Transformation of dehydroepiandrosterone and pregnenolone by Mucor piriformis

The mode of transformation of dehydroepiandrosterone (I, 3-hydroxyandrost-5-en-17-one) and pregnenolone (II, 3-hydroxypregn-5-en-20-one) was studied using Mucor piriformis. Biotransformation products formed from I were 3,17-dihydroxyandrost-5-ene (Ia), 3-hydroxyandrost-5-ene-7,17-dione (Ib), 3,17-dihydroxyandrost-5-en-7-one (Ic), 3,7-dihydroxyandrost-5-en-17-one (Ie). Biotransformation products formed from compound II were 3,7-dihydroxypregn-5-en-20-one (IIa) and 3,7,11-trihydroxypregn-5-en-20-one (IIb). The organism did not carry out isomerization of the 5-en-3-ol to a 4-en-3-one system in the steroid molecules tested. In addition, it failed to carry out 14-hydroxylation possibly because of the lack of a 4-en-3-one system in I and II, and stereospecific hydroxylation at the C-7 position in I and II.     

Cytogenetic analysis of structural rearrangements in three varieties of common wheat,Triticum aestivum

Summary The winter wheat varieties Starke and Cappelle Desprez and the spring wheat Chinese Spring were analysed for structural chromosome rearrangements that resulted in the formation of multivalents in F₁ hybrids. The analyses were carried out using hybrids involving euploids, monosomic and ditelosomic stocks, and double-monotelodisomic constructs. The study confirmed that Cappelle Desprez differs from Chinese Spring in a reciprocal translocation between chromosomes 5B and 7B (Riley et al. 1967); a translocation involving chromosomes 3B and 3D could not be verified. Furthermore, the analysis showed that Starke differs from Chinese Spring in a reciprocal translocation between chromosomes 7A and 7D. Both translocations have a coefficient of multivalent realisation of about 0.84. Further multivalents in euploid Starke, in euploid and some aneuploid stocks of Cappelle Desprez, and in euploid as well as various types of aneuploid hybrids between all three varieties could nearly all be explained hypothesizing that chromosome 2B of both Starke and Cappelle Desprez is a duplication-deficiency chromosome. In the hypothesis a part of the long arm of 2B is missing and replaced by a duplicated part of the long arm of chromosome 2D. The multivalents of this rearrangement showed an average coefficient of realisation of about 0.09.Sven Ellerström died in December 1985     

Expression inEscherichia coli K12 strains of two synthetic human calcitonin genes differing in codon composition

Two genes coding for a Val⁸-variant of the human calcitonin (hCT) are synthesized on two different codon biases: the native codons for the hCT gene and the codons preferential forEscherichia coli. Both genes are fused to a synthetic human interferon-gamma (IF) gene [6] and expressed in various strains ofE. coli K12. It is found that, in all host strains used, the level of expression of both genes is similar and much lower (1/50–1/100) than that of the IF gene alone.     

Cyclic nucleotide phosphodiesterase and 5′-nucleotidase: A coupled system

Evidence is presented that multiple forms of cyclic nucleotide phophodiesterase (PDE) activity chromatographically separated from the soluble fraction of bovine hypothalamus are co-eluted with multiple forms of 5-nucleotidase (5N) activity. The enzymes could not be resolved from each other by anion-exchange chromatography on DEAE-TSK; by affinity chromatography on phenyl-, blue-, concanavalin A-, 5 AMP-sepharose, cAMP-silica gel; or by gel filtration on sephacryl S-200. The catalytic activities were found to be associated with the tetrameric, dimeric, and monomeric forms of the enzymes. The molecular weights determined by gel filtration or by SDS-gel electrophoresis were 220, 114, and 57 kDa, respectively. Kinetic analysis revealed that the first-order rate constant for 5 AMP hydrolysis measured in the reactions: cAMP5AMPadenosine was 100 times higher than that in the reaction: 5AMPadenosine. Thus, functional interrelation between PDE and 5N was expressed in drastic acceleration of the consecutive reactions: cAMP 5AMPadenosine. The results confirm the conclusion about the existence of a multienzyme system involving PDE and 5N or of a single bifunctional enzyme in brain tissue.This revised version was published online in June 2005 with corrections to the author name Gurvits.     

