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1. The study aimed to investigate the pharmacokinetics of thrombomodulin alpha (TM-alpha), human-soluble thrombomodulin in rats. 2. Intravenously administered TM-alpha was eliminated in two phases (T(1/2 alpha) = 0.2-0.3 h and T(1/2 beta) = 6-8 h), and the elimination curve was linear in a dose range of 10-250 microg kg(-1). Based on the results of tissue concentration studies after reaching the steady-state, the highest concentration of TM-alpha was seen in the plasma, suggesting the low levels of transfer to tissues (or = 22% of plasma levels). 3. In vivo metabolism of TM-alpha was also analysed using high-performance liquid chromatography. The main peak observed in the plasma was TM-alpha, and even 72 h after the last dose of repeated administrations, 80% or more was unchanged form. Approximately half of the radioactivity excreted in the urine was recovered as a peak corresponding to TM-alpha. 4. The results reveal that although plasma clearance was lower in the renally impaired rats, the decrease was not large, with a difference of only about 20%. As a result, although the cause remains unclear, it is considered unlikely that the plasma concentrations of TM-alpha will vary considerably in patients with renal impairment.

Hippocalcin is a member of the neuronal calcium sensor (NCS) protein family that is highly expressed in hippocampal pyramidal cells and moderately expressed in the neurons of cerebral cortex, cerebellum and striatum. Here we examined the physiological roles of hippocalcin using targeted gene disruption. Hippocalcin-deficient (−/−) mice displayed no obvious structural abnormalities in the brain including hippocampal formation at the light microscopic level. Deletion of hippocalcin did not result in up-regulation of the hippocalcin-related proteins; neural visinin-like Ca 2+ -binding proteins (NVP) 1, 2, and 3. The synaptic excitability of hippocampal CA1 neurons appeared to be normal, as estimated by the shape of field excitatory postsynaptic potentials elicited by single- and paired-pulse stimuli, and by tetanic stimulation. However, N -methyl- d -aspartate stimulation- and depolarization-induced phosphorylation of cAMP-response element-binding protein (CREB) was significantly attenuated in −/− hippocampal neurons, suggesting an impairment in an activity-dependent gene expression cascade. In the Morris water maze test, the performance of −/− mice was comparable to that of wild-type littermates except in the probe test, where −/− mice crossed the previous location of the platform significantly less often than +/+ mice. Hippocalcin-deficient mice were also impaired on a discrimination learning task in which they needed to respond to a lamp illuminated on the left or right side to obtain food reinforcement. No abnormalities were observed in motor activity, anxiety behavior, or fear learning. These results suggest that hippocalcin plays a crucial role in the Ca 2+ -signaling pathway that underlies long-lasting neural plasticity and that leads to spatial and associative memory.

Only one aldolase can be resolved from Chlamydomonas reinhardtii by anion-exchange chromatography on DEAE-cellulose. Western blots with specific antisera against plastid and cytosol aldolase of spinach and Northern blots with the respective cDNAs from spinach indicate that only one aldolase, the plastid enzyme, is expressed. Full-length cDNA clones for the plastidic aldolase were isolated from a cDNA library constructed from poly(A) + mRNA of C. reinhardtii with plastid-aldolase-specific probes from spinach. The clones contained a 1.7-kb insert with an open reading frame for 374 amino acid residues covering the mature chloroplast protein of 347 amino acids and a N-terminal transit peptide of 27 amino acids for chloroplast import. The calculated molecular mass of the mature protein is 37.6 kDa and that of the precursor protein is 40.3 kDa. The aldolase of C. reinhardtii shows amino acid similarity of 62 to 67% with the chloroplast aldolase and of 47 to 52% with the cytosolic enzymes of higher plants, respectively. An evolutionary tree with all known class I aldolases shows separate clusters for the chloroplast and for the cytosol aldolases in higher plants and green algae. The aldolase of C. reinhardtii connects at a basal position with the chloroplast aldolases of higher plants.

To clarify the mechanism of tenderness after bone injury, we investigated changes in the withdrawal threshold to mechanical stimuli, nerve distribution and nerve growth factor (NGF)-expression in a rat model of bone injury without immobilization for bone injury healing. Rats were divided into three groups as follows: (1) rats incised in the skin and periosteum, followed by drilling a hole in the tibia [bone lesion group (BLG)]; (2) those incised in the skin and periosteum without bone drilling [periosteum lesion group (PLG)]; and (3) those incised in the skin [skin lesion group (SLG)]. Mechanical hyperalgesia continued for 28 days at a lesion in the BLG, 21 days in PLG and 5 days in SLG after treatments, respectively. Endochondral ossification was observed on days 5-28 in BLG and on days 5-21 in PLG. Nerve growth appeared in deep connective tissue (DCT) at day 28 in BLG. Nerve fibres increased in both cutaneous tissue and DCT at day 7 in PLG, but they were not found at day 28. Mechanical hyperalgesia accompanied with endochondral ossification and nerve fibres increasing at the lesion in both BLG and PLG. NGF was expressed in bone-regenerating cells during the bone injury healing. Anti-NGF and trk inhibitor K252a inhibited hyperalgesia in the different time course. This study shows that localized tenderness coincides with the bone healing and involves NGF expression and nerve sprouting after bone injury. The findings present underlying mechanisms and provide pathophysiological relevance of local tenderness to determination of bone fracture and its healing.

The distribution of 125I-labelled recombinant mouse interferon-beta (rMuIFN-beta) in normal and glioma (203 glioma) bearing mice was studied by radioassay and macro-autoradiography at 15 and 30 min after a single intravenous injection. The level of rMuIFN-beta in the spleen was about 20-fold higher than in serum. Concentrations higher than the serum level was detected in the lung, liver and kidney. The concentration of rMuIFN-beta in the brain was 8% of the serum level and the concentration in the glioma 30 min after administration was about 10-fold higher than in normal mouse brain. Macro-autoradiographic study demonstrated a wide distribution range and selective uptake in glioma tissue. Furthermore, we found that mouse gliomas were sensitive to mouse IFN-beta. Our findings demonstrate that in the mouse glioma model, intravenously administered interferon reaches the tumour.

