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J Hakala

Publications and source records attributed to J Hakala.

18 recordsLinked to original sources
PubMed2006

First precision mass measurements of refractory fission fragments.

Atomic masses of 95-100Sr, 98-105Zr, and [corrected] 102-110Mo and have been measured with a precision of 10 keV employing a Penning trap setup at the IGISOL facility. Masses of 104,105Zr and 109,110Mo are measured for the first time. Our improved results indicate significant deviations from the previously published values deduced from beta end point measurements. The most neutron-rich studied isotopes are found to be significantly less bound (1 MeV) compared to the 2003 atomic mass evaluation. A strong correlation between nuclear deformation and the binding energy is observed in the two-neutron separation energy in all studied isotope chains.

Journal Article↗
PubMed2001

The entire nogalamycin biosynthetic gene cluster of Streptomyces nogalater: characterization of a 20-kb DNA region and generation of hybrid structures.

Fragments spanning 20 kb of Streptomyces nogalater genomic DNA were characterized to elucidate the molecular genetic basis of the biosynthetic pathway of the anthracycline antibiotic nogalamycin. Structural analysis of the products obtained by expression of the fragments in S. galilaeus and S. peucetius mutants producing aclacinomycin and daunomycin metabolites, respectively, revealed hybrid compounds in which either the aglycone or the sugar moiety was modified. Subsequent sequence analysis revealed twenty ORFs involved in nogalamycin biosynthesis, of which eleven could be assigned to the deoxysugar pathway, four to aglycone biosynthesis, while the remaining five express products with unknown function. On the basis of sequence similarity and experimental data, the functions of the products of the newly discovered genes were determined. The results suggest that the entire biosynthetic gene cluster for nogalamycin is now known. Furthermore, the compounds obtained by heterologous expression of the genes show that it is possible to use the genes in combinatorial biosynthesis to create novel chemical structures for drug screening purposes.

Antibiotics, Antineoplastic↗
PubMed2000

Modifications of aclacinomycin T by aclacinomycin methyl esterase (RdmC) and aclacinomycin-10-hydroxylase (RdmB) from Streptomyces purpurascens.

The genes rdmB and rdmC of Streptomyces purpurascens encoding aclacinomycin modifying enzymes RdmB and RdmC were expressed in Streptomyces lividans TK24. In contrast to the earlier suggestion that RdmC may be an esterase that causes the removal of the carbomethoxy group from the 10 position of aclacinomycins, RdmC functions as an aclacinomycin methyl esterase and catalyzes the removal of the methoxy group from the C-15 position of aclacinomycin T producing 15-demethoxyaclacinomycin T. RdmB acts upon C-10 of 15-demethoxyaclacinomycin T and is able to remove the carboxylic group from the C-10 position. It functions also as an aclacinomycin-10-hydroxylase being able to add a hydroxyl group at the same, C-10 position in vitro. Aclacinomycin methyl esterase was purified to apparent homogeneity from S. lividans carrying the rdmC and aclacinomycin-10-hydroxylase as a glutathione S-transferase fusion construct from Escherichia coli carrying the rdmB gene, respectively. Aclacinomycin methyl esterase functions as a monomer and aclacinomycin-10-hydroxylase as a tetramer. Aclacinomycin methyl esterase has an exceptionally high temperature stability and has an apparent K(m) for aclacinomycin T of 15.5 microM. The introduction of rdmC and rdmB in a Streptomyces galilaeus mutant HO38 produced the same modifications of aclacinomycin T in vivo as aclacinomycin methyl esterase and aclacinomycin-10-hydroxylase in vitro.

Aclarubicin↗
PubMed2000

A gene cluster from Streptomyces galilaeus involved in glycosylation of aclarubicin.

