ATP-citrate lyase as a substrate of protein histidine phosphatase in vertebrates

The first protein histidine phosphatase from vertebrates discovered recently was found in a variety of tissues, however, a physiological substrate protein was missing. Phosphorylation of liver extracts in the presence of EDTA, followed by SDS-PAGE and autoradiography showed labeling of three proteins. Acid- and alkaline-treatment revealed the existence of N-phosphates. Addition of histidine phosphatase exclusively resulted in dephosphorylation of a 110kDa protein (denaturing conditions). Gelfiltration revealed its native molecular mass of approximately 450kDa. That protein was purified and identified as ATP-citrate lyase. The results are in favor of histidine phosphatase playing an important yet unidentified role in metabolic processes.     

Hepatitis C Virus Genotype 1b Core Protein Does Not Exert Immunomodulatory Effects on Virus-Induced Cellular Immunity

The hepatitis C virus (HCV) core protein is among the most conserved proteins in HCV and is known to induce sensitization of cytotoxic T lymphocytes (CTL). Therefore, it is a prime candidate for a component of a potential HCV vaccine. The HCV core protein has, however, been reported to exert multiple effects on cell functions, raising questions as to its suitability for this purpose. This question was investigated here with mice into which replication-deficient adenoviruses expressing core protein of an HCV genotype 1b isolate were injected. We show that induction of cytokines in response to the infection, infiltration of lymphocytes into the infected liver, priming of virus-specific CTL, and liver injury are not modulated by expression of the core protein in the liver. Moreover, no changes in the sensitivity to tumor necrosis factor alpha- or Fas-mediated liver injury are demonstrable. A similar lack of demonstrable effects of the core protein on immune functions has also been obtained using transgenic mice expressing another HCV genotype 1b core protein. It is concluded that the HCV core protein of genotype 1b has no modulatory effects on induction of virus-specific immune responses and may therefore be a suitable component of an HCV vaccine.     

Phylogeny of the lizard subfamily Lygosominae (Reptilia: Scincidae), with special reference to the origin of the new world taxa

Phylogenetic relationships of the three lygosomine skink genera occurring both in the Old World and the New World (Mabuya, Scincella and Sphenomorphus) were inferred from mitochondrial DNA sequence of 12S and 16S rRNA genes. Results strongly suggested the non-monophyly for any of these three genera. Within the Mabuya group, Asian members appear to have diverged first, leaving the Neotropical and the Afro-Malagasy Mabuya as sister groups. These relationships, together with the absence of extant or fossil representatives of the Mabuya group from North America, strongly suggest the trans-Atlantic dispersals of Mabuya from Africa to Neotropics. Our results also indicated a closer affinity of the New World Scincella with the New World Sphenomorphus than with the Old World Scincella. Such relationships suggest the trans-Beringian dispersal of the common ancestor from Asia and its subsequent divergence into the North American Scincella and the Neotropical Sphenomorphus.     

Two novel series of allocolchicinoids with modified seven membered B-rings: design, synthesis, inhibition of tubulin assembly and cytotoxicity

Two new attractive series of allocolchicinoids were designed as inhibitors of tubulin assembly using the potent ketone 4 and the tetracyclic, pyrazole annulated NCME variant 7 (NCME = N-acetyl colchinol-O-methylether (2)) as lead structures. The first group of inhibitors of type 6 with novel oxepine and azepine B-ring structures belongs to the NCME-series and was synthesized via a multistep total synthesis starting from simple and cheap 3-methoxybenzaldehyde (12) and 3,4,5-trimethoxybenzaldehyde (13). Biaryl-coupling of the starting materials 12 and 13 was accomplished via Ziegler-Ullmann-reaction to furnish the biphenyl 11 equipped with two carbaldehyde functions. The subsequent Cannizzaro reaction of this dicarbaldehyde 11 proceeded with high regioselectivity to yield almost exclusively the key compound, the hydroxymethyl carboxylic acid 9. Ring closure to the o,o'-bridged biphenyls was accomplished by two routes: on the one hand, treatment of 9 with aqueous hydrochloric acid yielded the lactone 15. On the other hand, a four step sequence starting from the isomeric mixture 9/10 furnished the constitutionally isomeric lactams 23 and 24; these could be converted to the corresponding thiolactams 25 and 26 and to the tetrazole annulated NCME-type derivatives 27 and 28. The second series of bioactive compounds are congeners of allocolchicine (3). The well known desacetyl allocolchicine (29) was easily oxidized to the oxime 30, which was further transformed to the corresponding ketone 31. This served as key precursor for the syntheses of various tetracyclic allocolchicine modifications 33-36 annulated with a pyrazole, isoxazole, pyrimidine or 2-aminopyrimidine heterocycle, respectively. Unexpectedly, all the NCME-variants with a substituent in position 7 like in NCME (2) inhibited the tubulin assembly only moderately. In contrast, the new series of allocolchicine modifications proved to be highly potent antimicrotubule agents. Inhibition of tubulin assembly occurred at lower concentrations compared to those measured for the reference colchicine (1). Surprisingly, these promising results could not be confirmed in the cytotoxicity tests against the human MCF-7 breast cancer cell line, where an unexpected loss of effectiveness compared to the corresponding NCME-derivatives was observed.     

