Efficacy and safety of collagenase Clostridium histolyticum injection for Dupuytren's contracture in non-Caucasian Japanese patients (CORD-J Study): the first clinical trial in a non-Caucasian population.

To assess the efficacy, safety and pharmacokinetics of 0.58 mg collagenase Clostridium histolyticum injections for the treatment of Dupuytren's contracture in Japanese patients, we conducted a phase III, multicentre, uncontrolled, open-label clinical study in patients with Dupuytren's contracture. Of the 77 patients, 66 achieved clinical success in the primary treated joint (86%; 95% confidence interval: 76% to 93%), confirming the efficacy of collagenase Clostridium histolyticum injections. More improvement was seen in the metacarpophalangeal joints than in the proximal interphalangeal joints (94% versus 73%). The main adverse reaction was a local reaction in the injected hand. No tendon rupture or anaphylactic reactions were seen. The concentrations of collagenase Clostridium histolyticum were below the lower limit of quantification in plasma samples at all time points. As seen in global studies in Caucasian patients, a corrective effect on Dupuytren's contracture and good tolerance were observed in most non-Caucasian (Asian) Japanese patients. LEVEL OF EVIDENCE: Level 3.

Temporal and sequential changes of glial cells and cytokine expression during neuronal degeneration after transient global ischemia in rats.

BACKGROUND: How glial cells and cytokines are associated with the progression of delayed neuronal death induced by transient global ischemia is still unclear. To further clarify this point, we studied morphological changes in glial cells (microglial cells and astrocytes), and cytokine protein levels, during the progression of neuronal cell loss in CA1 (Cornu Ammonis 1) of the hippocampus after transient global ischemia. METHODS: Morphological changes in glial cells were studied immuno-histochemically. Nine cytokines (IL-1α, IL-1ß, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ and TNF-α) were simultaneously measured by a multiplexed bead-based immunoassay from 6 h to day21 after transient four vessel occlusion (4VO) in rats. RESULTS: During the process of neuronal loss, we observed four distinct phases: (1) lag phase day0-2 (no NeuN+ cell loss observed), (2) exponential phase day2-7 (NeuN+ cells reduced in number exponentially), (3) deceleration phase day7-14 (reduction rate of NeuN+ cells became low), (4) stationary phase day14 onward (NeuN+ cell loss progressed no longer). In the lag phase, activated glial cells were observed in the entire hippocampus but later were gradually restricted to CA1. Cytokine protein levels in the lag and exponential phases were lower than in the deceleration and stationary phases. IL-1α, IL-1ß, IL-4, IL-6 and IFN-γ in 4VO were significantly higher in all four phases than in sham. Compared with sham level, GM-CSF was significantly high in the deceleration phase. TNF-α was significantly high in both the deceleration and stationary phases. CONCLUSION: Ischemic stress in 4VO activated glial cells in areas beyond CA1 in the lag phase. Pyramidal neurons were injured in CA1 from the end of the lag phase and then neuronal cells reduced in CA1 in the exponential phase. After neuronal death began, the influence of dead cells on glial cells and cytokine expression gradually became stronger than the influence by ischemic stress. Therefore, from the deceleration phase, changes in glial cells and cytokine production were likely caused by dead cells. Cytokine interaction in the microenvironment may determine the functions of IL-1α, IL-1ß, IL-4, IL-6 and IFN-γ in all four phases. The function of GM-CSF and TNF-α in the deceleration phase may be neurotrophic.

The effects of MPTP on the activation of microglia/astrocytes and cytokine/chemokine levels in different mice strains.

The effects of MPTP on two mouse strains with different MPTP sensitivities and immunological backgrounds were compared: MPTP-sensitive C57BL/6 mice (B6) with a propensity for Th1 and less MPTP-sensitive BALB/c mice (BALB) with a propensity for Th2. It was found that acute MPTP treatment induced behavioral dysfunction, activated microglia/astrocytes, and increased the levels of IL-10, IL-12(p40) IL-13, IFN-gamma, and MCP-1 in CSF in B6, but not in BALB. This suggests that variances in immunological backgrounds might be a major contributing factor in sensitivity differences to MPTP.