β-Adrenergic receptors of frog erythrocytes

Following persistent stimulation of -adrenergic receptors of frog erythrocytes with (–)-isoproterenol, the cyclic adenosine 3,5-monophosphate-dependent protein kinase (cAMP-dependent protein kinase) (EC 2.7.1.37) was activated for several hours. This activation outlasted the duration of the increase of cAMP content. Following a persistant stimulation of -adrenergic receptors with isoproterenol, the phosphorylation of selective membrane proteins was increased. This increase in phosphorylation lasted longer than 4 hr but less than 12 hr. Between 2 and 4 hr after receptor stimulation the loss of -adrenergic receptor form plasma membrane was maximal, and the phosphorylation of two membrane proteins characterized by molecular weights of 60,000 and 38,000 daltons was selectively enhanced. In addition we found that isolated erythrocytes are capable of synthesizing RNA and polypeptides and that incubation with (–)-isoproterenol induces a longterm delayed increase of the synthesis of erythrocyte proteins. This increase in the synthesis of proteins appears to require new RNA synthesis. Thus the possibility can be entertained that this delayed increase in protein synthesis participates in the new synthesis of receptor and is operative in the termination of -adrenergic receptor subsensitivity elicited by a persistent stimulation with (–)-isoproterenol.     

Hexahydroindanone derivatives of steroids formed by Rhodococcus equi

Summary A mutant strain of Rhodococcus equi accumulates three metabolites from the androst-4-ene-3,17-dione or from its degradation intermediate, 3a-H-4(3'-propionic acid)-7a-methylhexahydro-1,5-indanedione (MEPHIP). These three metabolites are: 3a-H-4a(3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone--lactone (HIL); 3a-H-4(3'-trans acrylic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (^2'-5-hydroxy-MEPHIP); and 3a-H-4(3'-hydroxy-3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (3'-hydroxy-HIL). The behaviour of this mutant allows us to propose a pathway for degradation of the intermediates, methylperhydroindanone propionates. However, during this degradation, the side-chain propionate was eliminated by a-oxidation mechanism. Offprint requests to: A. Miclo     

C(alpha) chemical shift tensors in helical peptides by dipolar-modulated chemical shift recoupling NMR

The C chemical shift tensors of proteins contain information on the backbone conformation. We have determined the magnitude and orientation of the C chemical shift tensors of two peptides with -helical torsion angles: the Ala residue in G*AL (=–65.7°, =–40°), and the Val residue in GG*V (=–81.5°, =–50.7°). The magnitude of the tensors was determined from quasi-static powder patterns recoupled under magic-angle spinning, while the orientation of the tensors was extracted from C–H and C–N dipolar modulated powder patterns. The helical Ala C chemical shift tensor has a span of 36 ppm and an asymmetry parameter of 0.89. Its ₁₁ axis is 116° ± 5° from the C–H bond while the ₂₂ axis is 40° ± 5° from the C–N bond. The Val tensor has an anisotropic span of 25 ppm and an asymmetry parameter of 0.33, both much smaller than the values for -sheet Val found recently (Yao and Hong, 2002). The Val ₃₃ axis is tilted by 115° ± 5° from the C–H bond and 98° ± 5° from the C–N bond. These represent the first completely experimentally determined C chemical shift tensors of helical peptides. Using an icosahedral representation, we compared the experimental chemical shift tensors with quantum chemical calculations and found overall good agreement. These solid-state chemical shift tensors confirm the observation from cross-correlated relaxation experiments that the projection of the C chemical shift tensor onto the C–H bond is much smaller in -helices than in -sheets.     

