Detection of antibodies against Fasciola hepatica in cirrhotic patients from Peru.

The prevalence of Fasciola hepatica infection, in endemic countries, in patients with established cirrhosis is unknown. We hypothesized that, in endemic countries, the presence of fascioliasis may be detected in a serum pool of cirrhotic patients. Forty-four previously stored serum samples of patients with established liver cirrhosis, in the Hospital Nacional Cayetano Heredia in Lima, Peru, were collected from 1998 to 2003 and assessed for hepatitis B, C and fascioliasis antibodies (Fas2 ELISA). Hepatitis B surface antigen (HBsAg) was positive in 8.8% (n = 34), hepatitis B core antibody (anti-HBc) in 32.5% (n = 34), hepatitis C antibodies (anti-HCV) in 9.1% (n = 33), and 9.1% (n = 44) were Fas2 ELISA positive. This disease is an example of an emerging tropical infection which can be present in chronic liver diseases, requiring greater clinician awareness especially in endemic rural areas. Further clinical studies are warranted.

Precision measurement of neutrino oscillation parameters with KamLAND.

The KamLAND experiment has determined a precise value for the neutrino oscillation parameter Deltam21(2) and stringent constraints on theta12. The exposure to nuclear reactor antineutrinos is increased almost fourfold over previous results to 2.44 x 10(32) proton yr due to longer livetime and an enlarged fiducial volume. An undistorted reactor nu[over]e energy spectrum is now rejected at >5sigma. Analysis of the reactor spectrum above the inverse beta decay energy threshold, and including geoneutrinos, gives a best fit at Deltam21(2)=7.58(-0.13)(+0.14)(stat) -0.15+0.15(syst) x 10(-5) eV2 and tan2theta12=0.56(-0.07)+0.10(stat) -0.06+0.10(syst). Local Deltachi2 minima at higher and lower Deltam21(2) are disfavored at >4sigma. Combining with solar neutrino data, we obtain Deltam21(2)=7.59(-0.21)+0.21 x 10(-5) eV2 and tan2theta12=0.47(-0.05)+0.06.

La infección por fasciola hepática en el Perú: una enfermedad emergente / The infection Fasciola Liver in Peru: an emerging disease

Objetivos: El objetivo del presente estudio es reportar el número de casos humanos con la infección por fasciola hepática en el Perú desde 1963 al 2005. Métodos: Se realizó una búsqueda electrónica en las bases de datos bibliográficas de MEDLINE, LILACS, en bibliotecas de las Facultades de Medicina, Veterinaria y Zootecnia, Filosofía y Ciencias de las principales universidades e institutos del Perú. Se incluyeron referencias en revistas nacionales e internacionales que reporten casos peruanos. Resultados: Un total de 1701 personas (1-71 años) infectadas fueron reportadas en el Perú entre 1963 y 2005. El género femenino fue significativamente más frecuente que el masculino. Del total de casos, 191 eran casos agudos (11por ciento); 1313 en fase crónica (77.1 por ciento); y 167, crónicos asintomáticos (9.8 por ciento). Los casos infectados procedían de 17 departamentos del Perú lo cual representa 71 por ciento (n=24) del territorio nacional. El número de sujetos infectados se presentan por décadas apreciándose un paulatino aumento alcanzando a 54.1 casos por año en la última década analizada. Conclusiones: Debido al significativo incremento de casos reportados en las últimas 4 décadas, la fasciolosis humana es una enfermedad infecciosa parasitaria emergente en el Perú y urgen programas de prevención y control para esta zoonosis.

Objetives: The study is a recompilation of the reported human cases of Fasciola hepatica infection in Peru since 1963 to 2005. Methods: We review the electronic documentation of bibliographic resources in MEDLINE, LILACS, libraries of the medical, veterinary, philosophy and sciences faculties of the main universities and scientific institutions from Peru. We include all the references from national and international journals who report Peruvian cases of fasciolosis. Results: 1701 subjects in total were report in Peru between 1963 and 2005. The range of the age of the reported cases goes from 1 year to 71 years. Females were significative more common than males. 191cases were acute (11 per cent); 1313 chronic (77.1 per cent); y 167, chronic asymptomatic (9.8 per cent). The reported cases came from 17/24 departments of Peru that represent 71 per cent of the; Peruvian territory. The number the reported cases are increasing during the last decade to reach 54.1 cases in the last decade.Conclusions. Due to the significative increase of reported cases in the last 4 decades, human fasciolosis is an emergent parasitary infective disease in humans in Peru and we need preventive and control national health programs for this zoonosis.

