Serum complement proteins comprise an important system that is responsible for several innate and adaptive immune defence mechanisms. There were three well described pathways known to lead to the generation of a C3 convertase, which catalyses the proteolysis of complement component C3, and leads to the formation of C3 opsonins (C3b, iC3b and C3d) that fix to bacteria. A pivotal step in the complement pathway is the assembly of a C3 convertase, which digests the C3 complement component to form microbial-binding C3 fragments recognized by leukocytes. The spleen clears microorganisms from the blood. Individuals lacking this organ are more susceptible to Streptococcus pneumoniae. Innate resistance to S. pneumoniae has previously been shown to involve complement components C3 and C4, however this resistance has only a partial requirement for mediators of these three pathways, such as immunoglobulin, factor B and mannose-binding lectin. Therefore it was likely that spleen and complement system provide resistance against blood-borne S. pneumoniae infection through unknown mechanism. To better understand the mechanisms involved, we studied Specific intracellular adhesion molecule-grabbing nonintegrin (SIGN)-R1. SIGN-R1, is a C-type lectin that is expressed at high levels by spleen marginal-zone macrophages and lymph-node macrophages. SIGN-R1 has previously been shown to be the main receptor for bacterial dextrans, as well as for the capsular pneumococcal polysaccharide (CPS) of S. pneumoniae. We examined the specific role of this receptor in the activation of complement. Using a monoclonal antibody that selectively downregulates SIGN-R1 expression in vivo, we show that in response to S. pneumoniae or CPS, SIGN-R1 mediates the immediate proteolysis of C3 and fixation of C3 opsonins to S. pneumoniae or to marginal-zone macrophages that had taken up CPS. These data indicate that SIGN-R1 is largely responsible for the rapid C3 convertase formation induced by S. pneumoniae in the spleen of mice. Also, we found that SIGN-R1 directly binds C1q and that C3 fixation by SIGN-R1 requires C1q and C4 but not factor B or immunoglobulin. Traditionally C3 convertase can be formed by the classical C1q- and immunoglobulin-dependent pathway, the alternative factor-B-dependent pathway and the soluble mannose-binding lectin pathway. Furthermore Conditional SIGN-R1 knockout mice developed deficits in C3 catabolism when given S. pneumoniae or its capsular polysaccharide intravenously. There were marked reductions in proteolysis of serum C3, deposition of C3 on organisms within SIGN-$R1^+$ spleen macrophages, and formation of C3 ligands. The transmembrane lectin SIGN-R1 therefore contributes to innate resistance by an unusual C3 activation pathway. We propose that in the SIGN-R1 mediated complement activation pathway, after binding to polysaccharide, SIGN-R1 captures C1q. SIGN-R1 can then, in association with several other complement proteins including C4, lead to the formation of a C3 convertase and fixation of C3. Therefore, this new pathway for C3 fixation by SIGN-R1, which is unusual as it is a classical C1q-dependent pathway that does not require immuno globulin, contributes to innate immune resistance to certain encapsulated microorganisms.

The yeast Saccharomyces cerevisiae has been shown to contain three isoforms of citrate synthase (CS). The mitochondrial CS, Cit1, catalyzes the first reaction of the TCA cycle, i.e., condensation of acetyl-CoA and oxaloacetate to form citrate [1]. The peroxisomal CS, Cit2, participates in the glyoxylate cycle [2]. The third CS is a minor mitochondrial isofunctional enzyme, Cit3, and related to glycerol metabolism. However, the level of its intracellular activity is low and insufficient for metabolic needs of cells [3]. It has been reported that ${\Delta}cit1$ strain is not able to grow with acetate as a sole carbon source on either rich or minimal medium and that it shows a lag in attaining parental growth rates on nonfermentable carbon sources [2, 4, 5]. Cells of ${\Delta}cit2$, on the other hand, have similar growth phenotype as wild-type on various carbon sources. Thus, the biochemical basis of carbon metabolism in the yeast cells with deletion of CIT1 or CIT2 gene has not been clearly addressed yet. In the present study, we focused our efforts on understanding the function of Cit2 in utilizing $C_2$ carbon sources and then found that ${\Delta}cit1$ cells can grow on minimal medium containing $C_2$ carbon sources, such as acetate. We also analyzed that the characteristics of mutant strains defective in each of the genes encoding the enzymes involved in TCA and glyoxylate cycles and membrane carriers for metabolite transport. Our results suggest that citrate produced by peroxisomal CS can be utilized via glyoxylate cycle, and moreover that the glyoxylate cycle by itself functions as a fully competent metabolic pathway for acetate utilization in S. cerevisiae. We also studied the relationship between Cit1 and apoptosis in S. cerevisiae [6]. In multicellular organisms, apoptosis is a highly regulated process of cell death that allows a cell to self-degrade in order for the body to eliminate potentially threatening or undesired cells, and thus is a crucial event for common defense mechanisms and in development [7]. The process of cellular suicide is also present in unicellular organisms such as yeast Saccharomyces cerevisiae [8]. When unicellular organisms are exposed to harsh conditions, apoptosis may serve as a defense mechanism for the preservation of cell populations through the sacrifice of some members of a population to promote the survival of others [9]. Apoptosis in S. cerevisiae shows some typical features of mammalian apoptosis such as flipping of phosphatidylserine, membrane blebbing, chromatin condensation and margination, and DNA cleavage [10]. Yeast cells with ${\Delta}cit1$ deletion showed a temperature-sensitive growth phenotype, and displayed a rapid loss in viability associated with typical apoptotic hallmarks, i.e., ROS accumulation, nuclear fragmentation, DNA breakage, and phosphatidylserine translocation, when exposed to heat stress. Upon long-term cultivation, ${\Delta}cit1$ cells showed increased potentials for both aging-induced apoptosis and adaptive regrowth. Activation of the metacaspase Yca1 was detected during heat- or aging-induced apoptosis in ${\Delta}cit1$ cells, and accordingly, deletion of YCA1 suppressed the apoptotic phenotype caused by ${\Delta}cit1$ mutation. Cells with ${\Delta}cit1$ deletion showed higher tendency toward glutathione (GSH) depletion and subsequent ROS accumulation than the wild-type, which was rescued by exogenous GSH, glutamate, or glutathione disulfide (GSSG). Beside Cit1, other enzymes of TCA cycle and glutamate dehydrogenases (GDHs) were found to be involved in stress-induced apoptosis. Deletion of the genes encoding the TCA cycle enzymes and one of the three GDHs, Gdh3, caused increased sensitivity to heat stress. These results lead us to conclude that GSH deficiency in ${\Delta}cit1$ cells is caused by an insufficient supply of glutamate necessary for biosynthesis of GSH rather than the depletion of reducing power required for reduction of GSSG to GSH.

