Cef1p is a component of the Prp19p-associated complex and essential for pre-mRNA splicing

The Prp19p protein of the budding yeast Saccharomyces cerevisiae is an essential splicing factor and is associated with the spliceosome during the splicing reaction. We have previously shown that Prp19p is not tightly associated with small nuclear ribonucleoprotein particles but is associated with a protein complex consisting of at least eight protein components. By sequencing components of the affinity-purified complex, we have identified Cef1p as a component of the Prp19p-associated complex, Ntc85p. Cef1p could directly interact with Prp19p and was required for pre-mRNA splicing both in vivo and in vitro. The c-Myb DNA binding motif at the amino terminus of Cef1p was required for cellular growth but not for interaction of Cef1p with Prp19p or Cef1p self-interaction. We have identified a small region of 30 amino acid residues near the carboxyl terminus required for both cell viability and protein-protein interactions. Cef1p was associated with the spliceosome in the same manner as Prp19p, i.e. concomitant with or immediately after dissociation of U4. The anti-Cef1p antibody inhibited binding to the spliceosome of Cef1p, Prp19p, and at least three other components of the Prp19p-associated complex, suggesting that the Prp19p-associated complex is likely associated with the spliceosome and functions as an integral complex.     

Interaction of antioxidants and their implication in genetic anemia

The generation of reactive oxygen species (ROS) is a steady-state cellular event in respiring cells. Their production can be grossly amplified in response to a variety of pathophysiological conditions such as inflammation, immunologic disorders, hypoxia, hyperoxia, metabolism of drug or alcohol, exposure to UV or therapeutic radiation, and deficiency in antioxidant vitamins. Uncontrolled production of ROS often leads to damage of cellular macromolecules (DNA, protein, and lipids) and other small antioxidant molecules. A number of major cellular defense mechanisms exist to neutralize and combat the damaging effects of these reactive substances. The enzymic system functions by direct or sequential removal of ROS (superoxide dismutase, catalase, and glutathione peroxidase), thereby terminating their activities. Metal binding proteins, targeted to bind iron and copper ions, ensure that these Fenton metals are cryptic. Nonenzymic defense consists of scavenging molecules that are endogenously produced (GSH, ubiquinols, uric acid) or those derived from the diet (vitamins C and E, lipoic acid, selenium, riboflavin, zinc, and the carotenoids). These antioxidant nutrients occupy distinct cellular compartments and among them, there are active recycling. For example, oxidized vitamin E (tocopheroxy radical) has been shown to be regenerated by ascorbate, GSH, lipoic acid, or ubiquinols. GSH disulfides (GSSG) can be regenerated by GSSG reductase (a riboflavin-dependent protein), and enzymic pathways have been identified for the recycling of ascorbate radical and dehydroascorbate. The electrons that are used to fuel these recycling reactions (NADH and NADPH) are ultimately derived from the oxidation of foods. Sickle cell anemia, thalassemia, and glucose-6-phosphate-dehydrogenase deficiency are all hereditary disorders with higher potential for oxidative damage due to chronic redox imbalance in red cells that often results in clinical manifestation of mild to serve hemolysis in patients with these disorders. The release of hemoglobin during hemolysis and the subsequent therapeutic transfusion in some cases lead to systemic iron overloading that further potentiates the generation of ROS. Antioxidant status in anemia will be examined, and the potential application of antioxidant treatment as an adjunct therapy under these conditions will be discussed.     

Heart-type fatty acid binding proteins are upregulated during terminal differentiation of mouse cardiomyocytes,as revealed by proteomic analysis

At birth, the cardiomyocytes in the mouse neonatal heart still retain their ability to proliferate. However, this lasts only a few days and then the cardiomyocytes irreversibly lose their potential to divide. It is still not fully understood what factors are involved in the cessation of cardiomyocyte proliferation. Using proliferating cell nuclear antigen (PCNA) antibodies, we established that cardiomyocytes could divide extensively in 2-day-old mouse neonatal hearts and to a lesser extent in 6-day-old hearts. By 13 days, the cardiomyocytes have mostly stopped dividing. Comparative two-dimensional gel electrophoresis (2-DE) was performed on total proteins extracted from the 2-day- and 13-day-old hearts, in order to identify peptides that might be involved in the inhibition of cardiomyocyte proliferation. Using matrix-assisted laser desorption ionization mass spectroscopy (MALDI-TOF), we identified two protein spots that have the same molecular weight (approximately 14 kDa) but different pIs (5.9 and 6.1). Mass spectra analysis determined the proteins to be isoforms of the heart-type fatty acid binding protein (H-FABP). The pI 6.1 H-FABP is also known as mammary-derived growth inhibitor (MDGI; Specht et al. 1996). MGDI is a breast tumour growth suppressor gene capable of inhibiting tumour cell proliferation (Huynh et al. 1995). Both H-FABP isoforms were expressed in 2-day-old hearts but became strongly upregulated in 13-day-old hearts. We examined whether H-FABPs and PCNA were coexpressed in 2-, 6- and 13-day-old heart histological sections, using MDGI antibodies. The antibody could detect both forms of H-FABPs. It was established that there was a correlation between an increase in H-FABP expression and a decrease in PCNA expression. Hence, we tentatively propose that H-FABP isoforms are involved in regulating cardiomyocyte growth and differentiation in mouse neonatal hearts.This project was supported by a grant from the National Natural Science Foundation of China (Project No. 30340038).     

