Preparation of the 3-monosulphates of cholic acid, chenodeoxycholic acid and deoxycholic acid.

Extraction with butan-1-ol of freeze-dried microsomal fractions from livers of 3-methyl-cholarthrene-pre-treated hamsters removed about 90% of the total lipid content, but the lipid remaining proved sufficient for the cytochrome P-450 enzyme system to retain about 15-40% of its original catalytic activity for dimethylnitrosamine demethylation. Addition of butan-1-ol-extracted total phospholipid or phosphatidylcholine could not restore any activity, whereas the addition of the synthetic phospholipid dilauroyl phosphatidylcholine was able to restore almost complete activity. Synthetic dipalmitoyl or distearoyl phosphatidylcholine was ineffective in restoring the activity in this reconstituted system.     

Effect of hippuric acid on the gaschromatographic retention of S-phenylmercapturic acid

S-phenylmercapturic acid (PMA) is one specific urinary biomarker of low-level benzene exposure. It is used for biological monitoring of benzene-exposed workers in the petrochemical industry and normally ranges from non-measurable to 10 microg/l levels in non-exposed non-smoking subjects. Benzene-exposure caused by workplace or lifestyle sources is frequently accompanied by toluene exposure, which can cause the occurrence of high levels (from 10 mg/l to more than 2000 mg/l) of hippuric acid (HA) in urine. Both solvents are toxic, and benzene is classified as a human carcinogen. The biological monitoring of benzene and toluene is therefore required for preventive care of exposed workers health. In this study a GC-MS method was adopted for measuring urinary PMA, which involved liquid-liquid extraction (LLE) with ethyl acetate from acidified urine and esterification with 0.5 N hydrochloric acid in methanol. The method evidenced a GC effect in a conventional HP-5 (30 m x 0.25 mm i.d., 0.25 microm film-thickness) methyl-phenylsilicone capillary column produced by HA on PMA. The results demonstrate that HA at concentrations as low as 250 mg/l can delay the elution of PMA and labelled internal standard from the column. The recognition and discussion of this particular GC phase soaking effect may be of help for those who are occupied in the determination of PMA and of urinary acidic metabolites by GC.     

Amino acid neurotransmitters in the CNS. Characteristics of the acidic amino acid exchange

D-Aspartate exchange, defined as amino acid-stimulated D-[3H]aspartate efflux, was investigated in a preparation of rat brain synaptosomes. The efflux of radiolabelled D-aspartate was found to be enhanced by micromolar concentrations of externally added D- and L-aspartate, L-glutamate, L-cysteate and L-cysteinesulphinate. The stimulation of release by external amino acids followed Michaelis-Menten kinetics; the apparent Km values (in microM) were: 14.65 +/- 0.98 for D-aspartate; 8.00 +/- 1.5 for L-aspartate; 22.31 +/- 1.62 for L-glutamate; 6.76 +/- 0.3 for L-cysteate and 7.89 +/- 1.23 for L-cysteinesulphinate. The Vmax values for efflux were 2.16-4.06 nmol/min per mg protein. The exchange process was found to require external NaCl but was very little affected by increase in the external [K+]. The demonstration of exchange as a part of the transport process provides support for the suggestion that in synaptosomal preparations a substantial portion of influx and efflux of amino acid neurotransmitters occurs via a reversible membrane carrier.     

Response of the cockroach brain gamma-aminobutyric acid system to isonicotinic acid hydrazide and mercaptopropionic acid

The presence of gamma-aminobutyric acid (GABA) as well as glutamic acid decarboxylase (GAD) and GABA-transaminase (GABA-T) enzymes was demonstrated in the cockroach (Periplaneta americana) brain. Isonicotinic acid hydrazide (INH) in vivo (2.19 mumol/g) inhibited brain GAD activity, the inhibition lasted for about 2 hours and the normal activity levels reappeared at 4 h after INH administration. Brain GABA levels increased initially but then declined and were restored to normal levels at 4 h after INH administration. GABA-T activity was strongly inhibited by INH and a total 100% inhibition was observed at 2-3 h following INH treatment. The GABA-T activity, however, began to recover after 3 h but only 37% of the total enzyme activity was released from inhibition. Mercaptopropionic acid (MPA) in vivo (32 micrograms/g) inhibited brain GAD activity and depleted GABA level also. Results indicate that INH response of the cockroach brain GABA system is similar to that reported for the chick brain but differs from that of the mammalian brain.     