High efficiency transient and stable transformation by optimized DNA microinjection into Nicotiana tabacum protoplasts

An efficient system has been established that allows well controlledDNA microinjection into tobacco (Nicotiana tabacum) mesophyllprotoplasts with partially regenerated cell walls and subsequentanalysis of transient as well as stable expression of injectedreporter genes in particular targeted cells or derived clones.The system represents an effective tool to study parametersimportant for the successful transformation of plant cells bymicroinjection and other techniques. Protoplasts were immobilizedin a very thin layer of medium solidified with agarose or alginate.DNA microinjection was routinely monitored by coinjecting FITC-dextranand aimed at the cytoplasm of target cells. The injection procedurewas optimized for efficient delivery of injection solution intothis compartment. Cells were found to be at the optimal stagefor microinjection about 24 h after immobilization in solidmedium. Embedded cells could be kept at this stage for up to4 d by incubating them at 4 C in the dark. Within 1 h successfuldelivery of injection, solution was routinely possible into20–40 cells. Following cytoplasmic coinjection of FITC-dextran and pSHI913,a plasmid containing the neo (neomycin phosphotransferase II)gene, stably transformed, paromomycin-resistant clones couldbe recovered through selection. Transgenic tobacco lines havebeen established from such clones. Injection solutions containingpSHI913 at a concentration of either 50 µg ml^–1or 1 mg ml^–1 have been tested. With 1 mg ml^–1 plasmidDNA the percentage of resistant clones per successfully injectedcell was determined to be about 3.5 times higher. Incubationof embedded protoplasts at 4C before microinjection was foundto reduce the percentage of resistant clones obtained per injectedcell Protoplasts were immobilized above a grid pattern and the locationof injected cells was recorded by Polaroid photography. Thefate of particular targeted cells could be observed. Isolationand individual culture of clones derived from injected cellswas possible. Following cytoplasmic coinjection of FITC-dextranand 1 mg ml^–1 plasmid DNA on average about 20% of thetargeted cells developed into microcalli and roughly 50% ofthese calli were stably transformed. Transient expression ofthe firefly luciferase gene (Luc) was nondestructively analysed24 h after injection of pAMLuc. Approximately 50% of the injectedcells that were alive at this time point expressed the Luc genetransiently. Apparently, stable integration of the injectedgenes occurred in essentially all transiently expressing cellsthat developed into clones. Key words: DNA microinjection, firefly luciferase, FITCdextran, Nicotiana tabacum, protoplast transformation     

Proteomic analysis of peripheral blood mononuclear cells: selective protein processing observed in patients with rheumatoid arthritis

In a comparative proteome analysis of peripheral blood mononuclear cells (PBMCs), we analyzed 130 two-dimensional gels obtained from 33 healthy control individuals and 32 patients diagnosed with rheumatoid arthritis (RA). We found 16 protein spots that are deregulated in patients with RA and, using peptide mass fingerprinting and Western blot analyses, identified these spots as belonging to 9 distinct proteins. A hierarchical clustering procedure organizes the study subjects into two main clusters based on the expression of these 16 protein spots, one that contains mostly healthy control individuals and the other mostly RA patients. The majority of the proteins differentially expressed in RA patients when compared with healthy controls can be detected as protein fragments in PBMCs obtained from RA patients. This set of deregulated proteins includes several factors that have been shown to be autoantigens in autoimmune diseases.     

Glucose is a pH-Dependent Motor for Sperm Beat Frequency during Early Activation

To reach the egg in the ampulla, sperm have to travel along the female genital tract, thereby being dependent on external energy sources and substances to maintain and raise the flagellar beat. The vaginal fluid is rich in lactate, whereas in the uterine fluid glucose is the predominant substrate. This evokes changes in the lactate content of sperm as well as in the intracellular pH (pH(i)) since sperm possess lactate/proton co-transporters. It is well documented that glycolysis yields ATP and that HCO(3)- is a potent factor in the increase of beat frequency. We here show for the first time a pathway that connects both parts. We demonstrate a doubling of beat frequency in the mere presence of glucose. This effect can reversibly be blocked by 2-deoxy-D-glucose, dichloroacetate and aminooxyacetate, strongly suggesting that it requires both glycolysis and mitochondrial oxidation of glycolytic end products. We show that the glucose-mediated acceleration of flagellar beat and ATP production are hastened by a pH(i) ≥7.1, whereas a pH(i) ≤7.1 leaves both parameters unchanged. Since we observed a diminished rise in beat frequency in the presence of specific inhibitors against carbonic anhydrases, soluble adenylyl cyclase and protein kinase, we suggest that the glucose-mediated effect is linked to CO(2) hydration and thus the production of HCO(3)- by intracellular CA isoforms. In summary, we propose that, in sperm, glycolysis is an additional pH(i)-dependent way to produce HCO(3)-(,) thus enhancing sperm beat frequency and contributing to fertility.     

Fault detection and diagnosis in an industrial fed-batch cell culture process

A flexible process monitoring method was applied to industrial pilot plant cell culture data for the purpose of fault detection and diagnosis. Data from 23 batches, 20 normal operating conditions (NOC) and three abnormal, were available. A principal component analysis (PCA) model was constructed from 19 NOC batches, and the remaining NOC batch was used for model validation. Subsequently, the model was used to successfully detect (both offline and online) abnormal process conditions and to diagnose the root causes. This research demonstrates that data from a relatively small number of batches (approximately 20) can still be used to monitor for a wide range of process faults.