrate analysis and lectin binding of extracted glycoproteins revealed a decreased fucosylation accompanied by an increased exposure of galactose residues as well as a loss or a decreased complexity of N-glycosidically bound oligosaccharides in the tumour cell glycoproteins compared with those of normal cells. These findings are discussed with regard to disturbed growth regulation of leukosis tumour cells.     

Scanning electron microscopy of surface and internal features of developing perithecia of Neurospora crassa.

Stages in the development of perithecia of Neurospora crassa, designated by the time elapsed after crossing, were investigated with the scanning electron microscope, from protoperithecia through perithecia. The usual examination of external features of whole specimens with this instrument was augmented by a freeze-fracture technique which allowed the viewing of development internally as well. Rapid increases in perithecial size soon after crossing were followed by the appearance, in section, of a centrum, at first undifferentiated but subsequently developing ascogenous hyphae. The perithecial beak appeared as a compact mass easily distinguishable in whole specimens from the surrounding hyphae by means of texture as well as shape. Two ascospores were photographed during emergence from an ostiole, but ostioles were found more frequently closed than open.     

Purification of immunoglobulin heavy chain messenger RNA by immunoprecipitation from the mouse myeloma tumor, MOPC-31C.

Immunoglobulin heavy chain mRNA was purified from immunoprecipitated polysomes derived from the mouse myeloma tumor, MOPC-31C. The purified mRNA migrated predominantly as a single band upon polyacrylamide gel electrophoresis in 98% formamide and the molecular weight of this mRNA was calculated to be 700,000. This mRNA was as active as the purified light chain mRNA when it was employed as a template in a cell-free protein synthesizing system from wheat germ. The translation product had a molecular weight of 55,000 daltons, and migrated slightly faster than mature heavy chain upon polyacrylamide gel electrophoresis in sodium dodecylsulfate. The protein synthesized by the direction of this mRNA was shown to yield tryptic peptides corresponding to those derived from the mature heavy chain protein except that one missing peptide was replaced by another additional peptide. DNA complementary to the mRNA was synthesized by RNA-dependent DNA polymerase from avian myeloblastosis virus. Hybridization kinetic analysis between the heavy chain mRNA and its complementary DNA indicated that the RNA was essentially homogenous with rabbit globin mRNA as a standard.     

O2''-Methylinosine, a constituent of the ribosomal RNA of Crithidia fasciculata.

A novel nucleoside, O2'-methylinosine (Im), has been identified as a constituent of the ribosomal RNA of Crithidia fasciculata, a hemoflaggelate protozoan. The nucleoside is released as part of an alkali-stable dinucleotide, Im-Up, by alkaline hydrolysis of Crithidia rRNA, and as a 5'-nucleotide, pIm, by snake venom hydrolysis of the same RNA. The Im-containing derivatives isolated from Crithidia rRNA were characterized by comparison with marker compounds prepared by chemical deamination of the corresponding adenosine analogues. O2'-Methylinosine prepared from either natural Im-Up or natural pIm had the same ultraviolet absorption spectra and chromatographic properties as marker Im. Characterization of the base and sugar components of Im as hypoxanthine and 2-O-methylribose, respectively, provided final confimration of structure. Control experiments have eliminated the possibility that Im arises from O2'-methyladenosine (Am), a known constituent of ribosomal RNA, by chemical or enzymatic deamination during hydrolysis of Crithidia rRNA.     

<Emphasis Type="Italic">γ</Emphasis>-Fluorinated analogues of glutamic acid and glutamine

Gamma-fluorinated analogues of glutamic acid and glutamine are compounds of biological interest. Syntheses of such compounds are extensively reviewed in this article. 4-fluoroglutamic acid was prepared as a mixture of racemic diastereomers by Michael reaction, inverse-Michael reaction or by electrophilic / nucleophilic fluorination. Optically enriched 4-fluoroglutamic acids were obtained by several resolution techniques as well as by asymmetric methodologies using the chiral pool. 4-fluoroglutamine was prepared as a mixture of stereoisomers as well as in racemic erythro and threo forms from the corresponding 4-fluoroglutamic acids using aminolysis and conventional protection and deprotection strategies. Racemic 4,4-difluoroglutamic acid was synthesized by a nitroaldol reaction and its L-enantiomer obtained via three different asymmetric routes. Racemic 4,4-difluoroglutamic acid was converted into the corresponding 4,4-difluoroglutamine using a protection / aminolysis / deprotection sequence while N-Boc-L-4,4-difluoroglutamine was prepared directly from (R)-Garner's aldehyde using a Reformatsky reaction as the key step.     

