Cerebral Infarction and Myalgia in a 75-year-old Man with Eosinophilic Granulomatosis with Polyangiitis.

Eosinophilic granulomatosis with polyangiitis (EGPA) is a rare condition of systemic vasculitis of small to medium-sized blood vessels. We herein report the case of a 75-year-old man who presented with hemiplegia on his right side due to cerebral infarction following myalgia and a high fever. He had no history of asthma or allergic rhinitis. He was diagnosed with EGPA based on the presence of eosinophilia, sinusitis suggested by magnetic resonance imaging, and muscle pathology. His hemiplegia improved rapidly after corticosteroid therapy. This case suggests that EGPA should be a differential diagnosis of cerebral infarction with myalgia and eosinophilia.

An extracellular [NiFe] hydrogenase mediating iron corrosion is encoded in a genetically unstable genomic island in Methanococcus maripaludis.

Certain methanogens deteriorate steel surfaces through a process called microbiologically influenced corrosion (MIC). However, the mechanisms of MIC, whereby methanogens oxidize zerovalent iron (Fe0), are largely unknown. In this study, Fe0-corroding Methanococcus maripaludis strain OS7 and its derivative (strain OS7mut1) defective in Fe0-corroding activity were isolated. Genomic analysis of these strains demonstrated that the strain OS7mut1 contained a 12-kb chromosomal deletion. The deleted region, termed "MIC island", encoded the genes for the large and small subunits of a [NiFe] hydrogenase, the TatA/TatC genes necessary for the secretion of the [NiFe] hydrogenase, and a gene for the hydrogenase maturation protease. Thus, the [NiFe] hydrogenase may be secreted outside the cytoplasmic membrane, where the [NiFe] hydrogenase can make direct contact with Fe0, and oxidize it, generating hydrogen gas: Fe0 + 2 H+ → Fe2+ + H2. Comparative analysis of extracellular and intracellular proteomes of strain OS7 supported this hypothesis. The identification of the MIC genes enables the development of molecular tools to monitor epidemiology, and to perform surveillance and risk assessment of MIC-inducing M. maripaludis.

Keratin 13 gene is epigenetically suppressed during transforming growth factor-ß1-induced epithelial-mesenchymal transition in a human keratinocyte cell line.

Epithelial-mesenchymal transition (EMT) is a biological event in which epithelial cells lose their polarity and cell-cell adhesions and concomitantly acquire mesenchymal traits, and is thought to play an important role in pathological processes such as wound healing and cancer progression. In this study, we evaluated transforming growth factor (TGF)-ß1-treated human keratinocyte HaCaT cells as an in vitro model of EMT. HaCaT cells were changed into an elongated fibroblast-like morphology, which is indicative of EMT in response to TGF-ß1. Phalloidin staining demonstrated the formation of actin stress fibers in TGF-ß1-treated cells. Quantitative RT-PCR analysis revealed that TGF-ß1 increased the mRNA levels of EMT transcription factors (SNAI2, TWIST1, and ZEB1) and mesenchymal markers (CDH2, VIM, and FN1), while it decreased the transcripts of epithelial phenotypic genes (CLDN1, OCLN, KRT5, KRT15, KRT13, and TGM1). Furthermore, we found that KRT13 was drastically suppressed through the reduction of RNA polymerase II occupancy of its promoter, which was accompanied by a decrease in active histone marks (H3K4me3 and H3K27ac) and an increase in a repressive mark (H3K27me3) during EMT. These findings indicate that the TGF-ß1-induced EMT program regulates a subset of epithelial and mesenchymal marker genes, and that KRT13 is transcriptionally suppressed through the modulation of the chromatin state at the KRT13 promoter in HaCaT cells.

[Functional Expression of a Ca(2+)-activated Cl(-) Channel Modulator Involved in Ion Transport and Epithelial Cell Differentiation].

