Estimation of the pentose cycle in the perfused cow''s udder

1. The distributions of (14)C have been compared in the glucose and galactose moieties of lactose obtained from cows' udders perfused with blood containing [1-(14)C]-, [2-(14)C]- and [6-(14)C]-glucose. The (14)C of the glucose moiety was found in the same position as that of the administered glucose, but in the galactose moiety the (14)C from [2-(14)C]glucose was extensively randomized into positions 1 and 3. It is concluded that the glucose moiety arose from free glucose and the galactose moiety from hexose phosphate intermediates and that the latter reflected the randomization occurring through reactions of the pentose cycle. 2. The proportion of the glucose metabolized via the pentose cycle for those cells making lactose was estimated from the distribution of (14)C in the galactose moiety and found to be about 23% in one experiment and 30% in another experiment. 3. The yield and distribution of (14)C were determined in the glycerol of fat from the tissue in experiments with [2-(14)C]- and [6-(14)C]-glucose. There was a greater randomization of (14)C in the glycerol than in C-1, C-2 and C-3 of the galactose moiety of lactose. The ratio of the yield of (14)C in the glycerol from [2-(14)C]glucose to that of [6-(14)C]glucose was very low and from this ratio it was calculated that less than 10% of the glucose was metabolized by the Embden-Meyerhof pathway and approx. 60-70% was converted into lactose. 4. [6-(14)C]Glucose and [6-(3)H]glucose were used to determine whether the (3)H at the C-6 position remained stable during its conversion into glyceride of fat from the tissue. Twenty-seven per cent of the (3)H was labilized during this conversion. Therefore it was not possible to use [2-(14)C]glucose and [6-(3)H]glucose in a single experiment to measure the relative conversion of the C-2 and C-6 positions of glucose to glycerol.     

Mineralization of Polycyclic Aromatic Hydrocarbons by the White Rot Fungus Pleurotus ostreatus

The white rot fungus Pleurotus ostreatus was able to mineralize to (sup14)CO(inf2) 7.0% of [(sup14)C]catechol, 3.0% of [(sup14)C]phenanthrene, 0.4% of [(sup14)C]pyrene, and 0.19% of [(sup14)C]benzo[a]pyrene by day 11 of incubation. It also mineralized [(sup14)C]anthracene (0.6%) much more slowly (35 days) and [(sup14)C]fluorene (0.19%) within 15 days. P. ostreatus did not mineralize fluoranthene. The activities of the enzymes considered to be part of the ligninolytic system, laccase and manganese-inhibited peroxidase, were observed during fungal growth in the presence of the various polycyclic aromatic hydrocarbons. Although activity of both enzymes was observed, no distinct correlation to polycyclic aromatic hydrocarbon degradation was found.     

Absorption by rat fetal tissues of 14C-labeled phospholipids injected into the rat body in the late stages of pregnancy

The authors studied the possibility of 14C-phospholipid transplacental penetration after 15C-phospholipid injection into rats at the 20th day of pregnancy. The preparation of 14C-phospholipids (total phospholipids) was isolated by thin-layer chromatography from the liver of rats injected with 2-14C-sodium acetate. One hour after its injection into the rat, 14C-phospholipids were detectable in total phospholipids of the pulmonary and cerebral fetal tissues. It was discovered that specific radioactivity of phospholipids contained by these tissues was 2--5 times higher when 14C-phospholipids were injected subcutaneously as compared with intramuscular injection. It is concluded that exogenous phospholipids entrapped in the mother's circulation penetrate the placental barrier of the fetus and the blood-brain barrier of the mature fetus, being consumed by different fetal tissues for forming membrane structures of the fetal tissues.     

Mapping the heparin-binding site on the 13-14F3 fragment of fibronectin

Fibronectin, a multifunctional glycoprotein of the extracellular matrix, plays a major role in cell adhesion. Various studies have revealed that the human 13th and 14th fibronectin type III domains (labeled (13)F3 and (14)F3 here) contain a heparin-binding site. Mapping of the heparin-binding sites of (13-14)F3, (13)F3, and (14)F3 by NMR chemical shift perturbation, isothermal titration calorimetry, and molecular modeling show that (13)F3 provides the dominant heparin-binding site and that the residues involved are within the first 29 amino acids of (13)F3. Predictions from earlier biochemical and modeling studies as well as the x-ray structure of (12-14)F3 were tested. It was shown that the positively charged residues that project into the solvent from the ABE face of the triple-stranded beta sheet on (13)F3 are involved in binding, but (14)F3 does not appear to contribute significantly to heparin binding.     

