Sudden termination of G-CSF injection leads to apoptosis of a large proportion of increased granulocytes.

The effectiveness of granulocyte colony-stimulating factor (G-CSF) for granulocytopenia has been widely recognized. However, although granulocyte counts rapidly increase after a few injection of G-CSF, a large proportion of the increased granulocytes disappear from peripheral blood within a few days following G-CSF withdrawal. Where do they go? In this report, the answer can be seen at a glance. Using MitoCapture and a CCD camera-equipped fluorescence microscope, we succeeded in demonstrating that G-CSF withdrawal induced loss of mitochondrial membrane potential, i.e., an early stage of apoptosis, in human peripheral granulocytes increased by G-CSF.

Lipopolysaccharide alters ecto-ATP-diphosphohydrolase and causes relocation of its reaction product in experimental intrahepatic cholestasis.

Lipopolysaccharide (LPS), a bacterial endotoxin, exerts profound inflammatory actions toward various tissues and cells. We induced intrahepatic cholestasis in rats by administration of LPS and followed ecto-ATP-diphosphohydrolase (ecto-apyrase) activity in the liver. The activity of the enzyme had decreased to 77% 2 h after injection compared with the activity in control animals. The maximum decrease was detected 24 h after administration. The activity was found to have partially recovered 1 week after injection, but had yet to reach control levels. In contrast to the decrease in ecto-apyrase activity, there were increases in alkaline phosphatase activity and bilirubin concentration, markers of cholestasis. In response to LPS, the reaction product of ecto-apyrase was found to relocate from the canalicular domain of the plasma membrane of hepatocytes, its predominant localization in the liver of intact animals, to the basolateral and sinusoidal domains. The pattern of histochemical reaction indicated modulation of the enzyme activity and changes in trafficking of intracellular proteins. Taken together, our findings showed that LPS administration alters ecto-apyrase and causes relocation of its reaction product from the canalicular domain of the plasma membrane of hepatocytes in the rat. It is suggested that relocation of the reaction product may be a protective mechanism to enable the hepatocytes to withstand the cytokine-induced metabolic perturbations.

Radiation-induced apoptosis of human peripheral T cells: analyses with cDNA expression arrays and mitochondrial membrane potential assay.

We examined sequential changes in post-irradiated peripheral blood T cells taken from normal volunteers, by using targeted Atlas cDNA Expression Arrays and mitochondrial membrane potential assay. At 1 and 3 hours after 5 Gy irradiation, changes of gene expression were examined by targeted Atlas cDNA Expression Arrays using Apoptosis Array. The Atlas Human Apoptosis Array includes 205 key genes that are known to control apoptosis, including extracellular and cytoplasmic effectors. Concerning Fas, no significant changes of spot intensities were identified between irradiated T cells and non-irradiated ones at both 1 h and 3 h after 5 Gy irradiation. Caspase families, including caspases 9 and 3 also showed no changes between these two groups. An apoptosis regulator bclw showed a remarkable decrease in irradiated T cells. These results suggested that irradiation induced direct apoptosis of T cells by changing the membrane potential of mitochondria. Using a CCD camera-equipped fluorescence microscope and MitoCapture, a mitochondrial membrane potential indicator, we demonstrated 5 Gy radiation induced loss of membrane potential, i.e., an early stage of apoptosis, in human peripheral blood T cells at 10 hours after irradiation.

Evaluation of the process for superoxide production by NADPH oxidase in human neutrophils: evidence for cytoplasmic origin of superoxide.