Variation in chromatin structure in two cell types from the same tissue: a short DNA repeat length in cerebral cortex neurons

We have used micrococcal nuclease as a probe of the repeating structure of chromatin in four nuclear populations from three tissues of the rabbit. Neuronal nuclei isolated from the cerebral cortex contain about 160 base pairs of DNA in the chromatin repeat unit, as compared with about 200 base pairs for nonastrocytic glial cell nuclei from the same tissue, neuronal nuclei from the cerebellum and liver nuclei. All four types of nuclei show the same features of nucleosomal organization as other eucaryotic nuclei so far studied: nucleosomes liberated by digestion with micrococcal nuclease give a “core particle” containing 140 base pairs as a metastable intermediate on further digestion and a series of single-strand DNA fragments which are mutiples of 10 bases after digestion with DNAase I. Nuclei from cerebral cortex neurons, which have a short repeat, are distinct from the others in being larger, in having a higher proportion of euchromatin (dispersed chromatin) as judged by microscopy and in being more active in RNA synthesis in vitro.     

对(艹孟)烷-3,8-二醇的合成与应用研究进展

对(艹孟)烷-3,8-二醇是一种具有生物活性的化合物,主要来源于柠檬桉精油,可直接用于香水、杀虫剂和药品等.通常采用化学和生物化学的方法合成对(艹孟)烷-3,8-二醇,以替代从柠檬桉叶油分离得到的天然产品.以香茅醛为原料通过Prins环缩合和水解,或者通过Solanum aviculare悬浮培养方式进行生物转化得到对(艹孟)烷-3,8-二醇;通过薄荷醇的氧化,或者以2-丁烯醛为原料与甲基锂、NaClO2等试剂经过较长步骤也可合成对(艹孟)烷-3,8-二醇.对(艹孟)烷-3,8-二醇可应用于制造驱虫剂、凉味剂、药物或者化妆品的添加剂以及海水防护涂料添加剂等.     

Diaryl dimers of estradiol and of estrone may be formed as major metabolites by mouse mammary glands

The formation of a novel estrogen metabolite by mammary tissues was investigated. Polar and nonpolar metabolites of endogenous estrogens are formed in liver and other tissues. Polar products such as the catechol estrogens are implicated in tumorigenesis in breast tissue, whereas a nonpolar metabolite, 2-methoxyestradiol, may be protective. Diaryl ether dimers, as a novel form, have been reported as nonpolar products from liver microsomes. We have noted major amounts of nonpolar metabolites in other tissues that were neither 2-methoxyestrogens nor estrogen fatty acid esters. The possible formation of such novel metabolites by breast tissues from adult nulliparous mice with [³H]-labeled estrogens as substrates was considered. Steroids were recovered from media by solid-phase extraction and profiles were obtained from HPLC (acetonitrile:water). Saponification was done with an internal standard of estradiol stearate. Major amounts of nonpolar metabolites were formed in all instances, with one or two principal peaks. Alkaline hydrolysis had no effect on the nonpolar product(s) but released estradiol from its stearate. Strong acid treatment also had no effect as shown by HPLC. Thus, it is suggested that diaryl dimers of estrogens may be formed as major metabolites by mouse mammary glands.     

Proton transport through a peptide-tethered bilayer lipid membrane by the H(+)-ATP synthase from chloroplasts measured by impedance spectroscopy.

