Collagen and fibronectin synthesis by trisomic and triploid fibroblasts from human spontaneous abortuses

Summary Collagen and fibronectin synthesis by trisomic and triploid fibroblasts derived from human spontaneous abortuses was studied. It was demonstrated that the level of fibronectin and collagen production in fibroblasts with trisomy 7, trisomy 9, and triploidy was reduced as compared with diploid cells. A correlation between this observation and an increased rate of intracellular ¹⁴C-procollagen degradation was also established for the anomalous strains. No difference in hydroxylation of ¹⁴C-proline residues in 1() and 2() collagen chains and no fluctuation in the collagen type (): type ratio was found in the strains with the abnormal karyotypes. It was concluded that differentiation of the abnormal fibroblasts was impaired. The data also favour the hypothesis that the deficiency of the fibroblasts in producing proteins may account for a variety of anatomic abnormalities of embryos.     

Biochemical genetics of chromosome forms of Venezuelan spiny rats of the proechimys guairae and proechimys trinitatis superspecies

Spiny rats from Venezuela show an extensive karyotypic diversification (2n=24 to 2n=62) and little morphological differentiation. This study reports genetic distance, heterozygosity and polymorphism based upon 22 loci in semispecies and allospecies of the Proechimys guairae superspecies from N Central Venezuela, as compared with Proechimys urichi, a member of the Proechimys trinitatis superspecies from eastern Venezuela. Four chromosome forms of the P. guairae complex are included, each characterized by karyotypes of 2n=46 (Fundamental Number=72), 2n=48 (FN=72), 2n=50 (FN=72) and 2n=62 (FN=74). Proechimys urichi has a distinetive karyotype of 2n=62 (FN=88). The overall mean value of Nei's genetic identity index for all pair-wise comparisons is I=0.942±0.011. Mean identity within the P. guairae complex is =0.969±0.033. Mean identity between P. urichi and members of that complex is =0.889±0.011. Within the P. guairae complex, increased genetic divergence is correlated with higher karyotypic divergence. Heterozygosity varies from H=0.059 to H=0.153, with a mean value of H0.059. The mean percent of polymorphic loci is P=18.2±3.9 after the 0,95% polymorphism criterion, and P=20,5±5.2 after the 0.99% criterion. These results are compared with similar data from fossorial and non-fossorial rodents. Spiny rats are non-fossorial, forest-dwelling rodents which have undergone a speciation process with little genetic divergence and extensive chromosome rearrangements.     

Variation in the karyotype of three cultivars of Narcissus tazetta L. (Amaryllidaceae)

Three cultivated varieties of Narcissus tazetta, Grand Soleil d'Or, Chinese Sacred Lily and Cypri, are triploid (2n = 3x+30) with the basic number 10. Grand Soleil d'Or has three homomorphic sets, each comprising 2 long submetacentrics, 4 long acrocentrics and 4 short acrocentrics. Karyotypes of the other two varieties are heteromorphic. Both possess one telocentric satellited chromosome. In addition, Cypri shows translocation between two chromosomes belonging to the seventh and eighth triplets. The number of secondary constrictions varies between 3 (Chinese Sacred Lily) and 4 (Grand Soleil d'Or and Cypri) which is also the number of nucleoli observed in the respective varieties.     

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     

Purification and characterization of DNA polymerase alpha of chinese hamster ovary cells

Summary The major pol activity of CHO cells was purified 2 800-fold to near homogeneity and was characterized with respect to its physical and catalytic properties. The purified enzyme, upon analysis in denaturing activity gels, displayed a major, 120 kilodalton, catalytically active core and two minor, catalytically inactive components of 180 and 135 kilodaltons. The native form of the enzyme behaved in velocity sedimentation and gel permeation experiments as an asymmetric protein of an apparent Mr. of 515 kilodaltons. The purified enzyme displayed catalytic behavior and inhibitor sensitivity typical of that displayed by other mammalian pol alphas. Specifically, the enzyme: (1) was sensitive to n-ethylmaleimide and the pol -specific inhibitors, BuPdGTP and aphidicolin; (2) was subject to neutralization by specific monoclonal antibodies raised against human pol ; (3) was devoid of detectable 3 to 5 exonuclease activity, and (4) displayed a ribonucleotide-dependent DNA primase activity.     

