Mg2+ Induces Conformational Changes in the Catalytic Subunit of Phosphorylase Kinase, Whether by Itself or as Part of the Holoenzyme Complex

Phosphorylase kinase (PhK) from skeletal muscle is a structurally complex, highly regulated, hexadecameric enzyme of subunit composition ()₄. Previous studies have revealed that the activity of its catalytic subunit is controlled by alterations in quaternary structure initiated at allosteric and covalent modification sites on PhK's three regulatory subunits; however, changes in the conformation of the holoenzyme initiated by the catalytic subunit have been more difficult to document. In this study a monoclonal antibody (mAb 79) has been generated against isolated subunit and used as a conformational probe of that subunit. The epitope recognized by this antibody is within the catalytic core of the subunit, between residues 100 and 240, and monovalent fragments of the antibody inhibit the catalytic activity of the holoenzyme, the -calmodulin binary complex, and the free subunit. Activation of PhK by a variety of mechanisms known or thought to act through its regulatory subunits (phosphorylation, ADP binding, or alkaline pH) increased the binding of the holoenzyme to immobilized mAb 79, indicating that activation by any of these distinct mechanisms involves repositioning of the portion of the catalytic domain of the subunit containing the epitope for mAb 79. The activating ligand Mg²⁺ also stimulated the binding of the PhK holoenzyme to immobilized mAb 79, as well as the binding of mAb 79 to immobilized subunit. Thus, Mg²⁺ increases the accessibility of the mAb 79 epitope in both the isolated subunit and in the holoenzyme. Our results suggest that previously reported influences of Mg²⁺ on the quaternary structure of the PhK holoenzyme are directly mediated by the subunit.     

Morphology and structural organization of spiracles in femaleArgas (Persicargas) walkerae (Acari: Argasidae)

Scanning electron microscopical investigations of fractures and corrosion casts of the spiracles from femaleA. walkerae ticks revealed a four-part structure, consisting of spiracular plate, ostium and macula forming the external closure, followed by the subostial space and the vestibulum of regulable volume, as well as the atrial chamber as the innermost part from which the main tracheal trunks originate. On the average, the spiracular plate was 158 m long and 188 m at the broadest width. It consisted of a thin, highly perforated external and a thick internal layer, which enclosed the interpedicellar space with numerous stout pedicels. In its posterior region, the spiracular plate was covered by the macula, which was up to 80 m in length and 110 m in width. The interpedicellar cavity opened into the subostial space measuring 95.5 m in length and 159.6m in width, which proceeded into the 112-m long vestibulum. The roof of the vestibulum was flexible and could be everted and inverted. Inverted, the roof formed a quadratic bulge with numerous deep cuticular folds, which confined the lumen of the vestibulum either partially or completely. In corrosion casts, the roof was everted to a length of up to 89.3 m. In the posterior part of the vestibulum, as well as in the initial fourth of the artrial chamber, numerous anvil-, cone-or drop-like cuticular projections were arranged in wedge-like fashion. The atrial chamber was almost spherical with a diameter of 138.4 m. Five main tracheal trunks of different luminal diameter as well as numerous channels opened into the atrial chamber.     

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.     

Identification of a complete set of isogenic wheat/rye D-genome substitution lines by means of Giemsa C-banding

Summary A complete set of isogenic wheat/rye D-genome substitutions were produced by crossing an inbred line of spring rye Secale cereale L. cv. Prolific to a tetraploid wheat, the A-and B-genomes of which had previously been extracted from hexaploid wheat, Triticum aestivum L. em Thell. cv. Thatcher. After chromosome doubling, the derived hexaploid triticale (x Triticosecale Wittmack) was backcrossed to 6x Thatcher and selection for wheat/rye substitution lines was carried out in BCF₃ to BCF₆ families by using Giemsa C-banding. Five fertile disomic wheat/rye D-genome substitution lines were obtained and their chromosomal constitution was determined to be 1D/1R, 2D/2R, 7D/4R, 6D/6R, 7D/7R. The two remaining 3R and 5R substitutions are at the moment in a monosomic condition. Another 1D/7R substitution was detected but this plant was very weak and sterile, indicating that only substitutions between homoeologous chromosomes result in fertile, vigorous plants. Furthermore, many rye telocentrics as well as rye-rye and rye-wheat translocations were selected. Since all lines selected in this program share the same genetic background of Thatcher wheat, genetic heterogeneity is excluded. The material is very useful, therefore, for analyzing the effects of different rye chromosomes or chromosome segments in an otherwise homozygous background.Contribution No. 797     

