Analysis of the human intestinal microbiota from 92 volunteers after ingestion of identical meals.

The intestinal microbiota composition of 92 volunteers living in Japan was identified following the consumption of 'identical meals' (1,879 kcal/day) for 3 days. When faecal samples were analysed by terminal restriction fragment length polymorphism with several primer-restriction enzyme systems and then clustered, the patterns could be divided into 2 clusters. Contribution tests and partition modelling showed that OTU211 of the 35f-MspI system and OTU237 of the 35f-AluI system were key factors in the distribution of these groups. However, significant differences among these groups in terms of body mass index and age were not observed.

Choroidal Tuberculoma in a Patient with Acquired Immunodeficiency Syndrome.

Background: In Japan, since the introduction of antituberculosis chemotherapy, the typical choroidal tuberculoma has been considered uncommon. A patient with acquired immunodeficiency syndrome (AIDS), because of the suppression of cell mediated immunity, faces the risk of tuberculous infection.Case: A 30-year-old Malayan man had continuous cough for 6 months. He was diagnosed as having miliary tuberculosis of the lung and spine. Because the serum test of human immunodeficiency virus (HIV) was positive, he was also diagnosed as having AIDS.Findings: Fundus examination showed a yellow white swollen lesion of the choroid with distinct border in his right eye, probably caused by tuberculosis. After 3 months of therapy with antituberculosis and anti-HIV drugs, his systemic and ocular findings were markedly improved.Conclusion: Because of the recent increase in the incidence of tuberculosis with the epidemic of HIV prevailing in the world, the recognition of ocular tuberculosis is important.

[Choroidal tuberculoma in a patient with acquired immunodeficiency syndrome].

BACKGROUND: In Japan, since the introduction of antituberculosis chemotherapy, the typical choroidal tuberculoma has been considered uncommon. A patient with acquired immunodeficiency syndrome (AIDS), because of the suppression of cell mediated immunity, faces the risk of tuberculous infection. CASE: A 30-year-old Malayan man had continuous cough for six months. He was diagnosed as having miliary tuberculosis of the lung and spine. Because the serum test of human immunodeficiency virus (HIV) was positive, he was also diagnosed as having AIDS. FINDINGS: Fundus examination showed a yellow white swollen lesion of the choroid with distinct border in his right eye, probably caused by tuberculosis. After three months of therapy with antituberculosis and anti HIV drugs, his systemic and ocular findings were markedly improved. CONCLUSION: Because of the recent increase in the incidence of tuberculosis with the epidemic of HIV prevailing in the world, the recognition of ocular tuberculosis is important.

Characterization of a novel giant scaffolding protein, CG-NAP, that anchors multiple signaling enzymes to centrosome and the golgi apparatus.

A novel 450-kDa coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), was identified as a protein that interacted with the regulatory region of the protein kinase PKN, having a catalytic domain homologous to that of protein kinase C. CG-NAP contains two sets of putative RII (regulatory subunit of protein kinase A)-binding motif. Indeed, CG-NAP tightly bound to RIIalpha in HeLa cells. Furthermore, CG-NAP was coimmunoprecipitated with the catalytic subunit of protein phosphatase 2A (PP2A), when one of the B subunit of PP2A (PR130) was exogenously expressed in COS7 cells. CG-NAP also interacted with the catalytic subunit of protein phosphatase 1 in HeLa cells. Immunofluorescence analysis of HeLa cells revealed that CG-NAP was localized to centrosome throughout the cell cycle, the midbody at telophase, and the Golgi apparatus at interphase, where a certain population of PKN and RIIalpha were found to be accumulated. These data indicate that CG-NAP serves as a novel scaffolding protein that assembles several protein kinases and phosphatases on centrosome and the Golgi apparatus, where physiological events, such as cell cycle progression and intracellular membrane traffic, may be regulated by phosphorylation state of specific protein substrates.

[A case of AIDS complicated by progressive outer retinal necrosis].

BACKGROUND: The retina may be involved in patients with acquired immunodeficiency syndrome (AIDS). Progressive outer retinal necrosis (PORN) is a liability. CASE: A 46-year-old female had repeated exacerbations of pulmonary tuberculosis since two years before. Herpes zoster developed in her right trigeminal nerve area two weeks before, leading to a diagnosis of AIDS. She was referred to us for ophthalmological evaluation. FINDINGS: Both eyes showed numerous yellowish white patches in the deeper retinal layers. The anterior chamber and the vitreous were almost intact. Herpes zoster virus was identified in the acqueous by the polymerase chain reaction (PCR) method. Systemic acyclovir or ganciclovir failed to prevent rapid extension of fundus lesions, resulting in whole-layer necrosis of the retina. Retinal detachment with multiple breaks developed in both eyes whthin eleven days after the patient was first seen by us. The clinical course was different from acute retinal necrosis and was characteristic of PORN. CONCLUSION: This case illustrates that PORN may develop in patients affected by AIDS.

