Physicochemical characterization of gamma-crystallins from bovine lens--hydrodynamic and biochemical properties

A detailed hydrodynamic study has been made on the -crystallin of the bovine lens. Sedimentation study indicates that -crystallin shows a nearly gaussian peak throughout the course of sedimentation at high speed, using a synthetic boundary cell. The diffusion and sedimentation coefficients are 10.3×10^–7 cm²/sec and 2.51 S, respectively. The weight-average molecular weight of the unfractionated -crystallin calculated from sedimentation equilibrium is 21,800. The four major subfractions of -crystallin show similar hydrodynamic properties with an intrinsic viscosity of 2.50 ml/g and a Stokes radius of 21 Å. The distinct electrophoretic mobilities exhibited by the four subfractions show gel-concentration dependence and similar slopes in the Ferguson plot, indicative of being charge isomers of the same molecular species. Amino acid analysis of these four subfractions corroborated the conclusions that these -crystallin polypeptides are closely related and comprise a multigene family of crystallins. Based on the sedimentation and intrinsic viscosity data, -crystallin can be modeled as a prolate ellipsoid with an axial ratio of approximately 3.0 and a hydration factor of 0.27 g water per gram protein. The circular dichroism data for -crystallins showed a minimum at about 217 nm, characteristic of a -sheet conformation. These structural characteristics are in good accord with those derived from X-ray diffraction data for -crystallin II.     

Comparative residual toxicities of pesticides to the predator Agistemus industani (Acari: Stigmaeidae) on citrus in Florida

Residual toxicities of registered and selected experimental pesticides used on citrus against Agistemus industani Gonzalez (Acari: Stigmaeidae) were compared. Pesticides considered highly toxic to A. industani were: abamectin 0.15 EC at 731ml/ha+FC 435-66 petroleum oil at 46.8l/ha, pyridaben 75WP at 469g/ha, ethion 4EC at 7.01l/ha+FC 435-66 petroleum oil at 46.8l/ha, propargite 6.55 EC at 3.51l/ha, chlorfenapyr 2SC at 1.46l/ha applied alone or in combination with FC 435-66 petroleum oil at 46.8l/ha, sulphur 80DF at 16.81kg/ha, dicofol 4EC at 7.01l/ha, fenbutatin oxide 50WP at 2.24kg/ha, benomyl 50WP at 2.24kg/ha, benomyl 50WP at 1.68kg/ha+ferbam 76 GF at 5.60kg/ha, ferbam 76GF at 11.21kg/ha, neem oil 90EC at 46.8l/ha, and copper hydroxide DF (40% metallic copper) at 4.48kg metallic copper/ha+FC 435-66 petroleum oil at 46.8l/ha. Pesticides that were moderately to slightly toxic included: copper sulphate 98% at 4.48kg metallic copper/ha+FC 435-66 petroleum oil at 46.8l/ha, fenbuconazole 2F at 280ml/ha+FC 435-66 petroleum oil at 46.8l/ha, FC 435-66 petroleum oil applied alone at 46.8l/ha or 23.4l/ha, and diflubenzuron 25WP at 1.40kg/ha. Pesticides that were non-toxic included: fenbuconazole 2F at 585ml/ha, malathion 57EC at 5.85l/ha, FC 435-66 petroleum oil at 46.8l/ha, carbaryl 80S at 3.36kg/ha, chlorpyrifos 4EC at 4.68l/ha, and formetanate 92SP at 1.12kg/ha. Understanding the toxic effects of field weathered pesticides against key predacious mite species is important for effective IPM. The results of this study provide a comparison of direct and indirect toxic effects of various pesticides to A. industani under field conditions.     