Solution conformations of proline rings in proteins studied by NMR spectroscopy

Summary Three different conformations of proline rings in a protein in solution, Up, Down and Twist, have been distinguished, and stereospecific assignments of the pyrrolidine -, - and -hydrogens have been made on the basis of ¹H-¹H vicinal coupling constant patterns and intraresidue NOEs. For all three conformations, interhydrogen distances in the pairs -³, ³-³, ²-², ²-², and ³-³ (2.3 Å) are shorter than those in the pairs -², ²-³, ³-², ²-³, and ³-² (2.7–3.0 Å), resulting in stronger NOESY cross peaks. For the Up conformation, the ³-² and ²-³ spin-spin coupling constants are small (<3 Hz), and weak cross peaks are obtained in a short-mixing-time (10 ms) TOCSY spectrum; all other vicinal coupling constants are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. For the Down form, the -², ²-³, and ³-² vicinal coupling constants are small, leading to weak TOCSY cross peaks; all other couplings again are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. In the case of a Twist conformation, dynamically averaged coupling constants are anticipated. The procedure has been applied to bovine pancreatic trypsin inhibitor and Cucurbita maxima trypsin inhibitor-V, and ring conformations of all prolines in the two proteins have been determined.     

Intracellular sites of the synthesis of sweet potato mitochondrial F1ATPase subunits

Summary Five subunits (-, -, -, - and -subunits) of the six -and -subunits) in the F₁ portion (F₁ATPase) of sweet potato (Ipomoea batatas) mitochondrial adenosine triphosphatase were isolated by an electrophoretic method. The - and -subunits were not distinguishable immunologically but showed completely different tryptic peptide maps, indicating that they were different molecular species. In vitro protein synthesis with isolated sweet potato root mitochondria produced only the -subunit when analyzed with anti-sweet potato F₁ATPase antibody reacting with all the subunits except the -subunit. Sweet potato root poly(A)⁺RNA directed the synthesis of six polypeptides which were immunoprecipitated by the antibody: two of them immunologically related to the -subunit and the others to the - and -subunits. We conclude that the -subunit of the F₁ATPase is synthesized only in the mitochondria and the -, - and -subunits are in the cytoplasm.     

Involvement of Erks activation in cadmium‐induced AP‐1 transactivation in vitro and in vivo

Cadmium is a potent and effective carcinogen in rodents and has recently been accepted by IARC (International Agency for Research on Cancer) as a category 1 carcinogen. Cadmium-induced upregulation of intracellular signaling pathways leading to increased mitogenesis is thought to be a major mechanism for the carcinogenic activity following chronic cadmium exposure. In the present study, we found that exposure of cells to cadmium induced significant activation of AP1 and all three members of the MAP kinase family in mouse epidermal JB6 cells. The induction of AP1 activity by cadmium appears to involve activation of Erks, since the induction of AP1 activity by cadmium was blocked by pretreatment of cells with PD98058. Interestingly, the induction of AP1 by cadmium was greatly enhanced by the chemical tumor promoter, TPA and the growth factor EGF, but not by ultraviolet C radiation. In vivo studies demonstrated that cadmium could also induce transactivation of AP1 in AP1luciferase report transgenic mice. Considering the role of AP1 activation in tumor promotion, the results presented in this study provide a possible molecular mechanism for cadmiuminduced carcinogenesis.     

Increase of catalytic activity of α-chymotrypsin in organic solvent by co-lyophilization with cyclodextrins

-Chymotrypsin was lyophilized in the presence of 2,3,6-tri-O-methyl -cyclodextrin, and it displayed activity 40 fold higher than free -chymotrypsin for transesterification in acetonitrile. -Chymotrypsin which was co-lyophilized with hydroxypropylated - or -cyclodextrins retained more than 98% of its initial activity after 6 h incubation in acetonitrile.     

