M13 Bacteriophage-Based Self-Assembly Structures and Their Functional Capabilities.

Controlling the assembly of basic structural building blocks in a systematic and orderly fashion is an emerging issue in various areas of science and engineering such as physics, chemistry, material science, biological engineering, and electrical engineering. The self-assembly technique, among many other kinds of ordering techniques, has several unique advantages and the M13 bacteriophage can be utilized as part of this technique. The M13 bacteriophage (Phage) can easily be modified genetically and chemically to demonstrate specific functions. This allows for its use as a template to determine the homogeneous distribution and percolated network structures of inorganic nanostructures under ambient conditions. Inexpensive and environmentally friendly synthesis can be achieved by using the M13 bacteriophage as a novel functional building block. Here, we discuss recent advances in the application of M13 bacteriophage self-assembly structures and the future of this technology.

In vitro evaluation of new functional properties of poly-γ-glutamic acid produced by Bacillus subtilis D7.

We investigated the functionality of poly-γ-glutamic acid (γ-PGA), which is produced by Bacillus subtilis D7, for its potential applications in medicine and cosmetics. The γ-PGA had angiotensin-converting enzyme (ACE) inhibition activity. ACE inhibition activity was dependent on the γ-PGA concentration; the highest ACE inhibition activity was observed at 1.25 mg/l of γ-PGA. IC50 (0.108 mg/ml) of the γ-PGA was lower than that of standard ACE inhibitory drug, N-[(S)-mercapto-2-methylpropionyl]-L-proline (0.247 mg/ml). The γ-PGA also had water-holding capacity and hygroscopicity. Furthermore, the γ-PGA inhibited growth of some pathogenic bacteria, including Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, Klebsiella pneumonia and Esherichia coli. The γ-PGA exhibited a good metal adsorption capacity; Cr (VI) adsorption capacity of γ-PGA increased with decreasing pH, and the maximal adsorption was observed at pH 2. Our results suggest that γ-PGA may be expected to be widely applied in cosmetics, biomedical and environmental industries with the feature of being less harmful to humans and the environment.

Characterization of a multifunctional feather-degrading Bacillus subtilis isolated from forest soil.

In this study, we isolated and characterized a novel feather-degrading bacterium that shows keratinolytic, antifungal and plant growth-promoting activities. A bacterium S8 was isolated from forest soil and confirmed to belong to Bacillus subtilis by BIOLOG system and 16S rRNA gene analysis. The improved culture conditions for the production of keratinolytic protease were 0.1% (w/v) sorbitol, 0.3% (w/v) KNO(3), 0.1% (w/v) K(2)HPO(4), 0.06% (w/v) KH(2)PO(4) and 0.04% (w/v) MgCl(2)·6H(2)O (pH 8.0 and 30°C), respectively. In the improved medium containing 0.1% (w/v) feather, keratinolytic protease production was around 53.3 ± 0.3 U/ml at 4 day; this value was 10-fold higher than the yield in the basal feather medium (5.3 ± 0.1 U/ml). After cultivation for 5 days in the improved medium, intact feather was completely degraded. Feather degradation resulted in free -SH group, soluble protein and amino acids production. The concentration of free -SH group in the culture medium was 15.5 ± 0.2 µM at 4 days. Nineteen amino acids including all essential amino acids were produced in the culture medium; the concentration of total amino acid produced was 3360.4 µM. Proline (2809.9 µM), histidine (371.3 µM) and phenylalanine (172.0 µM) were the major amino acids released in the culture medium. B. subtilis S8 showed the properties related to plant growth promotion: hydrolytic enzymes, ammonification, indoleacetic acid (IAA), phosphate solubilization, and broad-spectrum antimicrobial activity. Interestingly, the strain S8 grown in the improved medium produced IAA and antifungal activity, indicating simultaneous production of keratinolytic and antifungal activities and IAA by B. subtilis S8. These results suggest that B. subtilis S8 could be not only used to improve the nutritional value of feather wastes but also is useful in situ biodegradation of feather wastes. Furthermore, it could also be a potential biofertilizer or biocontrol agent applicable to crop plant soil.