Ocular trauma and disorders that lead to corneal blindness account for over 2 million new cases of monocular blindness every year. A popular ocular surface reconstruction therapy, amniotic membrane transplantation, has been shown to aid corneal wound repair. However, the success rates of the procedure are variable. Here, we proposed to bioengineer a novel synthetic material that would serve as a biomimetic corneal bandage. The PLGA-PEG-PLGA triblock copolymer was synthesised via ring-opening polymerisation. Thermoreversible gelation behaviour was investigated at different polymer concentrations (23%, 30%, 35%, 40%, 45%, w/v) at temperatures ranging between 5 and 60 degrees C. Viscoelastic properties were studied in dynamic mechanical analysis with 1 degrees C/min temperature ramp. Cryo-SEM revealed a porous hydrogel with interconnecting networks. No adverse cytotoxicity was observed with an in vitro scratch-wound assay and in in vivo biocompatibility tests. We have demonstrated that the PLGA-PEG-PLGA hydrogel possessed a suitable gelling profile and, for the first time, the biocompatibility properties for this application as a potential bandage for corneal wound repair.

We designed a new ophthalmic drug-delivery system for epidermal growth factor (EGF) from the biodegradable hydrogel of cationized gelatin. We placed a cationized gelatin hydrogel (CGH) with incorporated (125)I-labelled EGF in the conjunctival sac of mice and measured the residual radioactivity at different times to evaluate the in vivo profile of EGF release. Approximately 60-67% and 10-12% of EGF applied initially remained 1 and 7 days after application, respectively; whereas EGF delivered in topically applied solution or via EGF impregnation of soft contact lenses disappeared within the first day. We also placed CGH films with 5.0 mug of incorporated EGF on round corneal defects in rabbits to evaluate the healing process using image analysis software and to assess epithelial proliferation immunohistochemically by counting the number of Ki67-positive cells. The application of a CGH film with incorporated EGF resulted in a reduction in the epithelial defect in rabbit corneas accompanied by significantly enhanced epithelial proliferation compared with the reduction seen after the topical application of EGF solution or the placement of an EGF-free CGH film. The controlled release of EGF from a CGH placed over a corneal epithelial defect accelerated ocular surface wound healing.

A novel combretastatin A-4 derivative, AC7700, which is now in Phase I clinical trials under a new code, AVE8062, has shown strong antitumour effects against solid tumours in rodents because of its powerful and continued stanching of the tumour blood flow (TBF). Despite the strong tumour-suppressing qualities of AC7700, it does not produce an immediate reduction in tumour size. To elucidate the reason for this effect, we investigated the relationship between the change in tumour size in Sato lung carcinoma (SLC) and circulatory functions after therapy with AC7700, doxorubicin (Adriamycin [ADR]), or mitomycin C (MMC). To measure time-lapse changes in TBF with the hydrogen clearance method at the same site after drug administration, we developed a new apparatus for keeping electrodes within a tumour. AC7700 led to the destruction of both cancer cells and tumour vessels by interrupting the supply of nutrients. Intravenous (i.v.) administration of fluorescent dyes after AC7700 treatment revealed no fluorescence within the tumour vessels, which confirmed that the tumour microcirculation had been completely blocked. In contrast, ADR led to the destruction of SLC tumour cells, but did not have the same effect on tumour vessels. Intravenously administered fluorescent dyes immediately reached the tumour, which indicated that the tumour vasculature remained intact, and the TBF remained at the preadministration level, even 6 days after ADR treatment. In addition, although the size of the tumour increased slightly for 2 days with ADR treatment, possibly because of swelling of the cancer cells, thereafter it continued to decrease. MMC had virtually no effect on SLC tumour cells, tumour size or tumour vessels. We conclude that changes in tumour size brought about by cancer chemotherapy depend not only on the sensitivity of the cancer cells to the drug in question, but also on the nature of changes in the microcirculatory functions of the tumour brought about by the therapy. When both tumour cells and the tumour vasculature are destroyed, the effectiveness of therapy can not be determined from changes in tumour size alone.

Using several transplanted tumors in rats and mice, we have recently shown that the acute extensive necrosis of tumor nodules is caused by the systemic administration of AC7700, a novel combretastatin A-4 derivative, and that the necrosis can be attributed to irreversible stoppage of tumor blood flow (TBF). In this study, the antivascular and antitumor effects of AC7700 were tested on primary autochthonous tumor.Primary sarcomas were induced in Fischer F344 rats by a single s.c. inoculation of 3-methylcholanthrene (MC) (4 mg/0.5 ml/head). Changes in TBF due to AC7700 were measured by hydrogen clearance technique. Antitumor effects of AC7700 were evaluated by histology and tumor growth inhibition.The earliest primary tumor appeared 77 days after MC treatment and the last tumor appeared after 273 days (median, 140 days). Once tumors occurred, they continued to grow slowly and never regressed spontaneously. The mean doubling time of tumors (n = 34) during exponential growth was 12.6 +/- 9.0 days (mean +/- SD) (range, 5.4-55.9 days). Blood flow in these slow-growing autochthonous tumors decreased highly significantly from 25.2 +/- 15.5 to 4.4 +/- 3.2 ml/min/100 g (24 electrodes)(P0.001) within 30 min due to rapid i.v. injection (0.15 ml/min) of 10 mg/kg AC7700 and the decreased TBF did not recover during the experimental period of 6 h. On the other hand, TBF did not change significantly due to 0.9% NaCl solution (10 electrodes). In addition, TBF immediately began to decrease following slow infusion (0.005 ml/min) of 10 mg/kg AC7700 and decreased by approximately 80% (12 electrodes) at 20 min after the start of AC7700 infusion. To evaluate the antitumor effect of AC7700 on the primary tumors, 10 mg/kg AC7700 was injected i.v. to tumor-bearing rats. Although in the natural course, such tumors never stop growing, the tumor growth was strongly inhibited by AC700 (n = 5). In 2 cases, tumors never regrew for the observation period of 45 days. Biopsy and histological findings (n = 5) showed that tumors underwent extensive necrosis, as was observed previously for transplanted tumors following AC7700 administration.From the present experiment it was demonstrated not only in transplanted tumors but also in carcinogen-induced autochthonous primary tumors that acute and sustained stoppage of TBF led to strong antitumor effects. The novel anticancer compound AC7700 might become a very useful therapeutic strategy against refractory cancers.

To detect the changes in blood coagulation system and clarify the related mechanisms of chemotherapy-induced disseminated intravascular coagulation.Changes in blood coagulation system and immunohistochemistry for fibrinogen were investigated in six groups of rats designed for different purposes.Decreased platelet count, prolonged prothrombin time and active partial thromboplastin time, elevated fibrinogen level, and decreased antithrombin were observed in the rats receiving a newly developed chemotherapy (NDC group), in which mitomycin C was administered intravenously together with angiotensin. Accumulation of fibrinogen and microthrombi in the blood vessels of multiple organs were also found in the NDC group by immunohistochemistry and histopathological examination.Rapid reduction of tumor mass induced by an effective chemotherapy could cause hypercoagulable state and disseminated intravascular coagulation.

