Biomedical subjects

Y Okada

Publications and source records attributed to Y Okada.

At least 451 records · Page 25Linked to original sources

PubMed1998

Inhibition of membrane-type 1 matrix metalloproteinase by hydroxamate inhibitors: an examination of the subsite pocket.

The membrane-type 1 matrix metalloproteinase (MT1-MMP) has been reported to mediate the activation of pro-gelatinase A (proMMP-2), which is associated with tumor proliferation and metastasis. MT1-MMP can also digest extracellular matrix (ECM) such as interstitial collagens, gelatin, and proteoglycan and thus may play an important role in pathophysiological digestion of ECM. We studied the inhibitory effect of various hydroxamate MMP inhibitors, including known inhibitors such as BB-94, BB-2516, GM6001, and Ro31-9790, on a deletion mutant of MT1-MMP lacking the transmembrane domain (DeltaMT1) to further characterize the enzyme and develop a selective inhibitor for MT1-MMP. The evaluation of the inhibitory activities of various hydroxamates reveals general structural profiles affecting selectivities toward MMPs. In particular, a longer side chain at the P1' position is preferable for the binding to MMP-2, -3, and -9 and MT1-MMP. For the P2' position, an alpha-branched alkyl group is critical for the binding toward DeltaMT1, while the introduction of a bulky group at the alpha-position of hydroxamic acid seems to diminish the activity against DeltaMT1. Summation of the data on the sensitivity of DeltaMT1 to various hydroxamate inhibitors indicates that (1) the volume of the S1' subsite of DeltaMT1 is similar to that of MMP-2, -3, and -9, which is bigger than that of MMP-1, and (2) the S1 and S2' subsites are narrower than those in other MMPs. On the basis of these results, the hydroxamates with a P1' phenylpropyl and P2' alpha-branched alkyl group were synthesized and evaluated for inhibitory activity. These inhibitors (1h,i) showed strong activity against DeltaMT1 over MMP-1, but no selectivity between DeltaMT1 and MMP-9. These results are explained using molecular modeling studies conducted on MT1-MMP.

Amino Acid Sequence↗

PubMed1998

Activation of the precursor of human stromelysin 2 and its interactions with other matrix metalloproteinases.

Matrix metalloproteinases (MMP) are synthesized as inactive zymogens (proMMP) and subsequently activated by many factors to degrade the extracellular matrix (ECM). In the present study, we have examined the intermolecular activation mechanisms of proMMP by MMP-10 (stromelysin 2). ProMMP-10 was purified from the culture media of OSC-20 human oral squamous carcinoma cells stimulated with 12-O-tetradecanoylphorbol 13-acetate. The final products are partially activated (approximately 38% of the full activity) during the purification steps and contain proMMP-10 of Mr 56,000 with minor protein bands of Mr 47,000, 24,000 and 22,000. The zymogen is activated by 4-aminophenylmercuric acetate and processed to the active forms of Mr 47,000 and 24,000. The NH2-terminal sequence of the 47,000- and 24,000-Mr species is Phe82-Ser-Ser-Phe-Pro-Gly, which is identical to that of stromelysin 2. ProMMP-9 (progelatinase B) is activated by MMP-10 to its full activity and processed to the low-Mr species of Mr 81,000, 65,000, 57,000 and 55,000, the former two of which show proteolytic activity on a gelatin zymography. The NH2-terminal sequence analysis indicates that the 81,000-, 65,000- and 57,000-M, species have the identical sequence of Phe88-Gln-Thr-Phe-Glu-Gly, suggesting the cleavage of the Arg87-Phe88 peptide bond for activation and both NH2-terminal and COOH-terminal truncation in the 65,000- and 57,000-Mr forms. MMP-10 also activates proMMP-7 (promatrilysin) up to about 60% of the full activity and generates the same active species of Mr 19,000 as that obtained by activation with 4-aminophenylmercuric acetate. Incubation of proMMP-2 (progelatinase A) or proMMP-3 with MMP-10 does not result in activation of these proMMP. These results indicate that in addition to the previously reported activation of proMMP-1 (tissue procollagenase) and proMMP-8 (neutrophil procollagenase), MMP-10 can also activate proMMP-9 and proMMP-7, and suggest the possibility that MMP-10 may replace a role of MMP-3 in the ECM degradation in concert with other MMP under various pathological conditions.