[Fasciola hepatica infection in Peru: an emergent disease]. / La infección por Fasciola hepatica en el Perú: una enfermedad emergente.

OBJECTIVE: [corrected] The study is a recompilation of the reported human cases of Fasciola hepatica infection in Peru since 1963 to 2005. METHODS: We review the electronic documentation of bibliographic resources in MEDLINE, LILACS, libraries of the medical, veterinary, philosophy and sciences faculties of the main universities and scientific institutions from Peru. We include all the references from national and international journals who report Peruvian cases of fasciolosis. RESULTS: 1701 subjects in total were report in Peru between 1963 and 2005. The range of the age of the reported cases goes from 1 year to 71 years. Females were significative more common than males. 191cases were acute (11%); 1313 chronic (77.1%); y 167, chronic asymptomatic (9.8%). The reported cases came from 17/24 departments of Peru that represent 71% of the; Peruvian territory. The number the reported cases are increasing during the last decade to reach 54.1 cases in the last decade. CONCLUSIONS. Due to the significative increase of reported cases in the last 4 decades, human fasciolosis is an emergent parasitary infective disease in humans in Peru and we need preventive and control national health programs for this zoonosis.

Effect of asymmetric modification on the conformation of ascidiacyclamide analogs.

Ascidiacyclamide (ASC), cyclo(-Ile1-Oxz2-d-Val3-Thz4-)2 (Oxz=oxazoline and Thz=thiazole) has a C2-symmetric sequence, and the relationships between its conformation and symmetry have been studied. In a previous study, we performed asymmetric modifications in which an Ile residue was replaced by Gly, Leu or Phe to disturb the symmetry [Doi et al. (1999) Biopolymers49, 459-469]. In this study, the modifications were extended. The Ile1 residue was replaced by Gly, Ala, aminoisobutyric acid (Aib), Val, Leu, Phe or d-Ile, and the d-Val3 residue was replaced by Val. The structures of these analogs were analyzed by X-ray diffraction, 1H NMR and CD techniques. X-Ray diffraction analyses revealed that the [Ala1], [Aib1] and [Phe1]ASC analogs are folded, whereas [Val1]ASC has a square form. These structures are the first examples of folded structures for ASC analogs in the crystal state and are similar to the previously reported structures of [Gly1] and [Phe1]ASC in solution. The resonances of amide NH and Thz CH protons linearly shift with temperature changes; in particular, those of [Aib1], [d-Ile1] and [Val3]ASCs exhibited a large temperature dependence. DMSO titration caused nonlinear shifts of proton resonances for all analogs and largely affected [d-Ile1] and [Val3]ASCs. A similar tendency was observed upon the addition of acetone to peptide solutions. Regarding peptide concentration changes, amide NH and Thz CH protons of [Gly1]ASC showed a relatively large dependence. CD spectra of these analogs indicated approximately two patterns in MeCN solution, which were related to the crystal structures. However, all spectra showed a similar positive Cotton effect in TFE solution, except that of [Val3]ASC. In the cytotoxicity test using P388 cells, [Val1]ASC exhibited the strongest activity, whereas the epimers of ASC ([d-Ile1] and [Val3]ASCs), showed fairly moderate activities.

Microglial signaling by amyloid beta protein through mitogen-activated protein kinase mediating phosphorylation of MARCKS.

