Claudin promotes activation of pro-matrix metalloproteinase-2 mediated by membrane-type matrix metalloproteinases.

Genes associated with regulation of membrane-type matrix metalloproteinase-1 (MT1-MMP)-mediated pro-MMP-2 processing were screened in 293T cells by a newly developed expression cloning method. One of the gene products, which promoted processing of pro-MMP-2 by MT1-MMP was claudin-5, a major component of endothelial tight junctions. Expression of claudin-5 not only replaced TIMP-2 in pro-MMP-2 activation by MT1-MMP but also promoted activation of pro-MMP-2 mediated by all MT-MMPs and MT1-MMP mutants lacking the transmembrane domain (DeltaMT1-MMP). A carboxyl-terminal deletion mutant of pro-MMP-2 (proDeltaMMP-2) was processed to an intermediate form by MT1-MMP in 293T cells and was further converted to an activated form by introduction of claudin-5. In contrast to the stimulatory effect of TIMP-2 on pro-MMP-2 activation by MT1-MMP, activation of pro-MMP-2 by DeltaMT1-MMP in the presence of claudin-5 and proDeltaMMP-2 processing by MT1-MMP were both inversely repressed by expression of exogenous TIMP-2. These results suggest that TIMP-2 is not involved in cluadin-5-induced pro-MMP-2 activation by MT-MMPs. Stimulation of MT-MMP-mediated pro-MMP-2 activation was also observed with other claudin family members, claudin-1, claudin-2, and claudin-3. Amino acid substitutions or deletions in ectodomain of claudin-1 abolished stimulatory effect. Direct interaction of claudin-1 with MT1-MMP and MMP-2 was demonstrated by immunoprecipitation analysis. MT1-MMP was co-localized with claudin-1 not only at cell-cell borders, but also at other parts of the cells. TIMP-2 enhanced cell surface localization of MMP-2 mediated by MT1-MMP, and claudin-1 also stimulated it. These results suggest that claudin recruits all MT-MMPs and pro-MMP-2 on the cell surface to achieve elevated focal concentrations and, consequently, enhances activation of pro-MMP-2.

Interrelation between oxygen tension and nitric oxide in the respiratory system.

To understand the relationship between oxygen tension and nitric oxide (NO) function, one animal and two human studies were designed. In the animal study, the effect of NO in inducing the relaxation of aortic specimens was significantly lower by 68% under 480 mm Hg of oxygen tension than under 28 mm Hg, indicating that oxygen tension has an important role in determining the biological effects of NO. In a clinical analysis with nonsmokers (n = 23), the alveolar-to-arterial difference for oxygen (A-aDO(2)) was reciprocally correlated with exhaled NO concentrations (r = 0.53). Because NO concentration in the lower respiratory zone depends partly on the amount of inspirable NO originating in the upper airway, a well-ventilated area, requiring much perfusion, could receive greater amounts of NO than a poorly ventilated one. Thus, the reciprocal relation of A-aDO(2) with the concentration of exhaled NO is not necessarily incompatible with the effect of hypoxic pulmonary vasoconstriction in ventilation-to-perfusion (V'A/Q') imbalance. In our third experiment, with nonsmokers (n = 21), pure oxygen inhalation during mechanical ventilation significantly decreased the concentration of exhaled NO and enhanced A-aDO(2), indicating a relationship between NO and oxygen similar to that observed in the animal experiment. These findings led us to conclude that a positive relation between exhaled NO and blood oxygenation efficiency exists in the respiratory system, and further, that oxygen might affect this relationship. Thus, the relative balance of NO and oxygen concentrations may be another factor for consideration in respiratory function.

Mutation induction by N-propyl-N-nitrosourea in eight MutaMouse organs.

As a part of the 2nd Collaborative Study for the Transgenic Mouse Mutation Assay, we studied the organ specificity and the temporal changes in mutant frequency (MF) of the lacZ gene following intraperitoneal injection of 250 mg/kg N-propyl-N-nitrosourea into male MutaMouse. We used a positive selection system and examined eight organs, i.e., bone marrow, liver, kidney, lung, spleen, brain, heart, and testis. The chemical caused a significant increase in MF in all organs except for brain, and the bone marrow was the most sensitive organ, exhibiting a MF on day 7 that was 10 times that of the control. The MF increased from day 7 to day 28 in liver, kidney, and testis, while it decreased in bone marrow. The relationship between the results of this study and the target organs of carcinogenesis, and the cause of the temporal changes in MF, are discussed.

