Low temperatures stabilize interferon α-2 against proteolysis in Methylophilus methylotrophus and Escherichia coli

Summary The accumulation of interferon (IFN) -2 in transformed strains of Escherichia coli and Methylophilus methylotrophus was greater at 25° C than at 37° C. Interferon -2 catabolism was followed by measuring the change in IFN titre (measured immunoreactively) with time at temperatures between 25° C and 37° C in chloramphenicol-treated cells. The IFN -2 titre remained constant at 29° C and below, while at higher temperatures the titres declined. The t _1/2 values for IFN -2 decreased with increasing incubation temperature. Pulse-chase studies using [³⁵S]methionine, sodium dodecyl sulphate-gel electrophoresis and autoradiography demonstrated that IFN -2 was subjected to degradation at 37° C while at 25° C it was stable. It is proposed that the susceptibility of IFN -2 to degradation in both E. coli and M. methylotrophus is affected by incubation temperature and 30° C may be a transition temperature above which the conformation of the molecule is recognised by the bacterial proteases.     

Characterization of a new Bacillus stearothermophilus isolate: a highly thermostable α-amylase-producing strain

A novel strain of Bacillus stearothermophilus was isolated from samples of a potato-processing industry. Compared to known -amylases from other B. stearothermophilus strains, the isolate was found to produce a highly thermostable -amylase. The half-time of inactivation of this -amylase was 5.1 h at 80°C and 2.4 h at 90°C. The temperature optimum for activity of the -amylase was 70°C; the pH optimum for activity was relatively low, in the range 5.5–6.0. -Amylase synthesis was regulated by induction and repression mechanisms. An inverse relationship was found between growth rate and -amylase production. Low starch concentrations and low growth temperatures were favourable for enzyme production by the organism. At the optimal temperature for growth, 65°C, the -amylase was a growth-associated enzyme. The optimal temperature for -amylase production, however, was 40°C, with -amylase increasing from 3.9 units (U)/ml to 143 U/ml when lowering the growth temperature from 65°C to 40°C. Maximal -amylase production in a batch fermentor run at 65°C was 102 U/ml, which was 26-fold higher than in erlenmeyer flasks at 65°C. The dissolved O₂ concentration was found to be a critical factor in production of the -amylase.     

Isolation and characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by the mating hormone a-factor

Summary Nine independent mutants which are supersensitive (ssl ^–) to G1 arrest by the mating hormone a-factor were isolated by screening mutagenized Saccharomyces cerevisiae MAT cells on solid medium for increased growth inhibition with a-factor. These mutants carried lesions in two complementation groups, ssl1 and ssl2. Mutations at the ssl1 locus were mating type specific: MAT ssl1 ^– cells were supersensitive to -factor but MAT ssl1 ^– were not supersensitive to -factor. In contrast, mutations at the ssl2. locus conferred supersensitivity to the mating hormone of the opposite mating type on both MAT, and MATa cells. The -cell specific capacity to inactivate externally added a-factor was shown to be lacking in MAT ssl1 ^– mutants whereas MAT ssl2. cells were able to inactivate a-factor. Complementation analysis showed that ssl2 and sst2, a mutation originally isolated as conferring supersensitivity to -factor to MATa cells, are lesions in the same gene. The ssl1 gene was mapped 30.5 centi-Morgans distal to ilv5 on chromosome XII.     

A note on dimensionless life histories for birds versus mammals

Summary Offspring production over the adult lifespan (b/M whereb is the yearly fledgling or offspring production and 1/M is the mean adult lifespan) is an approximate invariant within both birds and mammals. The two taxa differ, however, in that mammals have bothM and b as invariants (b/M = b/M) while birds do not ( is the age at first breeding). Birds have a surprising cancellation in that bothM andb are ^–0.25.     