Homologues of a vacuolar processing enzyme that are expressed in different organs in Arabidopsis thaliana

Vacuolar processing enzymes (VPEs) are responsible for the maturation of seed proteins. These processing enzymes belong to a novel group of cysteine proteinases with molecular masses of 37 to 39 kDa. We isolated two genes of VPEs from a genomic library of Arabidopsis. The gene products were designated -VPE and -VPE, and they were 56% identical in terms of amino acid sequence. The amino acid sequences of -VPE and -VPE were also 55% and 67% identical to that of castor bean VPE, respectively. The gene for -VPE had 7 introns, while that of -VPE had 8 introns. Northern blot analysis revealed that -VPE is expressed in rosette leaves, cauline leaves and stems of Arabidopsis, while -VPE is predominantly expressed in the flowers and buds. Neither -VPE nor -VPE is expressed in the siliques. This result strongly suggests that the isolated genes encode isozymes of VPE that are specific to vegetative organs.     

The mitochondrially encoded "phantom ATPase" of S. cerevisiae. II. A heterogeneous mitochondrial ATPase population in situ.

Summary The mitochondrial ATPase from a PHO 1 mutant (OLI 2, PHO 1, OLI 4 region on mit DNA of S. cerevisiae) was further examined. A new purification method using Lysolecithin instead of Triton allowed us to solubilize and separate a heterogeneous ATPase population from PHO 1-mitochondria: the major abnormal fraction had extremely low oligomycin-sensitivity (but normal specific immunological reactivity), while a minor normal fraction (representing about 20% of the initial mitochondrial ATPase activity) had high sensitivity and affinity for oligomycin.Moderate urea treatment of PHO 1-mitochondria leads to partial loss of ATPase activity and a concomitant increase of oligomycin-sensitivity, suggesting that a heterogeneous ATPase population exists in situ in the mitochondrial membrane: part of the major abnormal ATPase fraction is selectively inactivated by urea, producing a concomitant enrichment in the initially minor normal ATPase fraction.If the minor normal ATPase fraction is the only one capable of in vivo ATP synthesis, the deficient but oligomycin-sensitive cell growth and oxidative phosphorylation in vitro are readily explained.Further structural studies are under way to ascertain whether the minor normal ATPase fraction is strictly identical to the wild type, in which case PHO 1 is a regulatory gene, or not, in which case PHO 1 is a structural gene.     

Causes,proximate and ultimate

Within evolutionary biology a distinction is frequently made between proximate and ultimate causes. One apparently plausible interpretation of this dichotomy is that proximate causes concern processes occurring during the life of an organism while ultimate causes refer to those processes (particularly natural selection) that shaped its genome. But ultimate causes are not sought through historical investigations of an organisms lineage. Rather, explanations referring to ultimate causes typically emerge from functional analyses. But these functional analyses do not identify causes of any kind, much less ultimate ones. So-called ultimate explanations are not about causes in any sense resembling those of proximate explanations. The attitude, implicit in the term ultimate cause, that these functional analyses are somehow superordinate to those involving proximate causes is unfounded. Ultimate causes are neither ultimate nor causes.     

Nuclear Overhauser effect investigation on GM1 ganglioside containingN-glycolyl-neuraminic acid (II3Neu5GcGgOse4Cer)

The conformational properties of the oligosaccharide chain of GM1 ganglioside containingN-glycolyl-neuraminic acid, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer, were studied through NMR nuclear Overhauser effect investigations on the monomeric ganglioside in dimethylsulfoxide, and on mixed micelles of ganglioside and dodecylphosphocholine in water. Several interresidual contacts for the trisaccharide core--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-were found to fix the relative orientitation of the three saccharides, while the glycosidic linkage of the terminal -Gal-was found to be quite mobile as the -Gal-(1-3)--GalNAc-disaccharide exists in different conformations. These results are similar to those found for two GM1 gangliosides containingN-acetyl-neuraminic acid and neuraminic acid [1].Abbreviations Ganglioside nomenclature is in accordance with Svennerholm [23] and the IUPAC-IUB Recommendations [24] GM3(Neu5Ac) II³Neu5AcLacCer, -Neu5Ac-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM3(Neu5Gc) II³Neu5GcLacCer, -Neu5Gc-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Ac) II³Neu5AcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Gc) II³Neu5GcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu) II³NeuGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu-(2-3)]--Glc-(1-1)-Cer - GD1a IV³Neu5AcII³Neu5AcGgOse₄Cer, -Neu5Ac-(2-3)--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GalNAc-GD1a IV⁴GalNAcIV³Neu5AcII³Neu5AcGgOse₄Cer, -GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - Neu neuraminic acid - Neu5Ac N-acetyl-neuraminic acid - Neu5Gc N-glycolyl-neuraminic acid - Cer ceramide     