Oral administration of dihomo-gamma-linolenic acid prevents development of atopic dermatitis in NC/Nga mice.

Disorders of the metabolism of essential fatty acids (EFAs) are related to atopic dermatitis (AD). Concentrations of dihomo-gamma-linolenic acid (DGLA), an EFA, in the serum of AD patients are lower than those in healthy volunteers. Recently we developed a fermented DGLA oil, and examined whether oral administration of DGLA prevents development of dermatitis in NC/Nga mice, which spontaneously develop human AD-like skin lesions. NC/Nga mice were fed a diet either containing or not containing DGLA for 8 weeks under in air-uncontrolled conventional circumstances. Clinical skin severity scores were significantly lower in mice fed DGLA than in mice not fed it. Scratching behavior and plasma total IgE levels were also reduced in the DGLA group, in association with histological improvement. DGLA suppressed clinical severity of skin lesions dose-dependently, with an increase in DGLA contents in phospholipids of skin, spleen, and plasma. Discontinuation of DGLA administration resulted in the onset of dermatitis and a decrease in DGLA contents in skin, spleen, and plasma. These findings indicate that oral administration of DGLA effectively prevents the development of AD in NC/Nga mice, and that DGLA in phospholipids is a compound of key importance in the development and prevention of dermatitis.

Long-lasting reactive changes observed in microglia in the striatal and substantia nigral of mice after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Parkinson's disease (PD) is an age-related movement disorder that progresses over a period of 10 to 20 years. The existence of microglia in a long-lasting activated state, expressing MHC II, has been thought to play an important role in the progression of PD. PD mouse models, induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), exhibit only transient PD-like movement dysfunction in contrast to MPTP-intoxicated monkeys which show progressive and permanent movement dysfunction. To understand the reasons why the progression does not occur in MPTP-treated mice, we used immunohistochemical analyses to study whether activated microglia in the striatum and/or substantia nigra persist long after MPTP treatment. Microglial changes in the striatum and substantia nigra of mice at 2 days and 6 months after MPTP treatment (four intraperitoneal injections of 20 mg/kg MPTP at two hour intervals) were examined. C57BL/6 mice (which are highly sensitive to MPTP) displayed transient movement dysfunction and highly activated microglia were observed at day two. In contrast, BALB/c mice (which are less sensitive to MPTP) exhibited no movement dysfunction and only slightly activated microglia were observed at day two. After 6 months, the microglia in the striatum and substantia nigra pars compacta of the treated C57BL/6 mice were still more hypertrophic compared with the control, although less hypertrophic than those observed at day two. In the treated BALB/c mice, the microglia were also hypertrophic compared with the control after 6 months. MHC II-positive microglia were undetectable at any time after MPTP treatment in both mice. These data show that MPTP administration results in the existence of persistent activated microglia that are not MHC II-positive, and is independent of the MPTP sensitivity of the mouse strain. These results suggest that long lasting MHC II-positive microglia might be required for PD progression. In MPTP-intoxicated mice, the absence of MHC II-positive microglia might explain why there is no progression of PD-like dysfunctional symptoms.

The intra-arterial injection of microglia protects hippocampal CA1 neurons against global ischemia-induced functional deficits in rats.