Myristoylated alanine-rich C kinase substrate (MARCKS), an acidic protein associated with cell motility and phagocytosis, is activated upon phosphorylation by protein kinase C (PKC) and proline-directed protein kinases. In Alzheimer disease (AD), activated microglia expressing MARCKS migrates around senile plaques. We reported that amyloid beta protein (A beta), a major component of senile plaques, activated MARCKS through a tyrosine kinase and PKC-delta. We have now identified another A beta signaling pathway through a mitogen-activated protein kinase (MAPK) involved in the phosphorylation of MARCKS and analysed cross-talk between PKC and MAPK pathways in primary cultured rat microglia. A selective inhibitor for MAPK kinase, PD098059, significantly inhibited the phosphorylation of MARCKS induced by A beta. Extracellulary regulated kinases, the activities of which were induced by A beta, directly phosphorylated a recombinant MARCKS in vitro. The MAPK pathway was sensitive to wortmannin, but not to a PKC inhibitor or to tyrosine kinase inhibitors. The activation of PKC by A beta was not sensitive to wortmannin. Our findings suggest involvement of the MAPK pathway through phosphoinositol 3-kinase in the phosphorylation of MARCKS in rat cultured microglia, an event may be associated with mechanisms activating microglia in AD.

Amyloid beta protein activates PKC-delta and induces translocation of myristoylated alanine-rich C kinase substrate (MARCKS) in microglia.

The increased accumulation of activated microglia containing amyloid beta protein (Abeta) around senile plaques is a common pathological feature in subjects with Alzheimer's disease (AD). Much less is known, however, of intracellular signal transduction pathways for microglial activation in response to Abeta. We investigated intracellular signaling in response to Abeta stimulation in primary cultured rat microglia. We found that the kinase activity of PKC-delta but not that of PKC-alpha or -epsilon is increased by stimulation of microglia with Abeta, with a striking tyrosine phosphorylation of PKC-delta. In microglia stimulated with Abeta, tyrosine phosphorylation of PKC-delta was evident at the membrane fraction without an overt translocation of PKC-delta. PKC-delta co-immunoprecipitated with MARCKS from microglia stimulated with Abeta. Abeta induced translocation of MARCKS from the membrane fraction to the cytosolic fraction. Immunocytochemical analysis revealed that phosphorylated MARCKS accumulated in the cytoplasm, particularly at the perinuclear region in microglia treated with Abeta. Taken together with our previous observations that Abeta-induced phosphorylation of MARCKS and chemotaxis of microglia are inhibited by either tyrosine kinase or PKC inhibitors, our results provide evidence that Abeta induces phosphorylation and translocation of MARCKS through the tyrosine kinase-PKC-delta signaling pathway in microglia.

Caged and clustered structures of endothelin inhibitor BQ123, cyclo(-D-Trp-D-Asp--Pro-D-Val-Leu-)-Na+, forming five and six coordination bonds between sodium ions and peptides.

BQ123 is a cyclic pentapeptide and a potent endothelin-1 inhibitor. The crystal structure of the BQ123 sodium salt was determined as the first example of an endothelin inhibitor. Four independent molecules and many solvent molecules were found in the asymmetric unit; the total weight was about 3000 Da. The precise structure including the solvent molecules was determined using high-resolution data collected on a synchrotron source. Sodium ions formed unique structures with five and six coordination bonds and their forms were distinguished into three classes. An ion was sandwiched by two BQ123 molecules. This peptide-sodium (2:1) complex showed a cage-like structure and octahedral coordination was observed. Sodium ions also formed a cluster composed of hydrated water molecules and peptides. Two sodium ions were contained in this cluster, making five coordination bonds. Despite having the same coordination numbers, these ions were distinguishable by differences in the polyhedra. One was trigonal bipyramidal (having six planes) and the other was square pyramidal (having five planes). Both shapes were very similar to each other, although the synchrotron data clearly revealed slight geometrical differences.

Effects of amino acids and chirality for molecular folding of desoxazoline-ascidiacyclamide derivatives: X-ray crystal structures of four cyclic octapeptides including unusual amino acids, cyclo(-Ile-aThr-D-Val-Thz-)(2), cyclo(-Ala-aThr-D-Val-Thz-Ile-aThr-D-Val-Thz-), cyclo(-Val-aThr-D-Val-Thz-Ile-aThr-D-Val-Thz-), and cyclo(-Ile-aThr-Val-Thz-Ile-aThr-D-Val-Thz-).