We present an up-to-date insight into the function of NADPH oxidase in human neutrophils, the signalling pathways involved in activation of this enzyme and the process of association of its components with the cytoskeleton. We also discuss the functional implications of morphological studies revealing localization of the sites of NADPH oxidase activity. An original model of the process of superoxide (O2*-) production in human neutrophils is shown. Organization of NADPH oxidase is associated with several components. Upon stimulation, tri-phox cytosolic components of NADPH oxidase (p40-phox, p47-phox and p67-phox) bind to actin filaments. This process involves other actin-binding proteins, such as cofilin and coronin. Activated protein kinase C, translocated from the plasma membrane, phosphorylates cytosolic components at a scaffold of cytoskeleton. Subsequently, p40-phox, responsible for maintaining the resting state of NADPH oxidase, is separated from other two cytosolic phox proteins following an attachment of the active form of small GTP-binding protein Rac to p67-phox. Cytosolic duo-phox proteins (p47-phox and p67-phox) conjugate with membrane components (gp91-phox, p22-phox and Rapla) of NADPH oxidase residing within membranes of intracellular compartments. This chain of events triggers production of O2*-. Then, oxidant-producing intracellular compartments associate with the plasma membrane. Eventually, intracellularly produced O2*- is released to the extracellular environment through the orifice formed by fusion of oxidant-producing compartments with the plasma membrane. Intracellular movement of the oxidant-producing compartments may be regulated by myosin light chain kinase. The review emphasizes that functional assembly of NADPH oxidase and, therefore, generation of O2*- is accomplished essentially within the intracellular compartments. Upon neutrophil stimulation, intracellularly generated O2*- is transported to the plasma membrane to be released and to ensure host defense against infection.

Suppressive effect of rebamipide, an antiulcer agent, against activation of human neutrophils exposed to formyl-methionyl-leucyl-phenylalanine.

Rebamipide, an antiulcer agent, has been shown to be able to prevent gastric mucosal injury resulting in part from activation of neutrophils. The mechanism of its suppressive action, however, remains to be established. The present study aimed to determine the effect of rebamipide on activation of isolated human neutrophils and to identify the signal transduction pathway involved in its regulation. In unstimulated cells, alkaline phosphatase activity was found residing in short rod-shaped intracellular granules. Upon stimulation with a chemotactic peptide formyl-methionyl-leucyl-phenylalanine, the granules fused to form elongated tubular structures and spherical vacuoles. Rebamipide inhibited reorganization of alkaline phosphatase-containing granules along with upregulation of alkaline phosphatase activity and CD16, a marker of the granules. It also suppressed chemotaxis, an increase in intracellular calcium ion concentration, and NADPH oxidase activation in cells stimulated with formyl-methionyl-leucyl-phenylalanine. In contrast, the drug showed no inhibitory action toward upregulation of alkaline phosphatase activity and CD16, and activation of NADPH oxidase in cells stimulated with phorbol myristate acetate, an activator of protein kinase C. These findings demonstrate that rebamipide exerts a broad spectrum of suppressive actions toward biological functions of human neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine, but not with phorbol myristate acetate, and suggest that the upstream point of protein kinase C is the signal transduction pathway involved in its regulation.

A simple approach for the analysis of intracellular movement of oxidant-producing intracellular compartments in living human neutrophils.

In human neutrophils, superoxide is generated primarily within specialized oxidant-producing intracellular compartments. The present study employs a simple methodological approach to evaluate the intracellular movement of these structures in living human neutrophils. Using a CCD camera system, we monitored fluorescence in cells loaded with the succinimidyl ester of dichlorodihydrofluorescein diacetate, which is nonfluorescent until oxidized by reactive oxygen species. Fluorescence-positive intracellular compartments became detectable after neutrophils were stimulated with phorbol myristate acetate for 1 min. Further stimulation increased the intracellular compartments in both number and size in a time-dependent manner. Upon stimulation with phorbol myristate acetate, no fluorescence was seen in intracellular compartments of neutrophils isolated from patients with X-linked chronic granulomatous disease lacking gp91-phox, a membrane component of NADPH oxidase. The method enables tracking of the movement of a single oxidant-producing intracellular compartment following cell stimulation and visualization of the intracellular structures formed by fusion of oxidant-producing intracellular compartments with endocytotic vesicles and phagosomes. Therefore, it is considered to be an informative tool for evaluation of the intracellular dynamics of oxidant-producing intracellular compartments in living human neutrophils and may have a diagnostic value.

Ultracytochemical study on the localization of superoxide producing sites in stimulated rat neutrophils.

