Specificity of a Nuclease from Penicillium citrinum

The enzyme with high milk clotting activity produced by Irpex lacteus was partially purified by a CM-cellulose chromatography. Throughout the over-all process, the enzyme was purified approximately 9-fold from a crude powder with about 22.8% recovery of the original activity. The MCA/PU ratio of this fraction was 2.51 and the specific milk clotting activity was 188.7.

The purified enzyme is a sort of acid protease with optimum pH of 2.5 for casein digestion and 4.0 for hemoglobin digestion. The Lineweaver-Burk plot, when casein was used as a substrate, showed that the Km value of the enzyme was about 0.07% and the V_max value was 0.4. The molecular weight of the enzyme is about 34,000, the isoelectric point is pH 5.2 and a ultraviolet absorption maximum is at 277 mμ. The enzyme has not yet been crystalized but seems to be a sort of glycoprotein, because the Molish reaction was positive at the present purification stage.

Some enzymological properties of the enzyme was studied and compared with those of a calf rennet and Mucor rennet. In some respects such as pH optima, pH stability, thermostability and temperature optima, the enzyme is Mucor rennet alike. On the other hand, as to the increase in activity along with decrease in pH of milk and the increase in activity along with the addition of Ca ion, the enzyme is not very different from the calf rennet. However, proteolysis of milk casein by the enzyme was fairly higher than by the calf rennet.

As to the production of enzymes, I. lacteus can produce at least three types of proteases into liquid media. When, for example, R medium was used, only one type of protease, that is the fraction A, could mainly be produced and it was this enzyme that assumed to be a rennet like enzyme.     

A novel model of ischemic enteritis induced in rats by stenosis of the superior mesenteric artery

AimsWe established a new model of ischemic enteritis in rats and evaluated its usefulness for screening prophylactic drugs.Main methodsMale SD rats were used after 18 h of fasting. Under ether anesthesia, the superior mesenteric artery (SMA) was exposed, and a calibrated stenosis was produced by placing a needle on a blood vessel, ligating both the vessel and needle, and then removing the needle from the ligature.Key findingsThe stenosis caused severe damage on the anti-mesenteric side of the small intestine within 3 days; the severity of the damage increased with the gauge of a needle. No damage occurred in the small intestine following the stenosis with a needle of less than 21 gauge. Multiple hemorrhagic lesions occurred at an incidence of 100% when a 23-gauge needle was used. The development of enteritis was accompanied by enterobacterial invasion in the mucosa, with an up-regulation of inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production. The ischemia-induced enteritis was significantly prevented by repeated treatment with aminoguanidine (a selective iNOS inhibitor), L-NAME (a nonselective NOS inhibitor), ampicillin, and aztreonam (a gram-negative bacterium antibiotic), but not vancomycin (a gram-positive bacterium antibiotic).SignificanceThese results showed that a novel model of ischemic enteritis is induced in rats by stenosis of the SMA, this model may be useful for screening drugs against ischemic enteritis, and gram-negative bacteria as well as iNOS/NO are involved in the pathogenesis of enteritis in this model.     

A Na+-dependent electrogenic glutamate transporter current in voltage-clamped cells of corpora allata in the cricket Gryllus bimaculatus