Influence of glycerol on production and structural-physical properties of cellulose from Acetobacter sp. V6 cultured in shake flasks.

Cost-effective production of bacterial cellulose (BC) by Acetobacter sp. V6 was investigated in shake culture using glycerol as carbon source and its structural and physical properties were determined. In medium containing 3% (w/v) glycerol, BC production was 4.98+/-0.03g/l after 7 days. This value was 3.8-fold higher than the yield in the glucose medium. FT-IR spectra revealed that all the BC samples were highly crystalline and were cellulose type capital I, Ukrainian. The crystallinity index value of the BC produced was 9% higher in the glycerol medium than in the glucose medium. Scanning electron micrographs showed that BC from the glycerol medium was more compact than that from the glucose medium. Water-holding capacity and viscosity of BC from the glycerol medium had 61.3% and 22.4% lower values than those from the glucose medium. These results suggest that glycerol could be a potential low-cost substrate for BC production by Acetobacter sp. V6, leading to the reduction in the production cost.

Keratinolytic activity of Bacillus megaterium F7-1, a feather-degrading mesophilic bacterium.

The aim of this study was to investigate environmental conditions affecting chicken feather degradation and keratinolytic enzyme production by Bacillus megaterium F7-1, a feather-degrading mesophilic bacterium. B. megaterium F7-1 degraded whole chicken feather completely within 7 days. The bacterium grew with an optimum at pH 7.0-11.0 and 25-40 degrees C, where maximum keratinolytic activity was also observed. The production of keratinolytic enzyme by B. megaterium F7-1 was inducible with feather. Keratinolytic enzyme production by B. megaterium F7-1 at 0.6% (w/v) skim milk was 468U/ml, which was about 9.4-fold higher than that without skim milk. The amount of keratinolytic enzyme production depended on feather concentrations. The degradation rate of autoclaved chicken feathers by cell-free culture supernatant was 26% after 24h of incubation, but the degradation of untreated chicken feathers was unsuccessful. B. megaterium F7-1 effectively degraded feather meal, duck feather and human nail, whereas human hair and sheep wool showed relatively low degradation rates. B. megaterium F7-1 presented high keratinolytic activity and was very effective in feather degradation, providing potential use for biotechnological processes of keratin hydrolysis.

Prevalence of gastroesophageal reflux disease symptoms and uninvestigated dyspepsia in Korea: a population-based study.

Various reports on the prevalence of gastroesophageal reflux disease (GERD) and uninvestigated dyspepsia have been conducted in Western countries. We sought to determine the frequency of GERD symptoms and uninvestigated dyspepsia in Korea. Telephone interviews were conducted with 1,044 individuals. Of all subjects, 7.1% reported that GERD symptoms were present at least once a week, and 3.8% at least twice a week. The prevalence of heartburn according to educational level and acid regurgitation according to age was significantly different (P < 0.05). The prevalence of uninvestigated dyspepsia was reported as 12.2%. Dyspepsia was divided into subgroups of 34% ulcer-like, 56% dysmotility-like, and 10% nonspecific. The occurrence of dyspepsia did not vary according to age, gender, educational level and household income. As frequency of GERD symptoms increased, quality of life significantly decreased. We concluded that GERD symptoms and uninvestigated dyspepsia were prevalent in Korea. The prevalence was similar to that of other Asian countries.

Cloning, expression, and characterization of a glycoside hydrolase family 50 beta-agarase from a marine Agarivorans isolate.