Thermally induced disintegration of the oligomeric structure of αB-crystallin mutant F28S is associated with diminished chapterone activity

B-crystallin, a member of the small heat-shock protein (hsp) family of proteins, is able to function as a molecular chaperone by protecting other proteins from stress-induced aggregation by recognizing and binding to partially unfolded species of damaged proteins. The present work has investigated the role of phenylalanine-28 (F28) of the ²²RLFDQFF²⁸ region of B-crystallin in maintaining chaperone function and oligomeric structure under physiological condition and under thermal stress. Bovine B-crystallin was cloned for the first time and the cDNA sequence revealed greater than 90% homology to that of human, rat and mouse B-crystallins. F28 was mutated to a serine followed by expression of the mutant F28S and the wild-type B (B-wt) in E. coli and subsequent purification of the protein by size-exclusion chromatography. Secondary and tertiary structure analyses showed some structural changes in the mutant. Chaperone activity and oligomeric size of the mutant was unchanged at 37°C whereas at 58°C the chaperone activity was significantly decreased and the oligomeric size ranged from low molecular weight to high molecular weight showing disintegration of the oligomeric structure. The data support the idea that the participation of large oligomeric structure rather than smaller units is required to have optimal chaperone activity and the hydrophobic F28 residue is needed for maintaining the native oligomeric structure under thermal stress.     

A novel wheat alpha-amylase gene (alpha-Amy3)

Summary A genomic clone of a wheat -amylase gene (Amy3/33) was identified, on the basis of hybridisation properties, as different from -Amy1 and -Amy2 genes which had been characterised previously. The nucleotide sequence revealed that this gene has the normal sequence motifs of an active gene and an open reading frame interrupted by two introns. The protein sequence encoded by this open reading frame is recognisably similar to that of -amylase from the -Amy1 and -Amy2 genes and there is high sequence homology in all three proteins at the putative active sites and Ca⁺⁺ binding region. In addition, the introns are at positions equivalent to the position of introns in the -Amy1 and -Amy2 genes. However, the sequence was less similar to -Amy1 and -Amy2 than these are to each other. Southern blot analysis showed that the Amy3/33 DNA is one of a small multigene family carried on a different chromosome (group 5) from either the -Amy1 or -Amy2 genes. A further difference from the -Amy1 and -Amy2 genes was the pattern of expression. Amy3/33 was expressed only in immature grains and, unlike the -Amy1 and -Amy2 genes, not at all in germinating aleurones. These data suggested therefore that this gene represents a third type of -amylase gene, not described before, which shares a common evolutionary ancestor with the -Amy1 and -Amy2 genes.     

Thermal response in murine L929 cells lacking αB-crystallin expression and αB-crystallin expressing L929 transfectants

We investigated the role of B-crystallin expression in the development of thermotolerance in murine L929 cells. An initial heat-shock of 10 min at 45°C induced thermotolerance in these cells to a heat challenge at 45°C administered 24 h later. The thermotolerance ratio at 10^–1 isosurvival was 1.7. Expression of B-crystallin gene was not detected during the 24 h incubation at 37°C following heat shock by either northern or western blots. In contrast, inducible HSP70 synthesis was observed during this time period. Thus, this cell line provided an unique system in which to examine the effects of transfected B-crystallin on thermoresistance and thermotolerance. Cells stably transfected with B-crystallin under the control of an inducible promoter did not show a significant increase in the ability to develop thermotolerance. However, a stably transfected L929 clone expressing high levels of constitutive B-crystallin exhibited an approximately 50% increase in thermal resistance over parental and control cells. Though expression of B-crystallin is not requisite for the development of thermotolerance in L929 cells, overexpression of transfected B-crystallin can contribute to increased thermoresistance.     

Secreted agglutinability-masking factors in Issatchenkia scutulata var. scutulata

The agglutinability-masking factors (AMFs) of a and mating types of Issatchenkia scutulata var. scutulata were prepared from culture fluids. AMFs masked the agglutinability of opposite mating-type cells sex-specifically, just like agglutination substances responsible for sexual cell agglutination. a AMF adsorbed to cells was eluted by incubating the cells at 60°C for 10 min. AMF was prepared directly from culture fluids of cells by DEAE-cellulose ion exchange chromatography. The active part of the AMFs is thought to be a peptidyl moiety because of the sensitivity to subtilisin. The pretreatment of cells with AMF of the opposite mating-type was shown to promote zygote formation. AMF slightly inhibited growth in a cells but not in cells, while a AMF did not show any growth-inhibitory effect on either a or x cells.     

