Adrenal oxidative enzymes in ox and sheep. The histochemical demonstration of some pathways involved in chromaffin granule synthesis

Summary Catechol amine secretion is achieved by exocytosis. In this, ATP and protein are also lost from the chromaffin cells. Histochemically various specific coenzyme linked dehydrogenases associated with ATP production have been demonstrated in the adrenals of ox and sheep. These included cytochrome oxidase, DPN and TPN diaphorases, isocitric dehydrogenase, malate dehydrogenase, glutamate dehydrogenase, succinic dehydrogenase, lactic dehydrogenase, alcohol dehydrogenase, -glycerophosphate dehydrogenase and -hydroxybutyrate dehydrogenase. Enzymes of the pentose shunt were found histochemically and biochemically. The RNA content of the adrenal medulla and cortex was also investigated.     

Mapping of genes involved in macromolecular synthesis on the chromosome of Streptomyces coelicolor A3(2).

The genes for the beta, beta', and seven sigma factor subunits of RNA polymerase, for elongation factors EF-Tu1 and EF-Tu3, and for six rRNA operons were mapped on the combined genetic and physical map of the Streptomyces coelicolor chromosome. Like the previously mapped tRNA genes, the RNA polymerase and rRNA genes map to scattered positions. The lack of rRNA operons in the immediate vicinity of the origin of replication (oriC) and the absence of tRNA genes in any of the rRNA operons are novel features of the Streptomyces chromosome.     

Adrenal medullary basophilia in ox,pig and sheep

Summary In ox, pig and sheep the adrenaline storing cells are intensely basophilic compared with the noradrenaline storing cells when aldehyde fixed tissue is stained with toluidine blue at pH 5.0 and above. This has been shown to be due to carboxyl groups from the glutamate rich chromaffin granule soluble protein. In isolated chromaffin granules adenosine nucleotides also bind the dye. Fixation of adrenal medulla in agents not containing aldehydes, or the use of cryostat sections results in equal basophilia in the adrenaline and noradrenaline storing cells. The probable mechanism of the differential basophilia of the two sorts of medullary cells following aldehyde fixation is discussed.     

Fresh approaches to antibiotic production

New antibiotics are needed, (a) to control diseases that are refractory to existing ones either because of intrinsic or acquired drug resistance of the pathogen or because inhibition of the disease is difficult, at present, without damaging the host (fungal and viral diseases, and tumours), (b) for the control of plant pathogens and of invertebrates such as helminths, insects, etc., and (c) for growth promotion in intensive farming. Numerous new antibiotics are still being obtained from wild microbes, especially actinomycetes. Chemical modification of existing compounds has also had notable success. Here we explore the uses, actual and potential, of genetics to generate new antibiotics and to satisfy the ever-present need to increase yield. Yield improvement has depended in the past on mutation and selection, combined with optimization of fermentation conditions. Progress would be greatly accelerated by screening random recombinants between divergent high-yielding strains. Strain improvement may also be possible by the introduction of extra copies of genes of which the products are rate-limiting, or of genes conferring beneficial growth characteristics. Although new antibiotics can be generated by mutation, either through disturbing known biosyntheses or by activating 'silent' genes, we see more promise in interspecific recombination between strains producing different secondary metabolities, generating producers of 'hybrid' antibiotics. As with proposals for yield improvement, there are two major strategies for obtaining interesting recombinants of this kind: random recombination between appropriate strains, or the deliberate movement of particular biosynthetic abilities between strains. The development of protoplast technology in actinomycetes, fungi and bacilli has been instrumental in bringing these idealized strategies to the horizon. Protoplasts of the same or different species can be induced to fuse by polyethylene glycol. At least in intraspecific fusion of streptomyces, random and high frequency recombination follows. Protoplasts can also be used as recipients for isolated DNA, again in the presence of polyethylene glycol, so that the deliberate introduction of particular genes into production strains can be realistically envisaged. Various kinds of DNA cloning vectors are being developed to this end. Gene cloning techniques also offer rich possibilities for the analysis of the genetic control of antibiotic biosynthesis, knowledge of which is, at present, minimal. The information that should soon accrue can be expected to have profound effects on the application of genetics to industrial microbiology.     