A lipid membrane was tethered to a gold film by a peptide spacer molecule terminated by a sulfhydryl group. Membranes were formed by fusion of liposomes prepared from egg phosphatidylcholine on self assembled monolayers of the thiolipopeptide Myr-Lys(Myr)-Ser-Ser-Pro-Ala-Ser-Ser-Ala-Ala-Ser-Ala-Cys-amide mixed with mercaptoethanol as a diluent molecule or lateral spacer. These mixed films, although not representing a perfect lipid bilayer, have been shown to retain the activity of incorporated H(+)-ATP synthases from chloroplasts in contrast to films prepared from the pure thiolipopeptide. The activity of the protein was demonstrated by impedance spectroscopy. The resistance decreased due to proton transport across the lipid film, which occurs as a consequence of adenosine triphosphate (ATP) hydrolysis. Several effects previously determined from kinetic measurements of the enzyme reconstituted in liposomes such as saturation with respect to the substrate (ATP), inhibition by venturicidin, activation by a positive potential pulse and increase of the proton current as a function of increasingly negative potentials have been confirmed also for this tethered membrane system. Changes in the impedance spectra due to the addition of ATP were fully reversible.     

Schizodeme and zymodeme characterization of Leishmania in the investigation of foci of visceral and cutaneous leishmaniasis

Leishmania parasites were isolated from humans and canines in foci of cutaneous and visceral leishmaniasis. After in vitro cultivation the parasites were examined by the following biochemical techniques: (i) restriction analysis of kinetoplast DNA (kDNA) also known as schizodeme analysis (Morel et al., 1980); (ii) zymodeme analysis (Barret et al., 1980); by agarose gel electrophoresis and (iii) isoelectricfocusing in polyacrylamide gels. The strains of cutaneous and visceralizing leishmanias studied could be differentiated by schizodeme analysis, using the endonuclease MspI, into three complexes agreeing with those accepted for human New World leishmaniasis. In the municipality of Rio de Janeiro, isolates from a focus of cutaneous leishmaniasis were identified as L. braziliensis braziliensis and from a focus of visceral leishmaniasis were identified as L. donovani by zymodeme characterization. Identical restriction enzyme profiles of kDNA from human and canine isolates indicated that in the cutaneous focus at Jacarepaguá, Rio de Janeiro, the same strain was probably circulating in both the canine and human populations. This suggests a possible role for dogs as a reservoir host for L. braziliensis braziliensis. In addition, our results confirm the importance of dogs as reservoirs in visceral leishmaniasis. The stability of the electrophoretic patterns of restriction digest ("fingerprints") of Leishmania kDNA as well as differences in the sensitivity of the techniques used were demonstrated. Strains from widely different geographical areas as well as strains maintained in vivo and in vitro showed identical kDNA restriction patterns, while strains showing similar banding patterns by enzyme electrophoresis could be differentiated by schizodeme analysis. These results demonstrate the usefulness of an integrated biochemical approach in the identification of Leishmania.     

THE TWO KINDS OF DEATH OF WILLIAM HARVEY

The determining factors in congestive heart failure as well as in shock are more often extracardiac than in the heart itself. Carbon dioxide tension in the blood is as important as the oxygen tension. Carbon dioxide is a hemodynamic agent of the first magnitude. It can be quickly increased or decreased by altering the ventilation of the lungs. It is a prime factor in determining whether the circulation fails from a lack or from suffocation by an excess.     

Modification of the Composition and Structure of the Yeast Cell Wall by Culture in the Presence of Sulfur Amino Acids

Growth of Candida utilis and Saccharomyces cerevisiae in a medium supplemented with sulfur amino acids led to synthesis and accumulation of S-adenosylmethionine, accompanied by a reduction in the cell yield, an increased sensitivity of the cell wall to snail gut enzymes (Helix pomatia), as judged by spheroplast formation, and by a modification of the chemical composition of both the intact cells and their isolated walls. Walls of supplemented cultures of C. utilis were three times as sensitive to enzymatic digestion as walls from nonsupplemented cultures. In contrast to C. utilis, walls isolated from supplemented cultures of S. cerevisiae were digested slightly more rapidly by the purified snail extract than those from nonsupplemented cultures. Chemical modifications of the cell wall are interpreted to explain the ease with which cells from sulfur amino acid-supplemented cultures are converted to spheroplasts.     

