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Biomedical subjects

T Kambara

Publications and source records attributed to T Kambara.

At least 73 records · Page 4Linked to original sources

The role of Pseudomonas aeruginosa elastase in corneal ring abscess formation in pseudomonal keratitis.

In order to identify the causative factors of ring abscess, which is the characteristic feature of pseudomonal keratitis, pseudomonal endotoxin, exotoxin A, and elastase were each separately injected into guinea pig cornea. There was no formation of ring abscess. Injection of living Pseudomonas aeruginosa strains IFO3455 and Takamatsu which produce all three molecules, clearly induced ring abscess. In contrast, when heat-killed bacteria strain IFO3455 or living bacteria of the non-elastase-producing strain PA103 were injected, ring abscess was not induced. Furthermore, when living bacteria strain IFO3455 were injected with anti-elastase antibody or a protease inhibitor, ovomacroglobulin, ring abscess formation was significantly inhibited. Histological examination demonstrated that the ring abscess was a dense accumulation and aggregation of polymorphonuclear leukocytes (PMN) with debris of cells and lamellae in the deep stroma at the corneal margins, suggesting prevention of PMN migration to the central lesion. The presence of anti-elastase antibody or a specific elastase inhibitor facilitated PMN migration towards living bacteria strain IFO3455 in an in vitro model. These results indicate that pseudomonal elastase is a necessary but not sufficient factor in the formation of ring abscess in pseudomonal keratitis.

ADP Ribose Transferases↗

The effect of age and dietary protein on immunity and resistance in lambs vaccinated with Trichostrongylus colubriformis.

Development of immunity and resistance to Trichostrongylus colubriformis was measured in sheep, 8-26 or 33-51 weeks of age, that were offered two levels of dietary protein (11 and 20%). Resistance to challenge infection was ascertained by measuring worm burden, faecal egg count and eggs in utero in the nematode. Development of immunity was measured by an in vitro lymphocyte blastogenesis test to T and B cell mitogens and to somatic antigen from infective (L3) larvae. Young lambs offered the low protein diet showed a significantly lower resistance to parasites than older animals. The young animals on the high protein diet developed better resistance. Age and dietary protein influenced in vitro T lymphocyte responses to parasite antigen and mitogens, which were particularly well developed in vaccinated lambs on the high protein diet. Lymphocyte responsiveness to L3 larval antigen was negatively correlated with parasite status in vaccinated older lambs, and may be important in the development of acquired resistance to T. colubriformis.

Aging↗

Model of chemically excitable membranes generating autonomous chaotic oscillations.

A simple mathematical model of the chemically excitable membranes leading to autonomous chaotic oscillations is presented. The model assumes two kinds of autocatalytic ion channels, one is for cations and the other is for anions. Self-consistency between the ion distributions and the electric potentials is taken into account by including the counter ions explicitly. Cations and anions pass through their own channels with their permeabilities changing nonlinearly with the densities of ions at the surfaces of the membrane. Cation and anion transport systems then form two subsystems that oscillate and interact with each other through the membrane potential. When the coupling strength between the two ion systems and adsorption rate of ions to channels are varied, various types of chaotic oscillations are generated autonomously, i.e., without a stimulating periodic force. Experimental evidence to the present model is discussed. It is suggested that endogenous chaos in biological systems may appear from the electric coupling among different kinds of ion transport systems.

Catalysis↗

Mechanism of the electric response of lipid bilayers to bitter substances.

In order to clarify by what mechanism the lipid bilayer membrane changes its potential under the stimulation of bitter substances, a microscopic model for the effects of the substances on the membrane is presented and studied theoretically. It is assumed that the substances are adsorbed on the membrane and change the partition coefficients of ions between the membrane and the stimulation solution, the dipole orientation in the polar head, and the diffusion constants of ions in the membrane. It is shown, based on the comparison of the calculated results with the experimental ones, that the response arises mainly from a change in the partition coefficients. Protons play an essential role in the membrane potential variation due to the change in their partition coefficients. The present model reproduces the following observed unique properties in the response of lipid bilayers to bitter substances, which cannot be accounted for by the usual channel model for the membrane potential: 1) the response of the membrane potential appears even under the condition that there is no ion gradient across the membrane, 2) the response remains even when the salt in the stimulating solution is replaced with the salt made of an impermeable cation, and 3) the direction of the polarization of the potential is not reversed, even when the ion gradient across the bilayer is reversed.

