Adventitious root formation of kiwifruit in relation to sampling date, IBA and Agrobacterium rubi inoculation

During the winters of 2001 and 2002, the effects of three strain of Agrobacterium rubi (A1, A16 and A18), a range of IBA concentrations (0, 2000, 4000 and 6000 ppm) alone and in combination with three strains of Agrobacterium rubi and date of cutting collection on the rooting of hardwood stem cuttings of kiwifruit cv. Hayward were evaluated. Treatments of hardwood stem cuttings of kiwifruit cv. Hayward with the bacteria, IBA and IBA plus bacteria were found to promote rooting. Highest rooting percentage was obtained from cuttings treated with 4000 ppm IBA plus A18 in cv. Hayward in both years. Higher rooting percentages were observed when shoots were collected in February rather than in January.     

pEg7, a New Xenopus Protein Required for Mitotic Chromosome Condensation in Egg Extracts

We have isolated a cDNA, Eg7, corresponding to a Xenopus maternal mRNA, which is polyadenylated in mature oocytes and deadenylated in early embryos. This maternal mRNA encodes a protein, pEg7, whose expression is strongly increased during oocyte maturation. The tissue and cell expression pattern of pEg7 indicates that this protein is only readily detected in cultured cells and germ cells. Immunolocalization in Xenopus cultured cells indicates that pEg7 concentrates onto chromosomes during mitosis. A similar localization of pEg7 is observed when sperm chromatin is allowed to form mitotic chromosomes in cytostatic factor-arrested egg extracts. Incubating these extracts with antibodies directed against two distinct parts of pEg7 provokes a strong inhibition of the condensation and resolution of mitotic chromosomes. Biochemical experiments show that pEg7 associates with Xenopus chromosome-associated polypeptides C and E, two components of the 13S condensin.     

Can we build synthetic,multicellular systems by controlling developmental signaling in space and time?

Using biological machinery to make new, functional molecules is an exciting area in chemical biology. Complex molecules containing both 'natural' and 'unnatural' components are made by processes ranging from enzymatic catalysis to the combination of molecular biology with chemical tools. Here, we discuss applying this approach to the next level of biological complexity -- building synthetic, functional biotic systems by manipulating biological machinery responsible for development of multicellular organisms. We describe recent advances enabling this approach, including first, recent developmental biology progress unraveling the pathways and molecules involved in development and pattern formation; second, emergence of microfluidic tools for delivering stimuli to a developing organism with exceptional control in space and time; third, the development of molecular and synthetic biology toolsets for redesigning or de novo engineering of signaling networks; and fourth, biological systems that are especially amendable to this approach.     

A new approach to sepsis treatment by rasagiline: a molecular,biochemical and histopathological study

Molecular Biology Reports - We aimed to investigate the effects of rasagiline on acute lung injury that develops in the sepsis model induced with the cecal ligation and puncture in rats. The rats...     

Evolutionary and biochemical differences between human and monkey acidic mammalian chitinases

Acidic mammalian chitinase (AMCase), an enzyme implicated in the pathology of asthma, is capable of chitin cleavage at a low pH optimum. The corresponding gene (CHIA) can be found in genome databases of a variety of mammals, but the enzyme properties of only the human and mouse proteins were extensively studied. We wanted to compare enzymes of closely related species, such as humans and macaques. In our attempt to study macaque AMCase, we searched for CHIA-like genes in human and macaque genomes. We found that both genomes contain several additional CHIA-like sequences. In humans, CHIA-L1 (hCHIA-L1) is an apparent pseudogene and has the highest homology to CHIA. To determine which of the two genes is functional in monkeys, we assessed their tissue expression levels. In our experiments, CHIA-L1 expression was not detected in human stomach tissue, while CHIA was expressed at high levels. However, in the cynomolgus macaque stomach tissue, the expression pattern of these two genes was reversed: CHIA-L1 was expressed at high levels and CHIA was undetectable. We hypothesized that in macaques CHIA-L1 (mCHIA-L1), and not CHIA, is a gene encoding an acidic chitinase, and cloned it, using the sequence of human CHIA-L1 as a guide for the primer design. We named the new enzyme MACase (Macaca Acidic Chitinase) to emphasize its differences from AMCase. MACase shares a similar tissue expression pattern and pH optimum with human AMCase, but is 50 times more active in our enzymatic activity assay. DNA sequence of the mCHIA-L1 has higher percentage identity to the human pseudogene hCHIA-L1 (91.7%) than to hCHIA (84%). Our results suggest alternate evolutionary paths for human and monkey acidic chitinases.     