The gene for a thermostable beta-agarase from Agarivorans sp. JA-1 was cloned and sequenced. It comprised an open reading frame of 2,988 base pairs, which encode a protein of 109,450 daltons consisting of 995 amino acid residues. A comparison of the entire sequence showed that the enzyme has 98.8% sequence similarities to beta-agarase from Vibrio sp. JT1070, indicating that it belongs to the family glycoside hydrolase (GH)-50. The gene corresponding to a mature protein of 976 amino acids was inserted and expressed in Escherichia coli. The recombinant beta-agarase was purified to homogeneity. It had maximal activity at 40 degrees C and pH 8.0 in the presence of 1 mM NaCl and 1 mM CaCl(2). The enzyme hydrolyzed agarose as well as neoagarohexaose and neoagarotetraose to yield neoagarobiose as the main product. Thus, the enzyme would be useful for the industrial production of neoagarobiose.

Solubilization of insoluble inorganic phosphates by a novel salt- and pH-tolerant Pantoea agglomerans R-42 isolated from soybean rhizosphere.

To develop environment-friendly biofertilizer solubilizing insoluble phosphates, salt- and pH-tolerant, insoluble inorganic phosphate-solubilizing bacterium was isolated from soybean rhizosphere. On the basis of its physiological characteristics and Vitek analysis, this bacterium was identified as Pantoea agglomerans. The optimal medium composition and cultural conditions for the solubilization of insoluble phosphate by P. agglomerans R-42 were 3% (w/v) of glucose, 0.1% (w/v) of NH4NO3, 0.02% (w/v) of MgSO4 x 7H2O, and 0.06% (w/v) of CaCl2 x 2H2O along with initial pH 7.5 at 30 degree C. The soluble phosphate production under optimal condition was around 900 mg/l, which was approximately 4.6-fold higher than the yield in the MPVK medium. The solubilization of insoluble phosphate was associated with a drop in the pH of the culture medium. P. agglomerans R-42 showed resistance against different environmental stresses like 5-45 degrees C temperature, 1-5% salt concentration and 3-11 pH range. Insoluble phosphate solubilization was highest from CaHPO4 (1367 mg/l), hydroxyapatite (1357 mg/l) and Ca3(PO4)2 (1312 mg/l). However, the strain produced soluble phosphate to the culture broth with the concentrations of 28 mg/l against FePO4, and 19 mg/l against AlPO4, respectively.

Expression of connective tissue growth factor, a biomarker in senescence of human diploid fibroblasts, is up-regulated by a transforming growth factor-beta-mediated signaling pathway.

Molecular changes associated with cellular senescence in human diploid fibroblasts (HDF), IMR-90, were analyzed by two-dimensional differential proteome analysis. A high percentage of replicative senescent cells were positive for senescence-associated beta-galactosidase activity, and displayed elevated levels of p21 and p53 proteins. Comparison of early population doubling level (PDL) versus replicative senescent cells among the 1000 spots resolved on gels revealed that the signal intensities of six spots were increased fivefold, whereas those of four spots were decreased. Proteome analysis data demonstrated that connective tissue growth factor (CTGF) is an age-associated protein. Up-regulation of CTGF expression in senescent cells was further confirmed by Western blotting and RT-PCR. We postulate that CTGF expression is controlled, in part, by transforming growth factor-beta (TGF-beta), in view of the high levels of TGF-beta isoforms as well as type I and II receptors detected only in late PDL of HDF cells. To verify this hypothesis, we stimulated early PDL cells with TGF-beta1 as well as stress inducing agents such as hydrogen peroxide. As expected, CTGF expression and Smad protein phosphorylation were dramatically increased up to observed levels in normal replicative senescent cells. In vivo experiments disclosed that CTGF, pSmad, and p53 were constitutively expressed at basal levels in up to 18-month-old rat liver, and expression was significantly up-regulated in 24-month-old rat tissue. However, expression patterns were not altered at all periods examined in livers of caloric-restricted rats. In view of both in vitro and in vivo data, we propose that the TGF-beta/Smad pathway functions in the induction of CTGF, a novel biomarker protein of cellular senescence in human fibroblasts.