Use of isoelectric focusing to determine the isoeletric point of bovine serum albumin after treatment with various common fixatives

Synopsis Isoelectric focusing was used to determine the effects of fixation on the isoelectric point of bovine serum albumin. Aqueous solutions of formaldehyde, -hydroxyadipaldehyde, osmium tetroxide and potassium dichromate produced no change or a relatively small decrease. Glutaraldehyde produced two fractions of fixed protein, the larger with a considerably lowered isoelectric point. The results are interpreted as suggesting that glutaraldehyde reacts rapidly and largely irreversibly, whereas the other fixatives react more slowly and reversibly with bovine serum albumin.     

Organisation and functions of the actV A region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor

Summary Sequence analysis of the actVA region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor revealed a succession of six open reading frames (ORFs), all running in the same direction and extending over 5.32 kb. The protein product of actVA-ORF1 strongly resembles that of another gene, elsewhere in the act cluster (actII-ORF2), which codes for a trans-membrane protein previously implicated in actinorhodin export from the mycelium. This suggests that the two gene products may co-operate in actinorhodin export, perhaps being sufficient for self-protection of the organism against suicide. At least four of the other five ORFs are implicated in the control of the C-6 and C-8 ring-hydroxylation reactions, lacking in actVA mutants, that occur at middle to late stages in the actinorhodin biosynthetic pathway. This conclusion was reached by genetic mapping of actVA mutants to actVA-ORF3 and-ORF5 (and perhaps -ORF4), and by the finding of strong resemblances between the protein products of actVA-ORF2 and -ORF6 and the products of genes of the oxytetracycline or tetracenomycin gene clusters that have been implicated in ring-hydroxylation reactions in the biosynthesis of these other aromatic polyketide antibiotics.     

Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome). An intermediate clinical phenotype caused by substitution of valine for glycine at position 137 of arylsulfatase B.

The Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI) is a lysosomal storage disease with autosomal recessive inheritance caused by deficiency of the enzyme arylsulfatase B. Severe, intermediate, and mild forms of the disease have been described. The molecular correlate of the clinical heterogeneity is not known at present. To identify the molecular defect in a patient with the intermediate form of the disease, arylsulfatase B mRNA from his fibroblasts was reverse-transcribed, amplified by the polymerase chain reaction, and subcloned. Three point mutations were detected by DNA sequence analysis, two of which, a silent A to G transition at nucleotide 1191 and a G to A transition at nucleotide 1126 resulting in a methionine for valine 376 substitution, were polymorphisms. A G to T transversion at nucleotide 410 causing a valine for glycine 137 substitution (G137V) was identified as the mutation underlying the Maroteaux-Lamy phenotype of the patient, who was homozygous for the allele. The kinetic parameters of the mutant arylsulfatase B enzyme toward a radiolabeled trisaccharide substrate were normal excluding an alteration of the active site. The G137V mutation did not affect the synthesis but severely reduced the stability of the arylsulfatase B precursor. While the wild type precursor is converted by limited proteolysis in late endosomes or lysosomes to a mature form, the majority of the mutant precursor was degraded presumably in a compartment proximal to the trans Golgi network and only a small amount escaped to the lysosomes accounting for the low residual enzyme activity in fibroblasts of a patient with the juvenile form of the disease.     

Human N-acetylgalactosamine-4-sulphate sulphatase. Purification, monoclonal antibody production and native and subunit Mr values.