Identification of interacting hot spots in the beta3 integrin stalk using comprehensive interface design

Protein-protein interfaces are usually large and complementary surfaces, but specific side chains, representing energetic "hot spots," often contribute disproportionately to binding free energy. We used a computational method, comprehensive interface design, to identify hot spots in the interface between the stalk regions of the β3 and the complementary αIIb and αv integrin subunits. Using the Rosetta alanine-scanning and design algorithms to predict destabilizing, stabilizing, and neutral mutations in the β3 region extending from residues Lys(532) through Gly(690), we predicted eight alanine mutations that would destabilize the αIIbβ3 interface as well as nine predicted to destabilize the αvβ3 interface, by at least 0.3 kcal/mol. The mutations were widely and unevenly distributed, with four between residues 552 and 563 and five between 590 and 610, but none between 565 and 589, and 611 and 655. Further, mutations destabilizing the αvβ3 and αIIbβ3 interfaces were not identical. The predictions were then tested by introducing selected mutations into the full-length integrins expressed in Chinese hamster ovary cells. Five mutations predicted to destabilize αIIb and β3 caused fibrinogen binding to αIIbβ3, whereas three of four predicted to be neutral or stabilizing did not. Conversely, a mutation predicted to destabilize αvβ3, but not αIIbβ3 (D552A), caused osteopontin binding to αvβ3, but not fibrinogen binding to αIIbβ3. These results indicate that stability of the distal stalk interface is involved in constraining integrins in stable, inactive conformations. Further, they demonstrate the ability of comprehensive interface design to identify functionally significant integrin mutations.     

Interactions mediated by the N-terminus of fibrinogen's Bbeta chain

Specific molecular interactions mediated by the N-terminus of fibrinogen's Bbeta chain were revealed using laser tweezers-based force spectroscopy. We examined interactions between fibrinogen fragments representing the center of the molecule, NDSK, desA-NDSK, and desAB-NDSK, and two recombinant fibrinogens, gammaD364H and gammaD364A, which have nonfunctional gamma-chain polymerization sites to prevent the dominant knob-hole binding. Interactions between desA-NDSK, where the N-terminus of the Bbeta chain is present, and the fibrinogen variants showed a complex spectrum of rupture forces which disappeared with desAB-NDSK, lacking both FpA and FpB. The interactions between desA-NDSK and gammaD364H or gammaD364A were inhibited by addition of soluble FpB, but not FpA or the polymerization inhibitor peptides GPRP and GHRP. When gammaD364H fibrinogen was replaced with its X-fragment lacking alphaC- domains or with fragment D, the strongest component of the rupture force spectrum disappeared, suggesting interactions between the uncleaved FpB and the alphaC-domain. Electron microscopy confirmed the binding of desA-NDSK to either D or E regions of fibrinogen as well as to alphaC-domains. The data demonstrate the existence of weak transient interactions within and between fibrin molecules mediated by the N-terminus of the fibrinogen Bbeta chain.     

On the mechanism of αC polymer formation in fibrin

Our previous studies revealed that the fibrinogen αC-domains undergo conformational changes and adopt a physiologically active conformation upon their self-association into αC polymers in fibrin. In the present study, we analyzed the mechanism of αC polymer formation and tested our hypothesis that self-association of the αC-domains occurs through the interaction between their N-terminal subdomains and may include β-hairpin swapping. Our binding experiments performed by size-exclusion chromatography and optical trap-based force spectroscopy revealed that the αC-domains self-associate exclusively through their N-terminal subdomains, while their C-terminal subdomains were found to interact with the αC-connectors that tether the αC-domains to the bulk of the molecule. This interaction should reinforce the structure of αC polymers and provide the proper orientation of their reactive residues for efficient cross-linking by factor XIIIa. Molecular modeling of self-association of the N-terminal subdomains confirmed that the hypothesized β-hairpin swapping does not impose any steric hindrance. To "freeze" the conformation of the N-terminal subdomain and prevent the hypothesized β-hairpin swapping, we introduced by site-directed mutagenesis an extra disulfide bond between two β-hairpins of the bovine Aα406-483 fragment corresponding to this subdomain. The experiments performed by circular dichroism revealed that Aα406-483 mutant containing Lys429Cys/Thr463Cys mutations preserved its β-sheet structure. However, in contrast to wild-type Aα406-483, this mutant had lower tendency for oligomerization, and its structure was not stabilized upon oligomerization, in agreement with the above hypothesis. On the basis of the results obtained and our previous findings, we propose a model of fibrin αC polymer structure and molecular mechanism of assembly.