Desoxazoline derivative of ascidiacyclamide (1), cyclo(-L-Ile-L-allo-threonine-D-Val-thiazole-)(2), was modified to disturb the C(2)-symmetry. An Ile(1) residue of 1 was replaced for Ala (2) or Val (3), and the D-Val(3) residue was replaced for Val (4). The crystal structures of 1-4 were analyzed by x-ray diffraction methods. The molecules of all compounds were folded and this type of structure was not observed in x-ray structures of ascidiacyclamide derivatives so far except for patellamide D. The folding patterns of 1-4 were similar to each other and resembled that of patellamide. The asymmetric modifications at position 1 caused the conformational changes at local area, and these were related with the peptide-peptide and peptide-solvent interactions. Despite the diverse backbone conformation by the epimeric modification at position 3, the entire molecule of 4 was folded. These results mean that (1) the desoxazoline-ascidiacyclamides favored the folded structures and (2) the modifications of the side chain size at position 1 and the chirality at position 3 brought the local conformational changes to derivatives, suggesting that (3) the lack of the oxazoline block leads to conformational flexibility of 1-4, which accepts the conformational change with no drastic change on the entire structure.

Phosphorylation of tau is regulated by PKN.

For the phosphorylation state of microtubule-associated protein, tau plays a pivotal role in regulating microtubule networks in neurons. Tau promotes the assembly and stabilization of microtubules. The potential for tau to bind to microtubules is down-regulated after local phosphorylation. When we investigated the effects of PKN activation on tau phosphorylation, we found that PKN triggers disruption of the microtubule array both in vitro and in vivo and predominantly phosphorylates tau in microtubule binding domains (MBDs). PKN has a catalytic domain highly homologous to protein kinase C (PKC), a kinase that phosphorylates Ser-313 (= Ser-324, the number used in this study) in MBDs. Thus, we identified the phosphorylation sites of PKN and PKC subtypes (PKC-alpha, -betaI, -betaII, -gamma, -delta, -epsilon, -zeta, and -lambda) in MBDs. PKN phosphorylates Ser-258, Ser-320, and Ser-352, although all PKC subtypes phosphorylate Ser-258, Ser-293, Ser-324, and Ser-352. There is a PKN-specific phosphorylation site, Ser-320, in MBDs. HIA3, a novel phosphorylation-dependent antibody recognizing phosphorylated tau at Ser-320, showed immunoreactivity in Chinese hamster ovary cells expressing tau and the active form of PKN, but not in Chinese hamster ovary cells expressing tau and the inactive form of PKN. The immunoreactivity for phosphorylated tau at Ser-320 increased in the presence of a phosphatase inhibitor, FK506 treatment, which means that calcineurin (protein phosphatase 2B) may be involved in dephosphorylating tau at Ser-320 site. We also noted that PKN reduces the phosphorylation recognized by the phosphorylation-dependent antibodies AT8, AT180, and AT270 in vivo. Thus PKN serves as a regulator of microtubules by specific phosphorylation of tau, which leads to disruption of tubulin assembly.

Rapamycin and FK506 induce long-term potentiation by pairing stimulation via an intracellular Ca(2+) signaling mechanism in rat hippocampal CA1 neurons.

Immunophilin-CsA and -FK506 complexes bind to calcineurin (CaN) and inhibit its phosphatase activity leading to enhancement of neuronal activities. However, inhibition of CaN activity is not the mediator of modulatory activity for IP3 and ryanodine receptors and does not mediate the neurotrophic actions of FK506. FK506 binding protein (FKBP)-12 also binds rapamycin, another immunosuppressant which does not affect CaN activity. Using whole-cell patch clamp techniques, excitatory postsynaptic currents (EPSCs) were recorded and we analyzed the effect of immunosuppressants on the synaptic potentiation induced by pairing weak presynaptic stimulation with postsynaptic depolarization in CA1 neurons of rat hippocampal slices. We found that postsynaptic application of rapamycin or FK506, at low concentrations, but not cyclosporin A, in conjunction with weak pairing stimulation, induced NMDA-dependent long-term potentiation (LTP). The rapamycin-induced LTP was blocked by chelating intracellular Ca(2+) or by inhibiting the intracellular Ca(2+) release. Thus, Ca(2+) release from intracellular Ca(2+) stores is required for the induction of LTP by weak pairing stimulation in the presence of rapamycin or FK506 at postsynaptic sites. We propose that postsynaptic FKBP-12 regulates synaptic transmission by stabilizing the postsynaptic Ca(2+) signaling mechanism in rat hippocampal CA1 neurons.

