Expression of three isoprenoid biosynthesis genes and their effects on the carotenoid production of the zygomycete Mucor circinelloides

The zygomycete Mucor circinelloides accumulates β-carotene as the main carotenoid compound. In this study, the applicability of some early genes of the general isoprenoid pathway to improve the carotenoid production in this fungus was examined. The isopentenyl pyrophosphate isomerase gene (ipi) was cloned and used together with the genes encoding farnesyl pyrophosphate synthase (isoA) and geranylgeranyl pyrophosphate synthase (carG) in overexpression studies. Transformation experiments showed that the first bottleneck in the pathway, from the aspect of carotenoid production, is the step controlled by the carG gene, but overexpression of the ipi and isoA genes also contributes to the availability of the precursors. Transformations with these isoprenoid genes in combination with a bacterial β-carotene ketolase gene yielded Mucor strains producing canthaxanthin and echinenone.     

Characteristics of bovine early puerperal uterine contractility recorded under farm conditions

A non-invasive, digital technique was used to measure and quantify intrauterine pressure (IUP) changes in early postpartum dairy cows kept under farm conditions in order to document physiological changes in uterine contractility after uncomplicated calvings. In addition, possible relationships between characteristics of uterine contractility and blood ionized calcium (Ca(2+))-concentrations were investigated. Recordings of uterine contractility were made by using a transcervically inserted open tip catheter in 12 healthy cows during their first 48h after calving. The IUP recording technique appeared easily applicable under farm conditions. Although mean frequency (FREQ), amplitude (AMP) and area under the curve (AUC) of the myometrial contractions significantly decreased due to time, untreated early postpartum cows showed a high variability in characteristics of uterine contractility. There was no correlation between blood Ca2+ -concentrations and any of the contractility parameters.     

Role of Caelyx in second line treatment of ovarian cancer

The authors published their own experiences and results of Caelyx treatment based on 40 ovarian cancer patients treated in 9 different institutions. Patients had been treated with platinum based or platinum-taxol combination chemoterapy. Their average age was 57.2 years (35-80). The average time to progression was 3.8 months (1-8). The effects of the therapy were assessed on 36 patients and the results were 3 CR, 8 PR, 7 SD and 18 PD. Summarised the Caelyx therapy caused improvement in 30.55% of the patients and stabilisation in 19.44%. Supportive therapy was needed in 8 cases.     

The effect of solvent environment on the conformation and stability of human polyclonal IgG in solution.

Stability of therapeutic IgG preparations is an important issue as adequate efficacy and safety has to be ensured throughout a long shelf life. To this end, denaturation and aggregation have to be avoided. In many cases sugars are applied for stabilizing IgG in relatively high concentration (5-10%). However, certain sugars (sucrose, maltose) are responsible for adverse effects including renal failure. In this work we reassessed the effect of pH and stabilizers to optimize the solvent environment and minimize the amount of additives without endangering quality and stability. Since both biological function and aggregation depend on the conformational properties of individual IgG molecules, two sensitive and rapid physical methods were introduced to assess conformational changes and structural stability as a function of pH and addition of standard stabilizers. It was observed that the conformational stability decreases with decreasing pH, while the resistance against aggregation improves. The optimum pH range for storage is 5.0-6.0, as a compromise between conformational stability and the tendency for oligomerization. Intriguingly, additives in physiologically acceptable concentration have no effect on the thermal stability of IgG. On the other hand, glucose or sorbitol, even at a concentration as low as 1%, have significant effect on the tertiary structure as revealed by near-UV-CD spectroscopy, reflecting changes in the environment of aromatic side-chains. Although, 0.3% leucine does not increase conformational stability, it decreases the aggregation tendency even more efficiently than 1% glucose or sorbitol. Both pH and storage temperature are decisive factors for the long-term stability of IgG solutions. An increase in the dimer content was observed upon storage at 5 degrees C which was partly reverted upon incubation at 37 degrees C. Storage at temperatures higher than 5 degrees C may help to maintain an optimal proportion of dimers. Regarding the known side effects, and their limited stabilizing capacity at low concentration, it is advisable to omit sugars at intravenous immunoglobulin (IVIG) formulation. Hydrophobic amino acids give promising alternatives.     