Cl(-)-permeable channels and transporters expressed on the cell membranes of various mammalian cell types play pivotal roles in the transport of electrolytes and water, pH regulation, cell volume and membrane excitability, and are therefore expected to be useful molecular targets for drug discovery. Both TMEM16A (a possible candidate for Ca(2+)-regulated Cl(-) channels recently identified) and cystic fibrosis transmembrane conductance regulator (CFTR) (or cAMP-regulated Cl(-) channels) have been known to be involved in Cl(-) secretion and reabsorption in the rat salivary gland. Crosstalk between two types of regulatory pathways through these two types of channels has also been described. Previously, we demonstrated that CLCA, a Ca(2+)-activated Cl(-) channel modulator, was involved in Cl(-) absorption in rat salivary ducts. In addition to Ca(2+), basal NF-κB activity in a mouse keratinocyte line was shown to be involved in the transcriptional regulation of CLCA. Conversely, a truncated isoform of CLCA was found in undifferentiated epithelial cells present in the rat epidermal basal layers. Under regulation by Ca(2+) and PKC, the surface expression of ß1-integrin and cell adhesion were decreased in the CLCA-overexpressing cells. Knockdown of this isoform elevated the expression of ß1-integrin in rat epidermis in vivo. These results indicate that the specific differentiation-dependent localization of CLCA, and transcriptional regulation through Ca(2+), are likely to affect ion permeability and the adhesive potential of epithelial cells. In summary, these types of Cl(-) channels and their modulators may function in a coordinated manner in regulating the functions of epithelial cells under different physiological conditions.

Idiopathic collapsing focal segmental glomerulosclerosis in an 81-year-old Japanese woman: a case report and review of the literature.

Focal segmental glomerulosclerosis (FSGS) is classified into five variants, with the collapsing variant being the most rare. Collapsing FSGS is characterized by a black racial predominance and is often associated with human immunodeficiency virus-associated nephropathy. However, the number of idiopathic cases is increasing and the presentation of non-black patients becoming more routine. Our analysis of 15 previous reports investigating FSGS variants shows that the collapsing variant accounts for 10.6 % of FSGS cases and its average age of onset is 32 years old. The current case is one of the oldest cases of idiopathic collapsing FSGS identified, being an 81-year-old Japanese woman. She presented with severe renal insufficiency (serum creatinine 7.9 mg/dL, albumin 1.5 g/dL) and so underwent hemodialysis immediately. Urinalysis demonstrated 3+ proteinuria and 3+ hematuria and the serological work up was all negative. Renal biopsy showed wrinkling of capillary walls with collapse lumens in every glomerulus, without endothelial tubuloreticular inclusions. Combined treatment with steroids, cyclosporine and low-density lipoprotein apheresis increased urine output slightly but she was unable to withdraw from hemodialysis and died 3 months later. This variant is reported to have the highest rate of progression to end-stage renal disease, regardless of the therapeutic intervention. However, there are also examples of cases with partial or complete remission in the literature. Progressive cases, like the current case, seem to be difficult to induce remission in, so it is important to diagnose idiopathic collapsing FSGS at an early stage by performing a renal biopsy, even in elderly patients.

3-Deazaneplanocin A suppresses aggressive phenotype-related gene expression in an oral squamous cell carcinoma cell line.

In tumor tissues, alterations of gene expression caused by aberrant epigenetic modifications confer phenotypic diversity on malignant cells. Although 3-deazaneplanocin A (DZNep) has been shown to reactivate tumor suppressor genes in several cancer cells, it remains unclear whether DZNep attenuates the malignant phenotypes of oral squamous cell carcinoma (OSCC) cells. In this study, we investigated the effect of DZNep on the expression of genes related to aggressive phenotypes, such as epithelial-mesenchymal transition, in OSCC cells. We found that DZNep reduced the cellular levels of polycomb group proteins (EZH2, SUZ12, BMI1, and RING1A) and the associated trimethylation of Lys27 on histone H3 and monoubiquitination of Lys119 on histone H2A in the poorly differentiated OSCC cell line SAS. Immunocytochemical staining demonstrated that DZNep induced the reorganization of filamentous actin and the membrane localization of E-cadherin associated with cell-cell adhesions. We also found an inhibitory effect of DZNep on cell proliferation using a WST assay. Finally, quantitative RT-PCR analysis demonstrated that genes involved in the aggressive phenotypes (TWIST2, EGFR, ACTA2, TGFB1, WNT5B, and APLIN) were down-regulated, whereas epithelial phenotype genes (CDH1, CLDN4, IVL, and TGM1) were up-regulated in SAS cells treated with DZNep. Collectively, our findings suggest that DZNep reverses the aggressive characteristics of OSCC cells through the dynamic regulation of epithelial plasticity via the reprogramming of gene expression patterns.

