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.     

A novel genomics approach for the identification of drug targets in pathogens, with special reference to Pseudomonas aeruginosa

Complete genome sequences of several pathogenic bacteria have been determined, and many more such projects are currently under way. While these data potentially contain all the determinants of host-pathogen interactions and possible drug targets, computational tools for selecting suitable candidates for further experimental analyses are currently limited. Detection of bacterial genes that are non-homologous to human genes, and are essential for the survival of the pathogen represents a promising means of identifying novel drug targets. We have used three-way genome comparisons to identify essential genes from Pseudomonas aeruginosa. Our approach identified 306 essential genes that may be considered as potential drug targets. The resultant analyses are in good agreement with the results of systematic gene deletion experiments. This approach enables rapid potential drug target identification, thereby greatly facilitating the search for new antibiotics. These results underscore the utility of large genomic databases for in silico systematic drug target identification in the post-genomic era.     

Natal homing in juvenile loggerhead turtles (Caretta caretta)

Juvenile loggerhead turtles (Caretta caretta) from West Atlantic nesting beaches occupy oceanic (pelagic) habitats in the eastern Atlantic and Mediterranean, whereas larger juvenile turtles occupy shallow (neritic) habitats along the continental coastline of North America. Hence the switch from oceanic to neritic stage can involve a trans-oceanic migration. Several researchers have suggested that at the end of the oceanic phase, juveniles are homing to feeding habitats in the vicinity of their natal rookery. To test the hypothesis of juvenile homing behaviour, we surveyed 10 juvenile feeding zones across the eastern USA with mitochondrial DNA control region sequences (N = 1437) and compared these samples to potential source (nesting) populations in the Atlantic Ocean and Mediterranean Sea (N = 465). The results indicated a shallow, but significant, population structure of neritic juveniles (PhiST = 0.0088, P = 0.016), and haplotype frequency differences were significantly correlated between coastal feeding populations and adjacent nesting populations (Mantel test R2 = 0.52, P = 0.001). Mixed stock analyses (using a Bayesian algorithm) indicated that juveniles occurred at elevated frequency in the vicinity of their natal rookery. Hence, all lines of evidence supported the hypothesis of juvenile homing in loggerhead turtles. While not as precise as the homing of breeding adults, this behaviour nonetheless places juvenile turtles in the vicinity of their natal nesting colonies. Some of the coastal hazards that affect declining nesting populations may also affect the next generation of turtles feeding in nearby habitats.     

A potential prodrug for a green tea polyphenol proteasome inhibitor: evaluation of the peracetate ester of (-)-epigallocatechin gallate [(-)-EGCG

Green tea has been shown to have many biological effects, including effects on metabolism, angiogenesis, oxidation, and cell proliferation. Unfortunately, the most abundant green tea polyphenol (-)-epigallocatechin gallate or (-)-EGCG is very unstable in neutral or alkaline medium. This instability leads to a low bioavailability. In an attempt to enhance the stability of (-)-EGCG, we introduced peracetate protection groups on the reactive hydroxyls of (-)-EGCG (noted in text as 1). HPLC analysis shows that the protected (-)-EGCG analog is six times more stable than natural (-)-EGCG under slightly alkaline conditions. A series of bioassays show that 1 has no inhibitory activity against a purified 20S proteasome in vitro, but exhibits increased proteasome-inhibitory activity in intact leukemic cells over natural (-)-EGCG, indicating an intercellular conversion. Inhibition of cellular proteasome activity by 1 is associated with induction of cell death. Therefore, our results indicate that the protected analog 1 may function as a prodrug of the green tea polyphenol proteasome inhibitor (-)-EGCG.     

Toll-like receptor 3 mediates a more potent antiviral response than Toll-like receptor 4

We have recently described an IFN regulatory factor 3-mediated antiviral gene program that is induced by both Toll-like receptor (TLR)3 and TLR4 ligands. In our current study, we show that activation of IFN/viral response gene expression in primary macrophage cells is stronger and prolonged with TLR3 stimulation compared with that of TLR4. Our data also reveal that the cytoplasmic tails of both TLR3 and TLR4 can directly interact with myeloid differentiation factor 88 (MyD88). However, although Toll/IL-1 receptor homology domain-containing adaptor protein/MyD88 adaptor-like is able to associate with TLR4, we were unable to detect any interaction between Toll/IL-1 receptor homology domain-containing adaptor protein/MyD88 adaptor-like and TLR3. By using quantitative real-time PCR assays, we found that TLR3 expression is inducible by both TLR3 and TLR4 ligands, while TLR4 expression is not inducible by these same stimuli. Furthermore, using cells derived from mice deficient in the IFN-alphabetaR, we show that both TLR3 and TLR4 require IFN-beta autocrine/paracrine feedback to induce TLR3 expression and activate/enhance genes required for antiviral activity. More specifically, a subset of antiviral genes is initially induced independent of IFN-beta, yet the cytokine further enhances expression at later time points. This was in contrast to a second set of genes (including TLR3) that is induced only after IFN-beta production. Taken together, our data argue that, despite both TLR3 and TLR4 being able to use IFN-beta to activate/enhance antiviral gene expression, TLR3 uses multiple mechanisms to enhance and sustain the antiviral response more strongly than TLR4.     

