Gene expression profiles of the heterotrophic microalga Chlorella pyrenoidosa F-9.

A strain of the microalga Chlorella pyrenoidosa F-9 in our laboratory showed special characteristics when transferred from autotrophic to heterotrophic culture. In order to elucidate the possible metabolic mechanism, the gene expression profiles of the autonomous organelles in the green alga C. pyrenoidosa under autotrophic and heterotrophic cultivation were compared by suppression subtractive hybridization technology. Two subtracted libraries of autotrophic and heterotrophic C. pyrenoidosa F-9 were constructed, and 160 clones from the heterotrophic library were randomly selected for DNA sequencing. Dot blot hybridization showed that the ratio of positivity was 70.31% from the 768 clones. Five chloroplast genes (ftsH, psbB, rbcL, atpB, and infA) and two mitochondrial genes (cox2 and nad6) were selected to verify their expression levels by real-time quantitative polymerase chain reaction. Results showed that the seven genes were abundantly expressed in the heterotrophic culture. Among the seven genes, the least increment of gene expression was ftsH, which was expressed 1.31-1.85-fold higher under heterotrophy culture than under autotrophy culture, and the highest increment was psbB, which increased 28.07-39.36 times compared with that under autotrophy conditions. The expression levels of the other five genes were about 10 times higher in heterotrophic algae than in autotrophic algae. In inclusion, the chloroplast and mitochondrial genes in C. pyrenoidosa F-9 might be actively involved in heterotrophic metabolism.

Preconditioning with Porphyromonas gingivalis lipopolysaccharide may confer cardioprotection and improve recovery of the electrically induced intracellular calcium transient during ischemia and reperfusion.

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis lipopolysaccharide (LPS) is a ligand for cell surface toll-like receptors (TLR), TLR2 and TLR4 while stimulation of either leads to cardioprotection. We hypothesized that: (1) pretreatment with P. gingivalis LPS at appropriate concentrations would induce cardioprotection against injury induced by ischemia and reperfusion; and (2) P. gingivalis LPS pretreatment at cardioprotective concentrations may reduce Ca(2+) overload, which is a precipitating cause of injury, and improve recovery of contractile function. MATERIAL AND METHODS: Male Sprague-Dawley rats were randomly selected to receive intraperitoneal saline or hot phenol-water-extracted P. gingivalis LPS at 0.2, 0.5, 1.0, 2.0 or 4.0 mg/kg 24 h before the experiment. The hearts were isolated and subjected to regional ischemia by coronary artery ligation followed by reperfusion. In isolated rat ventricular myocytes, the cytosolic Ca(2+) level and the electrically induced intracellular calcium (E[Ca(2+)](i)) transient, which reflects contractile function, were determined after pretreatment with a cardioprotective dose of P. gingivalis LPS. RESULTS: Pretreatment with 0.5 mg/kg P. gingivalis LPS significantly reduced, while pretreatment with 1.0-4.0 mg/kg significantly increased infarct size. The Ca(2+) overload induced by ischemia-reperfusion was attenuated in myocytes from rats pretreated with 0.5 mg/kg P. gingivalis LPS. Pretreated myocytes also showed an increased amplitude of the E[Ca(2+)](i) transient, no prolongation of the time to reach the peak E[Ca(2+)](i) transient and shorter 50% decay time during reperfusion. CONCLUSION: At a dosage of 0.5 mg/kg, P. gingivalis LPS confers cardioprotection against ischemia-reperfusion-induced injury and improved intracellular E[Ca(2+)](i) transient recovery, hence improving myocyte contractile recovery.

Protegrin-1 enhances bacterial killing in thermally injured skin.

OBJECTIVE: Septic complications and the emergence of drug-resistant microbes represent serious risks to patients. Recently, naturally occurring peptides have been discovered that possess potent and broad-spectrum antimicrobial activity. Protegrin-1 is particularly attractive for clinical use in human wounds because, unlike defensins, protegrin-1 retains broad antimicrobial and antifungal activity at physiologic salt concentration and in the presence of serum. The objective of this study was to examine the efficacy of protegrin-1 in killing multiple drug-resistant microbes isolated from human burn patients. DESIGN: For thein vitroexperiment, bilayer radial diffusion was performed comparing standard antibiotics with protegrin-1 on multiple-drug-resistant microbial organisms isolated from infected burn wounds. In vivo, rats received a 20% total body surface area partial-thickness burn by immersion in 60 degrees C water for 20 secs followed by wound seeding with 106 colony forming units of Silvadene-resistant Pseudomonas aeruginosa. SETTING: University of Michigan research laboratory. SUBJECTS: Adult, male Sprague-Dawley rats. INTERVENTIONS: Rats were randomized into three groups: those receiving synthetic protegrin-1, acetic acid (carrier), or gentamicin (positive control). Protegrin-1 was administered by topical application or intradermal injection. Wound tissues were harvested aseptically at different time points for quantitative bacterial counts. MEASUREMENTS AND MAIN RESULTS: In vivo and in vitro experiments revealed rapid and significant decreases in bacterial counts for protegrin-1-treated groups compared with controls. CONCLUSIONS: This study shows that protegrin-1 potentially may be used as an alternative or adjunct therapy to standard agents used to treat wound infections.

Potent fibrinolytic enzyme from a mutant of Bacillus subtilis IMR-NK1.