Long Range Force between Pre-Replication Complexes (Pre-RC) in DNA Controls Replication and Cell Cycle Progression

A nonstationary interaction, that controls DNA replication and the cell cycle, is derived from a manybody physics model in a chemically open T cell. The model predicts a long range force F()=-(/2) (1-)(2-) between the pre-replication complexes (pre-RCs) bound by DNA, =/N being the relative displacement of preRCs, the number of pre-RCs, N the threshold for initiation, and the compressibility modulus in thelattice of pre-RCs which behaves like an elastically braced string. Initiation of DNA replication is induced by a switch of sign of F(), from attraction (-)and assembly in the G ₁ phase (0 < < N), to repulsion (+) and partialdisassembly in the S phase (N < < 2N), with release of licensing factors from the pre-RCs, thus explaining prevention of re-replication. Replication is terminated by a switch of sign of F at = 2N, when all primed replicons are duplicated once, and F(0)=0 corresponds to a resting cell in absence of driving force at = 0. The switch of sign of force at = N also explains the dynamic instability in growing microtubules (MTs), as well as switch in the interleukin-2 (IL2) interaction with its receptor in late G ₁, at the restriction point. Shape, slope and scale of the response curves derived agree well with experimental data from dividing T cells and polymerizing MTs, the variable length of which is due to anonlinear dependence of the growth amplitude on the initial concentrations of tubulin dimers and guanosine-tri-phosphate (GTP).     

The problem of transition from the chemical to the biological evolution: Some possible solutions

On the basis of evidence that several low-molecular-weight substances as well as enzymes are compartmentalised within the socalled soluble phase of the cell, and other considerations, it is argued that DNA may not contain information for certain types of organisation found in living cells. It may be necessary for a cell to possess the non-DNA-controlled organisation for performance of its minimum functions; such organisation would then also serve as a template for its appearance in the daughter cell. The problem of transition from chemical to biological evolution (that is, the formation of the first cell) may be essentially the problem of emergence of such intracellular organisation for which information may not reside in DNA. Two possible mechanisms through which this may have happened are stated.     

Binding of the galactose-specificPseudomonas aeruginosa lectin,PA-I,to glycosphingolipids and other glycoconjugates

The carbohydrate-binding specificity ofPseudomonas aeruginosa lectin I (PA-I) in iodinated or biotinylated form was studied. A large number of glycosphingolipids, as well as some glycoproteins and neoglycoproteins were used as ligands. Also, inhibition by free saccharides of PA-I binding to glycosphingolipids was tested. It was found that the lectin binds most strongly to terminal and nonsubstituted Gal3Gal- or Gal4Gal-structures.Abbreviations PA-I Pseudomonas aeruginosa lectin I - Cer ceramide - lactosylceramide Gal4GlcCer - iso globotriaosylcerami Gal3Gal4GlcCer - globotriaosylceramide Gal4Gal4GlcCer - globoside or globotetraosylceramide GalNAc3Gal4Gal4GlcCer - Forssman glycolipid GalNAc3GalNAc3Gal4Gal4GlcCer - P1 glycolipid Gal4Gal4GlcNAc3Gal4GlcCer - lactoneotetraosylceramide Gal4GlcNAc3Gal4GlcCer - B5 glycolipid Gal3Gal4GlcNAc3Gal4GlcCer - gangliotetraosylceramide Gal3GalNAc4Gal4GlcCer - GM1 Gal3GalNAc4(NeuAc3)Gal4GlcCer - RBC red blood cells - BSA bovine serum albumin - PBS phosphate-buffered saline - SDS sodium dodecyl sulfate - TLC thin-layer chromatography - HPLC high pressure liquid chromatography - MS mass spectrometry - FAB fast-atom bombardment - EI electron impact     

DNA repair kinetics after exposure to X-irradiation and to internalβ-rays in CHO cells

Summary DNA strand breaks induced by X- or internal-rays were measured using the alkaline unwinding technique. For either type of radiation, repair kinetics were found to be best described by three exponential components, the half-times of which are 2 min, 17 min and 200 min, respectively. These values are the same for X- and internal,-irradiation but the initial fractions of the components are different.     