Differential expression of transforming growth factor-beta in the interstitial tissue of testis during aging.

Transforming growth factor-betas (TGF-betas) have significant effects on testis development. The pattern of TGF-beta expression in aging testis has not been established to date. We examined age-related changes in the expression of TGF-beta and its receptors in the testis using Western blot analysis. TGF-beta1 expression increased continuously in aging rat testis, whereas no age-associated changes were observed for TGF-beta3. Strong expression of TGF-beta2, as well as type I and II receptors was observed in 12-month-old testis, but following this time, expression decreased dramatically. Interestingly, TGF-beta2 and -beta3 displayed strong and similar expression patterns in liver, regardless of age, suggesting that the down-regulation of TGF-beta2 is testis-specific. We observed significant induction of p53 and p21WAF1 in 18-month-old testis that appeared to correspond with aging. Moreover, caloric restriction (CR) prevented age-related decrease in TGF-beta2 expression. Using immunohistochemistry, we showed that all TGF-beta1, -beta2, and -beta3 proteins are expressed primarily in interstitial cells, which are located in the space between adjoining seminiferous tubules. Our data collectively indicate that aging in the testis is regulated by differential expression of TGF-beta proteins, and decreased levels of TGF-beta2 contribute to the aging process.

[The significance of urine sodium measurement after furosemide administration in diuretics-unresponsive patients with liver cirrhosis].

BACKGROUND/AIMS: The diagnosis of refractory ascites means a poor prognosis for patients with liver cirrhosis. The definition of refractory ascites has already been established, but using the dosage of diuretics that correlates with the definition of refractory ascites in an out-patient department will lower the compliance of the patient, as well as causing serious complications, such as hepatic encephalopathy and hyponatremia, as the dosage of diuretics is increased. Due to this fact, it is very difficult to apply this definition of refractory ascites to patients in a domestic out-patient department. In this study, in situations where there are difficulties in applying the diuretics dosage according to definition of refractory ascites, we tried to find out whether measuring the value of urine sodium after the administration of intravenous furosemide can be the standard in early differentiation of the response to diuretics treatment. METHODS: We reviewed 16 cases of liver cirrhosis with ascites and classified them into two groups by the response to diuretics. The diuretics-responsive ascites group was 8 cases and the diuretics-unresponsive ascites group consisted of 8 cases. After admission, we examined the patients' CBC, biochemical liver function test, spot urine sodium, and 24 hour creatinine clearance. After the beginning of the experiment, all diuretic therapy was stopped for 3 days. Daily we examined the patients' CBC, biochemical liver function test, and in the 3rd experiment day, we measured 24-hour urine volume and sodium. In the 4th experiment day, after sampling for ADH, plasma renin activity and plasma aldosterone level, we administrated the furosemide 80 mg I.V, and measured the amount of 8 hour urine volume and sodium. RESULTS: The plasma aldosterone level was significantly higher in the diuretics- unresponsive ascites group than in the diuretics-responsive ascites group. In the 4th experiment day, the amount of urine volume and sodium was very significantly lower in the diuretics-unresponsive ascites group than in the diuretics-responsive ascites group (1297.5 +/-80.9 vs 2003.7 +/-114.6 ml, p<0.005, 77.3 +/-8.2 vs 211.8 +/-12.6 mEq, p<0.001). CONCLUSIONS: In out-patient departments, the measurement of urine sodium 8 hours after administrating 80 mg of intravenous furosemide, will help in differentiating ascites patients with lower treatment response to diuretics.

Analysis of CYP2E1 polymorphism for the determination of genetic susceptibility to gastric cancer in Koreans.