Biophysical Phenomena↗

A model for self-sustained potential oscillation of lipid bilayer membranes induced by the gel-liquid crystal phase transitions.

To clarify the mechanism of self-sustained oscillation of the electric potential between the two solutions divided by a lipid bilayer membrane, a microscopic model of the membrane system is presented. It is assumed, on the basis of the observed results (Yoshikawa, K., T. Omachi, T. Ishii, Y. Kuroda, and K. liyama. 1985. Biochem. Biophys. Res. Commun. 133:740-744; Ishii, T., Y. Kuroda, T. Omochi, and K. Yoshikawa. 1986. Langmuir. 2:319-321; Toko, K., N. Nagashima, S. liyama, K. Yamafuji, and T. Kunitake. Chem. Lett. 1986:1375-1378), that the gel-liquid crystal phase transition of the membrane drives the potential oscillation. It is studied, by using the model, how and under what condition the repetitive phase transition may occur and induce the potential oscillation. The transitions are driven by the repetitive adsorption and desorption of proton by the membrane surface, actions that are induced the periodic reversal of the direction of protonic current. The essential conditions for the periodic reversal are (a) at least one kind of cations such as Na+ or K+ are included in the system except for proton, and the variation of their permeability across the membrane due to the phase transition is noticeably larger than that of proton permeability; and (b) the phase transition has a hysteresis. When these conditions are fulfilled, the self-sustained potential oscillation may be brought about by adjusting temperature, pH, and the cation concentration in the solutions on both sides of the membrane. Application of electric current across the membrane also induces or modifies the potential oscillation. Periodic, quasiperiodic, and chaotic oscillations appear especially, depending on the value of frequency of the applied alternating current.

Biophysical Phenomena↗

Deoxyribonuclease treatment prevents blood-borne liver metastasis of cutaneously transplanted tumour cells in mice.

Murine L5178Y-ML cells, when transplanted subcutaneously into the flank of (BALB/c x DBA/2)F1 mice, grew locally and always formed spontaneous metastases in the liver. Even after surgical removal of the primary tumour mass 5 or 7 days after tumour cell inoculation, all mice died due to liver metastases within 18 days. Using this model of tumour metastasis, we examined whether serine protease or deoxyribonuclease I (DNase I) would affect metastasis. Spontaneous liver metastasis of L5178Y-ML cells was enhanced by systemic administration of alpha-chymotrypsin at 3, 4 and 5 days or at 5, 6 and 7 days after tumour cell inoculation. This result was consistent with a previous report on blood-borne lung metastasis. In contrast, systemic administration of DNase I at 3, 4 and 5 days or at 5, 6 and 7 days after tumour cell inoculation inhibited liver metastasis. Neither treatment affected primary tumour growth. An influence of DNase I on tumour cell arrest in the microvasculature of the liver was suggested by scanning electron microscopy. DNase I treatment resulted in a statistically significant prolongation of the survival period, however, the effect was not satisfactory. A more striking anti-metastatic treatment resulting in a greater prolongation of the survival period was achieved by combining surgical removal of the primary tumour mass with DNase I treatment. These results suggest that DNase I could be a potential therapeutic agent used in conjunction with surgery to prevent clinical blood-borne metastasis.

Animals↗

T-cell chemotactic activity of cytokine LD78: a comparative study with interleukin-8, a chemotactic factor for the T-cell CD45RA+ phenotype.

The recombinant LD78 cytokine (rLD78) promoted migration of human peripheral T lymphocytes at concentrations from 10(-11) M to 10(-7) M, with a peak of activity at 10(-8) M. There was no difference in migration promoting activity between LD78 alpha and its variant LD78 beta. Checkerboard analysis indicated that the migration of T cells attracted by rLD78 was chemotaxis but not chemokinesis. The chemotactic activity of rLD78 was apparently ten-fold lower than that of recombinant inteleukin-8 (rIL-8) when peripheral T cells were used as the indicator cells. Subset analysis on T cells responding to rLD78 or rIL-8 indicated that rIL-8 was more chemotactic for the CD45RA+ T cell subset than the CD45RO+ subset, but did not distinguish between CD4+ and CD8+ T cell subsets. In contrast, rLD78 apparently distinguished neither between CD4+ and CD8+ T cell subsets nor between CD45RA+ and CD45RO+ subsets.

Cell Movement↗

Thrombin generates monocyte chemotactic activity from complement factor H.