Amphipathic structure of theonellapeptolide-Id, a hydrophobic tridecapeptide lactone from the Okinawa marine sponge Theonella swinhoei.

Theonellapeptolide-Id (TNLP), a cyclic tridecapeptide lactone, was crystallized from dimethylformamide-water solution. In the asymmetric unit, two peptide molecules were combined with solvent molecules, and the total molecular weight was over 3000 Dalton. The crystal structure including solvent molecules was finally determined at 0.80 A resolution using synchrotron radiation. The conformations of two independent molecules were similar to each other and were also similar to the previously reported structure (Doi, Ishida, Kobayashi, Deschamps and Flippen-Anderson, 1999, Acta Crystallogr Sect C, 55, 796-798). About 13 hydrated water molecules were found at disordered 19 sites; they were located at a certain region to avoid contact with aliphatic side-chains of peptolide in the crystal. The spatial disposition of the solvent molecules and peptides subsequently caused the formation of the amphipathic layer.

Strongyloides stercoralis: formas clínicas severas asociadas a infección por HTLV-1 / Strongyloides stercoralis: clinical severe forms associated to HTLV-1 infection

Se condujo un estudio para determinar la tasa de infección por HTLV-1 en pacientes con diversos cuadros clínicos de strongyloidiasis. El estudio incluyó 21 pacientes con hiper-infestación por Strongyloides stercoralis (St St) (Grupo 1) y un grupo apareados por edad y sexo, sanos con heces negativas (Grupo Control-2). Se incluyó un tercer grupo de "probable hiper-infestación" de 33 pacientes (Grupo 3) y se incluyó un grupo de 63 pacientes con strongyloidiasis intestinal sin evidencia de enfermedad sistémica (Grupo 4). Se analizó el suero de cada paciente con método ELISA para HTLV-I y II; a los casos positivos se confirmaron con Western Blot. La tasa de infección en hiperinfestación fue significativamente mayor 85.7 por ciento (18/21) en relación al grupo control 4.7 por ciento (1/21) p<0.001. Asimismo, el grupo de "probable hiper-infestación" fue significativamente más elevado 69.7 por ciento (23/33) que el control p<0.01. El grupo de strongyloidiasis intestinal fue de 10 por ciento (6/62) fue más baja que los casos de hiper-infestación (p<0.05) pero este grupo no fue diferente al grupo control (>0.005), 3/6 tuvieron HTLV-I positivo en este último grupo, en el seguimiento posterior hicieron formas de hiper-infestación. En conclusión, las formas severas de St St se asocian en forma significativa con infección por HTLV-I en adultos y en niños mayores de 5 años. No se detectó ningún caso de HTLV-II.

Amyloid beta protein (25-35) phosphorylates MARCKS through tyrosine kinase-activated protein kinase C signaling pathway in microglia.

Myristoylated alanine-rich C kinase substrate (MARCKS) is a widely distributed specific protein kinase C (PKC) substrate and has been implicated in membrane trafficking, cell motility, secretion, cell cycle, and transformation. We found that amyloid beta protein (A beta) (25-35) and A beta (1-40) phosphorylate MARCKS in primary cultured rat microglia. Treatment of microglia with A beta (25-35) at 10 nM or 12-O-tetradecanoylphorbol 13-acetate (1.6 nM) led to phosphorylation of MARCKS, an event inhibited by PKC inhibitors, staurosporine, calphostin C, and chelerythrine. The A beta (25-35)-induced phosphorylation of MARCKS was inhibited by pretreatment with the tyrosine kinase inhibitors genistein and herbimycin A, but not with pertussis toxin. PKC isoforms alpha, delta, and epsilon were identified in microglia by immunocytochemistry and western blots using isoform-specific antibodies. PKC-delta was tyrosine-phosphorylated by the treatment of microglia for 10 min with A beta (25-35) at 10 nM. Other PKC isoforms alpha and epsilon were tyrosine-phosphorylated by A beta (25-35), but only to a small extent. We propose that a tyrosine kinase-activated PKC pathway is involved in the A beta (25-35)-induced phosphorylation of MARCKS in rat microglia.