Estimated molecular structure of a carbon nanotube molecular heater based on binding properties to a target protein.

BACKGROUND: Carbon nanotubes exhibit strong absorbance in the near-infrared (NIR) region and are considered as potent candidates for hyperthermic therapy because they generate significant amounts of heat upon excitation with NIR light. We prepared a single-walled carbon nanotube (SWNT)/IgG complex to use as a "smart molecular heater" for hyperthermic therapy. PURPOSE: The aim of the present study was to assess the binding efficiency of DNA-functionalized SWNT/IgG complexes to a target protein. METHODS: 3 types of complexes with different lengths of spacer arm chain (13.5, 29, and 56 Å) linked to biotinylated IgG were prepared, and we evaluated the effect of the spacer arm length on the specificity, affinity, and capacity of binding to a target protein. RESULTS: Complexes with longer spacer lengths showed increased binding affinity to a target protein. This could be due to a reduction in steric hindrance by increasing the segmental flexibility of the spacer arm. CONCLUSIONS: The results of this study suggested that DNA-functionalized SWNT/IgG complexes could act as a heating nano-device for hyperthermic cancer therapy, and the complexes can bind various types of tumor by modifiying the specific antibody.

NF-κB-regulated transcriptional control of CLCA in a differentiated mouse keratinocyte line.

BACKGROUND: CLCA was postulated to be a calcium-activated chloride channel accessory protein. Recent reports indicate that CLCA isoforms are likely to be expressed in different layers of the stratified epithelium of the skin. OBJECTIVE: The present study investigated the transcriptional mechanism by which murine CLCA2 (mCLCA2) is expressed in the transformed keratinocyte line Pam212 that can differentiate. METHODS: A luciferase reporter assay, chromatin immunoprecipitation (ChIP) assay, reverse transcription-PCR, and immunocytochemistry were performed using Pam212 cells. RESULTS: Promoter activity of mCLCA2 was inhibited profoundly by site-directed mutagenesis of a putative nuclear factor-κB (NF-κB) binding site and by treatment with siRNA against p65. ChIP and transcription factor assays showed the specific association of endogenously activated p65 protein with the NF-κB binding domain. As confirmed by the nuclear translocation of p65, tumor necrosis factor α and caffeic acid phenethyl ester (CAPE) increased and decreased mCLCA2 promoter activity, respectively, but exhibited modest effects on endogenous mCLCA2 expression in cells in culture medium containing 0.05 mM Ca(2+). When the Ca(2+) concentration was raised to 1.0mM, the mRNA and protein levels of mCLCA2 increased as well as those of the differentiation markers keratin 1 (K1) and K10. CAPE profoundly suppressed only the Ca(2+)-triggered expression of mCLCA2, not K1 or K10. Immunohistochemistry of native skin and organotypic 3D cultures confirmed the distribution of the CLCA2 homolog in differentiated cells. CONCLUSION: The present study revealed for the first time that basal NF-κB activity is involved in the Ca(2+)-dependent regulation of mCLCA2 expression in a mouse keratinocyte line.

Integrin α6ß4 and TRPV1 channel coordinately regulate directional keratinocyte migration.

The directional migration of epithelial cells is crucial for wound healing. Among integrins, a family of cell adhesion receptors, integrin ß4 has been assumed to be a promigratory factor, in addition to its role in stable adhesion. In turn, Ca(2+) signaling is also a key coordinator of migration. Keratinocytes reportedly express transient receptor potential vanilloid channels (TRPV1); however, the function of these channels as a regulator of intracellular Ca(2+) level in cell migration has remained uncharacterized. In the present study, we investigated the role of TRPV1 in directional migration related to integrin ß4 using a scratch wound assay on a confluent monolayer sheet of murine keratinocytes (Pam212 cells). Double immunofluorescence staining revealed the de novo expression of integrin ß4 and TRPV1 in migrating cells at the wound edge in response to scratch wounding, and both expression levels were almost matched. Epidermal growth factor (EGF) not only promoted keratinocyte migration, but also caused the further up-regulation of both integrin ß4 and TRPV1. In addition, the knockdown of the integrin ß4 or TRPV1 gene significantly impeded wound closure. The TRPV1 agonist capsaicin significantly promoted migration, while a selective TRPV1 antagonist inhibited it. The gene knockdown of TRPV1 inhibited the expression of the integrin ß4 gene and that of ß4 protein in migrating cells. These findings suggest that TRPV1 may stimulate directional migration directly by eliciting a Ca(2+) signal or indirectly via integrin ß4 expression.