Initial purification of N-acetylgalactosamine-4-sulphate sulphatase from human liver homogenates containing approx. 1 mg of enzyme in 26 g of soluble proteins was achieved by a six-column chromatography procedure and yielded approx. 40 micrograms of a single major protein species. Enzyme thus prepared was used to produce N-acetylgalactosamine-4-sulphate sulphatase-specific monoclonal antibodies. The use of a monoclonal antibody linked to a solid support facilitated the purification of approx. 0.5 mg of N-acetylgalactosamine-4-sulphate sulphatase from a similar liver homogenate. Moreover the enzyme isolated contained a single protein species, shown by SDS/polyacrylamide-gel electrophoresis to have an Mr of 57,000, which dissociated into subunits of Mr 43,000 and 13,000 in the presence of reducing agents. Essentially identical enzyme preparations were isolated from homogenates of human kidney and lung and from concentrated human urine. The native protein Mr of enzyme from human liver and kidney was assessed by gel-permeation chromatography to be 43,000 on Ultrogel AcA and Bio-Gel P-150. The liver N-acetylgalactosamine-4-sulphate sulphatase was shown to have pH optima of approx. 4 and 5.5 with the oligosaccharide substrate (GalNAc4S-GlcA-GalitolNAc4S) and fluorogenic substrate (methylumbelliferyl sulphate) respectively. Km values of 60 microM and 4 mM and Vmax. values of 2 and 20 mumol/min per mg were determined with the oligosaccharide and fluorogenic substrates respectively.     

Human liver iduronate-2-sulphatase. Purification, characterization and catalytic properties.

Human iduronate-2-sulphatase (EC 3.1.6.13), which is involved in the lysosomal degradation of the glycosaminoglycans heparan sulphate and dermatan sulphate, was purified more than 500,000-fold in 5% yield from liver with a six-step column procedure, which consisted of a concanavalin A-Sepharose-Blue A-agarose coupled step, chromatofocusing, gel filtration on TSK HW 50S-Fractogel, hydrophobic separation on phenyl-Sepharose CL-4B and size separation on TSK G3000SW Ultrapac. Two major forms were identified. Form A and form B, with pI values of 4.5 and less than 4.0 respectively, separated at the chromatofocusing step in approximately equal amounts of recovered enzyme activity. By gel-filtration methods form A had a native molecular mass in the range 42-65 kDa. When analysed by SDS/PAGE, dithioerythritol-reduced and non-reduced form A and form B consistently contained polypeptides of molecular masses 42 kDa and 14 kDa. Iduronate-2-sulphatase was purified from human kidney, placenta and lung, and form A was shown to have similar native molecular mass and subunit components to those observed for liver enzyme. Both forms of liver iduronate-2-sulphatase were active towards a variety of substrates derived from heparin and dermatan sulphate. Kinetic parameters (Km and Kcat) of form A were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparan sulphate, heparin and dermatan sulphate. Substrate with 6-sulphate esters on the aglycone residue adjacent to the iduronic acid 2-sulphate residue being attack were hydrolysed with catalytic efficiencies up to 200 times above that observed for the simplest disaccharide substrate without a 6-sulphated aglycone residue. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure, substrate concentration, buffer type and the presence of other proteins. Sulphate and phosphate ions and a number of substrate and product analogues were potent inhibitor of form A and form B enzyme activities.     

Directed Transfer of Large DNA Fragments between Streptomyces Species

The biosynthesis of complex natural products in bacteria is invariably encoded within large gene clusters. Although this facilitates the cloning of such gene clusters, their heterologous expression in genetically amenable hosts remains a challenging problem, principally due to the difficulties associated with manipulating large DNA fragments. Here we describe a new method for the directed transfer of a gene cluster from one Streptomyces species to another. The method takes advantage of tra gene-mediated conjugal transfer of chromosomal DNA between actinomycetes. As proof of principle, we demonstrate transfer of the entire ~22-kb actinorhodin gene cluster, and also the high-frequency cotransfer of two loci that are 150 to 200 kb apart, from Streptomyces coelicolor to an engineered derivative of Streptomyces lividans.