Characterization of sarcosylsarcoursodeoxycholic acid formed during the synthesis of sarcoursodeoxycholic acid

This report describes the isolation of sarcosylsarcosine conjugate of ursodeoxycholic acid (UDCA) formed during the synthesis of sarcoUDCA by the mixed anhydride method. The compound was characterized by its chemical ionization mass spectrum. The diamino acid conjugate was formed only when the free amino acid was used for conjugation. This was confirmed by the isolation of glycylglycoUDCA during the conjugation of UDCA with free glycine but not with glycine ethyl ester hydrochloride. Pure sarcoUDCA was prepared by conjugation of UDCA with sarocisine methyl ester hydrochloride while sarcoUDCA on further reaction with the protected sarcosine derivative gave pure sarcosylsarcoUDCA in 52% yield.     

The use of tannic acid for the ultrastructural visualization of hyaluronic acid

Summary This study describes a method, which makes use of tannic acid (2%) as a component of a paraformaldehyde-glutaraldehyde based fixative, to reveal the presence and ultrastructure of glycosaminoglycans in the extracellular matrix. The ultrastructure of the extracellular matrix in the stage 24 chick embryo wing is examined after fixation by several procedures. After fixation in the absence of tannic acid, the intercellular spaces contain little extracellular matrix, except for occasional fibrils (collagen?). On the other hand, when tannic acid is included in the primary fixative, the intercellular spaces contain considerable amounts extracellular matrix which includes 3±0.5 nm filaments, ±30 nm granules, as well as putative collagen fibrils. The 3±0.5 nm diameter fibrils are not observed when the limbs had been injected in ovo with Streptomyces hyaluronidase (specific for hyaluronic acid) prior to fixation. Furthermore, the 3±0.5 nm fibrils resemble authentic hyaluronic acid that had been fixed by the same procedure in the presence of tannic acid. Limbs treated with tannic acid after osmication contained only small amounts of extracellular material, which was confined largely to cell surfaces. These results demonstrate that the use of tannic acid in the primary fixative can serve as a useful method for the ultrastructural visualization of several extracellular matrix materials, including hyaluronic acid.This study was supported by NIH grant HD 05505     

Studies on the structure of hyaluronic acid. Characterization of the product formed when hyaluronic acid is treated with ascorbic acid

Physical and chemical methods were used to characterize hyaluronic acid before (fraction HAIIBI) and after (fraction HA-AA) treatment with ascorbic acid. Fraction HA-AA was recovered with an almost quantitative yield and was shown to be chemically identical with fraction HAIIBI by all the methods used. These two materials, however, differed markedly in their molecular sizes and degree of polydispersity. By using sedimentation, diffusion and sedimentation-equilibrium analyses, weight-average molecular weights of about 1.2x10(6) and 6.5x10(4) respectively were obtained for fractions HAIIBI and HA-AA. It is concluded from these results that hyaluronic acid has a molecular weight of about 65000 and that the polysaccharide chain of this molecule is not depolymerized by ascorbic acid. It is further proposed that hyaluronic acid molecules in the matrix of connective tissues are present either in an aggregated form or as subunits of heterogeneous macromolecules, and that it is the linkages responsible for the organization of these structures which are broken by ascorbic acid.     

Novel phosphinic and phosphonic acid analogues of the anticonvulsant valproic acid

1-Propylbutylphosphinic acid 2, (1-propylbutyl)methylphosphinic acid 3 and 1-propylbutylphosphonic acid 4 have been synthesized as bioisosteres of the corresponding carboxylic acid valproate 1, which is a potent anticonvulsant. The novel phosphinic and phosphonic acids were tested for their anticonvulsant activity and were found to be inactive.     

Effect of dietary amino acid on the amino acid pool of Aedes aegypti

Ion-exchange chromatography analysis of whole body extracts of Aedes aegypti mosquitoes which had received amino acids in their diet revealed that generally there were changes in the titre of two or more amino acids. Cysteine produced the greatest number of changes and was toxic to the insect. Of the ten amino acids provided, none resulted in the significant change in the concentration of tyrosine following a blood meal as was observed in previous studies. Evidence is presented for the conversion of arginine to ornithine and for the synthesis of arginine from glutamic acid. The data presented tend to support the hypothesis of lysine synthesis from α-ketoglutarate and for the use of proline as an energy reserve in the insect.     