Epigenetic alterations of the keratin 13 gene in oral squamous cell carcinoma.

BACKGROUND: Epigenetic modifications play important roles in the regulation of gene expression determining cellular phenotype as well as various pathologies such as cancer. Although the loss of keratin 13 (KRT13) is reportedly linked to malignant transformation of oral epithelial cells, the molecular mechanisms through which KRT13 is repressed in oral squamous cell carcinoma (OSCC) remain unclear. The aim of this study is to identify the epigenetic alterations of the KRT13 gene in OSCCs. METHODS: We investigated KRT13 expression levels and chromatin modifications of the KRT13 promoter in the three OSCC cell lines (HSC4, HSC3, and SAS). The expression levels of KRT13 protein and mRNA were analyzed by western blotting and quantitative reverse-transcription polymerase chain reaction, respectively, and the localization of KRT13 protein was detected by immunofluorescence. DNA methylation and histone modifications in the KRT13 promoter were determined by bisulfite sequencing and chromatin immunoprecipitation (ChIP), respectively. For the pharmacological depletion of Polycomb repressive complex 2 (PRC2), cells were treated with 3-deazaneplanocin A (DZNep). RESULTS: KRT13 expression was transcriptionally silenced in the HSC3 and SAS cells and post-transcriptionally repressed in the HSC4 cells, while the KRT13 promoter was hypermethylated in all of the three OSCC cell lines. ChIP analysis revealed that PRC2-mediated trimethylation of Lys 27 on histone H3 (H3K27me3) was increased in the KRT13 promoter in the HSC3 and SAS cells. Finally, we demonstrated that the treatment of SAS cells with DZNep reactivated the transcription of KRT13 gene. CONCLUSIONS: Our data provide mechanistic insights into the epigenetic silencing of KRT13 genes in OSCC cells and might be useful for the development of diagnostic markers and novel therapeutic approaches against OSCCs.

Correlation between number of renal cysts and aortic circumferences measured using autopsy material.

Although the presence of renal cysts has been reported to be associated with aortic aneurysm or dissection by imaging studies, an autopsy study has not been performed. Therefore, in our institute, recent consecutive adult autopsy cases (n=108, 64 males and 44 females) were reviewed. The circumferences and atherosclerosis ratios of both thoracic and abdominal aorta were individually measured and graded. The number of renal cysts was scored and graded. Age of subjects along with histories of smoking, hypertension, and diabetes mellitus were confirmed. Multiple linear regression analyses demonstrated that severity of atherosclerosis and the number of renal cysts were correlated with thoracic aortic circumference, while only the number of renal cysts was correlated with abdominal aortic circumference (p<0.05), which was more predominant in female subjects (p<0.05). Microscopically, significantly more dilated renal tubules (by Student's t-test, p<0.05) along with decreased stainability of basement membrane by Periodic acid-Schiff staining and immunostaining of type IV collagen were noted in background renal tissues in cases with numerous renal cysts than in age- and sex-matched controls without renal cysts (n=10 vs. 10). The present study suggests that a syndrome that affects both aorta and renal tubules may exist.

CLCA splicing isoform associated with adhesion through ß1-integrin and its scaffolding protein: specific expression in undifferentiated epithelial cells.