[The relationship between respiratory function and the change in end-tidal nitrous oxide concentration].

The aim of the present study is to clarify the relationship between respiratory function and the rate of change in alveolar anesthetic concentration. We measured the concentration of end-tidal nitrous oxide (N2O) when 50% N2O was administered to 15 patients of ASA I possessing normal respiratory function during the course of propofol-100% oxygen anesthesia. All patients were ventilated at a rate of 8-10 ml.kg-1 x 8 times per minute using a conventional anesthetic ventilator with semi-closed circuit and 4 l.min-1 inflow of fresh gas. Arterial CO2 partial pressure was maintained at 36.2 +/- 1.8 mmHg and no significant circulatory change was observed while N2O was administered. The rate of increase of end-tidal N2O concentration in poor FEV1.0/FVC% group was significantly slower than that in high FEV1.0/FVC% group, while there was no relation between %VC and the end-tidal N2O concentration change. Since N2O is an inhaled anesthetic, it is well considered that the effect of FEV1.0/FVC% may be observed in other inhaled anesthetic although the magnitude of the effect may vary. The present result suggests that respiratory function, especially FEV1.0/FVC%, is an important factor affecting the rate of change in alveolar anesthetic concentration and, in lower FEV1.0/FVC% group, it takes more time to achieve the intended alveolar concentration.

Distribution of fallover in the carboxylase reaction and fallover-inducible sites among ribulose 1,5-bisphosphate carboxylase/oxygenases of photosynthetic organisms.

The biphasic reaction course, fallover, of carboxylation catalysed by ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) has been known as a characteristic of the enzyme from higher land plants. Fallover consists of hysteresis in the reaction seen during the initial several minutes and a very slow suicide inhibition by inhibitors formed from the substrate ribulose-1,5-bisphosphate (RuBP). This study examined the relationship between occurrence of fallover and non-catalytic RuBP-binding sites, and the putative hysteresis-inducible sites (Lys-21 and Lys-305 of the large subunit in spinach RuBisCO) amongst RuBisCOs of a wide variety of photosynthetic organisms. Fallover could be detected by following the course of the carboxylase reaction at 1 mM RuBP and the non-catalytic binding sites by alleviation of fallover at 5 mM RuBP. RuBisCO from Euglena gracilis showed the same linear reaction course at both RuBP concentrations, indicating an association between an absence of fallover and an absence of the non-catalytic binding sites. This was supported by the results of an equilibrium binding assay for this enzyme with a transition state analogue. Green macroalgae and non-green algae contained the plant-type, fallover enzyme. RuBisCOs from Conjugatae, Closterium ehrenbergii, Gonatozygon monotaenium and Netrium digitus, showed a much smaller decrease in activity at 1 mM RuBP than the spinach enzyme and the reaction courses of these enzymes at 5 mM RuBP were almost linear. RuBisCO of a primitive type Conjugatae, Mesotaenium caldariorum, showed the same linear course at both RuBP concentrations. Sequencing of rbcL of these organisms indicated that Lys-305 was changed into arginine with Lys-21 conserved.

Antimetastatic and antitumor effect of a recombinant human tissue inhibitor of metalloproteinases-2 in murine melanoma models.

Tumor metastasis into distinct organs and tissues, of which many patients with malignancies die, is regulated in multiple steps. Using a murine metastasis model, in which highly metastatic B16-BL6 melanoma cells were inoculated i.v. into syngeneic C57BL/6 mice, the administration of a recombinant human tissue inhibitor of metalloproteinases-2 (r-hTIMP-2) once a day on days -1 to 3 after the implantation significantly inhibited the formation of metastatic foci in the lungs. The antimetastatic effect of r-hTIMP-2 was detected irrespective of administration route [i.v., i.p., s.c., and i.m. routes] and in a dose-dependent manner. The i.m.-injection of r-hTIMP-2 during the early phase after tumor inoculation is suggested to be essential for antimetastasis. In another model using spontaneously metastasing B16-BL6 cells, multiple i.m.-injections of r-hTIMP-2 also resulted in a reduced but not statistically significant number of pulmonary metastases. In addition to these antimetastatic effects, a slight inhibitory effect on tumor cell growth was observed in vitro and in vivo. In conclusion, the antimetastasis by r-hTIMP-2 may be due to inhibition of the degradation of the extracellular matrix by matrix metalloproteinases (MMPs) and, in part, to the suppression of the tumor cell growth.