Prevalence rates and risk factors for H. pylori infection have been reported in many countries over the past year. Infection patterns in developed and developing countries are very different. Very high infection rates in developing countries with high incidence of gastric cancer were described, however, African enigma is still under controversy. The main risk factors for H. pylori infection are fecal-oral and oral-oral spread because H. pylori has not been detected to any extent in the environment. Recently, in childhood, main risk factors for transmission may be the contact with own father with H. pylori infection in Japan. Re-infection are very rare in adults, but not uncommon in childhood. Prevalence of H. pylori infection is reviewed.

To examine the retina of basigin (Bsg) knockout mice by electrophysiological and histologic methods and thereby to determine the possible function of Bsg in phototransduction and retinal development.Scotopic and photopic electroretinograms (ERGs) were recorded from 11 wild-type, 12 heterozygous, and 8 homozygous Bsg gene knockout mice of different ages. The retinas were also examined by histologic and immunolabeling methods.Bsg knockout mice of 5 to 41 weeks of age showed a decrease in the amplitude of all components of both the photopic and scotopic ERGs. In contrast, the fundus and the fluorescein fundus angiography and morphology of the retina at the light microscopic level appeared to be normal until 8 weeks of age in Bsg knockout mice. Thereafter, the length of outer segment and outer nuclear layers decreased with increasing age. Immunohistochemical analysis localized Bsg protein in a variety of cells in the retina, especially in the pigment epithelium, the upper outer plexiform layer and the inner segments of photoreceptor cells.The results demonstrated that both rod and cone function were severely affected from an early age by the targeted disruption of the Bsg gene. In spite of abnormal ERGs, the photoreceptor cells maintained normal morphology up to 8 weeks. Thereafter, the photoreceptor cells degenerated gradually and were almost ablated by 41 weeks.

Tumor tissue is composed of cancer cells (parenchyma) and tumor vessels (interstitium). Many investigators have pointed out that blood flow in tumors has a very inhomogenous distribution, and that this inhomogeneity in blood flow increases as tumors grew. This would be a certain cause of insufficient drug delivery to tumor tissues. Among the experimental evidence using Yoshida Sarcoma and Ascites Hepatomas, functional differences in microcirculation between tumor and normal tissues were found by Suzuki et al. (1977). Under hypertensive state induced by the continuous infusion of angiotensin II, tumor blood flow increased remarkably, while there was no change or decrease in blood flow in normal tissues such as the brain, bone marrow, liver and kidney. Moreover, the increase in blood flow in tumors was selective, as the mean blood pressure remained at the level of 150 mmHg. Increases were confirmed not only in many growing sites such as in the liver, muscle, subcutis, and even microfoci, but also in various kinds of xenografted human tumors and autochthonous tumors. Augmentation of the anti-tumor effects of angiotensin II-induced hypertension chemotherapy (IHC) for advanced gastric carcinoma was revealed in two randomized controlled trials (RCT-12) of collaborative study groups in Japan. The response rates were 42.9% vs 10.5% in RCT-1 and 31.3% vs 6.7% in RCT-2. The frequencies of toxicities were not statistically different. In the results of phase II studies from 1978 to 1994 (OPN-1) and 1995 to 1999 (OPN-2) for advanced gastric carcinoma (GC), the response rates were 37.9% and 35.7%. Down staging in which the conclusive stage score was lower than the score of the clinical stage, was observed in 8 out of 94 cases (19%) with primary lesions in total and in 30 patients (63%) receiving reduction surgery after IHC, since 1978. It is very important for exact evaluation after chemotherapy to understand or estimate the pathohistological changes in the tumor and its degenerated or repaired tissues, which present various clinical images. In the present study, the actual administered dose intensity of adriamycin (aDIadm) was 5.9 +/- 2.4 mg/sqm/w, and the ratio of aDIadm to the proposed DIadm of reported FAM/FAP schedules was 0.78 +/- 0.32. IHC with smaller DI could lead to a reduction in the accumulation of toxicities of anti-cancer drugs in the host. In conclusion, IHC might be applied to all kinds of tumors to enhance the chemotherapeutic effects through selective increase of drug delivery to tumors.

Under systemic hypertension induced by angiotensin II (AII) infusion, an attenuated vasoconstrictive response to the infusion in tumours was observed and a marked increase in tumour blood flow was observed in comparison with that in normal tissues. The results show a parallel circuit that connects the vascular bed of the pre-existing tissue to that of the tumour. The phenomenon was absent when hypertension was provoked by other vasoconstrictive agents such as norepinephrine or endothelin-1. However, the biological basis for this attenuated vasoconstrictive response to angiotensin II observed in tumours has not been fully elucidated.We assessed this response to characterise the angiotensin II receptor density and affinity in normal and tumour tissues. AH109A and LY80 tumour cell lines were transplanted to the skin in nude rats. Four weeks later, the rats were sacrificed. 125I-[Sar1, Ile8] angiotensin II was used to map its receptors in rat tissues using in vitro computerised autoradiography. Operated human gastric cancer tissues from a 49-year-old and a 66-year-old male patients were also investigated.The numbers of angiotensin II receptors were markedly reduced in tumour tissues without a change of affinity. The numbers in AII-R in tumours were shown to be mainly AT1 by the marked reduction in radioligand binding achieved by losartan but not by PD123177. The same results were observed in human gastric cancer.These results suggest that the decrease in angiotensin II receptors in tumours may explain the haemodynamic effect of angiotensin II-induced hypertension on tumour blood flow. This condition for drug delivery to tumour tissue may play a major role in enhancing the therapeutic effects of chemotherapy.

Most solid tumors possess unique pathophysiological characteristics that are not observed in normal tissues or organs, such as extensive angiogenesis and hence hypervasculature, defective vascular architecture, impaired lymphatic drainage/recovery system, and greatly increased production of a number of permeability mediators. The phenomenon now known as the enhanced permeability and retention (EPR) effect for lipid and macromolecular agents has been observed to be universal in solid tumors. Primarily, enhanced vascular permeability will sustain an adequate supply of nutrients and oxygen for rapid tumor growth. The EPR effect also provides a great opportunity for more selective targeting of lipid- or polymer-conjugated anticancer drugs, such as SMANCS and PK-1, to the tumor. In the present review, the basic characteristics of the EPR effect, particularly the factors involved, are described, as well as its modulation for improving delivery of macromolecular drugs to the tumor. Tumor-specific vascular physiology is also described.