Amino Acid Sequence↗

PubMed1998

The effects of glucose, mannose, fructose and lactate on the preservation of neural activity in the hippocampal slices from the guinea pig.

Using hippocampal slices from guinea pigs, we investigated the effect of different concentrations of glucose and replacement of glucose with mannose, fructose and lactate on neural activity. As an index of neural activity, the population spikes (PS) were recorded in the granule cell layer of the dentate gyrus (DG) and the pyramidal cell layer of the CA3 area in the hippocampal slices. Lowering the concentration of glucose from 10 mM to 5, 3, 2, 1 and 0 mM caused a reduction in the PS amplitude. There were differences in the decay times of the PS evoked in these two regions. PS evoked in CA3 region decayed faster even at a concentration of 3 mM glucose at which PS in granule cell layer was well maintained. The decay time of the PS in the CA3 region in the presence of glucose up to a concentration of 3 mM was shorter than that evoked in the DG. After the replacement of glucose with mannose, fructose or lactate, the PS disappeared within 35 min and there were no significant differences between the decay times in the two regions of slices incubated in the same medium. ATP, creatine phosphate (CrP) and lactate levels in each slice were determined. To investigate whether mannose and fructose could be metabolized or not in the tissue slice, anaerobic production of lactate from glucose, mannose and fructose were measured during oxygen and glucose deprivation. Under anaerobic conditions for 60 min, the levels of high-energy phosphates decreased to 50% of the initial level and lactate was produced from glucose, mannose or fructose. However, there were significant differences in the rate of lactate production between the DG and CA3 areas during application of 3 mM glucose, 10 mM mannose and 10 mM fructose. These results indicate that mannose, fructose and lactate can be metabolized and are available for maintaining the levels of high-energy phosphates but not for neural activity in the tissue slices and that the presence of glucose is indispensable for the maintenance of neural activity.

Adenosine Triphosphate↗

PubMed1998

The levels of adenosine and its metabolites in the guinea pig and rat brain during complete ischemia-in vivo study.

Changes in levels of adenosine, inosine, hypoxanthine and ATP during complete ischemia after decapitation were determined in various areas of the guinea pig and rat brain using an HPLC method. These results were compared with levels in brains fixed by microwave irradiation. The levels of adenosine during 60 min of complete ischemia were extremely high and unevenly distributed while levels in the microwaved brains were very low and evenly distributed. The ratios of inosine plus hypoxanthine levels to adenosine which indicate the rate of metabolic degradation from adenosine into inosine and hypoxanthine, were also unevenly distributed during complete ischemia in the cerebellum, superior colliculus, cerebral cortex and hippocampus of the guinea pig and rat, and the highest ratio was observed in the cerebellum of the guinea pig and the superior colliculus of the rat. The activities of adenosine deaminase (ADA), one of the enzymes involved in adenosine metabolism, were measured in the four regions of the guinea pig. The ADA activities were unevenly distributed and the highest ADA activity was found in the cerebellum. These regional differences in ADA activities are in good agreement with the regional differences in the ratio of inosine plus hypoxanthine levels to adenosine during complete ischemia. Furthermore, the administration of EHNA [erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride] (10 mg/kg, i.p.), an ADA inhibitor, caused a significant increase of adenosine and decrease of inosine formation in all four regions and a drastic effect on the cerebellum with high ADA activity compared with the other regions in the guinea pig brain. These results indicate that the changes in concentrations of adenosine and its metabolites (inosine and hypoxanthine) during complete ischemia depend on ADA activity in each brain region.