Action of rat liver Galβ1-4GlcNAc α(2-6)-sialyltransferase on Manβ1-4GlcNAcβ-OMe,GalNAcβ1-4GlcNAcβ-OMe,Glcβ1-4GlcNAcβ-OMe and GlcNAcβ1-4GlcNAcβ-OMe as synthetic substrates

Incubation of synthetic Man\1-4GlcNAc-OMe, GalNAc1-4GlcNAc-OMe, Glc1-4GlcNAc-OMe, and GlcNAc1-4GlcNac-OMe with CMP-Neu5Ac and rat liver Gal1-4GlcNAc (2-6)-sialyltransferase resulted in the formation of Neu5Ac2-6Man1-4GlcNAc-OMe, Neu5Ac2-6GalNAc1-4GlcNAc-OMe, Neu5Ac2-6Glc1-4GlcNAc-OMe and Neu5Ac2-6GlcNAc1-4GlcNAc-OMe, respectively. Under conditions which led to quantitative conversion of Gal1-4GlcNAc-OEt into Neu5Ac2-6Gal1-4GlcNAc-OEt, the aforementioned products were obtained in yields of 4%, 48%, 16% and 8%, respectively. HPLC on Partisil 10 SAX was used to isolate the various sialyltrisaccharides, and identification was carried out using 1- and 2-dimensional 500-MHz¹H-NMR spectroscopy.Abbreviations 2D 2-dimensional - CMP cytidine 5-monophosphate - CMP-Neu5Ac cytidine 5-monophospho--N-acetylneuraminic acid - COSY correlation spectroscopy - DQF double quantum filtered - HOHAHA homonuclear Hartmann-Hahn - MLEV composite pulse devised by M. Levitt - Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid     

Polymorphisms in the human DNA polymerase β gene

Recently, evidence has accumulated that mutations in DNA repair genes might be associated with certain steps in carcinogenesis. The DNA polymerase gene is one of the DNA repair genes, and mutations in it have been detected in 83% of human colorectal cancers. To assess the involvement of polymerase gene mutations in the development of human prostate cancers, we performed sequence analyses of human DNA samples. Unexpectedly, we found six regions that were polymorphic. This information should be taken into consideration at the time of sequence analysis of the DNA polymerase gene.s     

Carcinogenic potential and genomic instability of beryllium sulphate in BALB/c‐3T3 cells

Occupational exposure to beryllium (Be) and Be compounds occurs in a wide range of industrial processes. A large number of workers are potentially exposed to this metal during manufacturing and processing, so there is a concern regarding the potential carcinogenic hazard of Be. Studies were performed to determine the carcinogenic potential of beryllium sulfate (BeSO₄) in cultured mammalian cells. BALB/c3T3 cells were treated with varying concentrations of BeSO₄ for 72 h and the transformation frequency was determined after 4 weeks of culturing. Concentrations from 50–200 g BeSO₄/ml, caused a concentrationdependent increase (9–41 fold) in transformation frequency. Nontransformed BALB/c3T3 cells and cells from transformed foci induced by BeSO₄ were injected into both axillary regions of nude mice. All ten Beinduced transformed cell lines injected into nude mice produced fibrosarcomas within 50 days after cell injection. No tumors were found in nude mice receiving nontransformed BALB/c3T3 cells 90 days postinjection. Gene amplification was investigated in Kras, cmyc, cfos, cjun, csis, erbB2 and p53 using differential PCR while random amplified polymorphic DNA fingerprinting was employed to detect genomic instability. Gene amplification was found in Kras and cjun, however no change in gene expression or protein level was observed in any of the genes by Western blotting. Five of the 10 transformed cell lines showed genetic instability using different random primers. In conclusion, these results indicate that BeSO₄ is capable of inducing morphological cell transformation in mammalian cells and that transformed cells induced by BeSO₄ are potentially tumorigenic. Also, cell transformation induced by BeSO₄ may be attributed, in part, to the gene amplification of Kras and cjun and some BeSO₄induced transformed cells possess neoplastic potential resulting from genomic instability.     