Molecular dynamics exposes α-helices in myelin basic protein

Molecular dynamics simulations of models of unmodified and deiminated MBP (myelin basic protein) have been performed on solvated structures with added counterions, for 10 ns using AMBER (assisted model building with energy refinement). The protein structures became extended, and a considerable number of -helical segments formed spontaneously. The degree of molecular extension was greater in the deiminated species, and the -helices were more transient. These structural disruptions may be operative in vivo during multiple sclerosis.Figure A model of the C1 isoform of myelin basic protein showing major -helical segments that were stable over the last 1 ns of a molecular dynamics simulation. The -helices are colored red, the -strands are colored yellow, the -turns are colored blue, and random coils are colored green     

Distance-constraint approach to higher-order structures of globular proteins with empirically determined distances between amino acid residues

An analysis of higher-order structures of globular proteins by means of a distance-constraint approach is presented. Conformations are generated for each of 21 test proteins of small and medium sizes by optimizing an objective functionf=w _ij(d _ij–d _ij)², whered _ij is a distance between residuesi andj in a calculated conformation, d _ij is an assigned distance to the (ij) pair of residues which is determined based on the statistics of known three-dimensional structures of 14 proteins in the earlier study, andw _ij is a weighting factor. d _ij involves information about hydrophobicity and hydrophilicity of each amino acid residue and about connectivity of a polypeptide chain. In these calculations, only the amino acid sequence is used as input data specific to a calculated protein. With respect to higher-order structures regenerated in the optimized conformations, the following properties are analyzed: (a) N14 of a residue, defined as the number of residues surrounding the residue located within a sphere of radius of 14 Å; (b) root-mean-square differences of the global and local conformations from the corresponding X-ray conformations; (c) distance profiles in the short and medium ranges; and (d) distance maps. The effects of supplementary information about locations of secondary structures and disulfide bonds are also examined to discuss the potential ability of this methodology to predict the three-dimensional structures of globular proteins.     

The effect of monoterpenes on astaxanthin production by a mutant ofPhaffia rhodozyma

Summary The total pigment and astaxanthin content ofPhaffia rhodozyma increased with increasing concentrations -pinene up to 500 l -pinene/l. Above this concentration the total pigment and astaxanthin content as well as the biomass production decreased. The addition of 500 l -pinene/l increased the total pigment content from 1652 g/g to 2201 g/g and the astaxanthin content from 1554 g/g to 1883 g/g. A sharp decrease in maximum specific growth rate occurred above 150 l -pinene/l.     

Effect of “fasting” and different concentrations of glucose on α-linolenic acid metabolism in HTC cells. Correlation with the ultrastructural study

Summary HTC cells are able to convert -linolenic acid into higher homologs by desaturating and elongating reactions. When the cells were cultured in a Krebs Ringer bicarbonate solution (fasted cells) a decrease in both biosynthetic reactions took place. Refeeding the cells with Swim's 77 medium without glucose enhanced the biosynthesis of polyunsaturated fatty acids from -linolenic acid family, but when glucose was added to the medium, -linolenic acid was preferably elongated rather than converted into eicose-pentaenoic acid.The ultrastructural study revealed HTC cells with a simple cytoplasmic organization, showing little evidence of their origin from hepatocytes. The cells cultured in a complete medium appeared well preserved and were similar to those fasted for 12 hours and refed for another 12 hours using Swim's 77 medium without serum. The amount of glucose in the medium plays a role in preserving the cell structure. This effect does not occur if glucose is added in the absence of aminoacids and vitamins.     

Kinetics of growth and lactic acid production in continuous and batch culture

Summary In a lactic acid fermentation by Streptococcus faecalis, the specific consumption rate of glucose (v) and the specific production rate of lactic acid () were represented by the following simple equations as functions of the specific growth rate (): 1/=(1/) + 1/ = (1/) + By use of data from a batch culture, these two equations were derived from enzyme kinetics of the product inhibition. These equations were successfully applied to the results of batch culture and chemostat culture. In addition, calculation of ATP yield by these equations agreed with the experimental results better than the conventional Leudeking-Piret type equation, which includes two terms associated with growth and not with growth. Correspondence to: H. Ohara     