Adenine↗

PubMed1998

The effects of simulated ischemia on the levels of adenosine and its metabolites in slices of cerebellum, superior colliculus and hippocampus in the guinea pig-in vitro study.

The purpose of this present study is to clarify adenosine (ADO) metabolism in guinea pig brain slices during simulated ischemia. In slice preparations after decapitation, ADO levels were lowest in slices of the cerebellum (1.2 nmol/mg protein), followed by the superior colliculus (3.4) and highest in the hippocampus (6.4), and the combined concentrations of inosine (Ino) and hypoxanthine (HX) were highest in the cerebellum (5.5), followed by the superior colliculus (3.5) and the hippocampus (1.5). After preincubation with standard medium with oxygen and glucose for 30 min, total ADO levels (tissue ADO plus ADO lost into medium during incubation) decreased to 0.3 in the cerebellum, to 1.3 in the superior colliculus and to 2. 9 in the hippocampus. On the other hand, levels of total Ino and HX increased to 21.1 in the cerebellum, to 14.3 in the hippocampus and to 12.0 in the superior colliculus. To investigate the effect of simulated ischemia on ADO metabolism, preincubated slices were exposed for 10 min in medium deprived of oxygen and glucose. The increases of ADO content after 10 min ischemia were 0.2 in the cerebellum, 1.0 in the superior colliculus and 1.3 in the hippocampus. In contrast, the increases of both Ino and HX concentrations were 2.9 in the cerebellum, 2.2 in the superior colliculus and 1.4 in the hippocampus. The total amount of the increase in ADO, Ino and HX was approximately 3 in all three regions. These results indicate that there are significant differences in the metabolic rate to degrade ADO into Ino and HX in various areas of brain possibly due to differences in adenosine deaminase activity in those areas.

Adenosine↗

PubMed1998

Development of an assay for bioactive insulin.

A method to detect the biological activity of serum insulin has been developed. This method, called a bioactive insulin assay, determines the ability of serum insulin to stimulate the autophosphorylation of insulin receptors in an intact cell system. For this, intact Chinese hamster ovary cells which overexpress the human insulin receptor are treated with serum and then lysed. Autophosphorylation of the insulin receptors is then measured by a two-site immunofluorometric assay using monoclonal anti-insulin receptor antibodies and europium-labeled anti-phosphotyrosine antibodies. The detection limit of this assay is 1 microU/ml of insulin. Dilution and recovery test inter- and intraassay coefficient variations are permissible. The amount of insulin determined by this assay correlated well with the amount of insulin detected by a traditional immunological assay for insulin (r = 0.94, P < 0.001). In the case of a mutant insulin, the insulin from a Wakayama subject, the biologically active insulin was found to constitute 9% of the immunologically reactive insulin. Since this assay specifically measures the amount of biologically active insulin present in serum, it should be particularly useful in monitoring active insulin in patients with various mutant insulins.

Animals↗

PubMed1998

Effects of extracellular calcium and magnesium on central respiratory control in the brainstem-spinal cord of neonatal rat.

The influence of extracellular Ca2+ and Mg2+ concentrations ([Ca2+]ECF and [Mg2+]ECF, respectively) on central respiratory control was analyzed using the isolated brainstem-spinal cord of the neonatal rat. Central respiratory activity was recorded from the C4 ventral roots. The depth profile of [Ca2+]ECF below the ventral medullary surface was measured with ion-sensitive electrodes. The gradient in [Ca2+]ECF disappeared about 1 h after changing superfusate Ca2+ ([Ca2+]CSF) from 2 to 0.5 mM, but not even in 2 h after switching to Ca2+-free superfusate. High [Ca2+]CSF (4 mM) or high [Mg2+]CSF (4, 8 mM) decreased respiratory frequency (fR), whereas low [Ca2+]CSF (0.5 mM) increased fR and augmented the respiratory CO2 responsiveness. High [Ca2+]CSF as well as low [Mg2+]CSF (0.5 mM) disturbed respiratory rhythm and pattern, which were markedly restored by high CO2. The depressing effect of high [Ca2+]ECF and the stimulating effect of low [Ca2+]ECF on the medullary neuronal activity were confirmed by perforated patch recordings. These results suggest that [Ca2+]ECF and [Mg2+]ECF determine the excitability of the respiratory neuron network by modulating the neuronal surface potential, transmembrane Ca2+ influx, Ca2+-sensitive cation channel gating, and synaptic transmission. Furthermore, some of these actions appear to be antagonized by CO2/H+.

