Structural insight into arginine degradation by arginine deiminase, an antibacterial and parasite drug target

l-Arginine deiminase (ADI) catalyzes the irreversible hydrolysis of arginine to citrulline and ammonia. ADI is involved in the first step of the most widespread anaerobic route of arginine degradation. ADI, missing in high eukaryotes, is a potential antimicrobial and antiparasitic drug target. We have determined the crystal structure of ADI from Pseudomonas aeruginosa by the multi-wavelength anomalous diffraction method at 2.45 A resolution. The structure exhibits similarity to other arginine-modifying or substituted arginine-modifying enzymes such as dimethylarginine dimethylaminohydrolase (DDAH), arginine:glycine amidinotransferase, and arginine:inosamine-phosphate amidinotransferase, despite the lack of significant amino acid sequence homology to these enzymes. The similarity spans a core domain comprising five betabetaalphabeta motifs arranged in a circle around a 5-fold pseudosymmetry axis. ADI contains an additional alpha-helical domain of novel topology inserted between the first and the second betabetaalphabeta modules. A catalytic triad, Cys-His-Glu/Asp (arranged in a different manner from that of the thiol proteases), seen in the other arginine-modifying enzymes is also conserved in ADI, as well as many other residues involved in substrate binding. Based on this conservation pattern and the assumption that the substrate binding mode is similar to that of DDAH, an ADI catalytic mechanism is proposed. The main players are Cys-406, which mounts the nucleophilic attack on the carbon atom of the guanidinium group of arginine, and His-278, which serves as a general base.     

Novel structure and nucleotide binding properties of HI1480 from Haemophilus influenzae: a protein with no known sequence homologues

The crystal structure of the Haemophilus influenzae protein HI1480 was determined at 2.1-A resolution. The amino acid sequence of HI1480 is unique, having no homology with other known protein sequences. The protein adopts a novel alpha+beta fold, and associates into a dimer of tightly associated dimers. The tight dimers are formed by intermolecular interactions that are mediated by an antiparallel beta-barrel involving both monomers. Helical regions of two dimers mediate the tetramer formation. The helical region contains a four-helix bundle that has been seen only in the anticodon binding domains of class I tRNA synthetases. A cluster of four residues, Tyr18, Arg134, Glu26, and Lys12 is located in a depression formed at the four-helix bundle/ beta-barrel interface. The arrangement is suggestive of an active center, possibly a catalytic site. The HI1480 gene is located within the Mu-like prophage region of H. influenzae, has no homology to bacteriophage genes, and is flanked by transposases. Hence, this is an example of horizontal transfer from an unknown organism. Gel mobility shift assays revealed that HI1480 binds DNA and RNA molecules. Double-stranded DNA is favored over single-stranded DNA, and longer DNA molecules are bound better than shorter ones.     

Epidemiological analysis of Staphylococcus aureus strains from nasal carriers in a teaching hospital

The present study was conducted to assess the epidemiological relation of Staphylococcus aureus isolates from nasal carriers of hospital staff. Nasal swabs were taken from each of 327 personnel. After culturing on blood agar for overnight, probable staphylococcal isolates were identified and subjected to tube coagulase test. After a two-week interval, second nasal swabs were taken from the subjects whose first cultures were positive for S. aureus. Nasal carriage was defined in 58 (17.7%) personnel with positive culture for both sampling time. Antibiogram typing and arbitrarily-primed polymerase chain reaction (AP-PCR) with M13 primer were used for typing of the strains. Antibiotyping distinguished seven types and three subtypes, and 85% of the isolates were clustered in one group. AP-PCR, in contrast, identified 12 distinct patterns with 13 variants. A specific profile was not found among the isolates obtained from the personnel in a particular clinic. These results indicate that antibiotyping has poor discrimination power and heterogeneity among the nasal S. aureus strains in the hospital personnel screened is high.     

TRAIL signaling promotes entosis in colorectal cancer

Entosis is a form of nonphagocytic cell-in-cell (CIC) interaction where a living cell enters into another. Tumors show evidence of entosis; however, factors controlling entosis remain to be elucidated. Here, we find that besides inducing apoptosis, TRAIL signaling is a potent activator of entosis in colon cancer cells. Initiation of both apoptosis and entosis requires TRAIL receptors DR4 and DR5; however, induction of apoptosis and entosis diverges at caspase-8 as its structural presence is sufficient for induction of entosis but not apoptosis. Although apoptosis and entosis are morphologically and biochemically distinct, knockout of Bax and Bak, or inhibition of caspases, also inhibits entotic cell death and promotes survival and release of inner cells. Analysis of colorectal cancer tumors reveals a significant association between TRAIL signaling and CIC structures. Finally, the presence of CIC structures in the invasive front regions of colorectal tumors shows a strong correlation with adverse patient prognosis.     