We previously found that a rat CLCA homologue (rCLCA-f) modulates Ca(2+)-dependent Cl(-) transport in the ductal cells of the rat submandibular gland. CLCA proteins have been shown to be multifunctional, with roles in, for example, cell adhesion. Here, we describe the mRNA and protein expressions of a splicing isoform of rat rCLCA (rCLCA-t). This isoform is a 514-amino acid protein containing a C-terminal 59-amino acid that is distinct from the rCLCA-f sequence. Immunohistochemistry revealed rCLCA-t to be located in the basal cells of the rat submandibular gland excretory duct and the stratum basale of rat epidermis, whereas rCLCA-f was detected in cells during the process of differentiation. In a heterologous expression system, rCLCA-t was found to be a membrane protein present predominantly in the perinuclear region, and not to be either present on the cell surface or secreted. rCLCA-t failed to enhance ionomycin-induced Cl(-) conductance (unlike rCLCA-f). When compared with rCLCA-f, it weakened cell attachment to a greater extent and in a manner that was evidently modulated by intracellular Ca(2+), protein kinase C, and ß(1)-integrin. rCLCA-t was found to associate with RACK1 (receptor for activated C kinase) and to reduce expression of mature ß(1)-integrin. Treatment of rat skin with rCLCA-t siRNA increased the expression of ß(1)-integrin in the stratum basale of the epidermis. These results are consistent with cell-specific splicing of rCLCA mRNA playing a role in the modulation of the adhesive potential of undifferentiated epithelial cells.

Preparation and binding study of a complex made of DNA-treated single-walled carbon nanotubes and antibody for specific delivery of a "molecular heater" platform.

Carbon nanotubes have been explored as heat-delivery vehicles for thermal ablation of tumors. To use single-walled carbon nanotubes (SWNT) as a "molecular heater" for hyperthermia therapy in cancer, stable dispersibility and smart-delivery potential will be needed, as well as lack of toxicity. This paper reports the preparation of a model complex comprising DNA-treated SWNT and anti-human IgG antibody and the specific binding ability of this model complex with the targeted protein, ie, human IgG. Treatment with double-stranded DNA enabled stable dispersibility of a complex composed of SWNT and the antibody under physiological conditions. Quartz crystal microbalance results suggest that there was one immobilized IgG molecule to every 21,700 carbon atoms in the complex containing DNA-treated SWNT and the antibody. The DNA-SWNT antibody complex showed good selectivity for binding to the targeted protein. Binding analysis revealed that treatment with DNA did not interfere with binding affinity or capacity between the immobilized antibody and the targeted protein. The results of this study demonstrate that the DNA-SWNT antibody complex is a useful tool for use as a smart "molecular heater" platform applicable to various types of antibodies targeting a specific antigen.

T-type voltage-activated calcium channel Cav3.1, but not Cav3.2, is involved in the inhibition of proliferation and apoptosis in MCF-7 human breast cancer cells.

T-type voltage-gated Ca2+ channels have unique electrophysiological properties, suitable for generating Ca2+ oscillations and waves and thus controlling the proliferation of various tumor cells. In the present study, we investigated the role of Cav3.1, a candidate tumor suppressor gene, in neoplastic processes, and compared the differences between Cav3.1 with Cav3.2 channels. While the overexpression of a full-length Cav3.1 clone suppressed cell proliferation, the knockdown of the Cav3.1 gene by siRNA, or treatment with ProTx-I, a relatively selective inhibitor for Cav3.1, promoted the cell proliferation of MCF-7 cells (a human breast adenocarcinoma cell line). Although Cav3.1 and Cav3.2 channels possess comparable biophysical properties and are often co-expressed in various tissues, gene knockdown or the overexpression of Cav3.2 channels exhibited no effect on cell proliferation. Using immunocytochemical co-staining, the Cav3.1 channels were specifically visualized in the plasma membranes of apoptotic cells, identified by Annexin V and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assays and nuclear condensation. On the contrary, Cav3.2 channels were expressed at the membrane of large portions of cells, with no likely relation to Cav3.1 expression or apoptosis. An apoptosis assay revealed that the overexpression of the Cav3.1 clone caused an increase in the number of apoptotic cells. Furthermore, Cav3.1 knockdown blocked cyclophosphamide-induced apoptosis. These results suggest that Cav3.1 channels may contribute to the repression of tumor proliferation and the promotion of apoptosis mediated via Cav3.1-specific Ca2+ influx.

Blockade of TRPM8 activity reduces the invasion potential of oral squamous carcinoma cell lines.