Animals↗

PubMed1998

Scavenging Effect of Water Soluble Proteins in Broad Beans on Free Radicals and Active Oxygen Species.

Water soluble proteins (WSP) in broad beans, Vicia faba, were purified, and their scavenging effects on free radicals and active oxygen species were investigated. The purification steps included ammonium sulfate precipitation followed by sequential chromatography on Sephadex G-75. The final gel filtration step yielded two peaks of scavenging activity, each containing M(r) values of 70 kDa (peak I) and 28 kDa (peak II). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of peak II fraction gave a single band with M(r) of 14 kDa, indicating that peak II protein is dimeric. WSP exhibited a marked scavenging effect on superoxide, and also an effect on hydrogen peroxide, but not so much on 1,1-diphenyl-2-picrylhydrazyl radical. WSP had only a small amount of sulfhydryl groups. Thus, the sulfhydryl groups are not responsible for the scavenging activity of WSP.

Journal Article↗

PubMed1998

Probing the water permeability of ROMK1 and amphotericin B channels using Xenopus oocytes.

Water permeability of ion channels in the plasma membrane of Xenopus oocytes was studied by simultaneously measuring the membrane conductance under two-electrode voltage-clamp and the cell size by video-imaging technique. The basal level of osmotic water permeability of oocyte plasma membrane was 15.9+/-0.98 microm/s (SE, n = 5). Extracellular application of pore-forming antibiotic amphotericin B at 5 microM developed macroscopic conductance of 995+/-70 microS (n = 5) and increased the osmotic water permeability of cell membrane by 44.9+/-4.1 microm/s. Meanwhile, after expressing ROMK1 channels, originally cloned from kidney, virtually no increase in the water permeability was observed even at the conductance level as high as 1113+/-47 microS (n = 5). This result suggests that even though potassium channels, like any others, are considered to be water-filled pores, K+-selective ion-transporting pathway remains virtually water-impermeable in physiological conditions, such as in kidney epithelia where huge water transport takes place at both apical and basolateral sides.

Amphotericin B↗

PubMed1998

Bioactive bone cement: comparison of apatite and wollastonite containing glass-ceramic, hydroxyapatite, and beta-tricalcium phosphate fillers on bone-bonding strength.

A study was conducted to compare the bone-bonding strengths of three types of bioactive bone cement, consisting of either apatite- and wollastonite-containing glass-ceramic (AW-GC) powder, hydroxyapatite (HA) powder, or beta-tricalcium phosphate (beta-TCP) powder as an inorganic filler and bisphenol-a-glycidyl methacrylate (Bis-GMA) based resin as an organic matrix. Seventy percent (w/w) filler was added to the cement. Rectangular plates (10 x 15 x 2 mm) of each cement were made and abraded with #2000 alumina powder. After soaking in simulated body fluid for 2 days, the AW cement (AWC) and HA cement (HAC) formed bonelike apatite over their entire surfaces, but the TCP cement (TCPC) did not. Plates of each type of cement were implanted into the tibial metaphyses of male Japanese white rabbits, and the failure loads were measured by a detaching test at 10 and 25 weeks after implantation. The failure loads of AWC, HAC, and TCPC were 3.95, 2.04, and 2.03 kgf at 10 weeks and 4.36, 3.45, and 3.10 kgf at 25 weeks, respectively. The failure loads of the AWC were significantly higher than those of the HAC and TCPC at 10 and 25 weeks. Histological examination by contact microradiogram and Giemsa surface staining of the bone-cement interface revealed that all the bioactive bone cements were in direct contact with bone. However, scanning electron microscopy and energy-dispersive X-ray microanalysis showed that only AWC had contacted to the bone via a Ca-P rich layer formed at the interface between the AW-GC powder and the bone, which might explain its high bone-bonding strength. Neither the HAC nor the TCPC contacted the bone through such a layer between each powder and the bone, although the HAC and TCPC directly contacted with bone. Our results indicate that all three types of abraded and prefabricated cement have bonding strength to bone, but AWC has superior bone-bonding strength compared to HAC and TCPC.