CD14 directly binds to triacylated lipopeptides and facilitates recognition of the lipopeptides by the receptor complex of Toll-like receptors 2 and 1 without binding to the complex

It has demonstrated that the recognition of triacylated lipopeptides by Toll-like receptor (TLR) 2 requires TLR1 as a coreceptor. In the NF-kappaB reporter assay system in which human embryonic kidney 293 cells were transfected with TLR2 and TLR1 together with an NF-kappaB luciferase reporter gene, S-(2,3-bispalmitoyloxypropyl)-N-palmitoyl-Cys-Lys-Lys-Lys-Lys (Pam(3)CSK(4)) and Pam(3)CSSNA were recognized by TLR2/TLR1, but the recognition level was unexpectedly very low. However, cotransfection of CD14 drastically enhanced the recognition of triacylated lipopeptides by TLR2/TLR1. The CD14-induced enhancement did not occur without cotransfection of TLR1. Both CD14(dS39-A48), a mutant with deletion of the part of possible N-terminal ligand-binding pocket, and anti-CD14 monoclonal antibody reduced the CD14-induced enhancement. Transfection of a TIR domain-deficient mutant of TLR2 (TLR2(dE772-S784)) or TLR1 (TLR1(dQ636-K779)) completely abrogated the CD14-induced enhancement. Soluble recombinant CD14 added extracellularly enhanced the recognition of Pam(3)CSSNA by TLR2/TLR1. Immunoprecipitation analysis demonstrated that CD14 was not associated with TLR2 but that TLR1 was associated with TLR2. In addition, surface plasmon resonance-based assay demonstrated that CD14 binds to Pam(3)CSK(4) at a dissociation constant of 5.7 microM. This study suggests that CD14 directly binds to triacylated lipopeptides and facilitates recognition of the lipopeptides by the TLR2/TLR1 complex without binding to the receptor complex.     

Studies on the biosynthesis of phenols in fungi. Conversion of [14C]orsellinic acid and [14C]orcinol into fumigatol by Aspergillus fumigatus I.M.I. 89353

1. Sodium [1-(14)C]acetate was incorporated into orsellinic acid and fumigatol by Aspergillus fumigatus. 2. [(14)C]Orsellinic acid was prepared biosynthetically. It was converted almost entirely into fumigatol and fumigatin within 2 days of supplementation of the medium. The apparent decrease in incorporation after a longer period of growth was due to decomposition of radioactive fumigatol and the production of relatively unlabelled material. The addition of orcinol to these cultures decreased the conversion of [(14)C]orsellinic acid into fumigatol. [(14)C]Orsellinic acid was incorporated into 3,4-dihydroxytoluquinol in both sets of cultures. 3. [(14)C]Orcinol was prepared from [(14)C]orsellinic acid after acid hydrolysis. It was also very effective as a precursor of fumigatol (60% incorporation). 4. The specific activity of fumigatin was lower than that of fumigatol at early stages of growth (4-5 days after inoculation) with all the labelled substrates that were tested. This indicated that fumigatin arose from fumigatol after oxidation in the medium. 5. The presence of orcinol in the medium greatly stimulated the incorporation of radioactivity (presumably derived from the (14)CO(2)H of orsellinic acid) into the isoprenoid compounds, ergosterol and ubiquinone, in the mycelium.     

Synthesis and properties of the 14-amino derivatives of daunorubicin and carminomycin]

Interaction of 14-bromine derivatives of daunorubicin and carminomycin, as well as their aglycones with secondary amines, such as piperidine, N-methylpiperazine and morpholine was studied with a purpose of preparing new potentially antitumor antibiotics. It was found that reaction in acetone or dioxan at a temperature of 50--60 degrees C resulted in formation of 14-amino derivatives of the respective bromides. 14-Piperidinyl- and 14-(N-methylpiperazinyl)-daunorubicin, 14-piperidinyl-daunorubicinon and 14-piperidinyl-carminomycinon were prepared. The structure of the new substances was confirmed by the IR, UV and NMR spectra. The antimicrobial activity of 14-amino derivatives amounted to 10--35 per cent of the activity of the initial antibiotics, i.e. daunorubicin and carminomycin.     