Genotype-environment interaction for awn development in isogenic lines of barley

Summary Awn length of four isogenic lines of barley differing by two genes for awn development (A andB) and their short iinkage blocks was evaluated at a wide range of plant densities (0.002 to 3.345 m²/plant) for two years. Awn development was reduced at high plant density. The quarter-awned genotype (aaBB) became phenotypically awnless (aabb) at high plant density. Similar results were obtained each year and the genotype x plant density effect was the major portion of the genotype-environment interaction variance. Additive ( _A , _B ) and additive x additive ( _AB ) gene effects were computed for each plant density for lateral and central floret awn length. For lateral awns _AB was not affected, but _A and _B increased with decreased plant density. In contrast, for central awns _A and _AB decreased and _B increased with decreased plant density.Central floret awns measured at each spike node showed that high plant density reduced awn development most in the lower half of the spike. This is the zone of most rapid awn differentiation and since culm elongation and spike growth rates were greatly increased by high plant density, it was suggested that rapid growth invoked a stress on awn development and differentially altered the expression ofA andB.

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Zusammenfassung An 4 isogenen Gerstenlinien, die sich durch zwei Gene für Grannenbildung (A undB) und entsprechende kurze Kopplungsblocks unterscheiden, wurde zwei Jahre lang die Länge der Grannen bei verschiedener Standdichte (0,002 bis 3,345 m² je Pflanze) untersucht. Bei dichtem Bestand ergab sich eine Beeinträchtigung der Grannenbildung, der viertelbegrannte Genotyp (aaBB) wurde phänotypisch grannenlos (aabb). Die Ergebnisse stimmten in beiden Jahren überein, der Effekt Genotyp x Standdichte hatte den Hauptanteil an der Interaktionsvarianz Genotyp: Umwelt. Additive ( _A , _B ) und additive x additive ( _AB ) Genwirkungen wurden bei jeder Standdichte für die Grannenlänge der Seiten-und Mittelährchen errechnet. Bei den seitlichen Grannen wurde _AB nicht beeinflußt, aber _A und _B erhöhten sich mit abnehmender Standdichte. Im Gegensatz dazu gingen bei den mittleren Grannen _A und _AB zurück, während für _B bei abnehmender Standdichte ein Ansteigen festzustellen war.Messungen der mittleren Grannen jeder Ähre zeigten, daß hohe standdichte der Pflanzen die Grannenbildung am meisten in der unteren Hälfte der Ähre reduzierte. Das ist die Zone, in der sich die Grannen am schnellsten differenzieren, und da die Halm- und Ährenwachstumsraten durch hohe Standdichte stark gesteigert wurden, scheint das schnelle Wachstum auf die Grannenentwicklung hemmend einzuwirken und die Manifestierung vonA undB unterschiedlich abzuändern.

     

Interaction of Duodenase with Serpins from Human Blood Serum

The interaction of duodenase, a new serine protease from a small group of Janus-faced proteases, with serpins, ₁-protease inhibitor (₁-PI) and antichymotrypsin (ACT) from human blood serum, was studied. The stoichiometry of the inhibition process was found to be 1.2 and 1.3 mol/mol for ₁-PI and ACT, respectively. The presence of a stable enzyme–inhibitory complex duodenase–₁-PI was confirmed by SDS-PAGE. The formation of the duodenase–ACT complex was not demonstrated; instead, the band of the cleaved inhibitor indicated the ACT hydrolysis. The suicide mechanism of the duodenase interaction with the human blood serpins was proved. The association rate constants (k × 10⁵, ^–1 s^–1) were 2.4 ± 0.3 × 10⁵ for ₁-PI and 3.0 ± 0.4 × 10⁵ for ACT. These results indicate the possibility of the regulation of duodenase activity by endogenous serpins.     

Regio- and stereo-selective synthesis of vinyl glucose ester catalyzed by an alkaline protease of Bacillus subtilis

The transesterification of -d-glucose with divinylsuccinate, divinyladipate and divinylsebacate in pyridine at 55 °C for 3 days was catalyzed by an alkaline protease from Bacillus subtilis to give corresponding 6-O-vinyl glucose esters at 30%, 53% and 35% yield, respectively. The stereo-selectivity of the alkaline protease toward the -anomer was affected by the acyl donor chain length. 6-O-Vinylsuccinyl-d-glucose was mixture of - and -anomers (/=44/56), the other two products were the pure -d-glucose derivatives.     