Analysis of the subcellular localization, function, and proteolytic control of the Arabidopsis cyclin-dependent kinase inhibitor ICK1/KRP1

Recent studies have shown that cyclin-dependent kinase (CDK) inhibitors can have a tremendous impact on cell cycle progression in plants. In animals, CDK inhibitors are tightly regulated, especially by posttranslational mechanisms of which control of nuclear access and regulation of protein turnover are particularly important. Here we address the posttranslational regulation of INHIBITOR/INTERACTOR OF CDK 1 (ICK1)/KIP RELATED PROTEIN 1 (KRP1), an Arabidopsis (Arabidopsis thaliana) CDK inhibitor. We show that ICK1/KRP1 exerts its function in the nucleus and its presence in the nucleus is controlled by multiple nuclear localization signals as well as by nuclear export. In addition, we show that ICK1/KRP1 localizes to different subnuclear domains, i.e. in the nucleoplasm and to the chromocenters, hinting at specific actions within the nuclear compartment. Localization to the chromocenters is mediated by an N-terminal domain, in addition we find that this domain may be involved in cyclin binding. Further we demonstrate that ICK1/KRP1 is an unstable protein and degraded by the 26S proteasome in the nucleus. This degradation is mediated by at least two domains indicating the presence of at least two different pathways impinging on ICK1/KRP1 protein stability.     

Time Course,Distribution and Cell Types of Induction of Transforming Growth Factor Betas following Middle Cerebral Artery Occlusion in the Rat Brain

Transforming growth factor-βs (TGF-β1–3) are cytokines that regulate the proliferation, differentiation, and survival of various cell types. The present study describes the induction of TGF-β1–3 in the rat after focal ischemia at 3 h, 24 h, 72 h and 1 month after transient (1 h) or permanent (24 h) middle cerebral artery occlusion (MCAO) using in situ hybridization histochemistry and quantitative analysis. Double labeling with different markers was used to identify the localization of TGF-β mRNA relative to the penumbra and glial scar, and the types of cells expressing TGF-βs. TGF-β1 expression increased 3 h after MCAO in the penumbra and was further elevated 24 h after MCAO. TGF-β1 was present mostly in microglial cells but also in some astrocytes. By 72 h and 1 month after the occlusion, TGF-β1 mRNA-expressing cells also appeared in microglia within the ischemic core and in the glial scar. In contrast, TGF-β2 mRNA level was increased in neurons but not in astrocytes or microglial cells in layers II, III, and V of the ipsilateral cerebral cortex 24 h after MCAO. TGF-β3 was not induced in cells around the penumbra. Its expression increased in only a few cells in layer II of the cerebral cortex 24 h after MCAO. The levels of TGF-β2 and -β3 decreased at subsequent time points. Permanent MCAO further elevated the levels of all 3 subtypes of TGF-βs suggesting that reperfusion is not a major factor in their induction. TGF-β1 did not co-localize with either Fos or ATF-3, while the co-localization of TGF-β2 with Fos but not with ATF-3 suggests that cortical spreading depolarization, but not damage to neural processes, might be the mechanism of induction for TGF-β2. The results imply that endogenous TGF-βs are induced by different mechanisms following an ischemic attack in the brain suggesting that they are involved in distinct spatially and temporally regulated inflammatory and neuroprotective processes.     

Genetic framework of cyclin-dependent kinase function in Arabidopsis

Cyclin-dependent kinases (CDKs) are at the heart of eukaryotic cell-cycle control. The yeast Cdc2/CDC28 PSTAIRE kinase and its orthologs such as the mammalian Cdk1 have been found to be indispensable for cell-cycle progression in all eukaryotes investigated so far. CDKA;1 is the only PSTAIRE kinase in the flowering plant Arabidopsis and can rescue Cdc2/CDC28 mutants. Here, we show that cdka;1 null mutants are viable but display specific cell-cycle and developmental defects, e.g., in S phase entry and stem cell maintenance. We unravel that the crucial function of CDKA;1 is the control of the plant Retinoblastoma homolog RBR1 and that codepletion of RBR1 and CDKA;1 rescued most defects of cdka;1 mutants. Our work further revealed a basic cell-cycle control system relying on two plant-specific B1-type CDKs, and the triple cdk mutants displayed an early germline arrest. Taken together, our data indicate divergent functional differentiation of Cdc2-type kinases during eukaryote evolution.     

Lead identification and optimization of novel collagenase inhibitors; pharmacophore and structure based studies

In this study, chemical feature based pharmacophore models of MMP-1, MMP-8 and MMP-13 inhibitors have been developed with the aid of HypoGen module within Catalyst program package. In MMP-1 and MMP-13, all the compounds in the training set mapped HBA and RA, while in MMP-8, the training set mapped HBA and HY. These features revealed responsibility for the high molecular bioactivity, and this is further used as a three dimensional query to screen the knowledge based designed molecules. These pharmacophore models for collagenases picked up some potent and novel inhibitors. Subsequently, docking studies were performed for the potent molecules and novel hits were suggested for further studies based on the docking score and active site interactions in MMP-1, MMP-8 and MMP-13.