Phenobarbital selectively modulates the glucagon-stimulated activity of adenylate cyclase by depressing the lipid phase separation occurring in the outer half of the bilayer of liver plasma membranes.

The pattern of incorporation of radioactivity from [1-14C]acetate and [2-14C]acetate into the polyprenyl side-chain of ubiquinones in bacteria (Azotobacter vinelandii, Pseudomonas sesami, Escherichia coli and Rhodopseudomonas capsulata) was studied. For this purpose, a new degradation method involving a modified Barbier-Wieland reaction of laevulinic acid was developed, and used along with the iodoform reaction. Both C-1 and C-2 of acetate were incorporated exclusively into C-2 of laevulinic acid suggesting that the well-known pathway through acetoacetyl-CoA ('acetoacetate pathway') was not operative in these bacteria. An alternative pathway ('acetolactate pathway'), starting with pyruvate and acetaldehyde as the distal precursors, and utilizing the reactions of leucine and valine metabolism, was postulated. It was also postulated that C-1 of acetate is incorporated not directly, but after oxidation to CO2. The pattern of incorporation of radioactivity from [U-14C]valine, [U-14C]alanine and NaH14CO3 into the side-chain of ubiquinone of R. capsulata was in agreement with the operation of the 'acetolactate pathway'.     

Biosynthesis of the pyrimidinyl amino acid lathyrine by Lathyrus tingitanus L.

The biosynthesis of the pyrimidinyl amino acid lathyrine by seedlings of Lathyrus tingitanus L. was shown to be stimulated by uracil. [6(-14)C]Orotate, [2(-14)C]uracil and [3(-14)C]serine were incorporated into lathyrine; the incorporation of [6(-14)C]orotate was substantially decreased in the presence of uracil. Chemical degradation to locate the 14C incorporated from labelled precursors showed that 90% of the radioactivity incorporated into lathyrine from [3(-14)C]serine could be recovered in the alanine side chain. Over 80% of the radioactivity incorporated from [2(-14)C]uracil was shown to be located in C-2 of lathyrine. It is concluded that under the conditions studied, lathyrine arises from a preformed pyrimidine arising via the orotate pathway. Paradoxically, it was also possible to confirm previous reports that radioactivity from L-[guanidino-14C]homoarginine is incorporated into lathyrine and gamma-hydroxyhomoarginine. However, as homoarginine and gamma-hydroxyhomoarginine are also both labelled by [2(-14)C]uracil, it is suggested that they are products of the ring-opening of lathyrine and that reversibility of this process accounts, at least in part, for their observed experimental incorporation into lathyrine.     

流行性乙型脑炎减毒活疫苗SA14-14-2E基因的稳定性

分别对SA14-14-2(PHK8代)疫苗株及其原野毒株SA14和另2个疫苗传代株[SA14-14-2(PHK17代)和SA14-14-2(鼠脑1代)]的E区基因进行了序列测定.结果表明,乙脑病毒减毒活疫苗SA14-14-2在PHK细胞上连传至17代时,发现2个氨基酸突变(E-331、E-398),但不是回复突变.虽然在毒力最容易返祖的乳鼠脑内传1代后发生E-107个氨基酸回复,但与野毒株毒力相比仍然相差很大,无毒力返祖现象.在疫苗的实际质控工作中,对上述与毒力相关的基因进行监测,可能有助于发现减毒活疫苗的毒力水平,为疫苗安全性提供更可靠的检测手段.     

Tricarboxylic acid-cycle metabolism in brain. Effect of fluoroacetate and fluorocitrate on the labelling of glutamate aspartate, glutamine and γ-amino butyrate

1. The effect of fluoroacetate and fluorocitrate on the compartmentation of the glutamate-glutamine system was studied in brain slices with l-[U-(14)C]glutamate, l-[U-(14)C]aspartate, [1-(14)C]acetate and gamma-amino[1-(14)C]butyrate as precursors and in homogenates of brain tissue with [1-(14)C]acetate. The effect of fluoroacetate was also studied in vivo in mouse brain with [1-(14)C]acetate as precursor. 2. Fluoroacetate and fluorocitrate inhibit the labelling of glutamine from all precursors but affect the labelling of glutamate to a much lesser extent. This effect is not due to inhibition of glutamine synthetase. It is interpreted as being due to selective inhibition of the metabolism of a small pool of glutamate that preferentially labels glutamine.     