A new agglutinin has been isolated from the red alga Gracilaria bursa-pastoris by affinity chromatography on a yeast mannan-Cellulofine column. This agglutinin was isolated as a monomeric glycoprotein with a relatively low molecular weight. It had an isoelectric point of 4.7 and contained large amounts of Gly, Asx and Glx. It agglutinated trypsin-treated rabbit erythrocytes at the low concentration of 30 ng/ml. The activity was inhibited only by glycoproteins bearing N-glycans. This agglutinin also showed mitogenic activity for mouse splenic lymphocytes.

Previous studies have suggested that angiotensin (ANG) synthesis, receptor activation, or both, in the median preoptic nucleus (MnPO) are responsible for initiating ANG-related salt appetite in normotensive rats. The present study was designed to test whether treatment with saralasin (Sar), an ANG II antagonist, in the MnPO area causes changes in saline (0.3 M NaCl solution) and water intakes in the spontaneously hypertensive rat (SHR), which is known to show greater salt appetite compared to normotensive Wistar-Kyoto (WKY) rats. Treatment with Sar in the MnPO area produced significantly attenuated saline intake, but had no effect on water intake. The results may support the importance of the ANG system in the MnPO area for salt appetite, and suggest that a disorder in the system may cause abnormal salt intake in SHR.

Na+,K+-ATPase activity is abundant on the basolateral infoldings of the strial marginal cells and contributes to the maintenance of the characteristic electrolyte composition of the endolymph. However, the stria vascularis of the cochlea is known not to be innervated. In order to clarify its humoral regulation by serotonin, the K+-p-nitrophenylphosphatase activity of strial marginal cells was investigated with a cerium-based method in normal guinea pigs and in guinea pigs treated with reserpine, 5-hydroxytryptamine or reserpine plus 5-hydroxytryptamine. K+-p-nitrophenylphosphatase activity was almost completely depressed 3-20 days after reserpine administration. Ten days after reserpinization, followed by repeated 5-hydroxytryptamine treatment, the enzyme activity was detectable. These results suggest that 5-hydroxytryptamine increases the phosphatase activity. Thus, the function of the stria vascularis in producing cochlear endolymph may be regulated by 5-hydroxytryptamine.

In cancer chemotherapy, selective enhancement of drug delivery to tumor tissue is essentially important for increase of chemotherapeutic effects. An attenuated vasoconstrictive response to angiotensin II (Ang II) in tumors and a marked increase in tumor blood flow were observed compared with normal tissues during systemic hypertension induced by Ang II infusion. The phenomenon was absent when hypertension was provoked by endothelin-1 (ET-1). We assessed this response to characterize ET receptor and Ang II receptor density and affinity in normal and tumor tissues. The tumor cell line LY80 was transplanted to the skin in nude rats. Four weeks later the rats were sacrificed. [ 125 I] ET-1 and [ 125 I Sar 1 , Ile 8 ]-Ang II were used to map the receptors for ET and Ang II in rat tissues using computerized in vitro autoradiography. A moderately high density of ET receptors, (ET B >ET A ) was found in tumors. The Ang II receptors were markedly reduced in tumor tissues without changes in the affinity. These results suggest that the decrease in Ang II receptors but not ET receptors in tumors may explain the hemodynamic effect of Ang II-induced hypertension and ET-induced hypertension on tumor blood flow.

Although the formalin test is widely used in rodents, the electrical response to this stimulus and the effect of isoflurane on this response, has not been well understood in cats. We have therefore examined the effect of isoflurane on spinal wide-dynamic-range neuronal activity evoked by a subcutaneous (s.c.) formalin injection in cats. In decerebrate, spinal cord-transected cats (L 1-2), a s.c. injection of formaldehyde solution (0.05 ml; 5%) was performed in the receptive fields of spinal wide-dynamic-range neurons at the hind paw. Isoflurane (1.5%) inhalation was given 20 mins prior to formalin injection or 5 mins after the formalin injection. The formalin injection elicited an immediate and continuous discharge or burst activity in the neurons that lasted at least 90 min. Inhaled isoflurane was found to markedly inhibit this neuronal discharge. At 30 mins after discontinuing of isoflurane, the neuronal activity in both groups recovered to approximately 55% of the control value. These results suggest that formalin injection will reliably produce a continuous burst of neuronal activity in the spinal dorsal horn of cats, and that this activity can be markedly reduced by isoflurane. The ability of isoflurane to inhibit this neuronal response was not temporally related to the formalin injection.

The effects of nitrous oxide (75%) on the spinal dorsal born wide dynamic range (WDR) neuronal activity were studied in either spinal cord intact or spinal cord-transected cats. Extracellular activity was recorded in the dorsal horn from single WDR neurons responding to noxious and non-noxious stimuli applied to the cutaneous receptive fields on the left bind foot pads of intact or decerebrate, spinal cord-transected (L 1-2) cats. The experiment was divided into four sections as follows: (1) When 10 micrograms of bradykinin (BK) was injected into the femoral artery ipsilateral to the recording site as the noxious test stimulus in the spinal cord-transected cat, all of 6 WDR neurons gave excitatory responses which were not depressed by 75% nitrous oxide. (2) When the injection of 10 micrograms of BK into the femoral artery ipsilateral to the recording site was used in the spinal cord-intact cat, 6 of 15 WDR neurons (40%) gave excitatory responses, which were significantly depressed by 75% nitrous oxide, and 9 of 15 WDR neurons (60%) gave inhibitory responses, which were not affected by 75% nitrous oxide. (3) When 10 micrograms of bradykinin (BK) was injected into the femoral artery contralateral to the recording site as the noxious test stimulus in the spinal cord transected cat, 6 of 12 WDR neurons gave excitatory reasons, which were not depressed by 75% nitrous oxide. (4) When the injection of 10 micrograms of BK into the femoral artery contralateral to the recording site was used in the spinal cord-intact cat, 6 of 6 WDR neurons (100%) gave responses, which were affected by 75% nitrous oxide. We have observed that nitrous oxide reduces the excitation and inhibition of dorsal born WDR neuronal activities induced by BK injection in spinal cord-intact cats, but does not reduce the excitation of those in spinal cord-transected cats. This finding confirmed that the antinociceptive effect of nitrous oxide might be modulated by supraspinal descending inhibitory control systems. In addition our result showed that the supraspinal effect of nitrous oxide was mediated not only by an increase but also a decrease in a supraspinal descending inhibition.