1H and13C-NMR analysis of four pentasaccharides from urine of blood-group A and B,Leb adults. Confirmation of the structure of two new oligosaccharides: Fuc(α1–2)[GalNAc(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc and Fuc(α1–2)[Gal(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc

The major pentasaccharides Fuc(1-2)[GalNAc(1-3)]Gal(1-4)[Fuc(1-3)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-4)[Fuc(1-3)]Glc, which are normally present in the urine of bloodgroup A Le^b and B Le^b healthy subjects, were each found to be contaminated by a minor component when analysed by¹H-NMR. The determination of these structures, Fuc(1-2) [GalNAc(1-3)]Gal(1-3)[Fuc(1-4)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-3)[Fuc(1-4)]Glc, was based on the results of methylation analysis and¹H/¹³C-NMR spectroscopy.Abbreviations HPLC high performance liquid chromatography - GLC gas liquid chromatography - NMR nuclear magnetic resonance - COSY correlation spectroscopy - Gal d-galactopyranose - GalNAc 2-acetamido-2-deoxy-d-galactopyranose - Glc d-glucopyranose - Fuc l-fucopyranose - LNDFH I lacto-N-difucohexaose I (Le^b determinant     

Effects of phytosulfokine-α on growth and tropane alkaloid production in transformed roots of Atropa belladonna

Phytosulfokine (PSK)- is a sulphated pentapeptide, isolated fromthe medium of cultured Asparagus officinalis mesophyllcells, that promotes cell proliferation. It is a putative key factor inconditioned medium required for the growth of low-density plant cell cultures.The present study investigates the effect of PSK- on growth and tropanealkaloid production in Atropa belladonna hairy rootstransformed with Agrobacterium rhizogenes (MAFF 03-01724). Although the growth rates ofhairy roots cultured in medium with orwithout PSK- for 4 weeks did not show any differences, the productivityof tropane alkaloids, especially of hyoscyamine, was enhanced by10^–7 or 10^–8 M PSK-. Inaddition, the content of tropane alkaloids in transformed roots treated withPSK- was 1.4 times higher than that of untreated roots after 4 weeks ofculture. The time course of growth and tropane alkaloid production inAtropa belladonna transformed roots suggested thatPSK- influenced the growth of transformed roots during the activegrowingphase, but not tropane alkaloid production.     

Combined use of 13C chemical shift and 1Hα−13Cα heteronuclear NOE data in monitoring a protein NMR structure refinement

Summary A large portion of the ¹³C resonance assignments for murine epidermal growth factor (mEGF) at pH 3.1 and 28°C has been determined at natural isotope abundance. Sequence-specific ¹³C assignments are reported for 100% of the assignable C, 96% of the C, 86% of the aromatic and 70% of the remaining peripheral aliphatic resonances of mEGF. A good correlation was observed between experimental and back-calculated C chemical shifts for regions of regular -sheet structure. These assignments also provide the basis for interpreting ¹H–¹³C heteronuclear NOE (HNOE) values in mEGF at natural isotope abundance. Some of the backbone polypeptide segments with high internal mobility, indicated by these ¹H–¹³C HNOE measurements, correlate with locations of residues involved in the putative mEGF-receptor binding site. Using four families of mEGF structures obtained over the last few years, we demonstrate that standard deviations between experimental and back-calculated C values can be used to monitor the refinement of this protein's structure, particularly for -sheet regions. Improved agreement between calculated and observed values of C is correlated with other measures of structure quality, including lowered values of residual constraint violations and more negative values of conformational energy. These results support the view that experimental conformation-dependent chemical shifts, C, can provide a reliable source of information for monitoring the process of protein structure refinement and are potentially useful restraints for driving the refinement.Abbreviations HSQC heteronuclear single-quantum coherence spectroscopy - PFG pulsed-field gradient - TOCSY ¹H-¹H total correlation spectroscopy - EGF epidermal growth factor - mEGF murine EGF - hEGF human EGF - hTGF human type- transforming growth factor - DIPSI spm-locking pulse sequence - NOE nuclear Overhauser effect - HNOE heteronuclear Overhauser effect     