Superoxide anion production in neutrophils plays an important role in the microbicidal defense system in the body. In this study, isolated rat neutrophils were stimulated experimentally and examined by electron microscopy to determine the site of superoxide production and its subsequent translocation during different cell stimulation time periods. Blood and peritoneal neutrophils were incubated for periods of 5, 10, and 15 min with phorbol 12-myristate 13-acetate (PMA), N-formyl-Met-Leu-Phe (fMLP), and combinations of PMA and cytochalasin B (CB) and fMLP and CB. Ultracytochemical detection of O(2)(-) was performed with the 3, 3'-diaminobenzidine-manganese (DAB/Mn) cytochemical method and cationized ferritin (CF) particles were added to stimulation media to monitor endocytotic events that occurred during neutrophil stimulation. Unstimulated neutrophils were devoid of O(2)(-) activity in cytoplasmic granules and at the plasma membrane surface. After 5 min stimulations with PMA, PMA + CB, or fMLP + CB, electron-dense DAB/Mn reaction product was detected in small, centrally located tubular compartments within the neutrophils. CF particles which were added to the stimulation media became internalized in endocytotic vesicles after 5 min stimulation; these vesicles were devoid of O(2)(-) activity. At 10 min stimulation with PMA, O(2)(-)-positive granules subsequently fused with each other and translocated to sub-plasma membrane regions where they either contacted the plasma membrane or fused with CF-containing endocytotic vesicles. Little reaction product was observed on the surface of the neutrophils. Spectrophotometric comparison of the stimulatory effects of PMA, fMLP, and fMLP + CB revealed different rates and yields of O(2)(-) production. Results from this study suggest that the O(2)(-)-producing sites of rat neutrophils originate intracellularly and translocate to the plasma membrane surface following stimulation with PMA, PMA + CB, and fMLP + CB, but not with fMLP or CB alone. Furthermore, these compartments appear to possess the ability to fuse with endocytotic vesicles, a process that may be linked to intracellular microbicidal activity in circulating and tissue neutrophils.

Novel insight into current models of NADPH oxidase regulation, assembly and localization in human polymorphonuclear leukocytes.

We review herein the definition of the NADPH oxidase-activating site in human neutrophils and eosinophils, together with the new biochemical findings of the assembly of NADPH oxidase components and the signal transduction for the activation of NADPH oxidase. The activation of this enzyme is associated with multiple interrelated signaling pathways. Upon cell stimulation, the second messengers act on the assembly of NADPH oxidase components. The cytosolic components are first phosphorylated, and then associated with the membrane components. Small GTP-binding proteins and cytoskeletal components also participate in the activation of the NADPH oxidase. The cytochemical findings demonstrate that the superoxide generated by NADPH oxidase activity is initially localized in distinct types of intracellular granules, and not at the plasma membrane as previously believed. Thus, the assembly of NADPH oxidase components possibly occurs at the limiting membrane of the intracellular compartments. The oxidant-producing compartments mobilize and become associated with the plasma membrane upon cell stimulation with soluble stimulants, or fuse to phagosomes upon stimulation with particulate stimulants. Accordingly, superoxide is released to the extracellular space and into phagosomes in proportion to the oxidant-producing intracellular granule association with the plasma membrane and with the phagosomal membrane, respectively.

Detection of oxidant producing-sites in glutaraldehyde-fixed human neutrophils and eosinophils stimulated with phorbol myristate acetate.