Glycosylated human interleukin-1α, neoglyco IL-1α, coupled with N-acetylneuraminic acid exhibits selective activities in vivo and altered tissue distribution

In order to study the effect of glycosylation on its biological activities and to develop IL-1 with less deleterious effects, N-acetylneuraminic acid (NeuAc) with C9 spacer was chemically coupled to human recombinant IL-1. NeuAc-coupled IL-1 (NeuAc-IL-1) exhibited reduced activities in vitro and receptor-binding affinities by about ten times compared to IL-1. In this study, we examined a variety of IL-1 activities in vivo. NeuAc-IL-1 exhibited a marked reduction in the activity to up-regulate serum IL-6, moderate reduction in the activities to up-regulate serum amyloid A and NOx. However, it exhibited comparable activities as IL-1 to down-regulate serum glucose and to improve the recovery of peripheral white blood cells from myelosuppression in 5-fluorouracil-treated mice. In addition, tissue level of NeuAc-IL-1 was high compared to IL-1. These results indicate that coupling with NeuAc enabled us to develop neo-IL-1 with selective activities in vivo and enhanced tissue level.     

Efficient determination of angles subtended by Cα-Hα and N-HN vectors in proteins via dipole-dipole cross-correlation§

The angle CH,NHN subtended by the internuclear vectors 13C-H and 15N-HN in doubly-labeled proteins can be determined by observing the effect of cross-correlation between the dipolar interactions on zero- and double-quantum coherences involving 13C and 15N. Two complementary 2D experiments with the appearance of 15N-HN correlation spectra yield signal intensities that depend on the rate of interconversion through cross-correlated relaxation of in-phase and doubly antiphase zero- and double-quantum coherences. The ratio of the signal intensities in the two experiments bears a simple relationship to the cross-correlation rate, and hence to the angle CH,NHN. Assuming planarity of the peptide bond, the dihedral angle (between C and C) can be determined from the knowledge of CH,NHN. The experiments are very time-effective and provide good sensitivity and excellent spectral resolution.     

High-level Expression of Maize <Emphasis Type="Italic">γ</Emphasis>-zein Protein in Transgenic Soybean (<Emphasis Type="Italic">Glycine max</Emphasis>)

Primers were developed for 118 microsatellites isolated from grape (Vitis vinifera) genomic libraries enriched for (AC)n repeats. Only one microsatellite sequence matched other grape SSR-sequences in the GeneBank database. Genotyping was carried out in the parental lines and four offspring of two pseudo-test-cross populations, Cabernet Sauvignon x Seyval and Chardonnay x Bianca, and a further six other grape genotypes (V. vinifera Sultanina, Merlot, Syrah, Müller-Thurgau, Vitis Regent and V. riparia Gloire de Montpellier). A total of 108 microsatellites showed easily scorable alleles and 100 of them segregated according to a configuration suitable for mapping in either cross. A further 8 SSRs, although unsuitable for mapping in those crosses, showed polymorphism in the other genotypes tested. This set of markers was used, along with 75 microsatellites of other repeat-types, to fingerprint 46 offspring of the cross Chardonnay x Bianca. For each full-sib, individual heterozygosity and distance in repeat units between pairs of alleles at each locus (mean d²) were calculated as a tool for predicting highly outbred recombinant individuals. Six microsatellites with segregation ratios significantly distorted towards the lack of homozygous sibs were identified and mapped to linkage groups LG 3 and LG 5. Estimation of heterozygosity at genome-wide level and genotyping at loci for which homozygous sibs are discriminated against are discussed for marker-assisted background selection in outcrossing grapevines.     