Animals↗

PubMed1998

Osteoclastic resorption of bone-like apatite formed on a plastic disk as an in vitro assay system.

We have investigated the applicability of a simple and inexpensive osteoclastic assay system using bone-like apatite-coated polyethyleneterephthalate (PET) disks. A 1 microm thick apatite layer, uniform and homogeneous bone-mineral-like with no organic components, was made on PET disks using a biomimetic process. As substrates for an osteoclastic assay, these coated disks were compared with dentine as well as with bone-like or heat-treated apatite of various thicknesses on apatite- and wollastonite-containing glass ceramic (A-W GC) disks. The unfractionated bone cells, including osteoclasts, of a neonatal rabbit were seeded onto these substrates. By scanning electron microscopic examination, the resorption lacunae of the thick bone-like apatite clearly showed track-like shapes at various depths, similar to those of dentine although the border between the A-W GC and the apatite was unclear. In contrast, those of heat-treated apatite showed small and shallow shapes with irregular margins, quite different from those of dentine. By reducing the thickness of bone-like apatite to 1 microm as well as using PET as its substrate, the margins of the resorption lacunae became quite clear, and with the use of phase-contrast microscopy during culture, osteoclasts and resorption pits could be precisely observed. The resorbed area, easily measured with the aid of bright-field microscopy and an image analyzer, was found to have increased in a time-dependent manner and at the end of 4 days of culture was not statistically different from that of dentine.

Animals↗

PubMed1998

Immunolocalization of prolyl 4-hydroxylase subunits, alpha-smooth muscle actin, and extracellular matrix components in human lens capsules with lens implants.

Lens capsules become fibrotic after the extraction of a cataract. To understand this phenomenon, we evaluated the immunolocalization of prolyl 4-hydroxylase (an enzyme involved in procollagen hydroxylation), and extracellular matrix components and cytoskeletal components in a normal human lens capsule and in others with intraocular lenses. Lens capsules containing intraocular lenses were removed from a patient with proliferative vitreoretinopathy and three with proliferative diabetic retinopathy during vitreous surgery. Two circular sections of the anterior capsules with lens epithelial cells were obtained by anterior capsulotomy during cataract surgery. In addition, a lens capsular bag was obtained immediately after phacoemulsification. The lens capsules were processed for light microscopic immunohistochemical detection of the alpha and beta subunits of prolyl 4-hydroxylase, extracellular matrix components (including collagen types, laminin and cellular fibronectin) or cytoskeletal components (such as cytokeratin, vimentin and alpha-smooth muscle actin). Monolayer lens epithelial cells were seen on the inner surface of the normal anterior capsules. Each intraocular lens was found to be fixed in the capsular bag. Light microscopic immunohistochemistry showed that these proliferating cells expressed vimentin and alpha-smooth muscle actin; in contrast, quiescent lens epithelial cells did not stain for alpha-smooth muscle actin. Marked immunostaining for subunits of prolyl 4-hydroxylase was detected in lens epithelial cells proliferating on the capsules, while no or only faint prolyl 4-hydroxylase immunoreactivity was detected in quiescent lens epithelial cells immediately after phacoemulsification. Collagen types I, III and VI and cellular fibronectin were observed diffusely in accumulated connective tissue on a capsule with an intraocular lens. Type IV collagen immunoreactivity was seen both in the capsules and in the connective tissue accumulation on the capsules. Collagen V and laminin were detected in association with cellular proliferation. Collagen VII and VIII and laminin 5 were not seen. We concluded that during wound healing of the lens capsule after cataract extraction, the lens epithelial cells that proliferate on the inner surface of the capsule transform it into a myofibroblastic phenotype, expressing prolyl 4-hydroxylase and alpha-smooth muscle actin. These proliferating cells are involved in the production of collagen on the lens capsule. This results in a postoperative fibrotic process and contraction of the lens capsule.