We have cloned and characterized a gene cluster for anthracycline biosynthesis from Streptomyces galilaeus. This cluster, 15-kb long, includes eight genes involved in the deoxyhexose biosynthesis pathway, a gene for a glycosyltransferase and one for an activator, as well as two genes involved in aglycone biosynthesis. Gene disruption targeted to the activator gene blocked production of aclacinomycins in S. galilaeus. Plasmid pSgs4, containing genes for a glycosyltransferase (aknS), an aminomethylase (aknX), a glucose-1-phosphate thymidylyltransferase (akn Y) and two genes for unidentified glycosylation functions (aknT and aknV), restored the production of aclacinomycins in the S. galilaeus mutants H063, which accumulates aklavinone, and H054, which produces aklavinone with rhodinose and deoxyfucose residues. Furthermore, pSgs4 directed the production of L-rhamnosyl-epsilon-rhodomycinone and L-daunosaminyl-epsilon-rhodomycinone in S. peucetius strains that produce epsilon-rhodomycinone endogenously. Subcloning of the gene cluster was carried out in order to further define the genes that are responsible for complementation and hybrid anthracycline generation.

Aclarubicin↗
PubMed2000

Identification of a cyclase gene dictating the C-9 stereochemistry of anthracyclines from Streptomyces nogalater.

Nogalamycin is an anthracycline antibiotic produced by Streptomyces nogalater. Its aglycone has a unique stereochemistry (7S, 9S, 10R) compared to that of most other anthracyclines (7S, 9R, 10R). The gene snoaL, encoding a nogalonic acid methyl ester cyclase for nogalamycin, was used to generate nogalamycinone, demonstrating that the single cyclase dictates the C-9 stereochemistry of anthracyclines.

Amino Acid Sequence↗
PubMed2000

Relationship between clinical signs and lung function in horses with recurrent airway obstruction (heaves) during a bronchodilator trial.

During a trial to determine the dose response to the beta2-adrenergic agonist pirbuterol, we judged the severity of airway obstruction by use of a clinical scoring system and compared this to objective data obtained by quantitative measures of lung function. Six horses affected by recurrent airway obstruction were used in this trial. Four hundred and sixty-eight measurements of lung function and clinical scores were obtained from 13 measurement periods when horses received each of 6 doses of pirbuterol. Scores of 1-4 were assigned to degree of nasal flaring and abdominal effort and summed for a total score. The veterinarian scoring the signs did not know the dose of pirbuterol received by the horse and was unaware of the lung function data. Nasal, abdominal and total scores were significantly related to changes in lung function and changes in breathing pattern. There were significant differences between total scores greater than 5 in indices that reflected changes in breathing strategy (peak inspiratory and expiratory flow), peripheral airway obstruction (dynamic elastance), and effort of breathing (maximal change in pleural pressure). Below a total score of 5, there were fewer significant differences in lung function even though measurements of pulmonary resistance and dynamic elastance indicated considerable airway obstruction. Failure of clinical score to reflect this low-grade airway obstruction suggests that airway disease is underdiagnosed and its detection would be helped by the availability of a convenient lung function test.

Airway Obstruction↗
PubMed1999

An efficient approach for screening minimal PKS genes from Streptomyces.

Degenerated oligonucleotide primers were designed to amplify fragments of ketosynthase genes from polyketide antibiotics producing Streptomyces spp. and bacterial strains enriched from soil samples. Cell lysates were used as templates in amplification, so time-consuming DNA purification was avoided. A phylogenetic tree constructed from the amino acid sequences of the amplified fragments shows a distribution of spore pigments and antibiotics in separate classes. In addition, several different subgroups form within the antibiotics group. Anthracyclines were divided into separate branches according to the starter unit used in biosynthesis.

Amino Acid Sequence↗
PubMed1999

Characterization of aklavinone-11-hydroxylase from Streptomyces purpurascens.

Aklavinone-11-hydroxylase (RdmE) is a FAD monooxygenase participating in the biosynthesis of daunorubicin, doxorubicin and rhodomycins. The rdmE gene encodes an enzyme of 535 amino acids. The sequence of the Streptomyces purpurascens enzyme is similar to other Streptomyces aromatic polyketide hydroxylases. We overexpressed the gene in Streptomyces lividans and purified aklavinone-11-hydroxylase to apparent homogeneity with four chromatographic steps utilizing a kinetic photometric enzyme assay. The enzyme is active as the monomer with a molecular mass of 60 kDa; it hydroxylates aklavinone and other anthracyclinones. Aklavinone-11-hydroxylase can use both NADH and NADPH as coenzyme but it is slowly inactivated in the presence of NADH. The apparent Km for NADPH is 2 mM and for aklavinone 10 microM. The enzyme is inactivated in the presence of phenylglyoxal and 2,3-butanedione. NADPH protects against inactivation of aklavinone-11-hydroxylase by phenylglyoxal.