We have recently found that the complement factor H (H) was the precursor of the major macrophage chemotactic factor in the delayed-type hypersensitivity (DTH) reaction site in the skin and was converted to the factor by an unidentified trypsin-like protease in plasma. Thrombin and plasmin are also present in the site, and we, therefore, examined the possibility that these proteases converted H to be monocyte chemotactic. Intact H caused no monocyte migration, although it was able to do so after incubation with thrombin, but not with plasmin. The activity was chemotactic rather than chemokinetic and was absorbed by an anti-H IgG-conjugated column. The generation of monocyte chemotactic activity from H was dependent on incubation time with thrombin and also the protease activity of thrombin, and the activity was seen at concentrations of H lower than 10(-8) M. The inhibitory activity of H for C3b-Bb was not affected by incubation with thrombin or plasmin. Incubation of H with thrombin, but not with plasmin, generated a hydrophobic molecule, in a time-dependent manner, which had monocyte chemotactic activity. These results show that H becomes a monocyte chemotactic factor due to cleavage by thrombin, which converts H to a more hydrophobic molecule and also suggest that thrombin-treated H induces monocyte migration in the DTH reaction site.

Chemotaxis, Leukocyte↗

Novel function of C4a anaphylatoxin. Release from monocytes of protein which inhibits monocyte chemotaxis.

The complement C4-derived anaphylatoxin, C4a, possesses a strong chemotaxis inhibitory capacity to blood monocytes at concentrations as low as 10(-16) mol/L. In our study, treatment with carboxypeptidase B to convert it to C4a des Arg77 decreased the inhibitory activity to less than 1/1,000. The extraordinary inhibitory capacity of C4a suggests the presence of an amplification mechanism in this inhibition. Indeed, we found that the conditioned media of peripheral blood mononuclear cells or monocyte/macrophage lineage cell lines (U937 and THP-1 cells) preincubated with 10(-16) mol/L C4a for 5 minutes or more at 37 C possessed the inhibitory capacity 100,000-fold stronger than the original activity of C4a. The monocyte-derived chemotaxis inhibitory factor seemed monocyte-specific. This cell-derived factor was sensitive to treatment with trypsin and chymotrypsin and immunologically distinct from C4a. The apparent molecular size of the monocyte factor was estimated to be approximately 20 kd by gel filtration. These results indicate that C4a anaphylatoxin induces the release from monocytes of a protein with inhibitory activity for monocyte chemotaxis.

Cell Line↗

Primary structure of guinea-pig Hageman factor: sequence around the cleavage site differs from the human molecule.

The guinea-pig and human Hageman factors differ in their sensitivity to activation by particular bacterial proteinases. To understand this difference, the primary structure and cleavage site on activation of the guinea-pig molecule were determined and compared with the human molecule. By the use of a synthetic oligodeoxyribonucleotide probe which encoded a part of human Hageman factor cDNA, a cDNA clone was isolated from a lambda gt11 cDNA library of guinea-pig liver and sequenced. The cDNA clone was identified as that of guinea-pig Hageman factor by the complete identity of the deduced amino-acid sequence with the actual sequence of the amino-terminal portion of guinea-pig Hageman factor molecule and the active form. The cDNA included part of a leader sequence and the entire coding region of the Hageman factor molecule. Guinea-pig Hageman factor was composed of the same domain structures as the human counterpart with an overall 72% homology in the amino-acid sequence. However, the sequences around the cleavage site were surprisingly different; -Met351-Thr-Arg-Val-Val-Gly-Gly-Leu-Val359-(human) and -Leu338-Ser-Arg-Ile-Val-Gly-Gly-Leu-Val346-(guinea-pig). The amino-acid substitutions around the cleavage site might explain the difference in sensitivity to activation between the human and guinea-pig molecules.

Amino Acid Sequence↗

A new biological activity of the complement factor H: identification of the precursor of the major macrophage-chemotactic factor in delayed hypersensitivity reaction sites of guinea pigs.

The guinea pig complement factor H(FH) and the plasma precursor(PMCFS-1) of the major monocyte-chemotactic factor(MCFS-1) found in the skin site of delayed hypersensitivity reaction(DHR) induced in the guinea pigs were compared in the antigenicity and the function. Both anti-FH-IgG and anti-MCFS-1-IgG formed a single precipitation line against FH, PMCFS-1, MCFS-1 and guinea pig plasma, and these lines fused one another without any spur formation. The inhibition activity of FH for C3bBb was absorbed by anti-MCFS-1-F(ab')2 in a dose-dependent manner. PMCFS-1 inhibited C3bBb activity dose-dependently as FH. These results show that FH is identical to PMCFS-1 and imply that FH, converted to MCFS-1 plays as a monocyte-chemotactic factor in the site of DHR.