To study evolutionary aspects of fructose-1,6-bisphosphate (Fru-1,6-P2) aldolase during deuterostomian evolution, we have purified and characterized aldolases from the muscle and liver of lamprey (Entosphenus japonicus). Aldolase from the skeletal muscle and liver was identified to be the muscle-type isozyme and the non-muscle-type isozyme that was encoded by cDNAs M8 and L3, respectively, as described previously (Zhang, R., Yatsuki, H., Kusakabe, T., Iwabe, Miyata, T., Imai, T., Yoshida, M., and Hori, K., J. Biochem. 117, 545-553, 1995). The muscle-type isozyme has properties similar to vertebrate aldolase A, while the non-muscle-type isozyme shows a similarity to bacterial class I aldolase and vertebrate aldolase C but not to aldolase B, the liver-type aldolase, in terms of kinetic parameters: the Kcat values toward Fru-1,6-P2 and Fru-1-P, the Fru-1,6-P2/Fru-1-P activity ratio, and the Km values toward these substrates. The two enzymes have tetrameric forms with a molecular mass of approximately 160,000 and have similar pH optimum. The muscle-type and non-muscle-type isozymes from the tissues show different electrophoretic mobility; the muscle-type isozyme moves much faster than the non-muscle-type isozyme toward anodic side. The recombinant muscle-type and non-muscle-type aldolases gave similar characteristics as those from the tissues. The results presented in this paper, together with the data presented in the previous paper, strongly suggest that in lamprey it is possible to have two types of aldolase isozymes rather than one or three isozymes.

Nine monoclonal mouse anti-human aldolase C antibodies, mAbs A4, A8, B4, B7, B8, C1, D9, E10, and H1, were isolated and characterized. These mAbs fall substantially into four groups according to their reactivity with antigens. (i) Human aldolase C-specific mAbs (B8, D9, and H1). (ii) Type C aldolase-specific mAbs (B4 and E10). (iii) Ubiquitous mAbs, which react with vertebrate aldolases irrespective of type of isozyme and species (A4 and B7). (iv) Sub-ubiquitous mAbs, which are closely similar to the ubiquitous mAbs but differ slightly in terms of antigenic specificity (A8 and C1). Aldolase C-specific mAbs B8, H1, B4, and E10, but not D9, have their epitopes on a region within amino acid positions 79-193 of antigens, where the type-C isozyme group-specific sequence-3 (IGS-3) is situated. In contrast, ubiquitous mAbs A4 and B7 and sub-ubiquitous mAb A8 may have their epitopes on the commonly conserved regions of the three isozyme groups. The epitope of sub-ubiquitous mAb C1 appears to be on the IGS-2/3 but this is yet to be resolved. These nine mAbs can be classified into two groups based on the mode of epitope recognition, which was determined by ELISA, immunoblotting, and immunoprecipitation assays: (i) primary sequence-epitope mAbs such as B4, E10, and B7; and (ii) conformation-epitope mAbs (B8, D9, H1, A4, A8, and C1). Among these mAbs, aldolase C-specific mAbs H1 and E10 appear to be useful as probes for detection of conformational change around the type-C IGS-3 motif of human aldolase C because, when assessed by immunoprecipitation assay, mAb H1 reacts only with human aldolase C but not with CA250 and CA306, while mAb E10 reacts with CA250 and CA306 but not with aldolase C, even though these antigens have a common type-C IGS-3 motif. Similarly, the ubiquitous mAb B7 should serve as a probe for general use to detect vertebrate aldolases irrespective of isozyme groups and species.

In order to understand the molecular mechanisms of the temporal and spatial differences of gene expression in higher organisms, rat aldolase A gene carrying two distinct promoters was introduced into fertilized eggs and the resulting transgenic mice were analyzed. The transgene expression is tissue-specific and is developmentally regulated. In addition, the expression is regulated in a copy-dependent manner irrespective of where the transgene is integrated, suggesting that a mechanism excluding the effect of the integration site exists within the transgene itself. To explore the conformational change of this gene in the genome, the DNase I hypersensitive sites of the gene were examined. Three sites (DHS-1,2, and 3) were identified upstream and downstream of the gene and these sites were retained in the transgene as well as in the gene observed endogenously.

Three isozymic forms, alpha, beta, and gamma, of Drosophila melanogaster aldolase are produced from a single gene by alternative usage of the triple exons 4 (4 alpha, 4 beta, and 4 gamma) [Shaw-Lee et al. (1992) J. Biol. Chem. 267, 3959-3967; Kim et al. (1992) Mol. Cell. Biol. 12, 773-783; Kai et al. (1992) J. Biochem. 112,677-688]. The expression plasmids for the respective isozymes were transfected into Escherichia coli cells, and the isozymes alpha and beta were purified to homogeneity by a simple procedure, though isozyme gamma was only partially purified. These isozymes are active towards two substrates, fructose-1,6-bisphosphate (Fru-1,6-P2) and fructose-1-phosphate (Fru-1-P), with a preference for Fru-1,6-P2 over Fru-1-P, but they have different kcat/Km values towards these two substrates; isozyme alpha shows the highest value for Fru-1,6-P2. These isozymes show similarity in optimal pHs, thermal stability, and Km values for both Fru-1,6-P2 and Fru-1-P. They are composed of four identical subunits of 40 kDa, forming a tetramer with a molecular weight of approximately 160 kDa. The three isozymes are different in primary structure only at the carboxyl-terminal region encoded by the respective exon 4. Therefore, this region should be primarily responsible for the distinct characteristics of these isozymes.

Isozymes are the different molecular forms of an enzyme that catalyze the same reaction. They have been known to show the different distribution and metabolic roles within one type of cell or in different types of cells. Three evolutionary strategies led to generate isozymic forms; (1) multiple forms encoded by separate genes, (2) those generated by allelic mutations, and (3) those generated from a single gene by either alternative usage of the protein-coding exons or alternative usage of multiple translation initiation sites. In general, genes encoding different types of isozyme show a similarity in the number and length of the protein-coding exons but not in the 5'- and 3'-non-coding exons and also in the introns. The expression of isozyme genes is regulated temporally or constitutively through the interaction of the cis-acting elements on regulatory regions of the genes with basal- and tissue-specific transcription factors.