Composition of poly-β-hydroxyalkanoate from Syntrophomonas wolfei grown on unsaturated fatty acid substrates

Poly--hydroxyalkanoate (PHA) from crotonate-grown cultures of Syntrophomonas wolfei contained only the d-isomer of -hydroxybutyrate. The PHA from cultures grown with trans-2-pentenoate or one of several hexenoates as the substrate also contained small amounts (5%) of -hydroxypentanoate or -hydroxyhexanoate, respectively. Thus, some PHA was synthesized without cleavage of the carbon skeleton of the substrate, but the predominant route for PHA synthesis was by the condensation and subsequent reduction of acetyl-coenzyme A (CoA). The ratio of the -hydroxypentanoate to the -hydroxybutyrate in PHA in pentenoate-grown cultures increased immediately after inoculation and then decreased as the amount of the -hydroxybutyrate in PHA increased. The amount of -hydroxypentanoate in the PHA did not markedly change throughout the remainder of growth. These data indicated that the unbroken carbon-chain was used for polymer production only in the early stages of growth and, later, polymer synthesis occurred by the condensation and reduction of acetyl-CoA molecules.     

Valine inhibition of β-isopropylmalate dehydrogenase takes part in the regulation of leucine biosynthesis inCandida maltosa

The -isopropylmalate (IPM) dehydrogenase (EC 1.1.1.85) ofCandida maltosa, the third pathway-specific enzyme of leucine biosynthesis, was purified, some properties of the enzyme were studied and a novel regulatory pattern was found. The K_m values of the enzyme were estimated to be 0.42 mM for -IPM and 0.34 mM for NAD⁺. It is demonstrated that the enzyme can be regulated by L-valine. The inhibition was competitive with respect to -IPM (K_i=1.84 mM) and non-competitive with respect to NAD⁺ (K_i=5.67 mM). Exogenous addition of L-valine toC. maltosa cells increased the intracellular pool of some intermediates of leucine biosynthesis (-ketoisovalerate, -IPM, -IPM), but has hardly influence on the leucine pool.     

Characterization of Brain β-Carboline-2-N-Methyltransferase,An Enzyme That May Play a Role in Idiopathic Parkinson's Disease

The activity of -carboline-2-N-methyltransferase results in the formation of neurotoxic N-methylated -carbolinium compounds. We have hypothesized that these N-methylated -carbolinium cations may contribute to the development of idiopathic Parkinson's disease. This report describes experiments undertaken to optimize assay conditions for bovine brain -carboline-2-N-methyltransferase activity. The activity of -carboline-2-N-methyltransferase is primarily localized in the cytosol, has a pH optimum of 8.5–9, and obeys Michaelis-Menten kinetics with respect to its substrates, 9-methylnorharman (9-MeNH) and S-adenosyl-L-methionine (SAM). Kinetic constants, K_M and V_max, with respect to 9-MeNH, are 75 M and 48 pmol/h/mg protein, respectively. The K_M for SAM is 81 M and the V_max is 53 pmol/h/mg protein. In addition, enzyme activity is inhibited by S-adenosyl-L-homocysteine (SAH) or zinc, and is increased 2-fold in the presence of iron or manganese. Enzyme characterization is a prerequisite to the purification of this N-methyltransferase from bovine brain as well as comparison of its activity in human brain from control and Parkinson's disease individuals.     