Selection of isoresponsive genotypes in toria (Brassica campestris L.) based on the pattern of response to environmental variations: a proposed method

Summary Thirty-one toria genotypes were compared with three well-established cultivars, Ludhiana Composite-2, K-1 and TCSU-2 (standard testers). The genotypes, which were almost identical to a standard tester in response to environmental variations and which also had other desirable characteristics, were considered to be acceptable for commercial cultivation. Using this criterion, TCSU-7, TH-5 and TH-4 were found to be acceptable for commercial cultivation. TH-4 and TCSU-7 were found superior to TH-5 if r² can be considered as a measure of the agronomical manipulations expected in environmental variations.     

Isolation of protoplasts by means of a “species-specific” autolysine inChlamydomonas

Summary Prior to fusion, gametes ofChlamydomonas reinhardii discard their cell walls. This naturally occuring phenomenon has provided the basis for a method of protoplast isolation from both gametes and vegetative cells within the genusChlamydomonas. When synchronized cultures of compatibleChlamydomonas gametes are mixed it is possible, after removal of the cells, to obtain a solution having a high cell wall lytic activity. That vegetative and gamete cells after treatment with this gamete-autolysine are indeed protoplasts has been proved by various light and electron microscopical methods.The species specifity of this autolysine, its difference to the previously described sporangialautolysine (Schlösser 1966) and furthermore its use in the large scale production of protoplasts is also described. Since this wall autolysine is a factor produced by the cells themselves at a particular stage in their life cycle it represents a non-foreign agent in contrast to all other enzymic methods previously employed for protoplast isolation.     

High expression of a recombinant human calcitonin precursor peptide in Escherichia coli

Human calcitonin (hCT) is a C-terminus -amidated peptide hormone consisting of 32 amino acids. The amidated structure is essential for its biological activities, and the C-terminal-glycine-extended precursor peptide, hCT[G], is converted to bioactive hCT by a C-terminus--amidating enzyme. An efficient production method is described for the hCT[G] peptide, as a part of the fusion protein consisting of a modified E. coli -galactosidase, linker amino acids and hCT[G]. Stable inclusion bodies of the fusion protein in E. coli were expressed by focusing on the amino acid charge, and the fusion protein was modified by inserting a basic amino acid sequence into its linker region. This modification greatly affected the formation of inclusion bodies. E. coli strain W3110/pG97S4DhCT [G]R4 could produce a large amount of stable inclusion bodies, and the hCT[G] peptide was released quantitatively from the fusion protein by S. aureus V8 protease. This enabled a large-scale production method to be established for the hCT[G] precursor peptide in E. coli to produce mature hCT.     

Some observations on cyclodextrin-mediated boosting of secreted amylolytic enzymes byLactobacillus amylovorus

The amounts of a 160-kDa amylase and a 140-kDa -amylase (A. Burgess-Cassler and S. H. Imam, Curr. Microbiol. 23:207–213, 1991) secreted into culture medium by the starchutilizingLactobacillus amylovorus were enhanced by the use of cyclodextrin (CD) as the carbon source. The levels of total extracellular -amylase obtained with glucose as the carbon source could be boosted severalfold by use of CD. The best enhancer was -CD, and the rank order of best to least effective was -CD>-CD=-CD>glucose.Another amylase, a 65-kDa -amylase, which degraded para-nitrophenyl-(1,4)-d-glucopyranoside, was also detected in this study. The most effective enhancer in this case was -CD, and the rank order was -CD>-CD>-CD glucose. Despite its ability to degradep-nitrophenylated glucose, this enzyme did not convert maltose to glucose. It showed a cleared zone on starch zymograms and did degrade short maltodextrins to maltose. Neither this new -amylase nor the 140-kDa -amylase exhibited any detectable ring-decyclizing (cyclodextrinase) activity against -or -CD.Other extracellular amylases (not characterized here) appeared to be similarly enhanced by CDs. Although the precise mechanism by which this effect is accomplished remains undefined, CDs can be useful inducing agents, boosting the expression and/or secretion of otherwise low-level enzymes, either as additives to growth media or as sole carbon source.     