Nearly full-length cDNA clones for muscle-type and non-muscle-type aldolase mRNAs were cloned from lambda gt10 cDNA libraries constructed from skeletal muscle and liver mRNAs of lamprey (Entosphenus japonicus). The cDNA-M8 has 2,240 bp carrying an open reading frame of 1,089 bp which encodes 362 amino acids without the amino terminal methionine, while the cDNA-L3 is 1,761 bp in length and has an open reading frame of 1,092 bp, which encodes 363 amino acids without the methionine. We designated the cDNA clones M8 and L3 as the muscle-type and non-muscle-type aldolase cDNAs, respectively. The entire amino acid sequences deduced from cDNA-M8 and -L3 show a high degree of identity to one another (76%) and also to vertebrate aldolases A (74-76%), B (68-70%), and C (71-76%) and Drosophila melanogaster aldolases alpha, beta, and gamma (66-67%). Northern blot analyses using the 3'-noncoding sequences of cDNA-M8 and -L3 as hybridization probes indicated that the muscle-type mRNA is expressed mainly in the skeletal muscle, heart muscle, brain, and some other tissues, but probably not in liver, while the non-muscle-type mRNA is expressed mainly in the liver and also in brain and other tissues, except for the heart muscle. Phylogenetic analyses showed that both muscle-type and non-muscle-type aldolases of lamprey resemble one another and might share a common ancestor with vertebrate aldolases A and C, but they are not direct ancestors of vertebrate aldolases.

To elucidate the microvascular mechanisms of change in tumor blood flow due to vasopressors (angiotensin II, epinephrine, methoxamine), we analyzed the site of vascular resistance (VR) increased by each vasopressor. Arteriolar vessels within a transparent rat chamber were classified centripetally (a2-a5) according to Strahler's nomenclature. Vessels that feed into the tumor microcirculation were a2 modified by the tumor (starting vessels). Under angiotensin II (A II)-induced hypertension, the pressure of all arteriolar vessels increased roughly in proportion to the increase in mean arterial blood pressure. The greatest pressure drop and hence the most resistance due to A II occurred across the a2. During epinephrine-induced hypertension, there were major pressure drops between a4 and a3, and between a3 and a2. The amount of contraction of arteriolar vessels due to methoxamine was much smaller than that due to epinephrine, and the pressure increase in a4 and a3 was also small. From the facts described above, we may conclude as follows: A II creates greater vascular resistance of a2 vessels to blood flow and also greater perfusion pressure of a5-a3 vessels, resulting in increased blood inflow into a starting vessel which then becomes a passive vessel. Epinephrine causes an increase in the resistance of a3, a4 and probably upstream from a4 arterioles to blood flow. Thus, tissue blood flow in subcutis and tumor almost always decreases together. The fact that tissue blood flow in normal subcutis and tumor did not change significantly under methoxamine-induced hypertension is probably due to the results that methoxamine had little effects on the vascular resistance of smaller arterioles to blood flow.

Adult T cell leukemia derived factor (ADF), originally defined as an interleukin-2 receptor inducer, is a human thioredoxin homolog. ADF/thioredoxin has several biologic functions, such as defense against cellular damage, activity to scavenge oxidative stress by hydrogen peroxide radicals and cytokine-like properties. We hypothesized that under certain conditions, ischemia followed by reperfusion and/or light exposure, ADF may be induced in the retina.To test this hypothesis, we prepared experimental animal models in which oxidative stress could be applied to the retina. In the present study, we examined ADF expression in the rat retina using temporary ischemia and reperfusion, and photodynamic retinal vascular thrombosis by immunohistochemical and Western blotting methods.ADF expression was strongly induced in the retinal pigment epithelial cells after 2 hours of ischemia followed by 2, 24, or 48 hours of reperfusion. ADF was also expressed in retinal pigment epithelial cells as early as 3 hours after light exposure followed by rose bengal injection, and this expression increased with time. Western blotting data showed that ADF expression increased in the retinal pigment epithelial cells in the experimental group, as compared with the control group. These results indicate that ADF is actively induced in retinal pigment epithelial cells upon oxidative stress.ADF induction in retinal pigment epithelial cells may be involved in the defense mechanism against cellular damage caused by active oxygen species generated during oxygen stress to the retina.

To elucidate the microvascular mechanisms of change in tumor blood flow elicited by vasopressors, a functional morphometric study of the s.c. microcirculation within a rat transparent chamber was performed. Arteriolar vessels were classified centripetally (a2-a5) according to Strahler's method. Arteriolar pressure in each segment both under normotension and under hypertension induced by angiotensin II, epinephrine, or methoxamine was measured using a microocclusion technique. Vasoconstriction was estimated by changes in vessel diameters. In addition, tissue blood flow of the subcutis and s.c. tumor (LY80, a variant of Yoshida sarcoma) under the same conditions was measured with the hydrogen clearance method. By comparing the sites of the greatest pressure drop and the vasoconstriction induced by each vasopressor, we assessed the sites of vascular resistance (VR) which showed increases due to these vasopressors. The greatest VR increase elicited by angiotensin II occurred across a2 vessels. On the other hand, the sites of VR increase due to epinephrine were in a3 vessels and larger vessels upstream from a3 arterioles. The VR increase induced by methoxamine was much smaller than that induced by epinephrine. We conclude that the fact that the sites of increased VR differ with each vasopressor is the primary reason that various vasopressors have been found to produce different changes in tumor blood flow.

Genomic clones encoding the Drosophila aldolase gene were isolated and the organization of the gene was determined. The protein-coding region spanning nearly 3.5 kb consists of five coding exons (exon 2, 3, 4 alpha, 4 beta, and 4 gamma). The insect exon 2 corresponds to exons 2 to 7 of vertebrate aldolase genes and thus appears to have been formed by the fusion of these 6 exons into a single exon during evolution. The Drosophila aldolase gene is predicted to generate mRNAs for three isozymes (alpha-, beta-, and gamma-types) from the primary transcripts by alternative usage of the final three exons. The reverse transcriptase-PCR assay revealed the occurrence of mRNAs for the three isozymic forms at different developmental stages, and tissue-specific expression was also found to occur in adult flies. In addition to the usual type mRNA species for the alpha-, beta-, and gamma-isozymes, two novel forms of mRNAs, alpha beta- and beta gamma-type mRNAs, were detected tissue-specifically in adult flies, although their functions are unpredictable. The alpha beta-mRNA is an alpha-type mRNA in which exon 4 beta remains unspliced, while the beta gamma-mRNA is a beta-type mRNA with the exon 4 gamma remaining unspliced. Recombinant enzymes expressed in Escherichia coli were all active and exhibited different enzymatic properties.