Enzymatic synthesis of the neuroexcitatory amino acid quisqualic acid by cysteine synthase

Purification of cysteine synthase from the leaves of Quisqualis indica var. villosa reveals the presence of two forms of this enzyme, separated by chromatography on DEAE-Sephadex A-50. Isoenzyme A was purified 10 000-fold and had a specific activity of 10.8 U/mg protein. Isoenzyme B was purified 460-fold with a specific activity of 0.49 U/mg protein. Both isoenzymes have the same M,s (58 000) and dissociate into identical subunits (M_r 29 000). The K_m value of isoenzyme A is 1.9 mM for O-acetyl-L-serine and 59 μM for sulphide, while that of isoenzyme B is 7.1 mM for O-acetyl-L-serine and 4.0 mM for 3,5-dioxo-1,2,4-oxadiazolidine. Both isoenzymes catalyse the formation of cysteine from O-acetyl-L-serine and hydrogen sulphide, but only isoenzyme B catalyses the formation of L-quisqualic acid. Other significant differences occur in the substrate specificity of the two isoenzymes. Some properties of the purified cysteine synthase isoenzymes are also described.     

Sialic acid interference with the thiobarbituric acid method for measuirng 3-deoxyoctulosonic acid.

Sialic acid produced an interfering color in an assay designed for measuring 3-deoxyoctulosonic acid. The color yield from sialic acid was not as high as that from 3-deoxyoctulosonic acid but was high enough to cause concern about problems arising when sialic acid-containing substances are present in preparations of bacterial lipopolysaccharides. The instabilityof 3-deoxyoctulosonic acid in acidic media led to reduced color yields. In general, the results suggested that data generated by the application of the thiobarbituric acid method to the measurement of 3-deoxyoctulosonic acid should be approached with caution.     

Studies on the stability of tritium-labeled polyuridylic acid and polyadenylic acid

Results are presented on the stability of high specific activity [uridylate-5,6-³H]polyuridylic acid and [adenylate-2,8-³H]polyadenylic acid stored under various conditions. Polyacrylamide disc gel electrophoresis in the presence of SDS was used to assess qualitatively the change in molecular weight distribution of the polynucleotides stored under different conditions. Products stored for a period of months in ethanol; water solution [1:1, $\text{v}\text{v}$] were found to have a significantly slower rate of decomposition than polynucleotides stored in frozen aqueous solution or as lyophilized solid.     

Evaluation of lactic acid bacteria autolysate for the supplementation of lactic acid bacteria fermentation

At the end of culture in a carbon-limited medium, i.e. the best conditions for subsequent autolysis, lactic acid bacteria were harvested and autolysed at 50 °C for 24 h. The resulting supernatant was then successfully tested as a substitute for industrial yeast extract for the supplementation of whey permeate and its conversion into lactic acid: for almost equivalent total nitrogen amounts of both supplements, the same growth and production rates were recorded.     

Inactivation of bacterial D-amino acid transaminases by the olefinic amino acid D-vinylglycine.

D-Vinylglycine (2-amino-3-butenoate) functions as a transamination substrate and irreversible inactivator of the homogeneous pyridoxal phosphate-dependent D-amino acid transaminases from Bacillus subtilis and Bacillus sphaericus. In the absence of alpha-ketoglutarate as co-substrate, vinyl-glycine causes little if any inactivation of either enzyme; in the presence of excess alpha-ketoglutarate, both enzymes are inactivated with pseudo-first order kinetics. The limiting rate constant for inactivation of the B. sphaericus enzyme is 1.9 min-1, for the B. subilis enzyme it is 0.36 min-1. The number of catalytic events before inactivation is about 450 for the B. sphaericus enzyme and about 800 for the B. subtilis enzyme; that is, about 0.2% inactivation in each catalytic cycle for the former enzyme and 0.15% for the latter. Comparisons are made with the L-aspartate amino-transferase from pig heart which is inactivated completely in one catalytic cycle and the L-alanine aminotransferase which is not inactivated in many cycles. Comparisons are also made between the likely mode of D-transaminase inactivation produced by vinylglycine and the mode of inactivation induced by beta-chloro-D-alanine.     