In the present study, we have attempted to elucidate the effects of the intra-arterial injection of microglia on the global ischemia-induced functional and morphological deficits of hippocampal CA1 neurons. When PKH26-labeled immortalized microglial cells, GMIR1, were injected into the subclavian artery, these exogenous microglia were found to accumulate in the hippocampus at 24 h after ischemia. In hippocampal slices prepared from medium-injected rats subjected to ischemia 48 h earlier, synaptic dysfunctions including a significant reduction of synaptic responses and a marked reduction of long-term potentiation (LTP) of the CA3-CA1 Schaffer collateral synapses were observed. At this stage, however, neither significant neuronal degeneration nor gliosis was observed in the hippocampus. At 96 h after ischemia, there was a total loss of the synaptic activity and a marked neuronal death in the CA1 subfield. In contrast, the basal synaptic transmission and LTP of the CA3-CA1 synapses were well preserved after ischemia in the slices prepared from the microglia-injected animals. We also found the microglial-conditioned medium (MCM) to significantly increase the frequency of the spontaneous postsynaptic currents of CA1 neurons without affecting the amplitude, thus indicating that MCM increased the provability of the neurotransmitter release. The protective effect of the intra-arterial injected microglia against the ischemia-induced neuronal degeneration in the hippocampus was substantiated by immunohistochemical and immunoblot analyses. Furthermore, the arterial-injected microglia prevented the ischemia-induced decline of the brain-derived neurotrophic factor (BDNF) levels in CA1 neurons. These observations strongly suggest that the arterial-injection of microglia protected CA1 neurons against the ischemia-induced neuronal degeneration. The restoration of the ischemia-induced synaptic deficits and the resultant reduction of the BDNF levels in CA1 neurons, possibly by the release of diffusible factor(s), might thus contribute to the protective effect of the arterial-injection of microglia against ischemia-induced neuronal degeneration.

Arundic acid (ONO-2506) ameliorates delayed ischemic brain damage by preventing astrocytic overproduction of S100B.

After focal cerebral ischemia, the infarct volume increases rapidly within acute infarct expansion (initial 12 to 24 h) and continues slowly during delayed infarct expansion (25 to 168 h). While acute infarct expansion represents progressive necrosis within the ischemic core, delayed infarct expansion starts as disseminated apoptotic cell death in a narrow rim surrounding the infarct border, which gradually coalesces to form a larger infarct. Discovery of a distinct correlation between reactive astrogliosis along the infarct border and delayed infarct expansion in the rodent ischemia model led us to investigate the possible causal relationship between the two events. Specifically, the calcium binding protein S100B exerts detrimental effects on cell survival through activation of various intracellular signaling pathways, resulting in altered protein expression. Arundic acid [(R)-(-)-2-propyloctanoic acid, ONO-2506] is a novel agent that inhibits S100B synthesis in cultured astrocytes. In the rodent ischemia model, this agent was shown to inhibit both the astrocytic overexpression of S100B and the subsequent activation of signaling pathways in the peri-infarct area. Concurrently, delayed infarct expansion was prevented, and neurologic deficits were promptly ameliorated. The results of subsequent studies suggest that the efficacy of arundic acid is mediated by restoring the activity of astroglial glutamate transporters via enhanced genetic expression.

Effects of dietary triglycerides on rheological properties of human red blood cells (abstract).

Atherogenic diets rich in saturated fat and cholesterol influence the blood viscosity and red blood cell (RBC) aggregability, the parameters associated with increased risk of circulatory disorders. However, little is known about the effect of triglycerides, which are the major dietary lipid form in humans, on blood rheology. Therefore, we studied the effects of postprandial plasma triglyceride levels on human RBC indices, hematological parameters, RBCs aggregation velocity and whole blood viscosity. For this purpose, whole blood was collected 2 hours after high-fat or low-fat meal. Proteins, triglycerides and cholesterol levels of plasma were analysed, and RBCs rouleaux formation rate was measured in 70% autologous plasma using a low-shear rheoscope. There were no significant differences in hematological parameters, RBC indices, whole blood viscosity, plasma protein and cholesterol content between high-fat and low-fat blood samples. However, a significant increase in rouleaux formation rate was observed in samples with high postprandial triglyceride levels, when compared with low-triglyceride samples. Plasma triglyceride levels correlated significantly with rouleaux formation rate. In conclusion, these results suggest that diet-dependent alterations of plasma triglyceride levels as well as possible changes in the cell membrane lipid composition lead to RBC hyperaggregability.