Since binding of an agonist to an ionotropic neurotransmitter receptor causes not only channel opening, but also desensitization of the receptor, inhibition of the receptor by the antagonist sometimes becomes very complicated. The transient state kinetics of ligand association and dissociation, and desensitization of the receptor were considered on the basis of the minimal model proposed by Hess' group, and the following possibilities were proposed. 1) When an agonist is simultaneously applied to the receptor with an antagonist whose affinity to the receptor is extremely strong and different from that of the agonist, it is usually impossible to estimate the real inhibition constant exactly from the responses because desensitization of the receptor proceeds before the equilibrium of the ligand binding. Simultaneous addition of the antagonist with strong affinity to the receptor may apparently accelerate inactivation (desensitization) of the receptor. The association rate constant of the antagonist can be estimated by analyses of the rate of the inactivation in the presence and the absence of the antagonist. 2) A preincubated antagonist with a slow dissociation rate constant, i.e., a very effective inhibitor, may cause apparent noncompetitive inhibition of the receptor, since the receptor is desensitized by an agonist as soon as the antagonist dissociates from the receptor and the dissociation of the antagonist from the receptor becomes the rate-determining step. A nicotinic acetylcholine receptor (nAChR) was expressed in Xenopus oocytes by injecting mRNA prepared from Electrophorus electricus electroplax and used for the experiments on inhibition by an antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

F-Actin was digested with alpha-chymotrypsin in 6 M urea, and two peptide fragments from subdomain 4 of actin molecule [Kabsch, W., Mannherz, H.G., Suck, D., Pai, E.F.,Holmes K.C. (1990) Nature 347, 37-44] were purified by reverse-phase HPLC and Sephadex G-50 gel filtration. The peptide fragments were identified as segments from Arg-177 to Tyr-198 (2.6-kDa peptide) and from Ser-199 to Tyr-279 (9.1-kDa peptide). Their effects on actin polymerization induced by 50 or 100 mM KCl were studied by measuring the increase in viscosity by the falling ball method. The 2.6-kDa peptide decreased the rate of actin polymerization and increased the critical concentration for the polymerization. Based on the atomic model of the actin filament [Holmes, K.C., Popp, D., Gebhard, W.,Kabsch, W. (1990) Nature 347, 44-49], the peptide is presumed to bind to the barbed end of the actin filament and inhibit the polymerization. By assuming that the peptide affected the rate of association of the actin monomer to the end of the actin filament, well-fitting curves for the polymerization kinetics were calculated. Computer-assisted results indicated that the dissociation constant of the 2.6-kDa peptide for F-actin is 200 to 260 microM. In contrast, the 9.1-kDa peptide only slightly inhibited actin polymerization. These results suggest that the actin-actin interface in the region between Arg-177 and Tyr-198 has a stronger interaction than those between Ser-199 and Tyr-279. The amino acid sequence L-T-D-Y-L present in the 2.6-kDa segment is homologous to a common sequence in the F-actin capping domain of various actin-binding proteins.

Circadian fluctuation in tumor blood flow of the rat subcutaneous tumor was investigated. Tumor tissue blood flows in the daytime zone (10 a.m. to 4 p.m.) and in the nighttime zone (10 p.m. to 4 a.m.) in both the first phase (doubling time of tumor volume = 1.7 days) and the second phase (doubling time of tumor volume = 5.7 days) of growth of the LY80 tumor in rats were measured using the hydrogen gas clearance technique. In the first phase of tumor growth, the tumor blood flow was 20.3 +/- 12.2 ml/min/100 g in the daytime zone (n = 22) and 46.6 +/- 19.3 ml/min/100 g in the nighttime zone (n = 22). In the second phase, tumor blood flow was 9.6 +/- 5.7 ml/min/100 g in the daytime zone (n = 45) and 19.4 +/- 8.2 ml/min/100 g in the nighttime zone (n = 38). Tumor blood flow in the nighttime zone was significantly higher than that in the daytime zone (first phase, P less than 0.001; second phase, P less than 0.001). However, there were no significant differences in the mean arterial blood pressure, tumor size, and body weight of rats between the daytime zone and the nighttime zone. There was also a marked difference in the effect of angiotensin II-induced hypertension on tumor blood flow between the daytime zone and the nighttime zone. These results suggest that circadian fluctuations in tumor blood flow should be carefully considered when developing strategies to maximize the effectiveness of cancer therapy in relation to the flow rate of circulating blood.

Three monoclonal antibodies (MAbs1A2, 3C5, and 4C2) for human aldolase A [EC 4.1.2.13] were established. MAbs1A2, 3C5, and 4C2 were shown to belong to subclasses IgM, IgG1, and IgG2a, respectively. None of the MAbs inhibits aldolase A activity. Their epitopes were mapped in detail on the molecule by examining the reactivities of the MAbs to chimeric proteins between aldolases A and B [Kitajima et al. (1990) J. Biol. Chem. 265, 17493-17498] in ELISA and to the CNBr-cleaved fragments of aldolase A in immuno-blotting. MAbs1A2 and 3C5 reacted with sites located within amino acid residues 306-363 at the C-terminal region of the enzyme. MAb4C2 recognized an epitope of the enzyme present within amino acid residues 34-108 at the N-terminal region. In a competitive binding assay, MAbs1A2 and 3C5 competed with each other for binding to the antigen and also interfered with the binding of MAb4C2, whereas MAb4C2 failed to inhibit the binding of MAbs1A2 and 3C5 to the antigen. MAb3C5 showed a species-specificity in the reaction with the antigen; it reacted with human and rabbit aldolase A with similar reactivity but not at all with the rat and mouse enzymes, which differ from the human and rabbit enzymes in two amino acid residues at positions 328 and 348. Reactivities of MAbs to aldolase A were further examined with engineered enzymes containing an amino acid substitution.(ABSTRACT TRUNCATED AT 250 WORDS)

To investigate the effects of aluminium hydroxide (AH), a phosphate binder, on the progress of chronic renal diseases, high doses (9.6 g/kg/day, group ADR-H), moderate doses (2.4 g/kg/day, group ADR-M), and low doses (1.2 g/kg/day, group ADR-L) of the compound, were orally administered for 34 weeks to rats with Adriamycin (ADR)-induced focal glomerular sclerosis. Serum creatinine and blood urea nitrogen were significantly lower in either group ADR-H or ADR-M than in group ADR at week 34. Urinary protein excretion was significantly lower in group ADR-H than in group ADR in all observation periods and was also lower in group ADR-M than in group ADR at weeks 28 and 32. Both glomerular sclerosis and tubular change were significantly less severe in group ADR-H, while tubular change was less marked in group ADR-M than in group ADR. These results revealed that phosphate depletion was induced, and progressive renal failure was ameliorated in proportion to the dosing of AH. On the other hand, body weight was significantly smaller in group ADR-H than in group ADR in more than half of the observation period. In conclusion, a high dose of AH may adversely affect the nutritional state irrespective of having an extremely protective effect on progress of renal dysfunction. Appropriate doses of AH should be used to prevent the renal deterioration.