Several members of the transient receptor potential (TRP)-channel family are expressed in cancer cells. One, cold/menthol-sensitive TRPM8, is reportedly an important player in carcinogenesis in human prostate cancer, although its involvement in oral squamous cell carcinoma (SCC) remains unclear. The present immunohistochemistry and RT-PCR results revealed intense TRPM8 expression in two SCC cell lines, HSC3 and HSC4, derived from the human tongue. Menthol, icilin, and a more specific TRPM8 agonist (WS-12) induced non-specific cation currents, with Ca2+ permeability being greater than that of Na+ or K+. The novel TRPM8 antagonist RQ-00203078 (RQ) profoundly reduced such agonist-induced cation currents. Intracellular Ca2+ imaging revealed that menthol induced both intracellular Ca2+ release and store-operated Ca2+ entry, with RQ inhibiting each effect. To assess the possible pathophysiological role of TRPM8 in oral SCC, we performed motility and invasion assays, and gelatin zymography. Menthol augmented the migration and invasion abilities of both HSC3 and HSC4 cells by potentiating MMP-9 activity. RQ suppressed all of these effects. These results may aid understanding of the pathophysiological implications of TRPM8 channels in the oral SCC cells, support TRP proteins as valuable targets for pharmaceutical intervention, and inform the targeting of oral SCC in which the prognosis is poor.

Dual signaling pathways of arterial constriction by extracellular uridine 5'-triphosphate in the rat.

We investigated actions of uridine 5'-triphosphate (UTP) in rat aorta, cerebral and mesenteric arteries, and their single myocytes. UTP (≥10 µM) elicited an inward-rectifying current strongly reminiscent of activation of P2X(1) receptor, and a similar current was also induced by α,ß-methylene adenosine 5'-triphosphate (ATP) (≥100 nM). UTP desensitized α,ß-methylene ATP-evoked current, and vice versa. The UTP-activated current was insensitive to G-protein modulators, TRPC3 inhibitors, or TRPC3 antibody, but was sensitive to P2-receptor inhibitors or P2X(1)-receptor antibody. Both UTP (1 mM) and α,ß-methylene ATP (10 µM) elicited similar conductance single channel activities. UTP (≥10 µM) provoked a dose-dependent contraction of de-endothelialized aortic ring preparation consisting of phasic and tonic components. Removal of extracellular Ca(2+) or bath-applied 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) (30 µM) or nifedipine (10 µM) completely inhibited the phasic contraction while only partially reducing the tonic one. The tonic contraction was almost completely abolished by additional application of thapsigargin (2 µM). Similar biphasic rises in [Ca(2+)](i) were also evoked by UTP in rat aortic myocytes. In contrast to the low expression of TRPC3, significant expression of P2X(1) receptor was detected in all arteries by RT-PCR and immunoblotting, and its localization was limited to plasma membrane of myocytes as indicated by immunohistochemistry. These results suggest that UTP dually activates P2X(1)-like and P2Y receptors, but not TRPC3.

Involvement of apical P2Y2 receptor-regulated CFTR activity in muscarinic stimulation of Cl(-) reabsorption in rat submandibular gland.

Previously, we presented in vivo evidence for a physiological significance of cAMP-regulated CFTR Cl(-) channels in Ca(2+)-activated Cl(-) reabsorption in the ductal system of the rat submandibular gland. Here, we address the mechanism by which basal CFTR activation contributes to the transepithelial Cl(-) movement evoked by muscarinic stimulation. The Cl(-) concentration ([Cl(-)]) was increased in the final saliva from rat submandibular gland during pilocarpine stimulation when a small interfering RNA for CFTR or a specific CFTR inhibitor, CFTR(inh)-172, was injected retrogradely into the gland's own duct, indicating that basal CFTR activation is involved in Cl(-) reabsorption. Systemically administered propranolol failed to alter the [Cl(-)], suggesting little involvement of a beta-adrenergic pathway in the Cl(-) movement that occurs through basal CFTR activation. Intraductal injection of suramin (a nonspecific P2-receptor antagonist) increased the salivary [Cl(-)], indicating the existence of endogenous purinergic activation. Upon separate intraductal injection, ATP and a P2Y(2)-receptor agonist, UTP, decreased the salivary [Cl(-)] almost equipotently. CFTR(inh)-172 and suramin each prevented these effects, whereas 2',3'-O-(4-benzoylbenzoyl)-ATP (Bz-ATP), a P2X(7) agonist, had no specific effect. Pilocarpine stimulation evoked ATP secretion into the salivary fluid. Immunohistochemistry revealed the partial coexistence of CFTR and P2Y(2) receptors on the luminal surface of epithelial cells in the striated ducts of this gland. These results raise the possibility that muscarinic stimulation-induced Cl(-) reabsorption occurs through basal CFTR activity and that this is regulated by P2Y(2) receptors in the ductal epithelium via stimulation by ATP secreted into the salivary fluid.