Interaction of PKN with alpha-actinin.

PKN is a fatty acid- and Rho-activated serine/threonine protein kinase, having a catalytic domain homologous to protein kinase C family. To identify components of the PKN-signaling pathway such as substrates and regulatory proteins of PKN, the yeast two-hybrid strategy was employed. Using the N-terminal region of PKN as a bait, cDNAs encoding actin cross-linking protein alpha-actinin, which lacked the N-terminal actin-binding domain, were isolated from human brain cDNA library. The responsible region for interaction between PKN and alpha-actinin was determined by in vitro binding analysis using the various truncated mutants of these proteins. The N-terminal region of PKN outside the RhoA-binding domain was sufficiently shown to associate with alpha-actinin. PKN bound to the third spectrin-like repeats of both skeletal and non-skeletal muscle type alpha-actinin. PKN also bound to the region containing EF-hand-like motifs of non-skeletal muscle type alpha-actinin in a Ca2+-sensitive manner and bound to that of skeletal muscle type alpha-actinin in a Ca2+-insensitive manner. alpha-Actinin was co-immunoprecipitated with PKN from the lysate of COS7 cells transfected with both expression constructs for PKN and alpha-actinin lacking the actin-binding domain. In vitro translated full-length alpha-actinin containing the actin-binding site hardly bound to PKN, but the addition of phosphatidylinositol 4, 5-bisphosphate, which is implicated in actin reorganization, stimulated the binding activity of the full-length alpha-actinin with PKN. We therefore propose that PKN is linked to the cytoskeletal network via a direct association between PKN and alpha-actinin.

Translocation of PKN from the cytosol to the nucleus induced by stresses.

Effects of environmental stresses on the subcellular localization of PKN were investigated in NIH 3T3, BALB/c 3T3, and Rat-1 cells. The immunofluorescence of PKN resided prominently in the cytoplasmic region in nonstressed cells. When these cells were treated at 42 degrees C, there was a time-dependent decrease of the immunofluorescence of PKN in the cytoplasmic region that correlated with an increase within the nucleus as observed by confocal microscope. After incubation at 37 degrees C following beat shock, the immunofluorescence of PKN returned to the perinuclear and cytoplasmic regions from the nucleus. The nuclear translocation of PKN by heat shock was supported by the biochemical subcellular fractionation and immunoblotting. The nuclear localization of PKN was also observed when the cells were exposed to other stresses such as sodium arsenite and serum starvation. These results raise the possibility that there is a pathway mediating stress signals from the cytosol to the nucleus through PKN.

PKN associates and phosphorylates the head-rod domain of neurofilament protein.

PKN is a fatty acid-activated serine/threonine kinase that has a catalytic domain highly homologous to that of protein kinase C in the carboxyl terminus and a unique regulatory region in the amino terminus. Recently, we reported that the small GTP-binding protein Rho binds to the amino-terminal region of PKN and activates PKN in a GTP-dependent manner, and we suggested that PKN is located on the downstream of Rho in the signal transduction pathway (Amano, M., Mukai, H., Ono, Y., Chihara, K., Matsui, T., Hamajima, Y., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996) Science 271, 648-650; Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N., Mukai, H., Ono, Y. Kakizuka, A., and Narumiya, S. (1996) Science 271, 645-648). To identify other components of the PKN pathway such as substrates and regulatory proteins of PKN, the yeast two-hybrid strategy was employed. By this screening, a clone encoding the neurofilament L protein, a subunit of neuron-specific intermediate filament, was isolated. The amino-terminal regulatory region of PKN was shown to associate with the head-rod domains of other subunits of neurofilament (neurofilament proteins M and H) as well as neurofilament L protein in yeast cells. The direct binding between PKN and each subunit of neurofilament was confirmed by using the in vitro translated amino-terminal region of PKN and glutathione S-transferase fusion protein containing the head-rod domain of each subunit of neurofilament. PKN purified from rat testis phosphorylated each subunit of the native neurofilament purified from bovine spinal cord and the bacterially synthesized head-rod domain of each subunit of neurofilament. Polymerization of neurofilament L protein in vitro was inhibited by phosphorylation of neurofilament L protein by PKN. The identification and characterization of the novel interaction with PKN may contribute toward the elucidation of mechanisms regulating the function of neurofilament.