Enhancement of red blood cell aggregation by plasma triglycerides.

The effects of plasma triglycerides level on human red blood cells (RBCs) indices, hematological parameters, RBCs aggregation velocity and whole blood viscosity were studied at 2 hours after high-fat or low-fat meal. Proteins, triglycerides and cholesterol levels of plasma were analysed. The RBCs rouleaux formation rate was measured in 70% autologous plasma (with 30% phosphate-buffered saline, PBS) or 1 g/dl dextran T70 solution (with 4 g/dl bovine serum albumin) in PBS, using a low-shear rheoscope. The results were grouped according to triglycerides content in plasma. No significant difference in whole blood viscosity, hematological parameters, RBC indices, protein and cholesterol content was observed between high-fat and low-fat blood samples. There was a significant increase in rouleaux formation rate of samples with high triglyceride levels, when measured in 70% autologous plasma, but it was not significant in dextran T70 containing medium. In conclusion, the results obtained suggest that alteration of plasma lipid levels as well as possible changes in the cell membrane lipid composition lead to enhanced RBC aggregation.

O(2) release from erythrocytes flowing in a narrow O(2)-permeable tube: effects of erythrocyte aggregation.

The effects of erythrocyte aggregation on O(2) release were examined using O(2)-permeable fluorinated ethylenepropylene copolymer tubes (inner diameter, 25 microm; outer diameter, 100 microm). Measurements were performed using an apparatus built on an inverted microscope that contained a scanning-grating spectrophotometer with a photon count detector connected to two photomultipliers and an image processor through a video camera. The rate of O(2) release from the cells flowing in the narrow tube was determined based on the visible absorption spectrum and the flow velocity of the cells as well as the tube size. When the tube was exposed to nitrogen-saturated deoxygenated saline containing 10 mM sodium dithionite, the flowing erythrocytes were deoxygenated in proportion to the traveling distance, and the deoxygenation at a given distance increased with decreasing flow velocity and cell concentration (hematocrit). Adding Dextran T-70 to the cell suspension increased erythrocyte aggregation in the tube, which resulted in suppressed cell deoxygenation and increased marginal cell-free-layer thickness. The deoxygenation was inversely proportional to the cell-free-layer thickness. The relation was not essentially altered even when the medium viscosity was adjusted with Dextran T-40 to remain constant. The rate of O(2) release from erythrocytes in the tube was discussed in relation to the O(2) diffusion process. We conclude that the diffusion of O(2) from erythrocytes flowing in narrow tubes is inhibited primarily by erythrocyte aggregation itself and partly by thickening of the cell-free layer.

Aggregation and sedimentation of mixtures of erythrocytes with different properties.

The rouleau formation of erythrocytes and the erythrocyte sedimentation were examined for mixture of different kinds of the cells suspended in isotonic phosphate-buffered saline containing 1 or 2 g/dl dextran T-70 (MW = 70,400) and 4 g/dl albumin, using a low shear rheoscope and the Westergren method, respectively. The deformability of cells was decreased by treating with diamide, diazene-dicarboxylic acid bis[N,N-dimethylamide], and the sialic acid content of cells, i.e., the surface negative charge, was reduced by treating with neuraminidase. (1) The rate of rouleau formation was decreased in cells with decreased deformability, while it was increased in cells with reduced sialic acid content. The rate changed in proportion to the ratio of the modified cells to normal cells. (2) The erythrocyte sedimentation was also decreased in cells with decreased deformability, while it was increased in cells with reduced sialic acid content. Furthermore, the erythrocyte sedimentation changed almost proportionally to the ratio of the modified cells to normal cells. (3) When normal deformable cells were mixed with cells with decreased deformability, the deformable cells seemed to settle faster than the less deformable cells, though the difference was not significant. (4) When normal cells were mixed with cells with reduced sialic acid content, the cells with less sialic acid settled significantly faster than those with more sialic acid. The present experiment may conclude that erythrocyte aggregation is induced preferentially among more deformable cells and/or among less negatively charged cells with weaker electrostatic repulsive force, and then the formed aggregates settle faster.