Identification and characterization of the functional alpha origin of DNA replication of the R6K plasmid and its relatedness to the R6K beta and gamma origins

Summary The functional R6K origin is composed of two DNA elements, one of 580 bp carrying the origin sequences and the other of 277 bp containing the seven 22 bp direct repeats previosly identified as also required for and origin activity. These two genetic elements are separated by approximately 3,000 bp of R6K sequences which are dispensable for origin activity. The function of the origin depends on the presence in cis of the 580 bp and the 277 bp fragments and requires that they be oriented as in the intact R6K. Activation of the origin depends on the R6K replication initiation protein .Within the 580 bp of the origin, there is a sequence of 98 bp which appears as an inverted repeat of 96 bp in the replicon. Deletion of the 96 bp or 98 bp results in inactivation of the and the origins respectively. These long repeats are palindromic and it is suggested that these may serve as the recognition signals for initiation of DNA replication in the and the origins of R6K. DNA homology analysis performed on , and origin sequences, also reveals 10–23 bp sequences in the and the origins that are related to the family of 22 bp direct repeats in the origin which were shown previously to be binding sites for the protein.     

Effect of androgens on histochemical fibre type

Summary The temporal muscles of the guinea pig show a sexual differentiation reflected in their histochemical enzyme pattern. Using histochemical methods for mitochondrial (SDH, -GPDH), and glycolytic enzymes (phosphorylase, LDH) it could be shown, that in adult animals the male muscle is a white muscle with marked activity of glycolytic enzymes, the female muscle a red muscle displaying high activity of mitochondrial enzymes. This differential enzyme pattern can be converted by the application of testosterone to the female type during the postnatal development. The male sex hormone thus affects the histochemical enzyme pattern of the muscle, converting the red, female into a white, male muscle in the female guinea pig.     

Short- and long-term effects of ACTH on the adrenal zona glomerulosa of the rat

Summary Short-term ACTH treatment provoked a decrease in volume of the lipid-droplet compartment in rat zona glomerulosa cells, and a rise in plasma and intracellular concentrations of corticosterone and aldosterone. It enhanced activities of 3-hydroxysteroid dehydrogenase (3HSD), 11-hydroxylase (11OH) and 18-hydroxylase (18OH). Long-term ACTH administration produced a hypertrophy of the zona glomerulosa and its parenchymal cells, a result of the increase in volume of the smooth endoplasmic reticulum and the mitochondrial compartment. The surface area per cell of mitochondrial inner membranes increased; the tubular cristae were transformed into a homogeneous population of vesicles. The plasma and intracellular concentrations of corticosterone further increased, whereas those of aldosterone fell below basal levels (the aldosterone-escape phenomenon). The activities of 3HSD and 11OH were enhanced, that of 180H decreased. Therefore, ACTH stimulates zona glomerulosa growth and transforms parenchymal elements into zona fasciculata celltypes. Cyanoketone nullified acute ACTH effects on plasma and intracellular concentrations of corticosterone and aldosterone, but did not affect the activities of 11OH and 18OH. Chronic ACTH treatment produced similar results, although 18OH activity was not suppressed. The mechanism underlying the aldosterone-escape phenomenon may thus involve a rise in the intracellular concentration of corticosterone, caused by the enhanced synthesis and activation of 3HSD and 11OH.     

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.     

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.     

Studies on aneuploids of Petunia

Summary The seven possible primary trisomics of Petunia (2 n= 14) located in the progenies of triploid, hypertriploid and hypotriploid plants were distinguished from one another and from diploid on the basis of cytological and morphological criteria. They were provisionally named as Oval, Semi, Slender, Pseudonormal, Arrow, Narrow and Giant. In three of the trisomics, the extra chromosome was identified for the first time at pachytene stage. Postpachytene studies revealed no precise relationship between the length of extra chromosome and the frequency of multiple association.     

