Refined structure of human carbonic anhydrase II at 2.0 Å resolution

The structure of human erythrocytic carbonic anhydrase II has been refined by constrained and restrained structure–factor least-squares refinement at 2.0 Å resolution. The conventional crystallographic R value is 17.3%. Of 167 solvent molecules associated with the protein, four are buried and stabilize secondary structure elements. The zinc ion is ligated to three histidyl residues and one water molecule in a nearly tetrahedral geometry. In addition to the zinc-bound water, seven more water molecules are identified in the active site. Assuming that Glu-106 is deprotonated at pH 8.5, some of the hydrogen bond donor–acceptor relations in the active site can be assigned and are described here in detail. The Oγ1 atom of Thr-199 donates its proton to the Oε1 atom of Glu-106 and can function as a hydrogen bond acceptor only in additional hydrogen bonds.     

Genetic and blood coagulation characterization of “Swedish” families with von Willebrand's disease types I and III: New aspects of heredity

Summary Twenty-five patients with von Willebrand's disease (vWD) type III were analysed with regard to blood coagulation variables and possible deletions. Nine of the probands and their families were further investigated with DNA linkage analyses. Different patterns of heredity can be suggested in our families with vWD type III, on the basis of blood coagulation analyses. The findings suggest homozygosity in five families and the possibility of compound heterozygosity or a new mutation in the proband in three families. The linkage analyses confirm the results of the coagulation analyses. The segregation of the von Willebrand factor (vWF) gene can be followed in the families, and carrier diagnosis can be made in several of the probands' relatives. The possibility of large deletions in the vWF gene of the probands and their parents was investigated with probes representing the whole vWF cDNA. No deletions were found.This study was approved by the Ethics Committee of the Karolinska Hospital (No. 84:1)     

Regeneration of transgenic plants of Prunus armeniaca containing the coat protein gene of Plum Pox Virus

Summary A system was developed which allows the transfer of foreign genes into apricot cultivars. We report the transformation and regeneration of Prunus armeniaca plants with Agrobacterium tumefaciens strain LBA 4404 containing various binary plasmids, pBinGUSint, carrying the marker gene ß-glucuronidase (GUS) and pBinPPVm, carrying the coat protein gene of Plum Pox Virus (PPV). The marker gene GUS was used for optical evaluation of the efficiency of the transformation system. The coat protein gene of PPV was used to introduce coat protein mediated resistance against one of the most important pathogens of stone fruit trees in Europe and the whole Mediterranean area. This is the first report of the successful integration of a viral coat protein gene into a fruit tree species, opening a new perspective on the control of the disease.Abbreviations GUS ß-glucuronidase - PPV Plum Pox Virus - BA 6-benzylaminopurine - NPTII neomycin phosphotransferase II - CP coat protein - CaMV Cauliflower Mosaic Virus - P35S 35S promoter - MS Murashige and Skoog - PCR polymerase chain reaction - P/C/I phenol/chloroform/isoamylalcohol - RNase ribonuclease - dNTP deoxyribonucleosidetriphosphate - DMSO dimethyl sulfoxide     

Methyljasmonate and α-linolenic acid are potent inducers of tendril coiling

A coiling-inducing factor was isolated from tendrils of Bryonia dioica Jacq. and identified by infrared, ¹H-, ¹³C-nuclear magnetic resonance and mass spectrometry as -linolenic acid. When applied to detached tendrils, exogenous -linolenic acid, but not linoleic acid or oleic acid, induced tendril coiling. Further investigations showed that metabolites of -linolenic acid, jasmonic acid and, even more so, methyljasmonate, are highly effective inducers of tendril coiling in B. dioica. Methyljasmonate was most active when administered by air and, in atmospheric concentrations as low as 40–80 nM, induced a full free-coiling response with kinetics similar to mechanical stimulation. Even atmospheric levels as low as 4–5 nM methyljasmonate were still found to be significantly active. Methyljasmonate could be one of the endogenous chemical signals produced in mechanically stimulated parts of a tendril and, being highly volatile, act as a diffusible gaseous mediator spreading through the intracellular spaces to trigger free coiling of tendrils.Abbreviations EI-MS electron impact-mass spectrometry - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - NMR nuclear magnetic resonance - TFA trifluoroacetic acid We are indebted to the Deutsche Forschungsgemeinschaft, Bonn and the Fonds der Chemischen Industrie, Frankfurt (literature provision) for their support and to Dr. C. Brückner, Halle, for jasmonic-acid determinations.     

A comparative study into the chemical constitution of cutins and suberins from Picea abies (L.) Karst., Quercus robur L., and Fagus sylvatica L.

The compositions of BF₃/CH₃OH depolymerisates of cutins and suberins from leaf and periderm samples from Picea abies [L.] Karst., Quercus robur L., and Fagus sylvatica L., respectively, were determined by quantitative capillary gas chromatography/mass spectroscopy. Long-chain monobasic, -hydroxymonobasic, dihydroxymonobasic, trihydroxymonobasic and epoxyhydroxymonobasic alkanoic acids constituted the major aliphatic monomers of leaf cutins. The total amounts of cutin monomers ranged from 629 mg · m^–2 (Fagus) to 1350 mg · m^–2 (Quercus). Cutin composition and amounts did not significantly differ between current year and three-year-old needles of Picea. Trans-esterification of periderm samples yielded a much greater variety of aliphatic monomers than obtained from cutins. In addition to the substance classes found with cutins, suberin depolymerisates also contained , -dibasic acids while dihydroxymonobasic acids were lacking. Depolymerisates from periderms taken from different locations on a Picea tree did not differ significantly in their relative composition. The results are discussed in terms of the distinctive characteristics of the aliphatic portions of cutins and suberins, respectively. Discriminant analysis is applied for formulating a quantitative and inarbitrary classification rule for cutins and suberins. The precision, statistical significance and robustness of this classification rule are tested by employing it to a large set of compositional data (70 plant species) from the literature. The relevance of data obtained by depolymerization methods for elucidating the physical structure of cutins and suberins in situ is evaluated.To whom correspondence should be addressedThe authors are indebted to Drs. J. Winkler and H. Krause (Laboratorium für Strukturchemie des Fachbereichs Chemie, Biologie und Geowissenschaften, Technische Universität München, Garching, FRG) for performing capillary gas chromatography-mass spectrometry and their valuable help in the identification of cutin and suberin constituents. The work was supported by grants from the Deutsche Forschungsgemeinschaft and the Bayerisches Staatsministerium für Unterricht, Kultus, Wissenschaft und Kunst.     