Kinetics of the binding 14C-DDT-and haemolymph lipoproteins of Periplaneta americana (author's transl)]

A study of the kinetics of the complexation of the 14C-DDT and lipoproteins of the haemolymph of Periplaneta americana (L.) was made, the method employed was that described by WINTER et alii (7). It was found that the above complex was unstable when kept at low temperatures (5 degrees) for more than twenty four hours; the incorporation of 14C-DDT in the proteic fraction increased with temperature up to 42 degrees C, and at least 6 hours of incubation were necessary to bind the 14C-DDT to the lipoprotein.     

Existence of glucocorticoid-specific receptors in the plasma membranes of rat liver

It was found that isolated plasma membranes whose purity was assayed by determinations of marker enzyme activities, specifically bind dexamethasone. The association constant and the number of binding sites were found to be equal to (7,03 +/- 4,05) . 10(9) M-1 and (1,6 +/- 0,18) . 10(-14) mol/mg protein, respectively. It was assumed that lipoprotein components of plasma membranes are involved in this binding.     

Studies of intermediary metabolism in germinating pea cotyledons. The pathway of ethanol metabolism and the role of the tricarboxylic acid cycle

1. The pathway of ethanol metabolism in cotyledons of 3-day-old pea seedlings has been examined by incubating tissue slices with [1-(14)C]ethanol and [2-(14)C]ethanol for periods up to 1hr. 2. Ethanol was rapidly incorporated into citrate and glutamate but relatively small amounts of (14)C were present in the evolved carbon dioxide even after 1hr. of ethanol metabolism. 3. Similar data were obtained from experiments in which [1,2-(14)C(2)]acetaldehyde and [(14)C]acetate were supplied. 4. The results are interpreted as indicating that ethanol is metabolized essentially via the reactions of the tricarboxylic acid cycle with a substantial drain of alpha-oxoglutarate to support the biosynthesis of glutamate. 5. It is concluded that oxaloacetate, required for the incorporation of ethanol into citrate, arises mainly from the transamination of aspartate and the fixation of carbon dioxide.     

Pathways of acetone's metabolism in the rat

Distributions of 14C were different from those of 13C in glucoses formed by livers of rats in diabetic ketosis and perfused with [2-14C]acetone and [2-13C]lactate. There was 32-73% of the 14C and 8-12% of the 13C in carbons 3 and 4 of the glucoses with the remaining 14C and 13C distributed about equally in the other carbons. Incorporations of 14C from [2-14C]acetone (14-39%) also exceeded those from [2-14C]pyruvate (8-10%) into carbons 3 and 4 of glucoses formed by hepatocytes from rats fed acetone or fasted. [2-14C]Acetone and [2-14C]pyruvate were infused into rats that were fed, fasted, given acetone in their drinking water, or in diabetic ketosis. Thirty-seven to 52% of the 14C in the glucoses formed was in their carbons 3 and 4 when the acetone was infused and 8 to 14% when the pyruvate was infused. [1,3-14C]Hydroxybutyrate was formed by the rats in diabetic ketosis given [2-14C]acetone. It is concluded that acetone is metabolized in rats to a large extent by a pathway in which lactate or its metabolic equivalent is not an intermediate and that pathway is via acetyl-CoA. via acetyl-CoA.     

流行性乙型脑炎病毒减毒株SA14-14-2E基因稳定性研究

为研究乙脑病毒减毒株SA14-14-2 E蛋白基因稳定性,将乙脑病毒减毒株SA14-14-2在原代地鼠肾细胞(PHK)上传至18代,应用RT-PCR分别扩增PHK6代、PHK7代、PHK8代、PHK13代、PHK18代E蛋白基因并测序后,与Genebank中乙脑病毒减毒株SA14-14-2(D90195)进行比较分析。PHK6、PHK7、PHK8代病毒与D90195 E蛋白核苷酸和氨基酸序列完全相同。PHK13、PHK18代病毒与D90195E蛋白核苷酸序列同源性分别为99.8%、99.7%,与D90195E蛋白氨基酸序列同源性分别为99.6%、99.4%。各代次病毒E蛋白与减毒相关氨基酸未发生改变,同时所有突变的氨基酸均非SA14原有的,故不是恢复性突变。结果表明乙脑病毒减毒株SA14-14-2的遗传学特性稳定,从分子水平证明乙脑病毒减毒株SA14-14-2及其生产的疫苗具有安全性。     