Spermatogonia and the cycle of the seminiferous epithelium in the nine-banded armadillo

Summary The cycle of the seminiferous epithelium of the nine-banded armadillo can be divided into ten stages. As in most mammals, only one stage is observed per tubular cross-section. The process of spermiogenesis can be divided into thirteen steps according to the development of the acrosomal system and the flagellum. Four generations of spermatogonia are observed in the germinal epithelium: 1) stem cells, 2) type A, 3) intermediate, and 4) type B spermatogonia. The stem cell is characterized by a highly irregular nucleus and the presence of glycogen in its cytoplasm. The type A spermatogonium contains an oblong nucleus with one or two shallow infoldings of the nuclear membrane. The intermediate spermatogonium contains an ovoid nucleus characterized by one or two nuclei and heterochromatin scattered in the nucleoplasm. The nucleus of the type B spermatogonium is more spherically shaped with a centrally placed nucleolus and heterochromatin associated with the nuclear envelope.The author wishes to acknowledge the technical assistance of Teri Lane     

Urease from Staphylococcus saprophyticus: purification,characterization and comparison to Staphylococcus xylosus urease

Urease from Staphylococcus saprophyticus was purified more than 800-fold by liquid chromatography reaching homogeneity, as shown by isoelectric focussing, at a maximum specific activity of 1979 U/mg. The molecular weight of the native enzyme was 420000; it consisted of subunits with molecular weights of 72400 (), 20400 (), and 13900 () in an estimated ()₄ stoichiometry. In native gradient polyacrylamide gel electrophoresis urease exhibited a multiple activity band pattern with molecular weights ranging from 420000 to 100000. In the native enzyme, 4.09 (±0.25) atoms of nickel per molecule were detected. The N-terminal amino acids of the urease subunits were identical to those from Staphylococcus xylosus, and amino acid analysis revealed high similarities in both enzymes; no cysteine was detected after acid hydrolysis of vinylpyridinylated urease. Electron micrographs of negatively stained urease specimens from both staphylococci showed identical size and structure.Abbreviation PAGE polyacrylamide gel electrophoresis     

Transforming growth factor-beta isoform expression in mature human healthy and carious molar teeth

Transforming growth factor (TGF)- isoforms have been implicated in cellular signalling during tooth development and repair, but little is known of their cellular localisation or distribution within the dental tissues in the mature tooth. This study investigated the presence of TGF-1, 2 and 3 isoforms in tissues of sound and carious human molar teeth, to understand better the expression of TGF-s during health and disease. In healthy tissues, odontoblasts, cells of the cell rich layer, pulpal fibroblasts and endothelial cells were stained to varying degrees for all isoforms, with TGF-3 showing the greatest intensity and TGF-1 the weakest intensity. Similar patterns of staining were observed in carious teeth; however, TGF-1 showed significantly increased staining intensity within odontoblasts and pulpal cells of carious teeth (p<0.001). Biochemical analysis showed greater amounts of TGF-1 in tertiary dentine than in primary dentine samples. The expression of TGF-s in odontoblasts and the increased presence of TGF-1 in tertiary dentine suggest that these isoforms may be important in odontoblast behaviour and the modulation of the tissue response to injury.     

Saccharomyces cerevisiae 2-mum DNA. An analysis of the monomer and its multimers by electron microscopy.

Summary The non-tandem inverted duplication in the 2-m DNA of Saccharomyces cerevisiae has a length of 0.19 m and is located asymmetrically along the molecule. The majority of the dumb-bell structures that are formed upon denaturation and selfannealing of the 2-m monomer consists of the renatured inverted duplication sequences as double stranded stem and two single stranded loops of 0.67 m±0.06 m (S-loop) and 0.86 m±0.05 m (L-loop) length. Two additional size classes which comprised 5–10% of the measured molecules had contour lengths of around 1.7 m and 2.1 m. The smaller dumb-bells contained two S-loops and the larger dumb-bells contained two L-loops as was shown by heteroduplex mapping with an HindIII fragment from the L-loop. Two models which assume illegitimate or site specific recombination, are presented to explain the generation of double S-loop and double L-loop molecules. At least part of the 4-m and 6- circular molecules present in the yeast supercoiled DNA fraction are shown to be dimers and trimers of 2-m monomers, but often with inverted loop segments most probably due to intramolecular recombination between sequences of the inverted duplication.2-m DNA is used to indicate the supercoiled DNA fraction although in our measurements the average monomeric length is 1.9 mPart of this work has been presented at the Conference: The Genetics and Biogenesis of Chloroplasts and Mitochondria, Munich, August, 1976