Genome delivery and ion channel properties are altered in VP4 mutants of poliovirus

During entry into host cells, poliovirus undergoes a receptor-mediated conformational transition to form 135S particles with irreversible exposure of VP4 capsid sequences and VP1 N termini. To understand the role of VP4 during virus entry, the fate of VP4 during infection by site-specific mutants at threonine-28 of VP4 (4028T) was compared with that of the parental Mahoney type 1 virus. Three virus mutants were studied: the entry-defective, nonviable mutant 4028T.G and the viable mutants 4028T.S and 4028T.V, in which residue threonine-28 was changed to glycine, serine, and valine, respectively. We show that mutant and wild-type (WT) VP4 proteins are localized to cellular membranes after the 135S conformational transition. Both WT and viable 4028T mutant particles interact with lipid bilayers to form ion channels, whereas the entry-defective 4028T.G particles do not. In addition, the electrical properties of the channels induced by the mutant viruses are different from each other and from those of WT Mahoney and Sabin type 3 viruses. Finally, uncoating and/or cytoplasmic delivery of the viral genome is altered in the 4028T mutants: the 4028T.G lethal mutant does not release its genome into the cytoplasm, and genome delivery is slower during infection by mutant 4028T.V 135S particles than by mutant 4028T.S or WT 135S particles. The distinctive electrical characteristics of the different 4028T mutant channels indicate that VP4 sequences might form part of the channel structure. The different entry phenotypes of these VP4 mutants suggest that the ion channels may be related to VP4's role during genome uncoating and/or delivery.     

Differential contribution of inhibitory phosphorylation of CDC2 and CDK2 for unperturbed cell cycle control and DNA integrity checkpoints

Inhibition of cyclin-dependent kinases (CDKs) by Thr14/Tyr15 phosphorylation is critical for normal cell cycle progression and is a converging event for several cell cycle checkpoints. In this study, we compared the relative contribution of inhibitory phosphorylation for cyclin A/B1-CDC2 and cyclin A/E-CDK2 complexes. We found that inhibitory phosphorylation plays a major role in the regulation of CDC2 but only a minor role for CDK2 during the unperturbed cell cycle of HeLa cells. The relative importance of inhibitory phosphorylation of CDC2 and CDK2 may reflect their distinct cellular functions. Despite this, expression of nonphosphorylation mutants of both CDC2 and CDK2 triggered unscheduled histone H3 phosphorylation early in the cell cycle and was cytotoxic. DNA damage by a radiomimetic drug or replication block by hydroxyurea stimulated a buildup of cyclin B1 but was accompanied by an increase of inhibitory phosphorylation of CDC2. After DNA damage and replication block, all cyclin-CDK pairs that control S phase and mitosis were to different degrees inhibited by phosphorylation. Ectopic expression of nonphosphorylated CDC2 stimulated DNA replication, histone H3 phosphorylation, and cell division even after DNA damage. Similarly, a nonphosphorylation mutant of CDK2, but not CDK4, disrupted the G2 DNA damage checkpoint. Finally, CDC25A, CDC25B, a dominant-negative CHK1, but not CDC25C or a dominant-negative WEE1, stimulated histone H3 phosphorylation after DNA damage. These data suggest differential contributions for the various regulators of Thr14/Tyr15 phosphorylation in normal cell cycle and during the DNA damage checkpoint.     

A rat cell line derived from DMBA-induced mammary carcinoma

A new cell line, designated UHKBR-01, was successfully established from a 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tumour. DMBA was administered orally at a dose of 4 mg/ml per rat on the first day of the experiment and thereafter at weekly intervals of same dosage, until the rats have reached a weight of around 150-200 g. The tumours grew rapidly after the injection, and were transplanted into nude mice one the harvest size (2.5 x 2 x 1 mm(3)) was reached, it was transplanted onto nude mice. We have developed a cell line from a portion of the DMBA-induced carcinoma of the nude mice. The UHKBR-01 cell exhibited a slow increase in growth rate during the time of culture and was highly tumourigenic in nude mice. The cells have been grown in culture for over 40 passages. Characterization of the cell line was performed. This included morphology by light and transmission electron microscopy, karyotype, growth rate, tumour antigen expression and xenograft implantation into nude mice. These cells exhibit ultrastructural and immunohistochemical features of epithelial cells of mammary origin. The above analyses also demonstrated that UHKBR-01 cells were oestrogen- and progesterone-receptor positive, in likeness to other established breast cancer cell lines such as MDA-MB-231 and MCF-7. The cell line grows as monolayers of oval-shaped cells with large folded nuclei accompanied by a rich supply of mitochondria. This report describes the first in vitro cell line from transplantable DMBA-induced mammary carcinoma of nude mice, which presents unique characteristics that may prove to be a good experimental model for investigating breast cancer biology.