Differential inactivation and methylation of a transgene in plants by two suppressor loci containing homologous sequences

In a previous study on doubly transformed tobacco plants, we observed the unexpected inactivation in trans of T-DNA-I (encoding Kan^rNOS) following the introduction into the same genome of an unlinked copy of T-DNA-II (encoding Hyg^rOCS). This inactivation, which probably resulted from interactions between homologous regions on each T-DNA, was correlated with methylation in the nos _pro, which controlled the expression of both the nptII and nos genes. In this paper, we show that the inactivation and methylation of the nos _pro nptII gene in the presence of a suppressor T-DNA-II locus can be either complete (epistasis) or partial (cellular mosaicism). In plants showing partial suppression, the strength of the Kan^r phenotype, which apparently reflected the proportion of cells expressing the nptII gene, was inversely correlated with the degree of methylation of the nos _pro. The extent of nos _pro methylation decreased progressively in successive generations as suppressor T-DNA-II loci were crossed out. The strength of the Kan^r phenotype was improved and nos _pro methylation was less extensive in first generation Kan^r progeny obtained from outcrossing with untransformed tobacco than from self-fertilization.     

Assignment by in situ hybridization of a fibroblast growth factor receptor gene to human chromosome band 10q26

Summary A 2.3-kb cDNA probe for the human bek fibroblast growth factor receptor was used to determine the chromosomal localization of the corresponding gene by in situ hybridization. The results show that this gene, a form of which is amplified in some poorly differentiated stomach cancers, is localized on chromosome region 10q26. The two previously identified fibroblast growth factor receptor genes are thus not on the same chromosome, as the related fig (fms-like gene) fibrovblast growth factor receptor gene has previously been mapped to human chromosome region 8p12.     

Regular and irregular divisions of Lepidoptera spermatocytes as related to the speed of meiotic prophase

Lepidoptera males bear two kinds of meiotic divisions. One is regular (eupyrene) and leads to nucleate, fertilizing spermatozoa. The other (apyrene) shows metaphase I chromosomes clumping together into irregular masses which later split forming daughter cells with unbalanced sets of chromosomes which are eventually extruded from the cells; hence, the spermatids develop into anucleate spermatozoa of unknown function. The apyrene divisions are induced by a haemolymph factor which becomes functional towards pupation. Using incorporation of tritiated thymidine at the premeiotic S-phase as a marker for timing, it was found that the prophase of the apyrene spermatocyte is shorter than that of the eupyrene spermatocyte. It is proposed that meiosis-specific proteins cannot be synthesized during the shortened apyrene prophase and that this is correlated with the irregular chromosome behaviour during the subsequent metaphase-telophase of these spermatocytes.     

Synthesis of sulfated proteoglycans throughout the cell cycle in smooth muscle cells from pig aorta

Cultured smooth muscle cells from pig aorta arrested in G0 phase by serum deprivation were stimulated to proliferate by replacing the medium with one containing 10% serum. Studies in DNA replication and proliferation of cells showed a relatively good synchrony: 90% of the cells were in G1 phase for 16 h after addition of serum; they entered S phase between 18 and 24 h, completed S phase and traversed G2 phase between 24 and 30–32 h; 75% of these cells multiplied after 30–32 h and the remainder were blocked at the end of G2 phase. The synthesis and secretion of sulfated proteoglycans were examined throughout a full cell cycle using metabolic labelling with [³⁵S]sulfate. Smooth muscle cells in G1 or G2 phase synthesized and secreted sulfated proteoglycans with a possible pause at the end of the G2 phase but at the beginning of the S phase and during mitosis the incorporation of [³⁵S]sulfate into these macromolecules stopped entirely. Structural characteristics of sulfated proteoglycans secreted into the medium during G1 phase and an entire cell cycle were investigated. The proportion of proteoglycan complexes and the relative hydrodynamic size of monomers and of constituent subunits of complexes were determined after chromatography on Sepharose CL-2B and CL-6B columns run under both associative and dissociative conditions. No significant differences were observed for the periods of the cell cycle that were studied:

1. 1. [³⁵S]Proteoglycan complexes represented at the end of G1 phase and of the cell cycle respectively 19 and 16% of the total [³⁵S]proteoglycans secreted into the medium.

2. 2. More than 90% of the subunits, obtained after dissociation of complexes, were characterized by a similar k_av after chromatography on Sepharose CL-2B columns eluted under dissociative conditions (k_av 0.68 at the end of G1 phase and 0.65 at the end of full cell cycle).

3. 3. About 95% of monomers synthesized at the two stages of the cell cycle were eluted at k_av 0.25 after chromatography on Sepharose CL-6B column run under associative conditions and were characterized by a similar glycosaminoglycan distribution. These results suggest that smooth muscle cells in culture liberate similar populations of proteoglycans into the medium during the G1 and G2 phases.