Effects of glycerol additions on post-thaw fertility of frozen rainbow trout sperm, with an emphasis on interaction between extender and cryoprotectant

The study was conducted to evaluate the effects of different extenders, cryoprotectants and glycerol additions on the post‐thaw fertility and interactions between extenders and cryoprotectants during cryopreservation. Semen was collected by abdominal stripping from 30 adult male rainbow trout (Oncorhynchus mykiss Walbaum, 1792) and diluted with three different extenders (Erdahl–Graham, Lahnsteiner, glucose‐based) containing 15% DMA, 15% DMSO, 15% DMA + 1% glycerol and 15% DMSO + 1% glycerol at a ratio of 1 : 2. Diluted samples were frozen as 0.1 ml pellets directly on dry ice (solid CO₂, −79°C). Eggs were pooled from 10 females. Fertilization was applied in plastic dishes and 600 eggs were used in each fertilization trial. Pellets were thawed in their own extenders (30°C) at a ratio of 1 : 10. 0.3% NaCl was used for activating motility. Sperm–egg ratio was approximately 3 × 10⁶ sp per egg. Experimental success was determined as the percentage of eyed‐eggs 25 days after fertilization. The highest eyed‐egg rate (49.3%) was obtained from semen frozen with glucose‐based extender containing 15% DMSO + 1% glycerol. Our results indicate that the glucose‐based extender containing DMSO is a useful combination, but that the addition of glycerol does not have a positive effect on post‐thaw fertility, and that interaction of the extender‐cryoprotectant is also important in the cryopreservation of rainbow trout semen.     

A genetic analysis of crystal growth

The regulation of crystal morphology by proteins is often observed in biology. It is a central feature in the formation of hard tissues such as bones, teeth and mollusc shells. We have developed a genetic system in the bacterium Escherichia coli to study the protein-mediated control of crystal growth. We have used the crystallization of gold as a model system and found polypeptides that control the morphology of the resulting gold crystals. Analysis of the crystallization process influenced by these polypeptides indicates they act catalytically by an acid mechanism. Our results suggest that the concepts and methods of microbial genetics are general and can be applied to substances not commonly found in biological systems.     

Glucagon-like peptide (GLP-1) is involved in the central modulation of fecal output in rats

In addition to its insulinotropic action, exogenously administered glucagon-like peptide (GLP-1) inhibits gastropancreatic motility and secretion via central pathways. The aims of the present study were to evaluate the effects of exogenous GLP-1-(7-36) amide on fecal output and to investigate the role of endogenous GLP-1 on stress-induced colonic activity. With the use of a stereotaxic instrument, adult male Sprague-Dawley rats weighing 200-250 g were fitted with stainless steel cerebroventricular guide cannulas under ketamine anesthesia. A group of rats were placed in Bollman-type cages to induce restraint stress. Fecal output monitored for 2 h was increased significantly by intracerebroventricular GLP-1 to 500, 1, 000, and 3,000 pmol/rat (P < 0.05-0.01), whereas intraperitoneal GLP-1 had no effect. Intracerebroventricular administration of the GLP-1 receptor antagonist exendin-(9-39) (10 nmol/rat) reversed the increases induced by GLP-1 (500 pmol/rat; P<0.01). Similar results were also observed with the injection of corticotropin-releasing factor receptor antagonist astressin (10 microg/rat icv). The significant increase in fecal pellet output induced by restraint stress was also decreased by both intracerebroventricular exendin (10 nmol/rat) and astressin (10 microg/rat; P<0.01-0.001). These results suggest that GLP-1 participates in the central, but not peripheral, regulation of colonic motility via its own receptor and that GLP-1 is likely to be a candidate brain-gut peptide that acts as a physiological modulator of stress-induced colonic motility.     

Insertion/deletion polymorphism of the angiotensin converting enzyme gene in coronary artery disease in southern Turkey

Genetic factors are important in the pathogenesis of coronary artery disease (CAD). Angiotensin converting enzyme (ACE) gene insertion(I)/deletion(D) polymorphism is one of the genetic factor found to be related with CAD. We investigated the association between I/D polymorphism of the ACE gene and the presence of CAD. Three hundred and seven patients (187 males and 120 females, aged between 35-80, mean 54.3 +/-9.8 years) who underwent diagnostic coronary angiography were included in the study. ACE I/D polymorphism was detected by polymerase chain reaction. Of the 307, 176 had CAD. The most frequently observed genotype in all subjects was ID (47.9 %). However, in patients with CAD the frequency of II genotype was lower whereas DD genotype was higher compared to the controls (p < 0.05). The number of D allele carrying subjects were also higher (p < 0.05) in CAD patients. The logistic regression analysis indicated that the ACE D allele is an independent risk factor (odds ratio = 1.48, 95 % CI = 1.01-2.18, p < 0.05). In conclusion, the I/D polymorphism of ACE gene (carrying D allele) is an independent risk factor for CAD in the studied Turkish population.     

Short term effects of dexamethasone on hyaluronidase activity and sperm characteristics in rams

The aim of this study was to determine the effects of dexamethasone on sperm characteristics and hyaluronidase activity of serum and semen. In this investigation, 14 healthy Akkaraman rams, at the age of 2 years and weighing between 50–60 kg, were used. The rams were randomly divided into two groups. After the last administration of dexamethasone intramuscularly at a dose of 0.25 mg/kg, semen and blood samples were taken at different times. The results showed that the serum hyaluronidase activity was increased significantly (p < 0.001) in the treatment group when compared with the control group except for the 1st hour. There was a significant difference (p < 0.001, 0.01, 0.05) in the hyaluronidase activity of semen between the treatment group and the control group. Furthermore, there was a significant difference (p < 0.01) in sperm concentration between both groups at all the times except the 96th hour. There were statistically significant (p < 0.05) differences in semen volume between the treatment and control groups. There were also significant differences (p < 0.05) in sperm motility between the treatment and control groups except for the 72 and 96th hours.

These findings indicate that dexamethasone increases hyaluronidase activity of serum and semen, but it decreases sperm concentration, semen volume and sperm motility in rams. Therefore the use of these drugs in breeding rams during breeding season is not suitable.