Effect of cell passage and density on protein kinase G expression and activation in vascular smooth muscle cells

It has been shown that rat aortic smooth muscle cells (AoSMCs) lost PKG-I expression when propagated repetitively or grown at low densities. Conversely, AoSMCs isolated from PKG-I deficient mice are indistinguishable from those isolated from normal mice in morphology and growth characteristics. In this study, human AoSMCs were grown from passage 9 (p9) to passage 15 (p15) and rat AoSMCs were isolated and cultured from p1 through p15. Western blotting and immunofluorescence microscopy showed little difference in PKG-I expression among different passages. Next, rat AoSMCs of p4 were grown and harvested at different cell densities. Western blotting again showed little difference among cells seeded or harvested at different densities. To test the effect of cell passage on PKG-I activation, rat AoSMCs of p4 and p11 were treated with cGMP and analyzed by Western blotting for phosphorylated vasodilator-stimulated phosphoprotein (P-VASP). The results showed that p4 had higher level of PKG-I activation than p11.     

Localization and activity of multidrug resistance protein 1 in the secretory pathway of Leishmania parasites

Upregulation of the multidrug resistance protein 1 (LeMDR1) in the protozoan parasite, Leishmania enriettii, confers resistance to hydrophobic drugs such as vinblastine, but increases the sensitivity of these parasites to the mitochondrial drug, rhodamine 123. In order to investigate the mechanism of action of LeMDR1, the subcellular localization of green fluorescent protein (GFP)-tagged versions of LeMDR1 and the fate of the traceable-fluorescent LeMDR1 substrate calcein AM were examined in both Leishmania mexicana and L. enriettii LeMDR1 -/- and overexpressing cell lines. The LeMDR1-GFP chimera was localized by fluorescence microscopy to a number of secretory and endocytic compartments, including the Golgi apparatus, endoplasmic reticulum (ER) and a multivesicular tubule (MVT)-lysosome. Pulse-chase labelling experiments with calcein AM suggested that the Golgi and ER pools, but not the MVT-lysosome pool, of LeMDR1 were active in pumping calcein AM out of the cell. Cells labelled with calcein AM under conditions that slow vesicular transport (low temperature and stationary growth) inhibited export and resulted in the accumulation of fluorescent calcein in both the Golgi and the mitochondria. We propose that LeMDR1 substrates are pumped into secretory compartments and exported from the parasite by exocytosis. Accumulation of MDR substrates in the ER can result in alternative transport to the mitochondrion, explaining the reciprocal sensitivity of drug-resistant Leishmania to vinblastine and rhodamine 123.     

A Simple Alternative Approach to Assessing the Fate of Absorbed Light Energy Using Chlorophyll Fluorescence

We propose a simplified alternative method for quantifying the partitioning of excitation energy between photochemistry, fluorescence and thermal dissipation. This alternative technique uses existing well-defined quantum efficiencies such as Phi(PS II), leaving no 'excess' efficiency unaccounted for, effectively separates regulated and constitutive thermal dissipation processes, does not require the use of F(o) and F'(o) measurements and gives very similar results to the method proposed by Kramer et al. [(2004) Photosynth Res 79: 209-218]. We demonstrate the use of the technique using chlorophyll fluorescence measurements in grapevine leaves and observe a high dependence on thermal dissipation processes (up to 75%) at both high light and low temperature.     

In vitro assay and light regulation of nitrate reductase in red alga Gracilaria chilensis

Nitrate reductase (NR) is the first enzyme in the nitrogen assimilation pathway. The in vitro NR activity of Gracilaria chilensis was assayed under different conditions to reveal its stability and biochemical characteristics, and an optimized in vitro assay is described. Maximal NR activities were observed at pH 8.0 and 15 degrees C. The apparent Km value for NADH was 8 microM and for nitrate 680 microM. Crude extracts of G. chilensis stored at 4 degrees C showed a 50% decrease of NR activity after 24 h. The highest NR activity value (253.20+/-2.60 x 10(-3) U g(-1)) was obtained when 100% von Stosch medium (500 microM NO3-) was added before extraction of apical parts. Algae under light:dark cycles of 12:12h exhibited circadian fluctuation of NR activity and photosynthesis with more than 2 times higher levels in the light phase. No evidence of endogenous diel rhythm controlling NR activity or photosynthesis was observed. Light pulses lasting 10 or 60 min during the darkness increased the NR activity by 30% and 45%, respectively. The results indicate that NR and photosynthesis are regulated mainly by light and not by a biological clock.     

PPD - Proteome Profile Database

With the complete sequencing of multiple genomes, there have been extensions in the methods of sequence analysis from single gene/protein-based to analyzing multiple genes and proteins simultaneously. Therefore, there is a demand of user-friendly software tools that will allow mining of these enormous datasets. PPD is a WWW-based database for comparative analysis of protein lengths in completely sequenced prokaryotic and eukaryotic genomes. PPD's core objective is to create protein classification tables based on the lengths of proteins by specifying a set of organisms and parameters. The interface can also generate information on changes in proteins of specific length distributions. This feature is of importance when the user's interest is focused on some evolutionarily related organisms or on organisms with similar or related tissue specificity or life-style. PPD is available at: PPD Home.     

Distributions of exons and introns in the human genome

The human genome is revisited using exon and intron distribution profiles. The 26,564 annotated genes in the human genome (build October, 2003) contain 233,785 exons and 207,344 introns. On average, there are 8.8 exons and 7.8 introns per gene. About 80% of the exons on each chromosome are < 200 bp in length. < 0.01% of the introns are < 20 bp in length and < 10% of introns are more than 11,000 bp in length. These results suggest constraints on the splicing machinery to splice out very long or very short introns and provide insight to optimal intron length selection. Interestingly, the total length in introns and intergenic DNA on each chromosome is significantly correlated to the determined chromosome size with a coefficient of correlation r = 0.95 and r = 0.97, respectively. These results suggest their implication in genome design.