Gamma-ray-irradiated red blood cells stored in mannitol-adenine-phosphate medium: rheological evaluation and susceptibility to oxidative stress.

BACKGROUND AND OBJECTIVES: To evaluate the rheological properties and the oxidative susceptibility of gamma-ray-irradiated red blood cells (RBCs). MATERIALS AND METHODS: RBCs in mannitol-adenine-phosphate (MAP) medium were irradiated with 35 Gy and stored at 4 degrees C for 4 weeks. The deformability of the RBCs was examined under shear flow in relation to the morphological and biochemical changes. The RBCs were further exposed to 1 mM FeSO(4) and 5 mM ascorbate to examine the oxidative susceptibility. RESULTS: The RBC deformability was decreased during storage, and the impairment was further enhanced by the irradiation, which promoted cell shrinkage and intracellular hemoglobin condensation accompanying potassium loss. Lipid peroxidation and protein aggregation of the RBC membrane as well as echinocytosis were not enhanced by the irradiation. The exposure to free iron did not stimulate the oxidation of the irradiated RBC membrane. CONCLUSION: The decreased deformability of gamma-ray-irradiated RBCs in MAP medium was mainly induced by dehydration due to potassium loss, and the membrane lipids and proteins were stably preserved against oxidative stress.

Decreased deformability of the X-ray-irradiated red blood cells stored in mannitol-adenine-phosphate medium.

X-ray irradiation of blood is an effective way to prevent transfusion-associated graft-versus-host disease. Red blood cells (RBCs) from normal donors suspended in mannitol-adenine-phosphate (MAP) medium were irradiated with X-ray of 15 and 35 Gy in minimum dose. The change of deformability of the RBCs during storage at 4 degrees C for 4 weeks was examined under shear stress of 13-130 dyn/cm2 using a rheoscope, in relation to the hematological and biochemical properties. (1) The deformability of RBCs was decreased during the storage, and it was further decreased by the irradiation. In addition, the number of undeformable RBCs against a given shear stress increased after the irradiation. (2) The cell volume gradually decreased, while the intracellular hemoglobin concentration increased. These changes were accelerated by the irradiation. The echinocytic transformation during the storage was not accelerated by the irradiation. (3) The content of aggregated proteins reducible with beta-mercaptoethanol in RBC membrane increased during the storage, but was not increased by the irradiation. Membrane lipid peroxidation was not increased during the storage and by the irradiation. (4) Leakage of potassium ions from RBCs during the storage was accelerated by the irradiation. In conclusion, shear-induced deformation of RBCs stored in MAP medium was impaired by X-ray irradiation, mainly due to dehydration caused by excess leakage of potassium ions from RBCs.

Histochemical investigation of ß-glucuronidase in culture cells and regenerated plants of Scutellaria baicalensis Georgi.

Strong activity of ß-glucuronidase first appeared in the epidermal and glandular hair cells of leaf primordia regenerated from callus of Scutellaria baicalensis Georgi. Leaf primordia matured rapidly in culture to form shoots within 1 month in which both the mesophyll cells and the glandular hairs were deeply stained. Leaves predominantly accumulated ß-glucuronidase in both glandular hair cells and mesophyll cells. ß-Glucronidase activity in leaves was higher in the summer and decreased in the winter. The stem section collected in the summer had a different ß-glucuronidase distribution pattern from that of the root in that in the former strong activity appeared in the periderm cells and collenchyma cells which was decreasingly dispersed into the phloem layer cells. In the winter, ß-glucronidase activity decreased compared to that in summer. It can be argued that the distribution of ß-glucuronidase in this plant is closely linked with the defense against pathogens: it is a starting key enzyme which may act together with the flavonoids, which play an important role as a proton donor for the detoxification metabolism of H2O2.