Coincubation of neutrophils with TNF inhibited the chemoattractant-directed migration of neutrophils under agarose and enhanced their migration in the multiwell chemotaxis chamber. To assess the physiological significance of these differing in vitro TNF effects, ex vivo and in vivo investigations were performed using animal models. Neutrophils from the peripheral blood of rabbits preadministered systemic TNF showed impaired ability to migrate toward chemoattractants in vitro. In addition, systemic TNF administration suppressed zymosan-activated plasma-induced local accumulation of leukocytes in mouse skin. The results indicate that circulating TNF may act as a suppressor for local inflammatory reaction.

The differing effects of angiotensin II (AT II) and five other vasoactive agents on the tissue blood flow in Yoshida rat ascites hepatoma AH 109 A were studied at the same electrode position by a hydrogen clearance method. The elevation of blood pressure by AT II resulted in a 3.4-fold increases in the tumor blood flow. However, when AT II was reinfused, it resulted in only 2.1, 2.2, 2.3, 1.9 and 2.4-fold increases, respectively, after infusion of methoxamine, norepinephrine, metaraminol, epinephrine, and phenylephrine. A pronounced reduction and further reducing tendency in the tumor blood flow were found after administration of norepinephrine and epinephrine. In addition, metaraminol, phenylephrine, and methoxamine did not change significantly the blood flow of the tumor. The results of this present blood flow study further suggest that AT II acts more peripherally in the host vascular bed from which newly growing tumor vessels bud than other vasoconstrictors do.

Using a rat transparent chamber and a device for measurement of microvascular pressure, we observed tumor vascularization process in vivo and measured the daily change in pressure of a "starting vessel" which supplies blood to the tumor vascular network. Results were summarized as follows: (1) The position from which tumor vessels originated was usually the terminal portion of a terminal arteriole (starting vessel). The frequency with which new capillaries originated from vein and venule was very low. (2) Pressure of a starting vessel increased from 30-40 cm H2O (22-30 mmHg) to 120-130 cm H2O (88-96 mmHg) with enlargement of the tumor vascular network. As soon as pressure of a starting vessel reached a plateau, no-flow vessels appeared in places within a tumor. (3) Pressure of a starting vessel was elevated by angiotensin II. In turn, the elevated pressure of a starting vessel brought about a marked increase in tumor blood flow, resulting in the blood flow in no-flow vessels.

Enzymatic studies on aldolase isozymes have been carried out by techniques of protein engineering. Site-directed mutagenesis helps us to verify the roles of amino acid residues in catalytic reactions. Chimeric fusion proteins give us information about the regions which specify the characteristics of the isozymes. The results are: (1) In aldolase A, COOH terminal Tyr and Lys-107 residues play important roles in catalysis, especially in binding of FDP. (2) Aspartic acid at the 128th residue in aldolase A is essential to thermostability; no other residue such as glutamic acid can substitute for it. (3) Studies on chimeric fusion proteins indicate that the C-terminal region (including C-terminus Tyr) or aldolase A is responsible for its substrate specificity, which is not seen in aldolase B. (4) A region near NH2 terminus in aldolase B determines its specific structure. (5) The region including His-107, Asp-128, and Tyr-137 (B-A junction of BA137) is located in a turn which is exposed outward (a model architecture by Sygusch et al [1987]). In BA137, this region would be constrained, and play a significant role in catalysis, thermostability, etc. (6) Tertiary structure of aldolase B seems to be dissimilar to that of aldolase A.

The effects of local and systemic administration of tumour necrosis factor (TNF) on the mouse-tail model of psoriasis were examined histologically at 4 days after administration. Subcutaneous injection of TNF at 1 x 10(1) to 1 x 10(4) units/mouse resulted in dose-dependent formation of a granular layer in the tail scales, which was quantified as the percentage of tail scales having granular layer formations extending over half or more of their surface and was larger than that occurring in untreated mice and in mice receiving injections of vehicle solution alone. Daily intraperitoneal injection of TNF at 1 x 10(3) or 1 x 10(4) units/mouse for 28 days resulted in similar granular layer formation and in a significantly reduced mitotic index of the tail keratinocytes, indicating an antiproliferative effect of TNF. The lowered mitotic index and the induction of granular layers, apparently attributable to a modulation of keratinocyte proliferation and differentiation by TNF, suggest that TNF may be effective as a drug for treatment of psoriasis.

A microcomputer system equipped with an image processing peripheral board was applied for detecting and controlling the pigeon's peck location. The peripheral board digitized a TV image of an experimental chamber at the instant of the occurrence of a response, and a simple machine language routine analyzed the digitized image to determine the location of pigeon's beak in real-time. Using this system, pigeons were trained to peck a target area of a long response key and their peck locations were analyzed.

1. 1. Digestive enzymes in the salivary gland of Stenotus binotatus were compared with those in the midgut. 2. 2. Amylase, α-glucosidase, pectinase and protease were detected both in the salivary gland and the midgut, while β-glucosidase, α-galactosidase and esterase were detected only in the midgut. 3. 3. Lactose-, cellulose-, inulin- and phlein-hydrolytic enzymes were found neither in the salivary gland nor in the midgut. 4. 4. The digestive enzymes were compared with those of other heteropterous insects.

Summary The chemiluminescent reactions of firefly luciferyl-adenylate and Renilla luciferin utilize molecular oxygen and produce CO2. If the reactions are allowed to proceed in the presence of 18O2, no incorporation of 18-oxygen is detected in the CO2. This is true over a wide range of substrate concentrations (0.03 μmoles – 6 μmoles). If H218O is added to the reaction medium there is incorporation of 1 oxygen-18 into the CO2. These results are consistent with the oxygen-18 labelling observed with the bioluminescent reactions.

1. 1. Carbohydrases in the salivary gland and midgut were compared among several phytophagous bugs with different feeding habits, Lygus disponsi, L. saundersi, Adelphocoris suturalis and Orthocephalus funestus (Miridae), Palomena angulosa and Eurydema rugosum (Pentatomidae) and Coreus marginatus (Coreidae). 2. 2. All the bugs had amylase, phleinase, α- and β-glucosidase and α- and β-galactosidase in the midgut. Carbohydrase variety in the salivary gland was poor. 3. 3. Salivary pectinase was detected in all the mirid bugs. 4. 4. In the salivary gland, C. marginatus had β-glucosidase, α- and β-galactosidase, P. angulosa α-galactosidase and O. funestus β-galactosidase. 5. 5. A distinct cellulolytic activity was detected in the midgut of P. angulosa, E. rugosum and C. marginatus. 6. 6. The relationship between the taxonomic position or the feeding habit of the bug and the presence of carbohydrase in the alimentary system is discussed.