Actins↗

PubMed1998

[Successful surgical correction for an incomplete endocardial cushion defect in an elderly patient].

A 72-year-old female who presented with symptoms of severe congestive heart failure, was subsequently diagnosed as having an incomplete endocardial cushion defect. A severe left-to-right shunt and mitral and tricuspid valve regurgitation were noted. Closure of the ostium primum defect, mitral valve replacement, and tricuspid valve annuloplasty were performed simultaneously. The postoperative course was uneventful. A review of literature revealed that this is the oldest such surgical patient described in Japan to date. We believe that surgical correction can be effective even in elderly patients.

Aged↗

PubMed1998

Fentanyl analgesia increases the incidence of postoperative hypothermia in neonates.

Postoperative hypothermia remains a clinical problem in neonates undergoing surgery. Intraoperative analgesia can blunt the metabolic and hormonal response to operative stress in neonates. However, its effects on heat production and thermoregulation are not known. The aim of this review was to characterise the effects of intraoperative analgesia on body temperature in neonates undergoing surgery. The case notes of 25 consecutive neonates who underwent major operations were retrospectively reviewed. Axillary temperature was measured before the operation, and postoperatively after returning to the neonatal intensive care unit (NICU). Patients were divided into groups based on the intraoperative analgesic used: (1) 9 neonates received fentanyl; (2) 5 received morphine; and (3) 11 received epidural bupivacaine. All groups were comparable in terms of conceptional age, postnatal age, body weight, duration of operation, and operative stress score. In all groups the body temperature was significantly lower at the time of returning to the NICU than preoperatively. Three patients (33%) who received fentanyl became hypothermic during the operation, whereas none of those who received either morphine or bupivacaine had hypothermia. The drop in temperature between preoperative and initial postoperative values was significantly greater in patients who received fentanyl intraoperatively (median drop 0.8 degreesC, range 0.6 - 2.4) when compared with patients who received morphine (P = 0.02) or epidural bupivacaine (P = 0.01). These data suggest that intraoperative fentanyl modulates the postoperative body temperature in neonates. We hypothesise that fentanyl blocks metabolic heat production, which results in a reduction in postoperative body temperature.

Analgesia↗

PubMed1998

Subretinal administration of tissue-type plasminogen activator to speed the drainage of subretinal hemorrhage.

BACKGROUND: Bleeding into the subretinal space in the vicinity of the macula is associated with age-related macular degeneration or retinal arterial macroaneurysm. The prognosis for restoration of vision is poor in the presence of blood clots. METHODS: Using a simple device composed of three disposable syringes we injected tissue-type plasminogen activator (tPA) into the subretinal space during conventional vitrectomy in six patients to assist the draining of subretinal clots. RESULTS: Four of six patients recovered their visual acuity postoperatively, while visual acuity in the other patients was stabilized. CONCLUSION: Early drainage of subretinal hemorrhage assisted by the introduction of tPA into the subretinal space led to uncomplicated surgery and favorable postoperative results.

Aged↗

PubMed1998

Immunohistochemical evaluation of cellular deposits on posterior chamber intraocular lenses.