Influence of gravity on flow distribution of red blood cells in microcirculation.

The influence of the gravity on flow distribution of erythrocytes in microcirculation was examined. We developed a new centrifuge system with a rotation disc. An observation system of blood flow in a micro-flow channel was arranged on the disc. Erythrocyte flow in the micro-flow tube was displaced under the gravity. This study suggests that the gravity affects the transfer of substances from blood vessels to tissues.

Increased chemokine receptor CCR7/EBI1 expression enhances the infiltration of lymphoid organs by adult T-cell leukemia cells.

Adult T-cell leukemia (ATL) is characterized by infiltration of various tissues by circulating ATL cells, a finding often associated with a poor prognosis. Leukocyte migration from the circulation into tissues depends on integrin-mediated adhesion to the endothelium, and integrins are tightly regulated by several factors, such as chemokines. In this study, we focused on the interaction between chemokines and chemokine receptors on ATL cells to understand factors involved in ATL cell infiltration of lymphoid organs. We compared freshly isolated ATL cells from patients with and without lymphoid organ involvement for the expression of the chemokine receptor CCR7/EBI1, the functional receptor for secondary lymphoid-tissue chemokine (SLC), which is expressed at high levels by high endothelial venules of lymph nodes and Peyer's patches. Reverse transcriptase-polymerase chain reaction and flow cytometric analysis, using anti-CCR7 monoclonal antibody (CCR7.6B3), revealed that ATL cells from patients with lymphoid organ involvement expressed significantly more CCR7/EBI1 than control CD4(+)CD45RO(+) T cells and ATL cells from patients without lymphoid organ involvement. Consequently, significantly more ATL cells from patients with lymphoid organ involvement than control CD4(+)CD45RO(+) T cells and ATL cells from patients without lymphoid organ involvement adhered to surfaces coated with ICAM-1 and SLC or EBI1-ligand chemokine (ELC), another ligand for CCR7/EBI1, under static and flow conditions and migrated toward SLC or ELC at a low concentration (30 ng/ml). These findings suggest that increased CCR7/EBI1 expression plays a role in lymphoid organ infiltration of ATL cells. (Blood. 2000; 30-38)

Distinctions between microglial cells and peripheral macrophages with regard to adhesive activities and morphology.

Activated microglial cells and peripheral macrophages are hardly distinguishable from the viewpoints of morphology and function. There are various immunological markers common to both microglial cells and peripheral macrophages. In the present study, however, we found that microglial cells have distinct characters in terms of adhesion and morphology. By using a "rheoscope," that is an apparatus to rheologically measure the strength of cell adhesion to substrates, rat microglial cells were found to attach to polystyrene dishes much more weakly than alveolar and peritoneal macrophages. Interferon-gamma (IFNgamma) strengthened the adhesion of alveolar and peritoneal macrophages, whereas it weakened that of microglial cells. Morphological changes of microglial cells induced by IFNgamma were also different from those of peripheral macrophages. Furthermore, alveolar and peritoneal macrophages produced NO in response to IFNgamma, while microglial cells did not. When cultured on astrocyte-derived extracellular matrix (AsECM) in serum-free medium, only microglial cells extended multiple ramified processes. Conversely, alveolar and peritoneal macrophages on AsECM shrunk their ruffling membrane and rounded up. These distinctions between microglial cells and macrophages may reflect differences in cell lineages as well as environments in which individual cells reside.

Identification and molecular characterization of novel peroxidase with structural protein-like properties.