The incorporation of amino acids and nucleic acid bases into the seedling,reproductive stage and young ear portion of rice plants

Summary The incorporation of C¹⁴-amino acids (aspartic acid, glutamic acid, threonine and proline) and C¹⁴-nucleic acid bases (adenine, guanine, cytosine and uracil) into the seedling, reproductive stage and young ear portion of rice plant was investigated. It was found that C¹⁴-aspartic acid was incorporated into the rice seedling more rapidly than C¹⁴-threonine or C¹⁴-proline; on the other hand C¹⁴-proline was found to be more rapidly incorporated than C¹⁴-aspartic acid into reproductive stage plant and young ear portion. Similarly C¹⁴-adenine was incorporated into the rice seedling more rapidly than other C¹⁴-labelled bases. On the other hand C¹⁴-uracil was preferentially incorporated to C¹⁴-adenine or C¹⁴-guanine into the reproductive stage plant and young ear portion. It is suggested from the results obtained that proline is polymerized into polypeptide or protein in the rice plant more rapidly at the reproductive stage than at the seedling stage and that a higher proportion of pyrimidine bases might be involved into the metabolic process at the reproductive stage of rice plant.     

Enzymic nature of the protein moiety of protochlorophyllide holochrome

The enzymic nature of the protein moiety of protochlorophyll(ide) holochrome was studied by following the fate of the [(14)C]protochlorophyll(ide) formed when dark-grown barley (Hordeum vulgare) or bean (Phaseolus vulgaris) leaves are incubated in the dark with 3 mm 4-delta-[(14)C]aminolevulinic acid. It was found that: [List: see text]Since turnover of protochlorophyll(ide) was not observed, these results show that there is a free exchange between the old "endogenous" and the new delta-aminolevulinic-acid-induced protochlorophyll(ide) molecules on the active site of the holochrome protein. These results are consistent with the hypothesis that the holochrome protein acts as an enzyme.     

Mitotic exit network controls the localization of Cdc14 to the spindle pole body in Saccharomyces cerevisiae

Budding yeast Cdc14 phosphatase plays essential roles in mitotic exit. Cdc14 is sequestered in the nucleolus by its inhibitor Net1/Cfi1 and is only released from the nucleolus during anaphase to inactivate mitotic CDK. It is believed that the mitotic exit network (MEN) is required for the release of Cdc14 from the nucleolus because liberation of Cdc14 by net1/cfi1 mutations bypasses the essential role of the MEN. But how the MEN residing at the spindle pole body (SPB) controls the association of Cdc14 with Net1/Cfi1 in the nucleolus is not yet understood. We found that Cdc14-5GFP was released from the nucleolus in the MEN mutants (tem1, cdc15, dbf2, and nud1), but not in the cdc5 cells during early anaphase. The Cdc14 liberation from the nucleolus was inhibited by the Mad2 checkpoint and by the Bub2 checkpoint in a different manner when microtubule organization was disrupted. We observed Cdc14-5GFP at the SPB in addition to the nucleolus. The SPB localization of Cdc14 was significantly affected by the MEN mutations and the bub2 mutation. We conclude that Cdc14 is released from the nucleolus at the onset of anaphase in a CDC5-dependent manner and that MEN factors possibly regulate Cdc14 release from the SPB.     

Compartmentation of glutamate metabolism in brain. Evidence for the existence of two different tricarboxylic acid cycles in brain

1. (14)C from [1-(14)C]glucose injected intraperitoneally into mice is incorporated into glutamate, aspartate and glutamine in the brain to a much greater extent than (14)C from [2-(14)C]glucose. This difference for [1-(14)C]glucose and [2-(14)C]glucose increases with time. The amount of (14)C in C-1 of glutamate increases steadily with time with both precursors. It is suggested that a large part of the glutamate and aspartate pools in brain are in close contact with intermediates of a fast-turning tricarboxylic acid cycle. 2. (14)C from [1-(14)C]acetate and [2-(14)C]acetate is incorporated to a much larger extent into glutamine than into glutamate. An examination of the time-course of (14)C incorporated into glutamine and glutamate reveals that glutamine is not formed from the glutamate pool, labelled extensively by glucose, but from a small glutamate pool. This small glutamate pool is not derived from an intermediate of a fast-turning tricarboxylic acid cycle. 3. It is proposed that two different tricarboxylic acid cycles exist in brain.