Strongyloides stercoralis hyperinfection associated with human T cell lymphotropic virus type-1 infection in Peru.

A study was conducted in Lima, Peru to determine if patients with Strongyloides hyperinfection had human T cell lymphotropic virus type-1 (HTLV-I) infection. The study included patients with Strongyloides hyperinfection and a control group consisted of sex- and age-matched asymptomatic healthy individuals whose stools were negative for Strongyloides. A third group included patients with intestinal strongyloidiasis. Sera from each study subject were tested for HTLV-1/2I by an ELISA and Western blot. The HLTV-1 infection rates (85.7%, 18 of 21) were significantly (P < 0.001) associated with Strongyloides hyperinfection compared with the control group (4.7%, 1 of 21). The HTLV-1 rate (10%, 6 of 62) for patients with intestinal strongyloidiasis was significantly (P < 0.001) lower than patients with Strongyloides hyperinfection, but did not differ significantly (P > 0.05) from the control group. The association of HTLV-1 infection was observed among 17 of 19 patients more than 20 years of age and one of two younger patients. None had HTLV-2 infection. In conclusion, Strongyloides hyperinfection among Peruvian patients was highly associated with HTLV-1 infection.

Regeneration of periodontal tissues by basic fibroblast growth factor.

Several growth factors (or cytokines) have recently received attention because of their ability to actively regulate various cellular functions of periodontal ligament (PDL) cells and the effects of topical application of such factor(s) on periodontal tissue regeneration has been evaluated. In this study, we examined the role of basic fibroblast growth factor (bFGF) in the wound healing and regeneration of periodontal tissues. Alveolar bone defects (such as 2-wall, 3-wall and furcation class II bone defects) were created surgically in beagle dogs and primates. Recombinant bFGF was topically applied to the artificial bony defects. Six or 8 wk after application, the periodontal regeneration was morphologically and histomorphometrically analyzed. In all sites where bFGF was applied, significant periodontal ligament formation with new cementum deposits and new bone formation was observed in amounts greater than in the control sites. We found it noteworthy that no instances of epithelial down growth, ankylosis or root resorption were observed in the bFGF sites. In vitro studies demonstrated that bFGF enhances the proliferative responses of human PDL cells, which express FGF receptor-1 and -2, but inhibits the induction of alkaline phosphatase activity and mineralized nodule formation by PDL cells. Interestingly, we observed that the mRNA level of laminin in PDL cells, which plays an important role in angiogenesis, was specifically upregulated by bFGF stimulation, but that of type I collagen was downregulated. The present study demonstrates that bFGF can be applied as one of the therapeutic modalities which actively induce periodontal tissue regeneration. The results of in vitro studies suggest that by suppressing the cytodifferentiation of PDL cells into mineralized tissue forming cells, bFGF may play important roles in wound healing by promoting angiogenesis and inducing the growth of immature PDL cells, and may in turn accelerate periodontal regeneration.

[STRONGYLOIDES STERCORALIS: CLINICAL SEVERE FORMS ASSOCIATED TO HTLV-1 INFECTION] / Strongyloides stercoralis: Formas Clínicas Severas Asociadas a Infección por HTLV-I.

A study was conducted in Lima to determine the HTLV-1 infection rate among patients with different clinical patterns of Strongyloidiasis. This study included 21 patients with Strongyloides Stercoralis (St St) hyperinfection (Group 1), and a group which consisted of sex and age matched asymptomatic healthy individuals whose stools were negative (Control Group Group 2). A third group included 33 patients with "probable hyperinfection", and another group of 63 patients with intestinal Strongyloidiasis, without evidence of systemic disease, was included. Serum from each studied individual was analized to find HTLV-1/II and ELISA; positive cases were confirmed by Western Blot. The hyperinfection rate was significantly higher [85.7% (18/21)] compared to the control group [4.7% (1/21)] p<0.001. Likewise, the "probable hyperinfection" group was considerably higher [69.7% (23/33)] in comparison to the control group p<0.01. The group with intestinal Strongyloidiasis was [10% (6/62)] lower than patients with Strongyloides hyperinfection, but did not differ significantly (p>0.05) from the control group. In this last group 3/6 were HTLV-1 positive, and their follow-up showed they developed hyperinfection forms. We conclude that severe forms of St St are considerably associated to HTLV-1 infection in adults and children over 5 years of age. No cases with HTLV-II were detected.