Biotechnological production of unnatural L-amino acids from D,L-5-monosubstituted hydantions. II. L-α- and L-β-naphthylalanine

Summary Resting cells ofArthrobacter sp. (DSM 3745) with the ability to form L-tryptophan from D,L-5-(3-indolylmethy)hydantoin were used for the bioconversion of D,L-5-- and D,L-5--naphthylmethylhydantoin (D,L-5-- and D,L-5--NMH) to the corresponding L-amino acids. Under the optimal reaction conditions of pH 9.7 and 40°C specific productivities of 0.2 (-naphtylalanine) and 0.6 (-naphtylalanine) mM amino acid x g cell dry mass^–1 x h^–1 were obtained in a 0.1 M Na₂CO₃/NaHCO₃-buffer in a strirred bioreactor.     

A dominant mutation (SAD) bypassing the requirement for the a mating type locus in yeast sporulation

Summary SAD (suppressor of a deficiencies) is a mutation that allows -mater diploids such as / or a1^-/ strains to sporulate. This mutation is unstable and reverts to wildtype (sad ⁺) even in strains homozygous for SAD. SAD is dominant to sad ⁺: / and a1^-/ sad ¹/SAD diploids are sporulation-proficient. SAD is located on chromosome III, 40 cM distal to the mating type locus, between THR4 and HMR a. The ability of SAD to support sporulation requires the presence of an mating type locus with an active 2 function. Possible models for the action of SAD are (1) SAD bypasses the need for a1 function in sporulation, and (2) SAD provides a1 function to MAT a1^- mutants by supplying a1 function itself, for example, by allowing expression of a silent copy of MAT a.     

Phylogeny and evolutionary rates of G protein α subunit genes

Summary Rooted phylogenetic trees for a total of 34 genes encoding the stimulatory (s), inhibitory (i), transducin (t), G_x (x), G_z (z), G₁₁ (11), G₁₂ (12), G₁₃ (13), G₁₆ (16), G_q (q), and other (o) G protein a subunits have been constructed. The analysis shows that the G₁₂ (12 and 13), G_q (11, 16, and q), and G_s (s genes) groups form one cluster, and the G_x (x and z genes), G_i (i genes), G_t (t1 and t2), and G_o (o genes) groups form another cluster. During mammalian evolution, the rates of synonymous substitutions for these genes were estimated to be between 1.77 × 10^–9/site/year and 5.63 × 10^–9/site/year, whereas those of nonsynonymous substitutions were between 0.008 × 10^–9/site/year and 0.067 × 10^–9/site/year. These evolutionary rates are similar to those for histone genes, suggesting equally important biological functions of the G protein a subunits. Offprint requests to: S. Yokoyama     

N-Carbamoyl-α-Amino Acids Rather than Free α-Amino Acids Formation in the Primitive Hydrosphere: A Novel Proposal for the Emergence of Prebiotic Peptides

Our previous kinetic and thermodynamic studies upon the reactional system HCHO/HCN/ NH₃ in aqueous solutions are completed. In the assumed prebiotic conditions of the primitive earth ([HCHO] and [HCN] near 1 g L^–1, T = 25 °C, pH = 8, [NH₃] very low), this system leads to 99.9% of -hydroxyacetonitrile and 0.1% of -aminoacetonitrile (precursor of the -amino acid). The classical base-catalyzed hydration of nitriles, slow and not selective, can not modify significantly this proportion. On the contrary, we found two specific and efficient reactions of -aminonitriles which shift the initial equilibrium in favor of the -aminonitrile pathway. The first reaction catalyzed by formaldehyde generates -aminoamides, precursors of -aminoacids. The second reaction catalyzed by carbon dioxide affords hydantoins, precursors of N-carbamoyl--aminoacids. In the primitive hydrosphere, where the concentration in carbon dioxide was estimated to be higher than that of formaldehyde, the formation of hydantoins was consequently more efficient. The rates of hydrolysis of the -aminoacetamide and of the hydantoin at pH 8 being very similar, the synthesis of the N-carbamoyl--amino acid seems then to be the fatal issue of the HCHO/HCN/NH₃ system that nature used to perform its evolution. These N-protected -amino acids offer new perspectives in prebiotic chemistry, in particular for the emergence of peptides on the prebiotic earth.