A mutant of Bacillus subtilis IMR-NK1, which is used for the production of domestic "natto" in Taiwan, produced high fibrinolytic enzyme activity by solid-state fermentation using wheat bran as medium. In addition, a strong fibrinolytic enzyme was purified from the cultivation media. The purified enzyme was almost homogeneous, as examined by SDS-PAGE and capillary electrophoresis. The enzyme had an optimal pH of 7.8, an optimal temperature of 55 degrees C, and a K(m) of 0.15% for fibrin hydrolysis. The molecular mass estimated by gel filtration was 31.5 kDa, and the isoelectric point estimated by isoelectric focusing electrophoresis was 8.3. The enzyme also showed activity for hydrolysis of fibrinogen, casein, and several synthetic substrates. Among the synthetic substrates, the most sensitive substrate was N-succinyl-Ala-Ala-Pro-Phe-pNA. PMSF and NBS almost completely inhibited the activity of the enzyme. These results indicate that the enzyme is a subtilisin-like serine protease, similar to nattokinase from Bacillus natto.

Infection with GB virus C and hepatitis C virus in hemodialysis patients and blood donors in Beijing.

RNAs of GB virus C (GBV-C) and hepatitis C virus (HCV) were sought by reverse-transcription polymerase chain reaction with nested primers deduced from the 5' untranslated region: 79 patients on maintenance hemodialysis, 205 commercial blood donors, and 205 voluntary donors in Beijing were studied. GBV-C RNA was detected in 43 (54%) patients and 17 (8%) commercial donors, and HCV RNA in 43 (54%) patients and 13 (6%) commercial donors, respectively. By contrast, GBV-C RNA was detected only in 2 (1%) and HCV RNA in none among 205 volunteer blood donors serving as controls. Thus both patients and commercial blood donors were at higher risk for infection with GBV-C (P < 0.001) than controls. HCV RNA was detected more often in patients with GBV-C RNA than without (29/43 or 67%, vs. 14/36 or 39%, P < 0.05) as well as in commercial donors with GBV-C RNA than without (5/17 or 29% vs. 8/188 or 4%, P < 0.01). A phylogenetic tree constructed on a sequence of 100 base pairs in the helicase region indicated that GBV-C isolates from Beijing are more similar to Japanese isolates than to isolates from the United States and Africa. Sequences from certain hemodialysis patients and those from some commercial donors were similar, suggesting nosocomial infection and spread among restricted groups.

Transmembrane residues of the parathyroid hormone (PTH)/PTH-related peptide receptor that specifically affect binding and signaling by agonist ligands.

Polar residues within the transmembrane domains (TMs) of G protein-coupled receptors have been implicated to be important determinants of receptor function. We have identified mutations at two polar sites in the TM regions of the rat parathyroid hormone (PTH)/PTH-related peptide receptor, Arg-233 in TM 2 and Gln-451 in TM 7, that caused 17-200-fold reductions in the binding affinity of the agonist peptide PTH-(1-34) without affecting the binding affinity of the antagonist/partial agonist PTH-(3-34). When mutations at the TM 2 and TM 7 sites were combined, binding affinity for PTH-(1-34) was restored to nearly that of the wild type receptor. The double mutant receptors, however, were completely defective in signaling cAMP or inositol phosphate production in response to PTH-(1-34) agonist ligand. The results demonstrate that Arg-233 and Gln-451 have important roles in determining agonist binding affinity and transmembrane signaling. Furthermore, the finding that residues in TM 2 and TM 7 are functionally linked suggests that the TM domain topology of the PTH/PTH-related peptide receptor may resemble that of receptors in the rhodopsin/beta-adrenergic receptor family, for which structural and mutagenesis data suggest interactions between TMs 2 and 7.

[The experiment research of OB glue used in tracheoanastomosis].

Resect a segment of canine cervical trachea. The length of resected trachea is from 1-4cm and 5-8cm. In the first group, one dog was used for each length and two dogs were used for each length in the second group, therefore twelve were used in the experiment. The upper and lower ends were matched and seved by three stitches, and OB glue was used to coat in the anastomosis crevice, and at last the cre-vice was wrapped with a sheet of loose tracheal sheath. After the operation, irritating cough, barking, swallowing and free movement of neck didn't cause fistula. Nakedeyes and tissue examination showed that the healing of the anastomostic orifice was well and no stenosis was found. The glue membrane was absorbed completely within 1-2 months. Outside body anti-pulling force experiment (for 8 cases) showed that the anastomostic line can hold 100cm H2O stress and 2kg pulling strength if only OB glue was used to connect the resected ends. Hence, this experiment shows that the usage of OB glue for tracheal anastomosis is a safety, simple and convenient method.

The long-term effects of recombinant human erythropoietin on the cardiovascular system.

Fifty-five hemodialysis patients (pts) received rHuEpo for 3-5 years (51 +/- 11 months, hematocrit 32.5 +/- 3.7). BP medication was required in 42% of pts prior to rHuEpo (Hct 20.8 +/- 3.5) and 69% (38 patients) now require such therapy. BP was controlled with single therapy in 16 pts and only 8 required 3 or more different BP drugs. Vascular access clotting episodes were rare in pts with autologous fistula (17 of 24 pts had no clotting), whereas access clotting episodes were 10 times more common in pts with AV grafts, yet 20% had no clotting after 3-5 years of rHuEpo. Heart size decreased in most who initially had cardiomegaly. Cardiovascular related and other deaths were decreased in this selected group when compared to other dialysis pts matched for age, race and type of renal disease.