Isolation and characterization of β-glucosidases from Aspergillus nidulans mutant USDB 1183

Two extracellular -glucosidases (cellobiase, EC 3.2.1.21), I and II, from Aspergillus nidulans USDB 1183 were purified to homogeneity with molecular weights of 240,000 and 78,000, respectively. Both hydrolysed laminaribiose, -gentiobiose, cellobiose, p-nitrophenyl--L-glucoside, phenyl--L-glucoside, o-nitrophenyl--L-glucoside, salicin and methyl--L-glucoside but not -linked disaccharides. Both were competitively inhibited by glucose and non-competitively (mixed) inhibited by glucono-1,5-lactone. -Glucosidase I was more susceptible to inhibition by Ag⁺ and less inhibited by Fe²⁺ and Fe³⁺ than -glucosidase II.     

Metabolism of a phenylcoumaran substructure lignin model compound in ligninolytic cultures of Phanerochaete chrysosporium

The degradation of the phenylcoumaran substructure model compound methyl dehydrodiconiferyl alcohol by the white-rot wood decay fungus Phanerochaete chrysosporium was investigated using culture conditions optimized for lignin oxidation. Initial attack was in the cinnamyl alcohol side chain, which was oxidized to a glycerol structure. This was subsequently converted by loss of the two terminal carbon atoms, C and C, to yield a C-aldehyde structure, which was further oxidized to the C-acid compound. The next detected intermediate, a phenylcoumarone, was produced by double bond formation between C and C, and oxidation of the C-alcohol to an aldehyde group. Further oxidation of C to an acid yielded the next intermediate. The final identified degradation product was veratric acid. No products from the 5-substituted aromatic ring, and no phenolic products, were found. The initial glycerol-containing intermediate was a mixture of the threo and erythro forms, and no optical activity could be found, suggesting that its formation might have involved nonstereospecific C-C epoxidation followed by non-enzymatic hydrolysis of the epoxide.Abbreviations TLC thin layer chromatography - LDA lithium diisopropyl amide - DDQ 2,3-dichloro-5,6-dicyanobenzoquinone - MS mass spectrometry - UV ultraviolet spectroscopy     

Alterations in protein kinase C isoenzyme expression and autophosphorylation during the progression of pressure overload-induced left ventricular hypertrophy

Cardiomyocytes express several isoenzymes of protein kinase C (PKC), which as a group have been implicated in the induction of left ventricular hypertrophy (LVH) and its transition to heart failure. Individual PKC isoenzymes also require transphosphorylation and autophosphorylation for enzymatic activity. To determine whether PKC isoenzyme expression and autophosphorylation are altered during LVH progression in vivo, suprarenal abdominal aortic coarctation was performed Sprague-Dawley rats. Quantitative Western blotting was performed on LV tissue 1, 8 and 24 weeks after aortic banding, using antibodies specific for total PKC, PKC and PKC, and their C-terminal autophosphorylation sites. Aortic banding produced sustained hypertension and gradually developing LVH that progressed to diastolic heart failure over time. PKC levels and autophosphorylation were not significantly different from sham-operated controls during any stage of LVH progression. PKC expression levels were also unaffected during the induction of LVH, but increased 3.2 ± 0.8 fold during the transition to heart failure. In addition, there was a high degree of correlation between PKC levels and the degree of LVH in 24 week banded animals. However, autophosphorylated PKC was not increased at any time point. In contrast, PKC autophosphorylation was increased prior to the development of LVH, and also during the transition to heart failure. The increased PKC autophosphorylation in 1 week banded rats was not accompanied by an increase in total PKC, whereas total PKC levels were markedly increased (6.0 ± 1.7 fold) in 24 week banded animals. Furthermore, both phosphorylated and total PKC levels were highly correlated with the degree of LVH in 24 week banded rats. In summary, we provide indirect evidence to indicate that PKC may be involved in the induction of pressure overload LVH, whereas both PKC and PKC may be involved in the transition to heart failure.