Anthracyclines↗
PubMed1999

Determinants of the maximal change in pleural pressure during tidal breathing in COPD-affected horses.

In six COPD-affected horses, we analysed the factors responsible for the changes in pleural pressure (delta Pplmax) that occur during tidal breathing. Four-hundred-and-sixty-eight measurements of each parameter: pulmonary resistance (RL), dynamic elastance (Edyn), air flow rates, and the timing of breathing, were gathered during a trial of the bronchodilator pirbuterol. Data were placed into seven ranks, based on the magnitude of delta Pplmax; rank 1: 5-15; rank 2: 15-25; rank 3: 25-35; rank 4: 35-45; rank 5: 45-55; rank 6: 55-65; and rank 7 > 65 cm H2O. Up to rank 4 (45 cm H2O), the increase in delta Pplmax was due to increases in RL, Edyn, and inspiratory and expiratory air flow rates. Further increases in delta Pplmax were due to continuing increases in Edyn and flow rates with little change in resistance. The increase in inspiratory flow rates was the result of a decrease in inspiratory time. The large increase in peak expiratory flow could not be explained by the small decrease in expiratory time and must therefore be due to a change in breathing strategy as delta Pplmax increased. Changes in air flow rates as well as changes in RL and Edyn must be considered as reasons for a change in delta Pplmax when evaluating horses with COPD.

Airway Resistance↗
PubMed1997

Characterization of Streptomyces nogalater genes encoding enzymes involved in glycosylation steps in nogalamycin biosynthesis.

The sno gene cluster in Streptomyces nogalater ATCC 27451 contains the nogalamycin biosynthesis genes. A set of plasmid constructions carrying fragments of the sno cluster that lie downstream of snoD were used to complement the S. galilaeus mutant H039, which is blocked in rhodosamine and 2-deoxyfucose biosynthesis in the aclacinomycin pathway. Sequence analysis of this cluster revealed three contiguous open reading frames (ORFs) that were designated snoF, snoG, and snoH. Only those plasmid constructs that expressed SnoG were able to complement H039. SnoG shows similarity to GalE, a UDP-glucose-4-epimerase catalyzing the epimerization of UDP-glucose to UDP-galactose. The putative SnoF protein is similar to 3,5-epimerases involved in rhamnose biosynthesis. The deduced product of snoH is a 489-amino acid polypeptide. It is similar to the product of dau ORF3 found in the daunomycin cluster. However its function is still unclear. Based on the complementation experiments and sequence analysis, this part of the sno cluster is suggested to be involved in the biosynthesis of the sugar portion of nogalamycin. Interestingly, SnoA, a transcriptional activator for the sno minimal polyketide synthase, is also needed to express this cluster.

Aclarubicin↗
PubMed1997

Folding of the polyketide chain is not dictated by minimal polyketide synthase in the biosynthesis of mithramycin and anthracycline.