Bacterial biofilms are analogous to multi-cellular organisms or to clonal communities of higher organisms. In this respect, it can be demonstrated that biofilms display the type of genetic variation associated with macroorganisms. The formation of genetic variants from biofilms is the result of internally produced and regulated signals and the appearance of these variants coincides with dispersal from the biofilm. Moreover, the generation of such variation, has similar outcomes for the bacterial community, where diversification of phenotypic traits ensures that the bacterial community optimizes its chances of success when dispersing or surviving when challenged with environmental stress. These observations increase the complexity with which we view bacteria and also suggest that microbial systems can serve as models for the testing of eukaryotic ecological theories.

A major hurdle to the development of RNA interference as therapy for HIV infection is the delivery of siRNA to T lymphocytes which are difficult cells to transfect even in vitro. We have employed a single chain antibody to the pan T cell surface antigen CD7 was conjugated to an oligo-9-arginine peptide (scFvCD7-9R) for T cell-specific siRNA delivery in NOD/SCIDIL2${\gamma}$-/- mice reconstituted with human peripheral blood lymphocytes (Hu-PBL). Using a novel delivery, we first show that scFvCD7-9R efficiently delivered CD4 siRNA into human T cells in vitro. In vivo administration to Hu-PBL mice resulted in reduced levels of surface CD4 expression on T cells. Mice infected with HIV-1 and treated on a weekly basis with scFvCD7-9R-siRNA complexes targeting a combination of viral genes and the host coreceptor molecule CCR5 successfully maintained CD4/CD3 T cell ratios up to 4 weeks after infection in contrast to control mice that displayed a marked reduction in CD4 T cell numbers. p24 antigen levels were undetectable in 3 of the 4 protected mice. scFvCD7-9R/antiviral siRNA treatment also helped maintain CD4 T cell numbers with reduced plasma viral loads in Hu-PBL mice reconstituted with PBMC from donors seropositive for HIV, indicating that this method can contain viral replication even in established HIV infections. Our results show that scFvCD7-9R could be further developed as a potential therapeutic for HIV-1 infection.

The phylogenetic diversity of microbial communities in calcareous mats from Spearfish Creek, a freshwater stream located in the Black Hills of South Dakota, was examined using PCR-based 16S rDNA sequence analysis. In this study, two types of calcareous mats were compared: mature mats formed on the natural substrate of rock surfaces and developing mats on an artificial substrate of glass slides. Among 63 unique isolates from a clone library of 16S rRNA genes from mature mat samples, there were 8 phyla of Bacteria represented. The predominant phylum was Proteobacteria (48%), with the $\beta$ subclass being the largest group. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes from slide samples collected at intervals for four months showed considerable diversity of the microbial community from the earliest stages of community development. Amplicons isolated from DGGE gels and sequenced indicated that community succession has occurred without increasing microbial diversity. However, light microscopic analysis revealed a significant increase in microbial cell density throughout the community development. Scanning electron microscopy of mat samples provides evidence that diatoms are also important members of calcareous mat communities.