Use of C-banding for identifying radiation induced micronuclei

Differentiation of micronuclei (MN) caused by ionizing radiation from those caused by chemicals is a crucial step for managing treatment of individuals exposed to radiation. MN in binucleated lymphocytes in peripheral blood are widely used as biomarkers for estimating dose of radiation, but they are not specific for ionizing radiation. MN induced by ionizing radiation originate predominantly as a result of chromosome breaks (clastogenic action), whereas MN caused by chemical agents are derived from the loss of entire chromosomes (aneugenic action). C-banding highlights centromeres, which might make it possible to distinguish radiation induced MN, i.e., as a byproduct of acentric fragments, from those caused by the loss of entire chromosomes. To test the use of C-banding for identifying radiation induced MN, a blood sample from a healthy donor was irradiated with 3 Gy of Co-60 gamma rays and cultured. Cells were harvested and dropped onto slides, divided into a group stained directly with Giemsa and another processed for C banding, then stained with Giemsa. The frequency of MN in 500 binucleated cells was scored for each method. In preparations stained with Giemsa directly, the MN appeared as uniformly stained structures, whereas after C banding, some MN exhibited darker regions corresponding to centromeres that indicated that they were not derived from acentric fragments. The C-banding technique enables differentiation of MN from acentric chromosomal material. This distinction is useful for improving the specificity of the MN assay as a biomarker for ionizing radiation.     

Isotopically sensitive branching in the formation of cyclic monoterpenes: proof that (-)-alpha-pinene and (-)-beta-pinene are synthesized by the same monoterpene cyclase via deprotonation of a common intermediate

To determine whether the bicyclic monoterpene olefins (-)-alpha-pinene and (-)-beta-pinene arise biosynthetically from the same monoterpene cyclase by alternate deprotonations of a common carbocationic intermediate, the product distributions arising from the acyclic precursor [10-2H3,1-3H]geranyl pyrophosphate were compared with those resulting from incubation of [1-3H]geranyl pyrophosphate with (-)-pinene cyclase from Salvia officinalis. Alteration in proportions of the olefinic products generated by the partially purified pinene cyclase resulted from the suppression of the formation of (-)-beta-pinene (C10 deprotonation) by a primary deuterium isotope effect with a compensating stimulation of the formation of (-)-alpha-pinene (C4 deprotonation). (-)-Pinene cyclase as well as (+)-pinene cyclase also exhibited a decrease in the proportion of the acyclic olefin myrcene generated from the deuteriated substrate, accompanied by a corresponding increase in the commitment to cyclized products. The observation of isotopically sensitive branching, in conjunction with quantitation of the magnitude of the secondary deuterium isotope effect on the overall rate of product formation by the (+)- and (-)-pinene cyclases as well as two other monoterpene cyclases from the same tissue, supports the biosynthetic origin of (-)-alpha-pinene and (-)-beta-pinene by alternative deprotonations of a common enzymatic intermediate. A biogenetic scheme consistent with these results is presented, and alternate proposals for the origin of the pinenes are addressed.     

Carbopol-Based Gels for Nasal Delivery of Progesterone

The purpose of this study was to investigate the nasal absorption of progesterone from carbopol-based nasal gels in rabbits. Progesterone nasal gels were prepared by dispersing carbopol 974 (1%, 1.5%, and 2%) in distilled water followed by addition of progesterone/progesterone–β cyclodextrin complex dissolved in propylene glycol then neutralization. The potential use of β cyclodextrin (CD) as nasal absorption enhancer by simple addition, as a physical mixture and as a complex with progesterone was investigated. The absolute bioavailability of progesterone from nasal gels in rabbits was studied by estimating the serum progesterone level by competitive solid-phase enzyme immunoassay in comparison to intravenous injection. The carbopol gel formulations produced a significant increase in bioavailability. CD complex promotes the nasal absorption of progesterone from carbopol gels as compared with gels where the CD is added by simple addition and gels which do not contain CD. This method of addition of CD as an inclusion complex in the gels could be considered as a preferred platform in nasal drug administration.