BACKGROUND: Mithramycin, nogalamycin and aclacinomycins are aromatic polyketide antibiotics that exhibit antitumour activity. The precursors of these antibiotics are formed via a polyketide biosynthetic pathway in which acetate (for mithramycinone and nogalamycinone) or propionate (for aklavinone) is used as a starter unit and nine acetates are used as extender units. The assembly of building blocks is catalyzed by the minimal polyketide synthase (PKS). Further steps include regiospecific reductions (if any) and cyclization. In the biosynthesis of mithramycin, however, ketoreduction is omitted and the regiospecificity of the first cyclization differs from that of anthracycline antibiotics (e.g. nogalamycin and aclacinomycins). These significant differences provide a convenient means to analyze the determinants for the regiospecificity of the first cyclization step. RESULTS: In order to analyze a possible role of the minimal PKS in the regiospecificity of the first cyclization in polyketide biosynthesis, we expressed the mtm locus, which includes mithramycin minimal PKS genes, in Streptomyces galilaeus, which normally makes aclacinomycins, and the sno locus, which includes nogalamycin minimal PKS genes, in Streptomyces argillaceus, which normally makes mithramycin. The host strains are defective in the minimal PKS, but they express other antibiotic biosynthesis genes. Expression of the sno minimal PKS in the S. argillaceus polyketide-deficient strain generated mithramycin production. Auramycins, instead of aclacinomycins, accumulated in the recombinant S. galilaeus strains, suggesting that the mithramycin minimal PKS is responsible for the choice of starter unit. We also describe structural analysis of the compounds accumulated by a ketoreductase-deficient S. galilaeus mutant; spectroscopic studies on the major polyketide compound that accumulated revealed a first ring closure which is not typical of anthracyclines, suggesting an important role for the ketoreductase in the regiospecificity of the first cyclization. CONCLUSIONS: These experiments clearly support the involvement of ketoreductase and a cyclase in the regiospecific cyclization of the biosynthetic pathway for aromatic polyketides.

Aclarubicin↗
PubMed1997

Isolation and characterization of 8-demethoxy steffimycins and generation of 2,8-demethoxy steffimycins in Streptomyces steffisburgensis by the nogalamycin biosynthesis genes.

Streptomyces steffisburgensis (NRRL 3193, ATCC 27466) is described as a steffimycin producer. Steffimycin belongs to the anthracycline group of aromatic polyketide antibiotics. The structural analysis of the products accumulated by the wild type ATCC 27466 strain revealed three different forms of 8-demethoxy steffimycin suggesting the loss of C-8 hydroxylation/methylation activity. In our approach to generate new anthracycline molecules, we used this strain as a host in gene cloning. The genes encoding the polyketide ketoreductase and aromatase enzymes of nogalamycin biosynthesis caused the production of 2-demethoxy steffimycins in S. steffisburgensis.

Anthracyclines↗
PubMed1996

Production of hybrid anthracycline antibiotics by heterologous expression of Streptomyces nogalater nogalamycin biosynthesis genes.

A cluster of anthracycline biosynthetic genes isolated from Streptomyces nogalater was expressed in Streptomyces lividans and in Streptomyces galilaeus. A 12 kb DNA fragment cloned from this cluster in pIJ486 caused the production of a novel compound when introduced into S. lividans. The compound is derived from nogalonic acid methyl ester, an early intermediate in nogalamycin biosynthesis. Complementation with the cloned 12 kb fragment of S. galilaeus mutants blocked in aclacinomycin biosynthesis caused the production of hybrid anthracyclines. Cloning of the nogalamycin gene cluster should make possible a detailed study of the biosynthesis of this interesting antibiotic, as well as the production of novel anthracyclines of potential value as cytostatic drugs.

Antibiotics, Antineoplastic↗
PubMed1996

Use of a hand-held, metered-dose aerosol delivery device to administer pirbuterol acetate to horses with 'heaves'.