Expression of a chimeric stilbene synthase gene in transgenic wheat lines

A chimeric stilbene synthase (sts)gene was transferred into wheat. Stilbene synthases play a role in the defence against fungal diseases in some plant species (e.g. groundnut or grapevine) by producing stilbenetype phytoalexins like resveratrol. Resveratrol is also claimed to have positive effects to human health. Embryogenic scutellar calli derived from immature embryos of the two commercial German spring wheat cultivars Combi and Hanno were used as target tissue for cotransformation by microprojectile delivery. The selectable marker/reporter gene constructs contained the bargene either driven by the ubiquitinpromoter from maize (pAHC 25, also containing the uidAgene driven by the ubiquitinpromoter), or by the actinpromoter (pDM 302) from rice. The cotransferred plasmid pStil 2 consisted of a grapevine stscoding region driven by the ubiquitin promoter. Eight transgenic Combi and one Hanno T_Oplant were obtained and, except one Combi T_Oplant, found to be cotransformants due to the integration of both the stsgene and the selectable marker or reporter genes. Expression of the stsgene was proven by RTPCR, and, for the first time, by detection of the stilbene synthase product resveratrol by HPLC and mass spectrometry. The stsgene was expressed in four of the seven transgenic Combi T_{_o}plants. Two of the respective T₁progenies segregated in a Mendelian manner were still expressing the gene. Investigations into methylation of the stsgene showed that in three nonexpressing progenies inactivation was paralleled by methylation.     

Modulation of natural killer activity by thymosin alpha 1 and interferon

Summary A single injection of -interferon (-IFN) (30 000 units/mouse), a major biological modifier of natural killer (NK) cytolytic activity, strongly stimulated NK activity in normal mice, as expected, while the same treatment did not statistically alter the NK response in cyclophosphamide (CY)-suppressed animals.We investigated the possibility of thymosin ₁ cooperating with -IFN in boosting NK activity in CY-suppressed animals.The results show that treatment with thymosin ₁ (200 g/kg) for 4 days, followed by a single injection of -IFN 24 h before testing, strongly restored NK activity in CY-suppressed mice. Thymosin ₁ was, moreover, able to accelerate the recovery rate of NK activity in bone marrow reconstituted murine chimeras.Taken together the data support the concept that the synergic effect between thymosin ₁ and -IFN could be the result of effects on differentiation of the NK lineage at different levels.     

Observations on the fine structure of the cercaria of Notocotylus attenuatus and formation of the cyst wall of the metacercaria

Summary The dorsal tegument of the mature cercaria of Notocotylus attenuatus is a syncytial, cytoplasmic layer, containing two types of secretory granule which are identifiable ultrastructurally. The type 1 secretory bodies are electron lucid, whereas most type 2 granules have a banded appearance. The ventral tegument contains granules which are secreted from the type 3 cells; the type 3 granules are membrane bound, electron dense, and consist of both an amorphous and a finely striated zone. The type 4 cells mainly contain cigar-shaped granules consisting of an amorphous core surrounded by concentric striations. The granules exhibit structural variability in shape and content. The type 4 cells undergo a cellular migration to the tegument during encystment. The structure of the posterior-lateral glands and mode of secretion of the granules are described. Possible functions of microtubules are discussed for each cell type. Details of some secretory processes involved in the formation of the hemispherical cyst wall are described. The layers of the cyst wall may be related to the granular contents of the various parenchymal cells of the cercaria. The tegument of the metacercaria originates primarily from the cytoplasm of the type 1, type 2, type 3 and type 4 cells.     

Transglycosylation reactions by exoglycosidases from the termite Macrotermes subhyalinus

The ability of four exoglycosidases (-galactosidase, -glucosidase, -glucosidase and invertase) from the termite Macrotermes subhyalinus to catalyse tranglycosylation reactions was tested using lactose, cellobiose, maltose and sucrose as glycosyl donors and 2-phenylethanol as glycosyl acceptor. The experimental conditions were optimized in relation to the time course of the reaction, pH and concentrations of glycosyl donor and acceptor. Whereas the hydrolytic activity was largely predominant over the transferase activity with -galactosidase and -glucosidase, the transglycosylation activity represented 68% with -glucosidase. In addition, as demonstrated by the transglycosylation product formed, the hydrolysis of sucrose was catalysed by -glucosidase and not by invertase. On the basis of this work, -glucosidase from M. subhyalinus appears to be a valuable tool for the preparation of neoglycoconjugates.     

Polygonal inequalities as a key to neuronic equations

Neuronic or decision equations, first proposed as a mathematical model of neural activity, have shown, after their exact, compact solution was found, typical behaviours that make them natural tools for General Systems studies. It is shown here that their mathematical investigation is remarkably furthered by generalizing the triangular inequality to polygonal ones. These permit the immediate computation of the tensorial expansion of linearly separable boolean functions, and exhibit clearly the connection between their continuous and discontinuous aspects.