Characterization of extracellular proteins produced by Aeromonas hydrophila AH-1

Aeromonas hydrophila is a ubiquitous Gram-negative bacterium which can cause motile aeromonad septicemia in both fish and humans. A. hydrophila secretes many extracellular proteins associated with pathogenicity and environmental adaptability. In this study, an extracellular proteome map of A. hydrophila AH-1 was constructed. The major extracellular virulence factors were characterized by comparing the proteomes of various deletion mutants with that of the wild type. The results suggested that serine protease was involved in the processing of a toxin and secreted enzymes such as hemolysin, glycerophospholipid-cholesterol acyltransferase and metalloprotease. We also showed that expressions of polar and lateral flagellins were under the control of temperature, FlhA, LafK, and RpoN. In addition, three novel proteins (potential effector proteins including one ExoT-like protein) were revealed to be secreted via the type III secretion system (TTSS) of A. hydrophila AH-1. Another novel finding was the demonstration of a crosstalk between the lateral flagellar system and the TTSS in A. hydrophila. These results showed that proteomics is a powerful tool for characterizing virulence factors. The construction of proteome maps will provide a valuable means of finding potential candidates for developing suitable diagnostics and therapeutics for this emerging pathogen.     

A role for Alström syndrome protein, alms1, in kidney ciliogenesis and cellular quiescence

Premature truncation alleles in the ALMS1 gene are a frequent cause of human Alstr?m syndrome. Alstr?m syndrome is a rare disorder characterized by early obesity and sensory impairment, symptoms shared with other genetic diseases affecting proteins of the primary cilium. ALMS1 localizes to centrosomes and ciliary basal bodies, but truncation mutations in Alms1/ALMS1 do not preclude formation of cilia. Here, we show that in vitro knockdown of Alms1 in mice causes stunted cilia on kidney epithelial cells and prevents these cells from increasing calcium influx in response to mechanical stimuli. The stunted-cilium phenotype can be rescued with a 5' fragment of the Alms1 cDNA, which resembles disease-associated alleles. In a mouse model of Alstr?m syndrome, Alms1 protein can be stably expressed from the mutant allele and is required for cilia formation in primary cells. Aged mice developed specific loss of cilia from the kidney proximal tubules, which is associated with foci of apoptosis or proliferation. As renal failure is a common cause of mortality in Alstr?m syndrome patients, we conclude that this disease should be considered as a further example of the class of renal ciliopathies: wild-type or mutant alleles of the Alstr?m syndrome gene can support normal kidney ciliogenesis in vitro and in vivo, but mutant alleles are associated with age-dependent loss of kidney primary cilia.     

Transgenesis and nuclear transfer using porcine embryonic germ cells

Embryonic germ (EG) cells are undifferentiated stem cells isolated from cultured primordial germ cells (PGC). Porcine EG cell lines with capacities of both in vitro and in vivo differentiation have been established. Because EG cells can be cultured indefinitely in an undifferentiated state, they may be more suitable for nuclear donor cells in nuclear transfer (NT) than somatic cells that have limited lifespan in primary culture. Use of EG cells could be particularly advantageous to provide an inexhaustible source of transgenic cells for NT. In this study the efficiencies of transgenesis and NT using porcine fetal fibroblasts and EG cells were compared. The rate of development to the blastocyst stage was significantly higher in EG cell NT than somatic cell NT (94 of 518, 18.2% vs. 72 of 501, 14.4%). To investigate if EG cells can be used for transgenesis in pigs, green fluorescent protein (GFP) gene was introduced into porcine EG cells. Nuclear transfer embryos using transfected EG cells gave rise to blastocysts (29 of 137, 21.2%) expressing GFP based on observation under fluorescence microscope. The results obtained from the present study suggest that EG cell NT may have advantages over somatic cell NT, and transgenic pigs may be produced using EG cells.     

The pathogenic fungus Cryptococcus neoformans expresses two functional GDP-mannose transporters with distinct expression patterns and roles in capsule synthesis

Cryptococcus neoformans is a fungal pathogen that is responsible for life-threatening disease, particularly in the context of compromised immunity. This organism makes extensive use of mannose in constructing its cell wall, glycoproteins, and glycolipids. Mannose also comprises up to two-thirds of the main cryptococcal virulence factor, a polysaccharide capsule that surrounds the cell. The glycosyltransfer reactions that generate cellular carbohydrate structures usually require activated donors such as nucleotide sugars. GDP-mannose, the mannose donor, is produced in the cytosol by the sequential actions of phosphomannose isomerase, phosphomannomutase, and GDP-mannose pyrophosphorylase. However, most mannose-containing glycoconjugates are synthesized within intracellular organelles. This topological separation necessitates a specific transport mechanism to move this key precursor across biological membranes to the appropriate site for biosynthetic reactions. We have discovered two GDP-mannose transporters in C. neoformans, in contrast to the single such protein reported previously for other fungi. Biochemical studies of each protein expressed in Saccharomyces cerevisiae show that both are functional, with similar kinetics and substrate specificities. Microarray experiments indicate that the two proteins Gmt1 and Gmt2 are transcribed with distinct patterns of expression in response to variations in growth conditions. Additionally, deletion of the GMT1 gene yields cells with small capsules and a defect in capsule induction, while deletion of GMT2 does not alter the capsule. We suggest that C. neoformans produces two GDP-mannose transporters to satisfy its enormous need for mannose utilization in glycan synthesis. Furthermore, we propose that the two proteins have distinct biological roles. This is supported by the different expression patterns of GMT1 and GMT2 in response to environmental stimuli and the dissimilar phenotypes that result when each gene is deleted.     