Human spermatozoa capacitated with progesterone or a long incubation show accelerated internalization by an alkyl ether lysophospholipid.

OBJECTIVE: To evaluate changes that occur in sperm plasma membranes during capacitation, the internalization of [(3)H]lyso-platelet activating factor ([(3)H]lyso-PAF) across the plasma membrane of human spermatozoa was measured as a function of incubation time or exposure to progesterone (P). DESIGN: In vitro cell culture study using human spermatozoa. SETTING: Department of Obstetrics and Gynecology, School of Medicine, the University of Tokushima, Japan. PATIENT(S): Semen were obtained from three fertile healthy volunteers. INTERVENTION(S): The internalization of [(3)H]lyso-PAF across the plasma membranes of human spermatozoa that were incubated for an extended period or exposed to P was measured at 5, 20, 60, and 120 minutes after the addition of the phospholipid probe using the modified albumin back-exchange method. MAIN OUTCOME MEASURE(S): The percentage of capacitated and acrosome-reacted sperm and the proportion of internalization of lyso-PAF across the plasma membrane. RESULT(S): A 6-hour incubation period significantly increased the percentage of capacitated spermatozoa and the proportion of internalization of [(3)H]lyso-PAF across the plasma membrane of human spermatozoa compared with controls (capacitated spermatozoa, 20.3 +/- 10.6% vs. 8.5 +/- 1.8%; internalization 120 minutes after the addition of the phospholipid probe, 25.6 +/- 2.5% vs. 11.6 +/- 3.0%) (mean +/- SEM). Exposure to P significantly increased the percentage of capacitated spermatozoa compared with controls (19.6 +/- 6.8% vs. 11.0 +/- 2.4%) and also significantly accelerated the internalization of [(3)H]lyso-PAF compared with controls (internalization 120 minutes after the addition of the phospholipid probe, 26.2 +/- 1.8% vs. 21.4 +/- 1.1%). CONCLUSION(S): The administration of P or a long incubation increased the proportion of internalization and consequently induced capacitation in human spermatozoa.

Regulation of RecA protein binding to DNA by opposing effects of ATP and ADP on inter-domain contacts: analysis by urea-induced unfolding of wild-type and C-terminal truncated RecA.

RecA protein requires ATP and its hydrolysis to ADP to complete the DNA strand-exchange reaction. We investigated how the nucleotides activate RecA by examining their effect on urea-induced unfolding, which could reflect domain-domain contact of protein. RecA is folded into three continuous domains: the N-terminal, central and C-terminal domains. The fluorescence of tyrosine residues, which lie mainly in the central domain, was modified in 1-3 M urea, while the red shift of fluorescence peak of the tryptophan residues located in the C-terminal domain occurred only in 3-6 M urea. Thus, the C-terminal domain of RecA is unfolded after the central part unfolds. The change in intensity of tryptophan fluorescence without a large shift in the peak at low concentrations of urea suggests that there are weak interactions between the central and C-terminal domains. This is supported by our observation that RecA protein lacking the C-terminal tail unfolded at lower concentrations of urea than the entire RecA, and with clear transitions, unlike the entire RecA. ATP and its unhydrolyzable analog (ATPgammaS), which enhance the binding of RecA to DNA, facilitated the urea-induced change in RecA tryptophan fluorescence, while ADP, an antagonist of ATP, prevented the change. ATP probably weakens the domain-domain contact and facilitates the DNA binding, while ADP stabilizes the contact and inhibits it. Supporting this conclusion, the binding of RecA lacking the C-terminal tail to DNA was not inhibited by ADP, while that of the intact RecA was.