C alpha and C beta carbon-13 chemical shifts in proteins from an empirical database

We have constructed an extensive database of 13C C and C chemical shifts in proteins of solution, for proteins of which a high-resolution crystal structure exists, and for which the crystal structure has been shown to be essentially identical to the solution structure. There is no systematic effect of temperature, reference compound, or pH on reported shifts, but there appear to be differences in reported shifts arising from referencing differences of up to 4.2 ppm. The major factor affecting chemical shifts is the backbone geometry, which causes differences of ca. 4 ppm between typical - helix and -sheet geometries for C, and of ca. 2 ppm for C. The side-chain dihedral angle 1 has an effect of up to 0.5 ppm on the C shift, particularly for amino acids with branched side-chains at C. Hydrogen bonding to main-chain atoms has an effect of up to 0.9 ppm, which depends on the main- chain conformation. The sequence of the protein and ring-current shifts from aromatic rings have an insignificant effect (except for residues following proline). There are significant differences between different amino acid types in the backbone geometry dependence; the amino acids can be grouped together into five different groups with different , shielding surfaces. The overall fit of individual residues to a single non-residue-specific surface, incorporating the effects of hydrogen bonding and 1 angle, is 0.96 ppm for both C and C. The results from this study are broadly similar to those from ab initio studies, but there are some differences which could merit further attention.     

Inverse relationship between poly (ADP-ribose) polymerase activity and 2′,5′-oligoadenylates core level in estrogen-treated immature rat

Summary The activity of poly (ADP-ribose) polymerase (ADPRP) and the content of 2,5-oligodenylates core (2,5A_n; n = 2,3 and 4) were measured in homogenates of the uterus and of the liver of immature rats immediately before (time 0) or at different times after injection of estradiol-valerate. ADPRP activity increased gradualy, starting 6 hours after estrogen injection, for about 4 days. Instead, the content of 2,5 A_n decreased by about 50% within 6 hours, and thereafter more slowly for 4 days to about 20% of starting values. Estrogen increased ADPRP activity and decreased 2,5A_n concentration also in the kidney and in the cardiac muscle of the same animals, but not in the skeletal muscle, where neither of the two parameters was affected. Injection of vehicle only (sesame oil) had no effect on ADPRP activity nor on 2,5A_n content of immature rat tissues.     

Genetic analysis of two wheat cultivars, ‘Sonalika’ and ‘WL 711’ for reaction to leaf rust (Puccinia recondita)

Summary Genetic analysis for leaf rust reaction of two widely adapted cultivars, Sonalika and WL 711, has been done using 21 near isogenic Lr lines and rust culture IL004 — avirulent on the two cultivars and all the Lr lines used. The segregation pattern in the F₂ generation indicated the presence of a recessive gene in Sonalika and of a dominant gene in WL 711. These genes in cultivars Sonalika and WL 711 have been identified as Lr 11 and Lr 13, respectively. Gene Lr 13 is no longer effective in WL 711 but it continues to give field resistance in the backgrounds of Chris, Prelude and Thatcher. There has been no significant change in the virulence spectrum of the leaf rust pathogen in India with the release of WL 711. High susceptibility of WL 711 seems to be due to the evolution of more aggressive forms of the pathogen to this cultivar. The gene Lr 11, which behaves as a recessive in Sonalika, was effective against leaf rust when this cultivar was released. The high susceptibility of Sonalika is probably due to an increase in the frequency of race 77 virulent on Lr 11. Lr 11 has shown a dominance reversal in the background of Sonalika. Present results suggest that interaction of resistance genes with the background genotype must be studied for their effective use in breeding programme.     