The aim of the present study was to detect oxidant-producing sites, and to elucidate their dynamic reorganization in human polymorphonuclear leukocytes (PMNs) fixed with glutaraldehyde which preserves cell structure. In biochemical analyses, the detectable O2- generation in unfixed PMNs upon stimulation with phorbol 12-myristate 13-acetate (PMA) in the presence of cytochalasin B was characterized by a lag period of approximately 10 sec followed by O2- production. The maximal rate reached was 3.18+/-0.07 nmol/min/l x 10(6) cells (mean+/-S.D.; n = 4) after 30 sec of stimulation. PMNs exposed to PMA and cytochalasin B followed by fixation with glutaraldehyde generated O2- without a lag period at a rate of 0.35+/-0.05 n mol/min/l x 10(6) cells (mean+/-S.D.) by the addition of NADPH as substrate to the cell suspension. In the cytochemical assays, we employed both cells exposed to PMA and cytochalasin B, and then fixed with glutaraldehyde followed by incubation in the cytochemical reaction medium (pre-fixed cells) and cells incubated in the medium containing PMA and cytochalasin B followed by fixation with glutaraldehyde (post-fixed cells). Oxidant reaction in the pre-fixed cells was detected by the addition of NADPH and FAD to the reaction medium. No oxidant-reaction product was seen in pre-fixed cells stimulated for 10 sec whereas the oxidant reaction was visualized in intracellular compartments of pre-fixed PMNs stimulated for 20 sec. The fact that the pre-fixed PMNs stimulated for 30 sec showed increased numbers of oxidant-producing structures compared to those seen in the pre-fixed cells stimulated for 20 sec, demonstrates that the amount of the reaction product and the number of oxidant-producing intracellular compartments increases between 20 and 30 sec after start of stimulation with PMA. These cytochemical results using the pre-fixed cells coincided with the findings obtained from the biochemical assays in the pre-fixed cells exposed to PMA and cytochalasin B. The oxidant reaction was observed in elongated tubular structures that were arranged in a radial fashion, and were associated with the plasma membrane in the pre-fixed PMNs, whereas post-fixed PMNs exhibited slender spherical or rod-shaped structures of various lengths. The present results indicate that the pre-fixed PMNs can be employed for elucidating the dynamic reorganization of oxidant-producing sites in human PMNs.

Ecto-ATPase activity in cerebellum: implication to the function of synaptic transmission.

The involvement of ATP in synaptic transmission was examined in synapses on granule cells of the rat cerebellum using ecto-ATPase activity. Reaction product was found in a majority but not all synapses between axodendritic, axoaxonic, and dendrodendritic appositions of granule cells and was associated with extracellular surface of both pre- and postsynaptic membranes. Specificity of the detection was justified by using diethyl pyrocarbonate, specific inhibitor of ecto-ATPase activity. These observations provide direct morphological evidence in support of the view that ATP participates in synaptic transmission and indicate functional heterogeneity of synapses in cerebellum.

Evaluation of neutrophil structure and function by electron microscopy: cytochemical studies.

Methods for studying human neutrophils at the ultrastructural level by enzyme cytochemistry and immunocytochemistry are presented. The focus of these methods is on the analysis of the alkaline phosphatase-positive secretory organelle of these cells. These methods provide unique information which, when coupled with biochemical studies, provide for the most complete analysis of neutrophil structure and function.

Dynamics of rat liver ecto-ATPase during development suggests its involvement in bile acid efflux. A cytochemical view.

We studied cytochemical localization of ectoadenosine triphosphatase in the rat liver during development from 15-day-old fetus to 4-week-old and adult animal. First signs of the enzyme activity were found in some of the primitive bile canaliculi of 15-day-old fetuses. The majority of canaliculi, however, did not reveal any reaction product. Although intensity of the cytochemical reaction increased at 20 days of gestation, it still remained relatively low. Intensity of the reaction increased significantly and its product became readily detectable in the liver of newborn rats. Liver of 1-, 2-, and 4-week-old animals showed strong reaction for ecto-ATPase at the luminal surface of the plasma membrane of the bile canaliculi. Liver of adult rats contained a prominent reaction product similar to that seen in newborns, 1-, 2-, and 4-week-old animals. At all stages of fetal development, as well as in postnatal and adult rats, reaction was found only within the hepatic bile canalicular system and exclusively at the luminal surface of the canalicular plasma membrane. Using diethyl pyrocarbonate (DEPC), a specific inhibitor of ecto-ATPase activity, cytochemical reaction was blocked in all examined samples. Results of the present study, taken together with established biochemical and immunological data, provide conclusive morphological evidence in support of the view that canalicular ecto-ATPase is involved in bile acid efflux.

Changes in telomerase activity of advanced cancers of oral cavity and oropharynx during radiation therapy: correlation with clinical outcome.