[Effect of controlled hypotension on cerebral oxygen delivery].

The margin of safety for controlled hypotension is still unclear especially in the central nervous system (CNS) which is one of the most sensitive organs to hypoxia and ischemia. Recently, cerebral optical spectroscopy in the infrared light range was developed as a useful tool which makes it possible to monitor cerebral oxygenation (rSO2) non-invasively and continuously during anesthesia. Resulting rSO2 mainly reflects oxygen extracts by cerebral tissue and then indicates cerebral oxygen delivery. We examined the limitation of controlled hypotension in the brain in 12 patients by monitoring rSO2 during anesthesia. rSO2 under room air breathing (control value as normal physiological condition) was 67 +/- 3% (mean +/- SEM). It significantly increased by 5.6 +/- 0.8% under 100% oxygen breathing, but decreased near to the control value under sevoflurane anesthesia (FIO2 1.0). During moderate controlled hypotension (70% of normal blood pressure) by prostaglandin E1 under sevoflurane anesthesia (FIO2 1.0). rSO2 remained at control value, indicating that cerebral oxygen delivery was still sufficiently maintained. However rSO2 decreased significantly by 9.0 +/- 1.1% in same controlled hypotension condition under FIO2 0.4. This decrease in rSO2 could be potentially harmful for CNS although any post-operative neurological disorder was not observed in our cases. We conclude that cerebral oxygen delivery may be insufficient even in the moderate controlled hypotension, and thus higher FIO2 is recommended in such procedures.

Determination of tissue inhibitor of metalloproteinases-2 (TIMP-2) in experimental animals using monoclonal antibodies against TIMP-2-specific oligopeptides.

We have developed a one-step sandwich enzyme immunoassay (EIA) using monoclonal antibodies against oligopeptides of the human tissue inhibitor of metalloproteinases-2 (TIMP-2) (Fujimoto et al. (1993) Clin. Chim. Acta 220, 31). The present studies further demonstrated that the antibodies cross-react with TIMP-2 species of experimental animals including mouse, rat, guinea pig and rabbit. The detection of the TIMP-2 species in our EIA system was verified using rat TIMP-2 and the EIA was subsequently used to measure the animal TIMP-2 in the sera. Using human TIMP-2 as a standard, TIMP-2 levels in the sera of mouse, rat, guinea pig and rabbit were approximately 80, 200, 270 and 25 ng/ml, respectively.

Lysine residues involved in the hysteresis and in the regulatory sites of spinach ribulose 1,5-bisphosphate carboxylase/oxygenase.

Lysine residues have been suggested to be involved in the hysteretic decrease of the activity of spinach ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and the binding of ribulose 1,5-bisphosphate to its regulatory sites [Yokota, A. & Tsujimoto, N. (1992) Eur. J. Biochem. 204, 901-909]. This work identifies the lysine residues and investigates the effects of their chemical modification on the course of RuBisCO reaction. The carbamylated form of RuBisCO reacted with trinitrobenzene sulfonate in three phases; an initial rapid, second slow, and final non-specific reaction. Lys-334 in loop 6, Lys-21, and Lys-128, all from the large subunits, were trinitrophenylated in the first 60-min reaction. Lys-305 of the large subunits was labeled in the next step. The modification of these residues was strongly suppressed in the enzyme form that had undergone hysteretic conformational change after binding 2-carboxyarabinitol 1,5-bisphosphate at its catalytic sites. Instead, Lys-450 of the large subunits and Lys-71 from the small subunits were newly modified in the quaternary complex. A higher concentration of 2-carboxyarabinitol 1,5-bisphosphate reduced the trinitrophenylation of the two residues to half. The modification of the carbamylated form of the enzyme for 30 min was expected to arylate Lys-21, Lys-128, and Lys-334 at random, and the course of the reaction of the partially modified enzyme was expected to deviate from that of the unmodified enzyme. Experimental results showed that this was the case.(ABSTRACT TRUNCATED AT 250 WORDS)