PKC and tyrosine kinase involvement in amyloid beta (25-35)-induced chemotaxis of microglia.

Microglia are activated by amyloid beta (Abeta) in vivo and in vitro, and Abeta-activated microglia may be involved in the pathogenesis of Alzheimer's disease (AD). We investigated the mechanism of microglial chemotaxis induced by Abeta (25-35), an active fragment of Abeta. Abeta (25-35) 0.1 and 1 nM stimulated microglial chemotaxis. The protein kinase C (PKC) inhibitors chelerythrine (0.5 and 2 microM), calphostin C (1 microM) and staurospine (10 nM) significantly inhibited the microglial chemotaxis induced by Abeta (25-35) (1 nM). The chemotactic effect of Abeta (25-35) on microglia was desensitized by pretreatment of microglia with 1 ng/ml 12-O-tetradecanoylphorbol 13-acetate (TPA). Pretreatment of cells with Abeta (25-35) (1 nM) also desensitized the chemotactic effect by Abeta (25-35) (1 nM). The desensitization by TPA or Abeta (25-35) was inhibited when staurosporine was present in the pretreatment media. The tyrosine kinase inhibitor herbimycin A (0.1 and 1 microM) significantly inhibited the microglial chemotaxis induced by Abeta (25-35) (1 nM). Based on these observations, it seems likely that PKC and tyrosine kinase are involved in the Abeta-induced chemotaxis of microglia.

Expression of receptors for basic fibroblast growth factor on human periodontal ligament cells.

Basic fibroblast growth factor (FGF-2; bFGF) is a major mitogen for connective tissue cells, and participates in the healing process. It has already been reported that FGF-2 could be applicable to enhance periodontal regeneration. In the present study, we examined FGF receptor (FGFR) expression on human periodontal ligament (PDL) cells. The binding of [125I]-labeled FGF-2 to human PDL cells was studied by radioreceptor assay. The binding of [125I]-FGF-2 to PDL cells reached a plateau after 2.5 h incubation at 4 degrees C and was inhibited by the addition of unlabeled FGF-2 and acidic FGF (FGF-1; aFGF), but not insulin-like growth factor-I, platelet-derived growth factor and transforming growth factor-beta 1. Scatchard analysis revealed the presence of approximately 1.0 x 10(5) FGF-2 binding sites per cell with an apparent Kd of 1.2 x 10(-10) M. Interestingly, the binding of [125I]-FGF-2 on PDL cells reached its maximum at d 6 of the culture and then gradually decreased. Scatchard analysis also demonstrated that the number of FGFRs on a PDL cell was altered during the course of the culture, while the affinity between FGF-2 and its receptor was not. The responsiveness of PDL cells to FGF-2, which was monitored by the inhibitory effect on alkaline phosphatase activity, was reduced in proportion to the decrease in the number of FGFRs on the PDL cells. The present study suggests that PDL cells alter the responsiveness to FGF-2 during the course of the culture by changing the density of its receptor, and that the density of FGFR expression might be a marker of the cytodifferentiation of PDL cells into mineralized tissue forming cells.

Single-channel activity of the Ca2+-dependent K+ channel is modulated by FK506 and rapamycin.

Single-channel patch clamp recordings were performed in primary cultured neurons from rat dorsal hippocampi. Ca2+-dependent and TEA-sensitive K+ current was recorded from the neurons. Application of immunosuppressants FK506 and rapamycin to the channel inside the plasma membrane of the neurons significantly prolonged the mean open time of the channel. Calcineurin autoinhibitory fragment and W-7 induced no significant alteration in the mean open time of the channel. These results suggest that modulation of the activity of the Ca2+-dependent K+ channel by FK506 and rapamycin is directly through association of immunosuppressants with FKBP12.