Elicitor treatment or mechanical damage to Scutellaria baicalensis Georgi (skullcap plants) callus causes an immediate insolubilization of a 36-kDa protein into cell walls. The 36-kDa protein was identified as peroxidase 1 by analysis of its internal amino acid sequence and by immunoblotting using affinity-purified anti-peroxidase 1. Insolubilized peroxidase 1 is cross-linked to lignin through covalent bonds, and the cross-linking is catalyzed in the presence of H(2)O(2) by peroxidase 1 itself. The properties of insolubilized peroxidase 1 resemble those of defense-related structural proteins (extensins and proline-rich proteins) cross-linked to cell wall. Although the isozymes peroxidases 2 and 3 have enzyme activities similar to peroxidase 1, they are not insolubilized by stress treatment. Molecular characterization established that peroxidase 1 contains regions characteristic of structural proteins, but peroxidases 2 and 3 do not have such regions. These results suggest that among the three isozymes, only peroxidase 1 has a structural protein-like function as well as an enzymatic function.

Design of PCR primers and gene probes for the general detection of bacterial populations capable of degrading aromatic compounds via catechol cleavage pathways.

For the general detection of bacterial populations capable of degrading aromatic compounds, two PCR primer sets were designed which can, respectively, amplify specific fragments from a wide variety of catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O) genes. The C12O-targeting primer set (C12O primers) was designed based on the homologous regions of 11 C12O genes listed in the GenBank, while the C23O-targeting one (C23O primers) was designed based on those of 17 known C23O genes. Oligonucleotide probes (C12Op and C23Op) were also designed from the internal homologous regions to identify the amplified fragments. The specificity of the primer sets and probes was confirmed using authentic bacterial strains known to carry the C12O and/or C23O genes used for the primer and probe design. Various authentic bacterial strains carrying neither C12O nor C23O genes were used as negative controls. PCR with the C12O primers amplified DNA fragments of the expected sizes from 5 of the 6 known C12O-carrying bacterial strains tested, and positive signals were obtained from 4 of the 5 amplified fragments on Southern hybridization with the C12Op. The C23O primers amplified DNA fragments of the expected size from all the 11 tested C23O-carrying bacterial strains used for their design, while the C23Op detected positive signals in the amplified fragments from 9 strains. On the other hand, no DNA fragments were amplified from the negative controls. To evaluate the applicability of the designed primers and probes for the general detection of aromatic compound-degrading bacteria, they were applied to wild-type phenol- and/or benzoate-degrading bacteria newly isolated from a variety of environments. The C12O and/or C23O primers amplified DNA fragments of the expected sizes from 69 of the 106 wild-type strains tested, while the C12Op and/or C23Op detected positive signals in the amplified fragments from 63 strains. These results suggest that our primer and probe systems can detect a considerable proportion of bacteria which can degrade aromatic compounds via catechol cleavage pathways.

Regulation of cytosol-nucleus pH gradients by K+/H+ exchange mechanism in the nuclear envelope of neonatal rat astrocytes.

In order to study the subcellular heterogeneity of intracellular H+ concentration in reactive astrocytes, the pH in the nucleus and cytosol of cultured astrocytes was measured using a confocal laser scanning microscope (CLSM) and pH indicator dye, 5'(and 6')-carboxyseminaphthofluorescein (carboxy SNAFL-1). The change in intracellular pH was indexed by the fluorescence ratio (F535/F610) at an excitation wavelength of 514.5 nm. The in vitro fluorescence ratio increased as pH decreased. This ratio in the nucleus was significantly lower than that in the cytosol of astrocytes when perfused by HEPES-buffered Hanks' balanced salt solution (HHBSS) at pH 7.4. Acid stimulations of cells (pH 5.0) raised the fluorescence ratio in both nucleus and cytosol. However, the increase in the fluorescence ratio of the nucleus was less than that of cytosol. Treatment with a K+/H+ ionophore, nigericin (20 microM), reversibly nullified this cytosol-nucleus pH gradient. These findings suggest that a buffering mechanism(s) for maintaining of intracellular pH exists between the nucleus and cytosol, and a K+/H+ exchanger may act on the nuclear envelope to eventuate intranuclear pH maintenance in the living cells.