Application of L-glutamate (1 mM) to corpora allata cells of the adult male cricket Gryllus bimaculatus caused a membrane depolarization of 5.9+/-0.3 mV (mean +/- SE) from a resting potential of -62.2+/-1.3 mV (n=57). The underlying mechanism for this depolarization was studied by applying the two-electrode voltage-clamp technique. Application of L-glutamate (1 mM) elicited an inward current that peaked at 8.1+/-0.7 nA (n = 73) at a holding potential of-50 mV. Both L- and D-aspartate also induced an inward current of almost the same amplitude as L-glutamate, whereas D-glutamate failed to induce an inward current. Glutamate receptor agonists, such as kainate, quisqualate, alpha-amino-3-hydroxy-5-methyl isoxazole-4-propionic acid, and N-methyl-D-aspartate, were ineffective in eliciting inward currents. The glutamate-induced inward current did not reverse even when the holding potential was set to +40 mV. The replacement of extracellular Na+ with choline+ eliminated the inward current. These results strongly suggest that the current induced by glutamate is mediated by a glutamate transporter rather than a glutamate receptor. We further examined the effects of 12 amino acid analogs which are known to be selective inhibitors of the mammalian excitatory amino acid transporters (EAATs) on the corpora allata transporter. From the effects of these inhibitors, we conclude that the glutamate transporter expressed in corpora allata cells of the cricket is similar to the high affinity glutamate transporters cloned from human brain, especially EAAT1 and EAAT3. Unlike mammalian transporters, however, serine-O-sulfate has the most potent action, suggesting the unique feature of the glutamate transporter expressed in the corpora allata.     

Eradication of intractable malignant ascites by abdominocentesis,reinfusion of concentrated ascites,and adoptive immunotherapy with dendritic cells and activated killer cells in a patient with recurrent lung cancer: a case report

Introduction

Dynamic retinal vessel analysis represents a well-established method for the assessment of vascular reactivity during both normal conditions and after various provocations. We present a case where the subject showed abnormal retinal vessel reactivity after fasting voluntarily for 20 hours.

Case presentation

A healthy, 21-year-old man who fasted voluntarily for 20 hours exhibited abnormal retinal vascular reactivity (dilation and constriction) after flicker provocation as measured using the Dynamic Retinal Vessel Analyser (Imedos, Jena, Germany).

Conclusion

The abnormal vascular reactivity induced by fasting was significant; abnormal levels of important nutrients due to fasting and dehydration could play a role through altering the concentration of vasoactive substances such as nitric oxide. This hypothesis needs further investigation.     

Ras/Raf1-dependent signal in sphingosine-1-phosphate-induced tube formation in human coronary artery endothelial cells

Since we recently reported that high density lipoprotein, which contains the bioactive lipid sphingosine-1-phosphate (S1P) [Arterioscler. Thromb. Vasc. Biol. 23 (2003) 802], induced human coronary artery endothelial cell (HCEC) tube formation mediated by a Ras/Raf/ERK (extracellular signal-activated kinase) pathway, we thought that it would be very important to evaluate whether the signal in S1P-induced tube formation is Ras-dependent or -independent. In an in vitro model of HCEC tube formation on a matrix gel, S1P-induced tube formation. ERK1/2 inhibitor (PD98059) and pertussis toxin (PTX) suppressed S1P-induced tube formation. S1P activated phospho(p)-ERK1/2, while dominant-negative RasN17 blocked S1P-induced p-ERK1/2. Moreover, RasN17 inhibited S1P-induced tube formation. S1P activated Ras/Raf1 by Ras pull-down assay and this effect was inhibited by PTX. These results demonstrate that Ras/Raf1-dependent ERK activation mediated by PTX-sensitive G protein-coupled receptors may be a potent signal in S1P-induced HCEC tube formation.     

The complete genomic sequence of Mycoplasma penetrans,an intracellular bacterial pathogen in humans

The complete genomic sequence of an intracellular bacterial pathogen, Mycoplasma penetrans HF-2 strain, was determined. The HF-2 genome consists of a 1 358 633 bp single circular chromosome containing 1038 predicted coding sequences (CDSs), one set of rRNA genes and 30 tRNA genes. Among the 1038 CDSs, 264 predicted proteins are common to the Mycoplasmataceae sequenced thus far and 463 are M.penetrans specific. The genome contains the two-component system but lacks the essential cellular gene, uridine kinase. The relatively large genome of M.penetrans HF-2 among mycoplasma species may be accounted for by both its rich core proteome and the presence of a number of paralog families corresponding to 25.4% of all CDSs. The largest paralog family is the p35 family, which encodes surface lipoproteins including the major antigen, P35. A total of 44 genes for p35 and p35 homologs were identified and 30 of them form one large cluster in the chromosome. The genetic tree of p35 paralogs suggests the occurrence of dynamic chromosomal rearrangement in paralog formation during evolution. Thus, M.penetrans HF-2 may have acquired diverse repertoires of antigenic variation-related genes to allow its persistent infection in humans.     