PURPOSE: We used immunohistochemistry to characterize cellular and proteinaceous deposits on the surfaces of explanted posterior chamber intraocular lenses (PC-IOLs). METHODS: A total of 30 PC-IOLs were immunostained for the alpha- or beta-subunits of prolyl 4-hydroxylase, which is involved in collagen biosynthesis; cellular fibronectin; alpha B crystalline; and CD68, a macrophage marker, to characterize the cellular deposits that adhere to the IOL surfaces and to evaluate the distribution of cells involved in the deposition of extracellular matrix on IOLs. RESULTS: Cellular or proteinaceous deposits were observed on all 30 PC-IOLs. Cells that showed positive staining for alpha B crystallin were classified as lens epithelial cells; CD68-positive cells were considered to be of macrophagic origin. Positivity for cellular fibronectin, including the macrophages and related cells, appeared to be responsible for the accumulation of fibronectin on the surfaces of PC-IOLs. Prolyl 4-hydroxylase-positive cells were involved in the deposition of collagen on PC-IOLs. CONCLUSION: Immunohistochemical study revealed that macrophages, foreign-body giant cells, and lens epithelial cells adhered to explanted PC-IOLs. Such adherent cells are responsible for the deposition of extracellular matrix on the surfaces of PC-IOLs and may regulate the assembly of the extracellular matrix, influencing the biocompatibility of PC-IOLs.

Adult↗

PubMed1998

Immunolocalization of proto-oncogene products in keratocytes after epithelial ablation, alkali burn and penetrating injury of the cornea in rats.

BACKGROUND: We examined the immunolocalization of proto-oncogene products, including c-Fos and c-Jun, in the rat cornea during epithelial and stromal wound healing after simple epithelial ablation, penetrating injury or alkali burn. METHODS: Eighty-four male Wistar rats were divided into three groups and subjected to treatments as follows: (a) ablation of central corneal epithelium leaving basement membrane intact, (b) alkali burn in the central cornea with 1 N NaOH and (c) penetrating injury at the central cornea. The affected eyes were then enucleated after various intervals of healing. The frozen sections were immunohistochemically stained with the antibodies against proto-oncogene products. RESULTS: c-Fos- and c-Jun-immunoreactive cells were detected in the epithelium around the epithelial defect from 60 to 120 min after these treatments. The distribution of these cells were varied in these three types of injury. The immunoreactivities for these proteins were also detected in keratocytes after epithelial ablation. In the corneas with alkali burn, the immunoreactivities were detected in the keratocytes in the whole corneal stroma, and these immunoreactions were stronger than those observed in simple epithelial ablation. In the corneas with penetrating injury, such immunoreaction was seen only in keratocytes around the wound. CONCLUSION: These findings indicate that activator protein 1-mediated transcriptional activation for epithelial migration is initiated in the early phase after each injury, and that stromal keratocytes are also transcriptionally activated not only by alkali burn or penetrating injury but also by simple epithelial ablation in which basement membrane was not affected.

Animals↗

PubMed1998

Cell surface measurements of ATP release from single pancreatic beta cells using a novel biosensor technique.

To examine the possibility that ATP modulates insulin secretion by an autocrine mechanism, we measured the local concentration of released ATP at the surface of a single pancreatic beta cell by a new biosensor technique, using PC12 cells expressing ligand-gated cation channels, P2X2 receptors. Upon application of glucose or glibenclamide, a series of current spikes, whose amplitude equates to an ATP concentration of over 25 microM, were recorded from a PC12 cell using the whole-cell patch-clamp technique, when placed near a rat pancreatic beta cell at 37 degrees C. The current response was inhibited by cooling (below 30 degrees C) or by applying an ATP-hydrolysing enzyme (apyrase) or a P2 receptor blocker (suramin). Thus, it is concluded that pancreatic beta cells secrete ATP in response to glucose stimulation, thereby increasing the ATP concentration close to the cell surface sufficiently high enough to enhance insulin secretion from the pancreatic beta cells.

Adenosine Triphosphate↗