Various biosensors were evaluated for identifying and detecting foodborne pathogens in a rapid and effective manner. First, five strains of Escherichia coli and six strains of Salmonella were identified using Fourier transform infrared spectroscopy and a statistical program. For doing this, lipopolysaccharides (LPSs) and outer membrane proteins (OMPs) were extracted from a cell wall of each bacterial strain. As a result, each strain was identifed at the level of 97% for E. coli and 100% for Salmonella. Second, E. coli O157:H7, S. Enteritidis, and Listeria monocytogenes were identified by multiplex PCR products from four specific genes of each bacteria using a capillary electrophoresis (CE). Also, ground beef for E. coli O157:H7, lettuce for S. Enteritidis, and hot dog for L. monocytogenes were used to determine the possibility of detecting pathogens in foods. Foods inoculated with respective pathogen were cultivated for six hours and multiplex PCR products were obtained and assessed. The minimum detection levels of tested bacteria were <10 cells/g, <10 cells/g, and $10^4$ cells/g for E. coli O157:H7, S. Enteritidis, and L. monocytogenes, respectively. Third, it was possible to detect S. Typhimurium in a pure culture and lettuce by a bioluminescence-based detection assay using both recombinant bacteriophage P22::luxI and a bioluminescent bioreporter. In addition, bacteriophage T4 was quantitatively monitored using E. coli including luxCDABE genes.

Methylophaga aminisulfidivorans $MP^T$, a restricted facultative marine methylotrophic bacterium, was able to utilize methanol as a sole carbon and energy source, and possessed a methanol dehydrogenase (MDH) that is a key enzyme in the process of methanol oxidation. During purification of MDH, three types of MDH (MDH I, II, and III) were obtained in the cell free extracts from $MP^T$ cells grown on methanol. When analyzed by SDS-PAGE and ESI-FT ICR MS, MDH I was confirmed to consist of two subunits and with molecular masses of ~66 and ~10 kDa, respectively, in a form of ${\alpha}_2{\beta}_2$. While MDH II and MDH III contained an additional ~30 kDa protein, designated ${\gamma}$, in a form of ${\alpha}_2{\beta}_2{\gamma}$ and ${\alpha}_2{\beta}_2{\gamma}_2$, respectively. MDH III showed 1.5.2.0 times higher activity than MDH II, while MDH I remained the lowest activity. Based on these observations and experimental data, it seems that the original MDH conformation is ${\alpha}_2{\beta}_2{\gamma}2$ within $MP^T$ growing on methanol, and subunit ${\gamma}$ keeps MDH in an active form, and/or makes MDH easily bind to the substrate, methanol.

The tidal flat of Korea is one of the most dynamic areas in terms of sediment erosion and deposition. Tidal flats provide important food resources, ecological niche for diverse organisms and have functional role of bioremediation by mineralizing organic matters. Generally, microbes are essential constituent for biochemical transformations, such as mineralization of organic matters and biodegradation of pollutants. Understanding microbes in tidal flat sediment is necessary to understand these processes. In order to understand role of microbes in tidal flat, this study investigated prokaryotic communities by several methods such as clone library, DGGE, and pyrosequencing. In addition, quantification of microorganisms and isolation biodegrading bacteria were investigated in tidal flats of Western Coast.

We monitored the occurrence of human enteric viruses in urban rivers by cell culture-PCR and RT-nested PCR. Water samples were collected monthly or semimonthly between May 2002 and March 2003 in four urban tributaries. Enteric viruses were detected by RT-nested PCR and cell culture-PCR based on a combination of Buffalo Green monkey kidney (BGMK) and A549 cell lines, followed by phylogenetic analysis of amplicons. By RT-nested PCR analysis, 45 (77.6%), 32 (55.2%), 32 (55.2%), 26 (44.8%), 12 (20.7%), 2 (3.4%), 4 (6.9%), and 4 (6.9%) of 58 samples showed positive results with adenoviruses, enteroviruses, noroviruses (NV) genogroup I (GI) and II (GII), reoviruses, hepatitis A viruses, rotaviruses and sapoviruses, respectively. Adenoviruses were most often detected and only eight (13.8%) samples were negative for adenoviruses and positive for other enteric viruses in the studied sites. Thirty-one (77.5%) of the 40 samples were positive for infectious adenoviruses and/or enteroviruses based on cell culture-PCR, and the frequency of positive samples grown on A549 and BGMK (65.0%) was higher than that grown on BGMK alone (47.5%). The occurrence of each enteric virus, except reoviruses and hepatitis A viruses was not statistically correlated with the water temperature and levels of fecal coliforms according to Binary logistic regression model. By sequence analysis, most strains of adenoviruses and enteroviruses detected in this study are similar to the causative agent of viral diseases in Korea and most NV GI- and GII-grouped strains were closely related to the reference strains from China and Japan, and GII/4-related strains had similar sequences to strains recognized as a worldwide epidemic outbreak. Our results suggested that monitoring human enteric viruses is necessary to improve microbial quality and cell culture-PCR using the combination of A549 and BGMK cells and the adenovirus detection by PCR could be useful for monitoring viral contamination in the aquatic environment.