Targeting bladder tumor cells in vivo and in the urine with a peptide identified by phage display

Bladder cancer is one of the most common tumors of the genitourinary tract. Here, we use phage display to identify a peptide that targets bladder tumor cells. A phage library containing random peptides was screened for binding to cells from human bladder tumor xenografts. Phage clones were further selected for binding to a bladder tumor cell line in culture. Six clones displaying the consensus sequence CXNXDXR(X)/(R)C showed selective binding to cells from primary human bladder cancer tissue. Of these, the CSNRDARRC sequence was selected for further study as a synthetic peptide. Fluorescein-conjugated CSNRDARRC peptide selectively bound to frozen sections of human bladder tumor tissue, whereas only negligible binding to normal bladder tissue was observed. When the fluorescent peptide was introduced into the bladder lumen, in a carcinogen-induced rat tumor model, it selectively bound to tumor epithelium. Moreover, when the peptide was intravenously injected into the tail vein, it homed to the bladder tumor but was not detectable in normal bladder and control organs. Next, we examined whether the peptide can detect tumor cells in urine. The fluorescent peptide bound to cultured bladder tumor cells but not to other types of tumor cell lines. Moreover, it bound to urinary cells of patients with bladder cancer, while showing little binding to urinary cells of patients with inflammation or healthy individuals. The CSNRDARRC peptide may be useful as a targeting moiety for selective delivery of therapeutics and as a diagnostic probe for the detection of bladder cancer.     

In situ proteolysis for protein crystallization and structure determination

We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.     

Effect of the engineered indole pathway on accumulation of phenolic compounds in Catharanthus roseus hairy roots

Catharanthus roseus has been well-known to contain indole alkaloids effective for treatment of diverse cancers. We examined the intracellular accumulation profiles of phenolic compounds in response to ectopic overexpression of tryptophan feedback-resistant anthranilate synthase holoenzyme (ASalphabeta) in C. roseus hairy roots. Among 13 phenolic compounds measured, 6 phenolic compounds were detected in late exponential phase ASalphabeta hairy roots. Uninduced and induced ASalphabeta hairy roots accumulated up to 1.2 and 4.5 mg/g DW over a 72-h period, respectively. Upon induction, in parallel with a rapid increase in tryptophan in the first 48 h, accumulation of phenolic compounds tended to increase to a maximum level (4.5 mg/g DW) at 48 h, after which phenolic levels decreased back to the uninduced level by 72 h. Naringin was a predominant form that comprised about 72% and 36% of the total content of phenolic compounds in the uninduced and induced lines, respectively. Upon induction, accumulation of catechin drastically increased with the highest level (3.6 mg/g) occurring at 48 h, whereas that of all others except for salicylic acid showed no statistical difference. Catechin is a final product of the flavonoid pathway, and thus metabolic flux into this pathway is transiently increased by overexpression of AS. Like catechin, salicylic acid is very sensitive to induction as it began to increase to 5-fold within 4 h of induction, but unlike catechin, no significant accumulation of salicylic acid was noted after 4 h of induction. The results suggest differential regulation of this particular biosynthesis branch within the phenolic pathway.     

Exercise improves glucose homeostasis that has been impaired by a high-fat diet by potentiating pancreatic beta-cell function and mass through IRS2 in diabetic rats.

In this study, we investigated the effects of a high-fat diet and exercise on pancreatic beta-cell function and mass and its molecular mechanism in 90% pancreatectomized male rats. The pancreatectomized diabetic rats were given control diets (20% energy) or a high-fat (HF) diet (45% energy) for 12 wk. Half of each group was given regular exercise on an uphill treadmill at 20 m/min for 30 min 5 days/wk. HF diet lowered first-phase insulin secretion with glucose loading, whereas exercise training reversed this decrease. However, second-phase insulin secretion did not differ among the groups. Exercise increased pancreatic beta-cell mass. This resulted from stimulated beta-cell proliferation and reduced apoptosis, which is associated with potentiated insulin or IGF-I signaling through insulin receptor substrate-2 (IRS2) induction. Although the HF diet resulted in decreased proliferation and accelerated apoptosis by weakened insulin and IGF-I signaling from reduction of IRS2 protein, beta-cell mass was maintained in HF rats just as much as in control rats via increased individual beta-cell size and neogenesis from precursor cells. Consistent with the results of beta-cell proliferation, pancreas duodenal homeobox-1 expression increased in the islets of rats in the exercise groups, and it was reduced the most in rats fed the HF diet. In conclusion, exercise combined with a moderate fat diet is a good way to maximize beta-cell function and mass through IRS2 induction to alleviate the diabetic condition. This study suggests that dietary fat contents and exercise modulate beta-cell function and mass to overcome insulin resistance in two different pathways.