Animals↗

Activation of human plasma prekallikrein by Pseudomonas aeruginosa elastase. II. Kinetic analysis and identification of scissile bond of prekallikrein in the activation.

Activation of human plasma prekallikrein by a bacterial metalloendopeptidase, Pseudomonas aeruginosa elastase, was reported (Shibuya et al. (1991) Biochim. Biophys. Acta 1097, 23-27). Details of the activation process were presently studied. The activation accompanied limited proteolysis of a peptide bond inside of a disulfide bridge of prekallikrein molecule. Amino acid sequencing analysis of the newly generated amino-terminal revealed that the cleavage site was Arg371-Ile372 bond which is the scissile bond in the activation of prekallikrein with trypsin-type proteinases. A pentapeptide substrate, 2-aminobenzoyl-Ser-Thr-Arg-Ile-Val-4- nitrobenzylamide, which contained the amino acid sequence identical to that around the scissile bond of prekallikrein was synthesized. Pseudomonal elastase, indeed, hydrolyzed the substrate at Arg-Ile bond with the kinetic parameters of Km = 118 microM, kcat = 1.56/s and kcat/Km = 1.33.10(4)/s M. These results indicated that the Arg371-Ile372 bond was sensitive not only to trypsin-type serine proteinases, but also a bacterial metalloproteinase. Kinetic analysis of the prekallikrein activation by pseudomonal elastase, however, revealed that the activation rate was slow, though the Km values was good enough to expect an occurrence of this activation in vivo (Km = 248 nM, kcat = 6.8.10(-4)/s, and kcat/Km = 2.7.10(3)/s M). The activation rate of prekallikrein by pseudomonal elastase in Hageman factor deficient plasma was remarkably improved when the plasma was reconstituted with purified Hageman factor molecule. From the results, a biological significance of the proteinase cascade in the plasma kinin generation was also indicated. The present in vitro study might support the hypothesis that the Hageman factor/kallikrein-kinin system plays an important role in bacterial infection including the pseudomonal one.

Amino Acid Sequence↗

The major plasma kallikrein inhibitor of guinea pig plasma.

A plasma kallikrein inhibitor in guinea pig plasma (KIP) was purified to homogeneity. KIP is a single chain protein and the apparent molecular weight is estimated to be 59,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In amino acid composition, KIP is similar to human and mouse alpha 1-proteinase inhibitors and mouse contrapsin. KIP forms an equimolar complex with plasma kallikrein in a dose- and time-dependent fashion. The association rate constants for the inhibition of guinea pig plasma kallikrein by KIP, alpha 2-macroglobulin, C1-inactivator and antithrombin III were 2.5 +/- 0.3.10(4), 2.4 +/- 0.4.10(4), 6.6 +/- 0.5.10(4) and 9.1 +/- 0.6.10(2), respectively. Comparison of the association rate constants and the normal plasma concentrations of the four inhibitors demonstrates that KIP is ten-times as effective as alpha 2-MG and other two inhibitors are marginally effective in the inhibition of kallikrein. KIP inhibits trypsin and elastase rapidly, and thrombin and plasmin slowly, but is inactive for chymotrypsin and gland kallikrein. These results suggest that KIP is the major kallikrein inhibitor in guinea pig plasma and the proteinase inhibitory spectrum is unique to KIP in spite of the molecular similarity to alpha 1-proteinase inhibitor.

Amino Acids↗

Factor XIII-dependent generation of 5th complement component(C5)-derived monocyte chemotactic factor coinciding with plasma clotting.