Monoclonal antibody specific for α2→8-linked oligo deaminated neuraminic acid (KDN) sequences in glycoproteins. Preparation and characterization of a monoclonal antibody and its application in immunohistochemistry

Two particular types of sialoglycoproteins have been detected in fish: polysialoglycoproteins containing 28-linked polysialic acid (8Neu5Gc2) _n present in unfertilized Salmonidae fish eggs, and glycoproteins bearing oligo/polymers of deaminated neuraminic acids (KDN) found in the vitelline envelope of the eggs and ovarian fluid. We report the preparation and characterization of a monoclonal antibody specifically recognizing oligo/polymers of KDN sequences in glycoproteins and its application in immunohistochemistry. Fusion of spleen cells from a BALB/c mouse immunized with a KDN-rich glycoprotein (KDN-gp) containing (8KDN2) _n 6(KDN23Gal13GlNAc13) GalNAc1 residues, with mouse myeloma cells yielded a hybrid cell line producing a monoclonal antibody that bound to KDN-gp, but not to KDN-gp depleted of KDN residues. The specificity of the monoclonal antibody, designated mAb.kdn8kdn, was determined by an enzyme-linked immunosorbent assay using KDN-gp samples that varied in KDN content. These antigens were prepared by the selective removal of KDN residues from the native KDN-gp. The mAb.kdn8kdn reacted most strongly with the intact KDN-gp and less strongly with KDN-gp samples containing decreased numbers of KDN residues. The mAb.kdn8kdn was shown specifically to recognize the 28-linked oligo/polyKDN sequences, (8KDN2) _n , and to be able to distinguish specifically (8KDN2) _n chains from (8Neu5Ac2) _n and (8Neu5Gc2) _n chains. The antibody was used successfully for the immunohistochemical detection of reactive KDN epitopes in sections of paraffin embedded rat pancreas. Several controls verified the specificity of the immunohistochemical staining, thus providing the first demonstration of (8KDN2) _n sequences in a mammalian tissue. The mAb.kdn8kdn can now be used to search further for glycoconjugates containing (8KDN2) _n chains and will facilitate studies on their biosynthesis, intracellular localization and function.     

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.     

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     

Quantitative utilization of selected grasses by fall armyworm larvae

Food utilization by larvae of the fall armyworm (Spodoptera frugiperda [J. E. Smith]) (Lepidoptera: Noctuidae), showed greater consumption of corn (Zea mays L.) than pinto bean diet, Tifton 10, or Coastal bermudagrass (Cynodon dactylon [L.] Pers.). Transfer of larvae from diet to susceptible grasses such as corn, Tifton 10 or Coastal produced differences in growth rates as a result of food consumption rates. Transfer of larvae from diet to resistant grasses such as common centipedegrass (Eremochola ophiuroides [Munro] Hack) Tifton 292 bermudagrass, and zoysiagrass (Zoysia sp.) reduced larval growth as a result of low consumption rates and/or greater metabolic expenditures. Larvae initially fed Tifton 10, Coastal, or centipedegrass before feeding on corn grew significantly faster than when they fed continuously on corn.

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Résumé Des chenilles de S. frugiperda ont consommé plus de maïs (Zea mays), que d'un régime à base de Phaseolus vulgaris de la variété Pinto, ou de Tifton 10, ou de Cynodon dactylon de la variété Coastal. Des chenilles, transférées du régime artificiel sur des plantes sensibles comme le maïs, le Tifton 10 ou le Coastal, ont présenté des taux de croissance différents provenant de modifications de leur consommation. Le transfert du régime à des plantes résistantes, telles que Eremochola ophiuroides, Tifton 292, C. dactylon et Zoysia sp., a entraîné une diminution de la croissance larvaire par suite d'une plus faible consommation et/ou de dépenses métaboliques plus élevées. Des chenilles ayant consommé du Tifton 10, du Coastal ou de l'E. ophiuroides avant de consommer du maïs, se sont développées significativement plus vite que celles qui s'étaient alimentées continuellement sur maïs.

     

Untersuchungen zum C′3-Polymorphismus (β 1c-Globulin)

Zusammenfassung Serumproben von 1322 Blutspendern aus Hessen, 40 Familien mit 89 Kindern, 20 Mutter-Kind-Kombinationen und 268 Sera einer Bantupopulation aus Portugiesisch Angola wurden mit einer modifizierten Technik der Hochspannungs-Dünnschichtelektrophorese auf Agarosegel hinsichtlich des C3-Polymorphismus untersucht. Die Genfrequenzen für Weiße (C3^S=0.779, C3^F=0.215) und Neger (C3^S=0.95, C3^F=0,048) stimmen gut mit den Werten anderer Autoren überein. Insgesamt ließen sich bei Weißen 9 Phänotypen sicher abgrenzen, bei Negern 3. Familienstudien bestätigten den für die Allele C3^S und C3^F angenommenen Vererbungsmodus (autosomal codominant) ausnahmslos. Die Frage der Lagerungsstabilität des C3 wurde abschließend untersucht.