BACKGROUND: Interindividual genetic differences in susceptibility to chemical carcinogens are among the most important host factors in human cancer. The present study was undertaken to reveal the association between the polymorphism of CYP2E1 (CYP2E1/PstI and CYP2E1/DraI) with genetic susceptibility to gastric cancer development in Koreans. METHODS: In the present study, 120 gastric cancer patients and 145 controls with no history of tumors were analyzed. CYP2E1 was determined by polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP), or PCR and direct gel electrophoresis. RESULTS: The overall genotype distribution of CYP2E1 was not significantly different from that of controls. However, the genotype distribution of the patient subgroups with a history of heavy cigarette smoking (>30 pack/year) in the CYP2E1/PstI and CYP2E1/DraI polymorphisms were significantly different from those of non-smoking patients (P = 0.0122 and P = 0.0029, respectively). The difference was also noticeable in the younger patient subgroup (aged

[Five-year follow-up of clinical and laboratory data of early liver cirrhosis patients confirmed by liver biopsy].

BACKGROUND/AIMS: It is important to evaluate the general status of the liver including the structural and inflammatory aspects, as well as the functional aspects, in order to determine a patient's treatment modality and prognosis. METHODS: 55 Child-Pugh class A liver cirrhosis patients confirmed by liver biopsy have been categorized into 4 groups based on the shunt index and p-value(Y= 3.3431-0.8160 ALT/AST ratio-0.0343 X prothrombin time+2.6963 X shunt index, p = e(y)/(e(y)+1)), which was obtained by Thallium- 201 scan; group I - shunt index less than 0.3 and p-value less than 0.7; group II - shunt index less than 0.3 and p-value more than 0.7; group III - shunt index more than 0.3 and p-value less than 0.7; and group IV - shunt index more than 0.3 and p-value more than 0.7. Statistical analyses used were ANOVA, paired t-test, and Chi-square test. RESULTS: 1. The laboratory data after a 5-year follow-up also showed a significant difference between four groups. 2. In group IV, the Child-Pugh class after 5 years worsened, and complications of liver cirrhosis such as esophageal varix, ascites, and hepatic encephalopathy occurred more frequently. 3. In group II, the laboratory data after a 5-year follow-up indicated some improvement. CONCLUSION: It can be seen that even early in patients with initially the same cirrhosis, the course of the illness can progress to a variety of different situations. The measurement of shunt index and the p-value of cirrhosis will be more helpful in the follow-up evaluation and predicting its prognostic index in liver cirrhosis patients.

[Relationship of propranolol pharmacokinetic parameters with portosystemic shunt in CCl4-induced cirrhotic rats].

BACKGROUND/AIMS: This study was designed to determine the relationship of propranolol pharmacokinetic parameters with portosystemic shunt in CCl4-induced cirrhotic rats. METHODS: Cirrhotic rats(n=6) were induced by intramuscular injection of CCl4 in olive oil(two time per weeks) for 12 weeks. Controls (n=6) were injected intramuscularly with the same dose of olive oil for 12 weeks. We evaluated the amount of portosystemic shunt by thallium-201 per rectal scintigraphy. After intravenous bolus injection of propranolol (2mg/kg) to rats, the serum propranolol concentrations were analyzed by a HPLC-fluorimetric detector system. Pharmacokinetic parameters such as C0, AUC, t1/2(beta), and CLp were determined in each group. Then, a small amount of heptic tissue was obtained and subjected to determination of the hepatic collagen content by quantitating 4-hydroxyproline and were inspected by microscope after hematoxylin and eosin stain. RESULTS: In liver biopsy, liver fibrosis progressed in CCl4-induced cirrhotic rats. The serum concentrations of propranolol were significantly (p < 0.01) elevated in CCl4-induced cirrhotic rats. Mean amount of 4-hydroxyproline, mean H/L ratio, and mean AUC in CCl4-induced cirrhotic rats was significantly (p < 0.01) higher than that in control rats. There was a relationship between AUC, H/L ratio, and amount of 4-hydroxyproline. CONCLUSION: H/L ratio may help in the selection of drug dosage (especially blood flow dependent drug) in pre-clinical studies for chronic liver disease during the drug development process.