Identification of Schizosaccharomyces pombe gene psk1+, encoding a novel putative serine/threonine protein kinase, whose mutation conferred resistance to phenylarsine oxide.

We have identified a novel putative protein kinase-encoding gene from Schizosaccharomyces pombe (Sp), designated psk1+, by using a highly conserved amino acid (aa) sequence motif to design amplification of DNA fragments using PCR. The putative translation product of psk1+ contains 436 aa, with a molecular mass of 49,317 Da. A single psk1+ was identified by genomic Southern blot analysis, and the genomic mapping indicated that psk1+ was localized in Sp chromosome III. Growth of wild-type Sp cells was inhibited by 0.5 microM phenylarsine oxide, a protein tyrosine phosphatase inhibitor, but psk1- cells were relatively resistant to this drug.

Functional characterization of the promoter region of the mouse protein kinase C gamma gene.

Promoter activity of protein kinase C (PKC) gamma gene was analysed by chloramphenicol acetyltransferase (CAT) assay using extracts from the cells transfected with various fusion constructs containing the 5'-flanking region of the mouse PKC gamma gene and CAT gene. Transient expression experiments in PC12 cells revealed that the upstream region of 87 bp from the transcriptional initiation site was sufficient for promoter activity. The region containing nucleotides 87 upstream from the transcriptional initiation site was shown to silence CAT activity in Balb/c3T3 cells, in which mRNA of PKC gamma was not detected, suggesting that this region might contain a transcriptional regulatory element for the cell type-specific expression of the PKC gamma gene.

Xenopus PKN: cloning and sequencing of the cDNA and identification of conserved domains.

cDNA clone encoding Xenopus laevis PKN has been isolated from Xenopus kidney library. Sequencing of this clone has revealed a single open reading frame encoding a protein of 901 amino acids. Immunoprecipitate from cytoplasmic fraction of COS7 cells transfected with this cDNA construct using antiserum against bacterially expressed Xenopus PKN revealed arachidonic acid-dependent autophosphorylation activity. Comparison of the closely related sequences of human and rat PKN with a protein from evolutionarily distant Xenopus, revealed several highly invariant domains in the NH2-terminal regulatory regions, suggesting that they participate in binding interaction with arachidonic acid.

Activation of PKN, a novel 120-kDa protein kinase with leucine zipper-like sequences, by unsaturated fatty acids and by limited proteolysis.

PKN, a novel protein kinase with catalytic domain homologous to PKC family and unique amino terminal leucine zipper-like sequences, was purified partially from COS7 cells transfected with the cDNA construct encoding human PKN for enzymatic characterization of the enzyme. Using serine containing synthetic peptides based on PKC pseudosubstrate sites as the phosphate acceptors, kinase activities estimated from partially purified PKN were not stimulated by Ca2+/phosphatidylserine/diolein but were activated several-fold to several tens-fold by 40 microM unsaturated fatty acids, such as arachidonic acid, linoleic acid, and oleic acid. Autophosphorylation of the immunoprecipitates using anti-PKN antiserum was also stimulated by various unsaturated fatty acids. Limited proteolysis of PKN with trypsin induced an enhancement of the peptide kinase activity that was almost independent of arachidonic acid.

[Non-granulomatous anterior uveitis and HLA-B27 antigen].

In Caucasians, there is close correlation between acute anterior uveitis and histocompatibility antigen HLA-B27. But in Japanese, this is not clear. Therefore, we examined 58 patients with non-granulomatous anterior uveitis (NGAU) about HLA typing in our uveitis clinic at Tokyo Women's Medical College Hospital. HLA-B27 was identified in 20 out of 58 patients (34.5%) with NGAU and it was statistically significant. We also studied the clinical features of patients with HLA-B27 positive NGAU. We found that in HLA-B27-positive NGAU, the visual acuity was more strongly affected during the attack and the duration of inflammation was longer than HLA-B27 negative NGAU patients. The duration of the first attack was longer than re-attack, and the duration of the first attack was longer than HLA-B27 negative NGAU patients. It was less commonly associated with systemic disorders in Japanese HLA-B27 positive anterior uveitis than in Caucasian. However, in Japanese NGAU patients without systemic disorders, there was the same tendency concerning the HLA-B27 positive rate as in Caucasians.