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Investigations on C3-polymorphism ( _1c-Globulin)Gene frequencies and family studies in blood donors from Hessen and a Bantu population

Summary Serum samples of 1322 unrelated individuals from Hessen (Germany), 40 families with 89 children, 20 mother-child-combinations and 268 sera of a Bantu population from Angola were examined for C3 polymorphism using a modified technique of high voltage agarose gel electrophoresis. The gene frequencies for Caucasians (C3^S=0.779, C3^F=0.215) and negroes (C3^S=0.95, C^F=0.048) are in good accordance with those obtained by other authors. In total 9 different phenotypes were observed in Caucasians, 3 phenotypes in negroes. Family studies verify the supposed way of inheritance (autosomal codominant for C3^S and C3^F) without exception. Finally the problem of C3-inactivation by storage was investigated.

     

Plant regeneration from Citrus protoplasts: Variability in methodological requirements among cultivars and species

Summary Nucellar callus lines were established from two orange cultivars (Nucellar Shamouti, Shamouti Landau), three mandarin cultivars (Murcott, Dancy, Ponkan) one grapefruit cultivar (Duncan) and sour orange (Citrus aurantium). These callus lines were initiated from in vitro cultured ovules of young fruits and maintained an embryogenic capacity. The plating efficiencies of protoplasts derived from these calli, as well as those of protoplasts from lemon (cv. Villafranca) nucellar callus were differentially affected by the maceration enzymes and by the sugars used as osmotic stabilizers. Plants with normal morphological features were regenerated from cultured protoplasts derived from each of the nucellar callus lines. The establishment of eight new protoplast systems in Citrus paves the way for cell genetics studies and for novel breeding approaches in these economically important orchard trees.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. E-290, 1981 series     

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.     

Chromosomal localization of genes required for the terminal steps of oxidative metabolism: α and γ subunits of ATP synthase and the phosphate carrier

The terminal steps of oxidative phosphorylation include transport of phosphate and ADP into the mitochondrial matrix, synthesis of ATP in the matrix, and transport of the product ATP into the cytosol where it can be utilized to perform cellular work. Three nuclear genome encoded membrane proteins, namely, the phosphate carrier (PHC), the adenine nucleotide carrier (ANT), and the ATP synthase complex, consisting of at least 13 individual subunits, catalyze these reactions. The locations of the and subunits of the mitochondrial ATP synthase complex and the mitochondrial phosphate carrier, PHC, on human chromosomes were determined using cloned rat liver cDNA as probes. Human homologues of the subunit are on chromosomes 9 and 18, the subunit are on chromosomes 10 and 14, and the PHC was localized to chromosome 12.     

Altered synthesis and stability of RNA polymerase holoenzyme subunits in mutants of Escherichia coli with mutations in the β or β′ subunit genes

Summary Bacteria with specific temperature sensitive lethal mutations in the gene for the subunit of RNA polymerase synthesize both the and subunits at a several fold higher rate at 42°C than wildtype cells relative to total protein. Synthesis of the and subunits proceeds at essentially the wild-type rates under these conditions. In contrast, a mutant with a temperature sensitive lethal mutation in the subunit gene synthesizes and at 42°C at slightly lower rates than wild-type, while and synthesis is not significantly altered. In all of the mutants at 42°C, newly synthesized subunits are stable, while the , and subunits are rapidly degraded. The apparent uncoupling of from subunit synthesis seen in the mutants at 42°C might suggest that the synthesis of these subunits is at least in part controlled by different mechanisms.