Down-regulation of Mcl-1 by inhibition of the PI3-K/Akt pathway is required for cell shrinkage-dependent cell death.

The anti-apoptotic effect of a chloride-bicarbonate exchange blocker has been previously examined in endothelial cells and cardiomyocytes. However, the anti-apoptotic effects of this blocker on epithelial cells and the mechanism of the anti-apoptotic effect remain unknown. We examined the anti-apoptotic effects of a chloride-bicarbonate exchange blocker in a renal epithelial cell line (MDCK cells). Changes in the expression of bcl-2 family proteins, which are known to have anti-apoptotic effects, were also examined. Staurosporine was used to induce apoptotic cell death in the MDCK cells. Staurosporine treatment was sufficient to induce apoptotic cell death, detected by propidium iodide and DNA ladder formation. A chloride-bicarbonate exchange blocker was added 24 h before the staurosporine treatment and during treatment. The chloride-bicarbonate exchange blocker inhibited the staurosporine-induced apoptosis in the MDCK cells in a dose-dependent manner. The expression of bcl-2 family gene products was detected by RT-PCR and Western blotting. No changes in the expression of Bax, Bid and Bik (pro-apoptotic proteins), or Bcl-2 (an anti-apoptotic protein) were detected. However, Mcl-1 expression was reduced by the staurosporine treatment, and this reduction was recovered when the chloride-bicarbonate exchange blocker was added. LY294002, a PI 3-kinase inhibitor, partially inhibited this anti-apoptotic effect. In conclusion, chloride-bicarbonate exchange blockers appear to offer cell-protective effects via Mcl-1 up-regulation.     

Enhanced secretion of tissue plasminogen activator by simultaneous use of retinoic acid and ascorbic acid from tissue cultured gastroepiploic artery

Tissue-type plasminogen activator (tPA) is a key enzyme in the fibrinolysis system and the regulation of its expression has been extensively studied in cultured vascular endothelial cells. Many kinds of supplements including growth factors are needed, however, to keep endothelial cells viable, which leads the culture condition far from the physiological milieu. Using a new device of amorphous calcium phosphate coated culture plate, we succeeded in culturing ring-cut gastroepiploic artery in a basic medium of RPMI 1640 containing 10% fetal calf serum. The overall normal vessel architecture and the antigenicity of von Willebrand factor, tPA and plasminogen activator inhibitor type 1 (PAI-1) were retained for at least 9 days. tPA was constantly secreted into the conditioned medium at least up to day 12. Employing this organ culture technique, we analyzed the effects of two well-known profibrinolytic vitamins of retinoic acid (Vit. A) and ascorbic acid (Vit. C) on the release of tPA and PAI-1. The cultured artery responded well and the tPA secretion was enhanced by factors of 1.5 fold by Vit. A, 1.7 fold by Vit C and 3.2 fold by their combination, whereas none of these stimuli increased PAI-1 secretion. These results suggested that the cultured ring-cut artery retained functional endothelial cells for at least 9 days and was suitable in analyzing the regulatory mechanism of protein synthesis and secretion from the vascular wall. Using this method, vitamins A and C were shown to lead the intravascular condition to a profibrinolytic state.     

Synthesis and biological evaluation of N-(7-indolyl)-3-pyridinesulfonamide derivatives as potent antitumor agents

We herein report the synthesis and antitumor activity of E7070 analogues containing a 3-pyridinesulfonamide moiety. E7070 was selected from our sulfonamide-based compound collections, currently undergoing Phase II clinical trials because of its tolerable toxicity profile and some antitumor responses in the Phase I setting. Of the analogues examined, ER-35745, a 6-amino-3-pyridinesulfonamide derivative, demonstrated significant oral efficacy against the HCT116 human colon carcinoma xenograft in nude mice.