Telomerase activity has been evaluated for many kinds of malignancies, and it has been clarified that the activity reflected the malignant potential of the tumor. With regard to radiation therapy for cancer, simple and reliable assays for the prediction of therapeutic effect have not been fully developed yet. Therefore, we aimed to determine whether telomerase activity is changed by radiotherapy for advanced head and neck malignancies, and to clarify the possible correlation of telomerase activity with the therapeutic effect of radiation therapy and patients' clinical outcomes. Twenty-five patients with advanced cancers of the oral cavity and oropharynx were examined. All the cancers were confirmed histopathologically as squamous cell carcinomas. Biopsies were performed before treatment and at doses of 4 and/or 10 Gy, and 20 Gy of radiotherapy. To semi-quantitate telomerase activity, a highly sensitive PCR-based telomeric repeat amplification protocol assay was performed. Nineteen of the 21 cancers (90.1%) showed telomerase activity of varied extents prior to radiation therapy. Nine of the 25 patients showed continuous elevations of telomerase activity (> 10 TPG units/microg protein) in their tumors during radiation therapy, and these nine showed 13.8-216.9 TPG units/microg protein at 20 Gy of radiotherapy. In eight of the nine patients, tumors did not respond well to radiotherapy and relapsed locally in a short period after the treatment. On the other hand, in another group of 11 patients, who showed low (< 10) and/or no activity of telomerase in their tumors at 20 Gy of radiotherapy, there were nine patients whose tumors responded well to radiotherapy (p < 0.025 by the chi2 test). None of them relapsed locally in the follow-up period of approximately 21 months, and the difference was statistically significant (p < 0.025 by the chi2 test). It is concluded that the high activity of telomerase in tumor tissue at 20 Gy of radiotherapy can predict a poor therapeutic effect of radiation therapy and unfavorable patients' outcomes for advanced cancers of oral cavity and oropharynx.

Intracellular dynamics of alkaline phosphatase-containing granules in electropermeabilized human neutrophils.

Human neutrophils possess alkaline phosphatase-containing intracellular granules which are upregulated to the cell surface upon stimulation. The mechanism that governs the intracellular dynamics of these granules is, however, poorly understood. The aim of the present study was to investigate the possible participation of GTP-binding proteins in the reorganization and exocytosis of the alkaline phosphatase-containing granules using electropermeabilized cells. Biochemical assays using intact neutrophils showed that the alkaline phosphatase activity was upregulated and exocytosed into the extracellular space upon stimulation with AIF4 and N-formyl peptide. This upregulation was inhibited by treatment of cells with pertussis toxin and botulinum toxin. Alkaline phosphatase activity was also upregulated in electropermeabilized cells stimulated with guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), but not with guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS). Cytochemically, alkaline phosphatase-containing granules were dispersed throughout the cytoplasm in unstimulated electropermeabilized neutrophils. Upon stimulation with GTPgammaS, but not with GDPbetaS, these granules fused to form elongated tubular structures which eventually became associated with the plasma membrane. Nocodazole disturbed the reorganization of the alkaline phosphatase-containing granules in cells stimulated with GTPgammaS. The results from this study indicate that GTP-binding proteins participate in the reorganization and exocytosis of alkaline phosphatase-containing granules associated with the microtubules in electropermeabilized human neutrophils.

Changes of mutant-type p53 expression in squamous cell carcinoma of the head and neck during radiation therapy and its clinical significance: comparison of an immunohistochemical method and PCR-SSCP assay.