Aerosol administration of bronchodilators to horses is recommended for treatment of certain airway diseases such as 'heaves'. We have developed a novel, hand-held, metered-dose inhaler and we sought to determine the bronchodilator efficacy of the beta 2 adrenoceptor agonist pirbuterol delivered by this device to horses affected with 'heaves'. To induce airway obstruction, 6 heaves-susceptible horses were stabled, bedded on straw and fed hay. When the maximum change in pleural pressure during tidal breathing (delta Pplmax) was greater than 20 cmH2O on 2 consecutive days, pulmonary function was measured before and 5, 10 and 30 min, as well as 1, 2, 3, 4, 5, 6 and 7 h after administration of aerosol pirbuterol. Pirbuterol was administered using a metered canister and the hand-held delivery device that was inserted into the left nostril. Either vehicle or pirbuterol acetate (400, 600, 800, 1200 or 1600 micrograms) was administered to each horse. Relief of airway obstruction indicated by changes in pulmonary function was observed within 5 min after administration of both vehicle and pirbuterol. Significant decreases in delta Pplmax and pulmonary resistance (RL) and an increase in dynamic compliance (Cdyn) persisted for the 7 h duration of the experiment. Comparison of the effect of vehicle and pirbuterol at each time period showed that pirbuterol decreased RL and delta Pplmax significantly for up to 1 h. The optimal dose was determined to be 600 micrograms. Immediate response to treatment, magnitude of drug effect and lack of side effects indicated that aerosol pirbuterol is an effective and safe bronchodilator in horses with 'heaves'. The hand-held, metered-dose aerosol delivery device was very convenient and extremely effective and is, therefore, recommended for delivery of therapeutic aerosols to horses.

Administration, Inhalation↗
PubMed1994

Hybrid anthracycline antibiotics: production of new anthracyclines by cloned genes from Streptomyces purpurascens in Streptomyces galilaeus.

A DNA segment cloned from Streptomyces purpurascens ATCC 25489 close to a region that hybridized to a probe containing part of the actinorhodin polyketide synthase caused S. galilaeus ATCC 31615 to produce new anthracyclines. When transformed with certain sub-clones of this segment, the host produced glycosides of epsilon-rhodomycinone, beta-rhodomycinone, 10-demethoxycarbonylaklavinone and 11-deoxy-beta-rhodomycinone in addition to those of aklavinone, the natural anthracyclines of S. galilaeus. The first two compounds are S. purpurascens products and the other two are novel compounds that conceptually are structural hybrids between S. galilaeus and S. purpurascens products. Three glycosides of one of the novel aglycones, 11-deoxy-beta-rhodomycinone, were purified and found to possess cytotoxic activity against L1210 mouse leukaemia cells. Separate regions of the cloned S. purpurascens DNA are responsible for modification of the S. galilaeus host product at the 10- and 11-positions.

Acyltransferases↗
PubMed1994

Isolation and characterization of aclacinomycin A-non-producing Streptomyces galilaeus (ATCC 31615) mutants.

Twelve mutants of Streptomyces galilaeus (ATCC 31615) blocked in the production of aclacinomycin A, an anthracycline antibiotic with significant antitumour activity, accumulated intermediates of the biosynthesis of aclacinomycins and several anthracyclines with variant sugar moieties. Three of these aklavinone glycosides have not been described before. Mutant strains H028, H061 and H036 were blocked before the formation of aklavinone, a common intermediate for most anthracyclines. Strain H039 accumulated aklavinone and H026, H035, H038 and H054 had mutations that changed glycosylation of aklavinone. Characterization of the mutants and their products is described.

Acetates↗
PubMed1988

Pancreatic digestive enzymes and ultrastructure after chronic alcohol intake in the rat.

The effects of chronic ethanol intake on the digestive enzyme content of rat pancreas, pancreatic juice and serum were studied by means of reversed-phase high performance liquid chromatography (HPLC), gel electrophoresis, isoelectric focusing and traditional enzymatic methods. The biochemical changes were further correlated to ultrastructural lesions caused by chronic alcohol consumption. 55 animals were given ethanol in their drinking water as 15% (w/v) solution up to 16 months. 49 control animals received water. The protein composition of the pancreatic juice was qualitatively similar in the alcoholic and control animals. Alcohol had a biphasic effect on pancreatic enzyme secretion. The protein and especially the trypsinogen concentrations of the pancreatic juice were increased after 8 months alcohol intake and diminished after 16 months. The changes in the acinar cell ultrastructure were marked only during the 2nd year of alcohol consumption. The amylase, lipase and especially the trypsinogen concentrations of pancreatic tissue were increased in the alcoholic animals. The results suggest that chronic ethanol consumption profoundly interferes with the pancreatic protein metabolism, especially that of trypsinogen and that the decrease in the enzyme secretion after 16 months alcohol intake may be due to acinar cell injury.

Amylases↗