CMP-sialic acid synthetase of the nucleus

Sialic acids of cell surface glycoconjugates play a pivotal role in the structure and function of animal cells and in some bacterial pathogens. The pattern of cell surface sialylation is species specific, and, in the animal, highly regulated during embryonic development. A prerequisite for the synthesis of sialylated glycoconjugates is the availability of the activated sugar-nucleotide cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc), which provides the substrate for sialyltransferases. Trials to purify the enzymatic activity responsible for the synthesis of CMP-NeuAc from different animal sources demonstrated that the major localisation of the enzyme is the cell nucleus. These earlier findings were confirmed when the murine CMP-NeuAc synthetase was cloned and the subcellular transport of recombinant epitope tagged forms visualised by indirect immunofluorescence. Today, the primary sequence elements that direct murine CMP-NeuAc synthetase into the cell nucleus are known, however, information regarding the physiological relevance of the nuclear destination is still not available. With this article, we provide a detailed review on earlier and recent findings that identified and confirmed the unusual subcellular localisation of the CMP-NeuAc synthetase. In addition, we take the advantage to discuss most recent developments towards understanding structure--function relations of this enzyme.     

Pattern of dissolution of deoxyribonucleic acid in trichloroacetic acid in the presence of protein

Hot TCA solutions are unsatisfactory for the solubilization of radioactive DNA from precipitates containing large amounts of protein. Under such conditions, only a portion of the radioactivity becomes solubilized initially. With longer heating, the radioactivity becomes reattached to the precipitate but eventually becomes partly or completely resolubilized when the protein precipitate itself dissolves as a result of the decomposition of the TCA. Hot PCA or HCl solutions are satisfactory for the solubilization of radioactive DNA in the presence of protein, but longer heating is required to effect solubilization than in the absence of protein.     

Thermodynamics of the conversion of aqueous L-aspartic acid to fumaric acid and ammonia

The thermodynamics of the conversion of aqueous L-aspartic acid to fumaric acid and ammonia have been investigated using both heat conduction microcalorimetry and high-pressure liquid chromatography. The reaction was carried out in aqueous phosphate buffer over the pH range 7.25-7.43, the temperature range 13-43 degrees C, and at ionic strengths varying from 0.066 to 0.366 mol kg(-1). The following values have been found for the conversion of aqueous L-aspartateH- to fumarate2- and NH4+ at 25 degrees C and at zero ionic strength: K = (1.48 +/- 0.10) x 10(-3), DeltaG degrees = 16.15 +/- 0.16 kJ mol(-1), DeltaH degrees = 24.5 +/- 1.0 kJ mol(-1), and DeltaC(p) degrees = -147 +/- 100 J mol(-1) K(-1). Calculations have also been performed which give values of the apparent equilibrium constant for the conversion of L-aspartic acid to fumaric acid and ammonia as a function of temperature, pH and ionic strength.     

Production of the polyunsaturated fatty acids arachidonic acid and eicosapentaenoic acid by the fungus Pythium ultimum

Several strains of species of the fungal genus Pythium, and of Phytophthora cinnamomi, were screened for content of the polyunsaturated fatty acids (PUFAs) arachidonic acid (AA) and eicosapentaenoic acid (EPA). The aim of the investigation was to establish alternative sources of these PUFAs, which are of importance in human nutrition. As a relatively prolific producer of EPA and AA, P. ultimum strain #144 was selected for a study of conditions that enhance their production over baseline levels that are present in the fungus when cultured for 6 d at 25 degrees C with rotary shaking (120 r.p.m.) in Vogel's medium containing sucrose as the carbon substrate. The levels of AA and EPA under these conditions were 133 +/- 27 and 138 +/- 25 mg l-1 (n = 5), respectively. Maximal production of these fatty acids was accomplished by the following sequence of steps. (1) Incubate the cultures for 6 d after inoculation under the conditions described above. Then (2) add glucose to the cultures (2%, w/v, final concentration) and incubate for a further 6 d at 13 degrees C. Under these conditions, the AA content of the mycelium was 205% higher than baseline levels and the EPA content was 198% higher. (3) Allow the cultures to remain stationary for 10 d which increases the AA content to 253% above baseline levels and the EPA content by 236%. Using such a procedure, 322 mg AA l-1 and 383 mg EPA 1-1 were produced.     

Algorithms for the search of amino acid patterns in nucleic acid sequences.

Some algorithms are described for the search of regions in a nucleic acid sequence that, when translated into amino acids, are homologous to a given amino acid pattern. All algorithms are modifications of the dynamic programming method for sequence comparison such that the translation of codons is taken into account. One of the algorithms has been implemented as a FORTRAN 77 program. The program operates on files that follow the format of the EMBL Nucleotide Sequence Data Library.