P53 has been reported to be one of tumor suppressor genes that play a major role in signal transduction following many kinds of stresses, including ionizing radiation. Changes in p53 expression during radiation therapy in tumor tissues have not yet been reported. We determined whether radiotherapy changes p53 expression in human squamous cell carcinomas of the head and neck, and established the possible correlations between p53 expression and the therapeutic effects of radiation therapy. 30 patients with tumors of the oral cavity, oropharynx, and maxillary sinus were examined, and all the tumors were confirmed as squamous cell carcinomas. Biopsies were performed on the cancer tissues before treatment and at doses of 4, 10, and 20 Gy of radiotherapy, and the specimens were preserved in liquid nitrogen for further examination. Samples were immunohistochemically stained using streptoavidin-biotin peroxidase method and a monoclonal antibody against p53 (Ab-3, mutant type). For all the samples p53 PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) assays were performed. 14 of the 30 patients with squamous cell carcinomas showed expression of p53 in their tumor cells before and/or at 4 Gy or 10 Gy of radiotherapy. Eleven of the 14 tumors showed high radiosensitivity. Results of the p53 PCR-SSCP assays revealed mutations of p53 in 13 of 30 patients examined, and percentages of mutated p53 DNA varied at radiation doses of 4 Gy and 10 Gy. Ten of 12 patients with mutated p53 in their tumors showed decreased percentages of mutated p53 DNA during radiotherapy. The relationship between the immunohistochemical findings and the antitumor effect of a radiation dose of 20 Gy was examined on the correspondent hematoxylin-eosin sections. In patients whose p53 expressions in tumor cells were grades + or ++ or before radiotherapy and/or at 4 Gy of radiotherapy, the tumors responded significantly well to radiation therapy but the patients responded with significantly unfavorable clinical courses. The high radiosensitivity of squamous cell carcinomas in our samples could be explained by an overexpression of mutant type p53 in the tumor cells, and these mutant type p53-positive tumor cells possibly showed radioresponsiveness.

Expression of beta3-adrenoceptors in rat detrusor smooth muscle.

PURPOSE: To investigate the expression of beta-adrenoceptor (AR) subtypes responsible for detrusor smooth muscle relaxation. MATERIALS AND METHODS: Isolated rat detrusor smooth muscle was examined by tension measurement and reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Norepinephrine (NE), epinephrine (EP) and isoproterenol (ISO) were found to relax the detrusor muscle pre-contracted by 6 x 10(-7) M carbachol in the presence of 10(-6) M phentolamine. NE relaxed the detrusor muscle as potently as EP. This potency order (NE=EP) thus indicated the beta-ARs of the rat detrusor muscle to possibly be a beta1 subtype. However, in the presence of 10(-6) M propranolol, beta1- and beta2- but not beta3-AR antagonist, NE showed a more potent relaxation than EP. This observation indicated that the rat detrusor muscle also possesses beta3-AR. RT-PCR revealed all three subtypes of beta-AR mRNA, namely beta1-, beta2- and beta3-AR mRNA, to be expressed in rat detrusor smooth muscle cells. CONCLUSION: We concluded that beta3-ARs exist in rat detrusor smooth muscle based on both pharmacological and molecular biological studies. Based on these findings, beta-ARs of rat detrusor smooth muscle are considered to be mixed populations consisting of three subtypes which play an important role in relaxing smooth muscle in response to catecholamines.

Induction of DNA fragmentation by total-body irradiation in murine liver.

Total-body irradiation (TBI) is an accepted modality to treat patients with disseminated tumors. The influence of the treatment on normal tissues is evaluated using mice by measuring the rate of the induction and distribution of apoptosis, as well as DNA fragmentation which occurs in the murine liver within hours of irradiation. Unanesthetized female C3H/He mice were exposed to gamma-ray TBI of 2, 7, and 20 gray (Gy) delivered from 60Co at a dose rate of 114 cGy/min. Frozen sections of livers which were excised from the animals at various times after irradiation were stained by hematoxylin-eosin (H-E) to count numbers of apoptotic cells, or were examined to detect DNA fragmentation. The percentages of apoptotic cells and length of the period during which the maximum levels of the percentages were exhibited showed a dose-dependent increase in the sections stained with H-E. No positive cells for 3'-OH ends of fragmented DNA were found in the liver before TBI, whereas positive cells were observed immediately after irradiation without dose-dependency, these positive cells returned to nearly basal levels after several hours. Positive cells were observed prior to showing apoptosis, suggesting that DNA fragmentation occurs immediately after TBI independent of apoptosis. The difference in the time courses between induction of DNA fragmentation and of apoptosis was not observed in other organs or in the samples treated with the detergent. These results suggested that the 3'-OH ends newly generated by TBI were masked by a detergent-soluble DNA-binding molecule which might be preferentially present in the murine liver.