Blood coagulation or plasma clotting caused generation of a monocyte chemotactic factor(s) in vitro. The chemotactic factor, of which the apparent molecular mass was 75 kDa, shared antigenicity with complement C5 and possessed the affinity to monocytes, but not to polymorphonuclear leukocytes. The generation of the chemotactic factor was hindered in the presence of a thiol enzyme inhibitor, p-chloromercuriphenyl sulfonic acid, at the concentration of 1 mmol/l, although the gelation of plasma was apparently completed. Furthermore, the generation of chemotactic factor was not observed when a plasma deficient in blood coagulation factor XIII, which is a precursor of a thiol enzyme, plasma transglutaminase, was used; and the activity normally appeared when the deficient plasma was reconstituted with purified factor XIII or with a tissue transglutaminase prior to clotting. When the human sera were injected into guinea pig skin, the serum derived from normal plasma or from the reconstituted factor XIII deficient one caused mononuclear cell infiltration, however, the serum from the deficient plasma without reconstitution infiltrated to a significantly smaller extent. These results indicated that the complement system was initiated somehow during the clotting process resulting in the generation of the C5-derived monocyte chemotactic factor in cooperation with factor XIIIa (activated factor XIII).

Animals↗

Role of thrombin and plasmin in development of delayed hypersensitivity reaction in guinea pig skin.

Induration is a prominent feature of delayed hypersensitivity reaction (DHR), and fibrin deposition is the central mechanism. We studied the effects of two inhibitors of DHR on the activities of thrombin and plasmin in the extract of the skin site of DHR and compared the two activities in the site of DHR with those in the site of the Arthus reaction (AR) that lacks induration. Warfarin, an anticoagulant, inhibited thrombin activity and induration, but not plasmin activity. Ferritin, a blocker of a macrophage-dependent reaction, inhibited the two activities and induration. The lesion of DHR had three to four times the thrombin activity of the lesion of AR, and the activity paralleled the development of induration. In contrast, plasmin activity of the site of DHR was lower than that of the site of AR and was associated with the reduction of induration. The two protease activities in the site of AR did not correlate with the development of the AR lesion. These results suggest that thrombin and plasmin mediate the development of induration and that induration is produced by a synergistic effect of high thrombin activity and low plasmin activity in the site of DHR.

Amino Acid Sequence↗

Substance P as a potent stimulator of sneeze responses in experimental allergic rhinitis of guinea pigs.

Substance P was examined for sneeze-inducing activity and its involvement of sneeze responses in experimental allergic rhinitis. Substance P, dripped into a nostril of guinea pigs, at concentrations of 100 pM and above induced sneezing in a dose-dependent fashion. The activity of substance P was not affected by the previous subcutaneous injections of capsaicin that depleted substance P in nerve fibers. Histamine induced sneezing at concentrations of 30 mM and above and the activity was reduced by capsaicin treatment. The frequency of antigen-induced sneezing was proportional to the substance P content in nasal mucosa of sensitized guinea pigs treated with increasing doses of capsaicin; correlation coefficient 0.91. These results suggest that substance P plays an important role as a stimulator of sneeze responses in experimental allergic rhinitis in guinea pigs.

Animals↗

Effect of opioids on delayed neuronal death in the gerbil hippocampus.

The effect of opioids on delayed neuronal death was evaluated in the gerbil hippocampus. Male Mongolian gerbils were subjected to transient forebrain ischemia and neuronal density was evaluated in the hippocampus 7 days following ischemia. When hypothermia during and after ischemia was prevented, treatment with morphine, U-50488H, or naloxone provided no significant protection. In contrast, a spontaneous drop in rectal temperature to 32 degrees C at the end of ischemia produced near-complete protection of CA1 pyramidal neurons. No opioids modulate the protective effect of hypothermia.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

Effect of capsaicin as a neuropeptide-releasing substance on sneezing reflex in a type I allergic animal model.

To elucidate the effect of capsaicin-sensitive nerve fibers, which are known to contain substance P (SP) and other sensory neuropeptides, on the sneezing reflex, we have investigated the effect of capsaicin on this reflex provoked in guinea pigs passively sensitized with anaphylactic antibody followed by specific antigen challenge. It has already been established that histamine released from mast cells is a reliable inducer of the sneezing reflex in type I allergy. Our experimental results indicated that the frequency of sneezing provoked by antigen challenge as well as histamine application was significantly reduced by pretreatment with capsaicin in a dose-dependent fashion. SP is considered to be one of the main neurotransmitters in sensory nerves. When the amount of SP in animal nasal mucosa was measured 12 h after capsaicin treatment, a marked reduction was noted. However, the histamine content in the nasal mucosa was not changed by capsaicin treatment. These data suggest that neuropeptides, especially SP, which are released or depleted from sensory nerves by capsaicin treatment, probably play an important role as neurotransmitters of the stimulant histamine in the development of sneezing in type I allergy.

Animals↗