CD34-positive cell yield in peripheral blood of cancer patients during radiation therapy.

For the purpose of evaluating the feasibility of inducing CD34-positive cells in peripheral blood by radiation therapy, we examined the changes in CD34-positive cells in 21 cancer patients (16 with lung cancer and five with esophageal cancer) during thoracic radiotherapy using flow cytometry and CD34 monoclonal antibody. Although assays of granulocyte-colony-forming units (G-CFU) and granulocyte-monocyte-colony-forming units (GM-CFU) were also performed for 16 of the patients during radiation therapy, in most of these cases there was approximately one G-CFU or GM-CFU for every 100 CD34-positive cells. Seven of the 21 cancer patients showed percentages of CD34-positive cells of more than 3% (more than mean + 2 S.D.; standard deviation) of mononuclear cells in peripheral blood in at least one of the examinations. However, six of these seven patients were in stage IV with distant metastases (four with lung cancer and two with esophageal cancer), and another lung cancer patient was in stage III. Therefore, CD-34 positive cells were shown to appear in peripheral blood especially in the patients with advanced stages of malignancy, and further examinations are needed to induce CD34-positive cells by radiation therapy for possible application of ultra-high dose chemotherapy supported by peripheral blood stem cell transplantation.

Expression and function of alpha1-adrenoceptor subtypes in the porcine renal artery.

We investigated the expression and function of alpha1-adrenoceptor subtypes in the porcine renal artery. Reverse transcription polymerase chain reaction (RT-PCR) and nucleotide sequencing indicated that the mRNAs for alpha1a- and alpha1b-adrenoceptors were expressed in the porcine renal artery. Chloroethylclonidine, an alpha1B- and alpha1D-adrenoceptor antagonist, partially inhibited the phenylephrine-induced contraction, while 3 nM BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]dec ane-7,9-dione dihydrochloride), an alpha1D-adrenoceptor antagonist, had no effect. In contrast, 5-methylurapidil, an alpha1A-adrenoceptor antagonist, induced a rightward shift of the phenylephrine concentration-response curve. The simultaneous measurement of cytosolic Ca2+ concentration ([Ca2+]i) and tension revealed that chloroethylclonidine pretreatment abolished the phenylephrine-induced increases in [Ca2+]i and tension in the Ca2+-free solution. The application of 5-methylurapidil (3 nM) to the chloroethylclonidine-pretreated strips completely inhibited the 3 microM phenylephrine-induced [Ca2+]i and tension increase in normal PSS. We concluded that both alpha1A- and alpha1B-adrenoceptors mediate the phenylephrine-induced contraction of the porcine renal artery accompanied by an increase in [Ca2+]i, and that alpha1A-adrenoceptors cause Ca2+ influx whereas alpha1B-adrenoceptors mainly mediate Ca2+ release.

An ultracytochemical study on the dynamics of alkaline phosphatase-positive granules in rat neutrophils.

Alkaline phosphatase (ALPase) activity was examined by cerium-based ultracytochemistry in isolated rat neutrophils following stimulation with phorbol myristate acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP). In control neutrophils, low levels of ALPase activity were detected in small tubular and spherical compartments distributed throughout the cytoplasm. Neutrophils stimulated for 2.5, 5, 15, and 30 min with 50 ng/ml PMA or 10(-7) M fMLP displayed a time-dependent increase in ALPase activity. At 2.5 min, an increase in activity was first identified in compartments that were aggregated in the central regions of the cell. By 15 min, a dense precipitate was seen in tubular or elongated bead-like structures that extended to and made contact with the plasma membrane. Large enzyme-positive vacuoles were also observed in regions near the plasma membrane. At the longer stimulation times, a fine precipitate was present on the cell surface of the neutrophil in regions where subplasmalemmal ALPase activity was present. The results of this study indicate that an increase in activity and a redistribution of ALPase-positive structures occurs in neutrophils in response to stimulation with PMA and fMLP. It is likely that these compartments are latent pools of ALPase which, upon stimulation, fuse and mobilize the enzyme activity to the cell surface.