Terminal restriction fragment length polymorphism analysis of the diversity of fecal microbiota in patients with ulcerative colitis

BACKGROUND: Terminal restriction fragment length polymorphism (T-RFLP) analysis is a powerful tool to assess the diversity of complexed microbiota. This permits rapid comparison of microbiota from many samples. In this study, we performed T-RFLP analysis of the fecal microbiota from patients with ulcerative colitis (UC). METHODS: Forty-four patients with UC (23 women and 21 men, median age 25 years) and 46 healthy individuals (25 women and 21 men, median age 34 years) were enrolled in this study. DNA was extracted from their stool samples, and the 16S rRNA genes were amplified by PCR. The PCR products were then digested with HhaI and/or MspI restriction enzymes, and the length of the T-RF was determined. RESULTS: The fecal microbial communities were classified in 8 clusters. Almost all the healthy individuals (39 of 46) were included in cluster I, and most of the UC patients could be divided into the other 7 clusters, indicating that fecal bacterial communities are different between healthy individuals and active UC patients. Some T-RFs, derived from the unclassified bacteria, Ruminococcus, Eubacterium, Fusobacterium, gammaproteobacteria, unclassified Bacteroides, and unclassified Lactobacillus, were detected in the UC patients, but not in the healthy individuals. The T-RFLP patterns were also different between the active patients and inactive (remission) patients. The T-RF derived from the unclassified bacteria, Ruminococcus and Eubacterium, and the T-RFs derived from the unclassified bacteria, Eubacterium, and Fusobacterium were predominantly detected in the active patients not the inactive patients. In contrast, the T-RFs derived from Lactobacillus and unclassified Lactobacillus were more predominant in the inactive (remission) patients. In 4 patients with proctitis, the pattern of fecal microbial diversity was very similar. CONCLUSIONS: T-RFLP analyses showed that the diversity of fecal microbiota in patients with UC was different from that in healthy individuals. Unclassified bacteria, as well as known bacteria, can contribute to alterations in the bacterial diversity of UC patients.     

Agitated depression successfully treated with electroconvulsive therapy combined with steroid cover in a patient with Addison's disease

We report the case of a 70-year-old man with Addison's disease who developed severe agitated depression resulting in life-threatening medical conditions. The depression was treated safely with electroconvulsive therapy (ECT) combined with steroid cover. Administration of steroid cover just before each ECT session may increase safety of the ECT procedure in psychiatric patients with Addison's disease.     

RANTES -28G delays and DC-SIGN - 139C enhances AIDS progression in HIV type 1-infected Japanese hemophiliacs

The relationships between host immune factors and HIV-1 disease progression are still in dispute. Unlike CCR5Delta32, which has been found to delay disease progression of HIV-1, there still remain several factors whose effect on the clinical course is unconfirmed. To clarify the relationships, we selected seven single-nucleotide polymorphisms (SNPs) out of the previously reported factors, namely, RANTES promoter -28G/-403A, RANTES In1.1C, SDF-1 3'A, IL-4 promoter -589T, and DC-SIGN promoter -139C/-336C, and examined these in Japanese HIV-1-infected hemophiliacs (n = 102). The genotypes were examined by the direct sequencing method, and the distributions of genotype and allelic frequencies were compared between two groups, slow progressors (n = 54) who did not develop AIDS more than 10 years after intravenous infection and others (progressors) (n = 48). The allelic frequency of RANTES -28G was significantly higher in slow progressors (0.185) than in the progressor group (0.074) [p = 0.023, OR = 0.35, 95% CI (0.142, 0.880)]. DC-SIGN promoter -139C, and appeared in progressors with significantly higher allelic frequency (0.333) than slow progressors [0.204, p = 0.040, OR = 1.95, 95% CI (1.039, 3.677)]. With RANTES -403A, RANTES In1.1C, SDF-1 3' A, IL-4 -589T, and DC-SIGN -336C, no significant difference was observed in allelic frequencies between the two groups. These results suggest that RANTES -28G was associated with delayed AIDS progression, while DC-SIGN -139C was associated with accelerated AIDS progression in HIV-1-infected Japanese hemophiliacs.     

Wnt-5a signaling is correlated with infiltrative activity in human glioma by inducing cellular migration and MMP-2

Wnts are secreted ligands that consist of 19 members in humans, regulate cell proliferation, differentiation, motility and fate in many stages including the embryonic stage and tumorigenesis. Wnts bind to cell surface receptors named Frizzleds and LRPs, and transduce their signals through β-catenin-dependent and -independent intracellular pathways. Gliomas are one of the most common intracranial tumors. Gliomas exhibit a progression associated with widespread infiltration into surrounding neuronal tissues. However, the molecular mechanisms that stimulate the invasion of glioma cells are not fully understood. We established two cell lines from human glioma cases and analyzed the expression of all Wnt and Frizzled members in these cell lines and other well-known glioma cell lines by real-time PCR study. The mRNA of Wnt-5a and -7b and Frizzled-2, -6 and -7 were overexpressed in glioma cells. The elevation of Wnt-5a expression was most remarkable. Although Wnt-5a is reported to have oncogenic and antioncogenic activity in several cancers, the role of Wnt-5a signaling in human glioma cells remains unclear. Immunohistochemical study also revealed high expression of Wnt-5a in 26 (79%) of 33 human glioma cases. The positivity of Wnt-5a expression was correlated with the clinical grade. Knockdown of Wnt-5a expression suppressed migration, invasion and expression of matrix metalloproteinase-2 of glioma cells. Reciprocally, treatment with purified Wnt-5a ligand resulted in stimulation of cell migration and invasion. MMP-2 inhibitor suppressed the Wnt-5a-dependent invasion of U251 cells. These results suggested that Wnt-5a is not only a prognostic factor but also a therapeutic target molecule in gliomas for preventing tumor cell infiltration.     

The role of ZIP8 down‐regulation in cadmium‐resistant metallothionein‐null cells

The mechanisms of cellular cadmium uptake in mammalian cells remain obscure. To solve this problem, we established cadmium‐resistant cells (A7 and B5) from metallothionein‐null mouse cells, and found that cadmium accumulation was markedly suppressed in these cells. DNA microarray and real‐time PCR analyses revealed that expressions of ZIP (Zrt‐, Irt‐related protein) 8 and ZIP14 were down‐regulated in A7 and B5 cells. In particular, both mRNA and protein levels of ZIP8 were markedly suppressed in A7 and B5 cells. Introduction of short hairpin RNA (shRNA) of ZIP8 into parental cells reduced the accumulation of cadmium to about 35% of that of mock‐transfected cells, whereas the introduction of shRNA of divalent metal transporter 1 hardly changed cadmium accumulation. Thus, the cadmium resistance in A7 and B5 cells may be conferred primarily by the down‐regulation of ZIP8. In mouse tissues, high expression of ZIP8 was noted in the liver, kidney, lung and testis. These data suggest that ZIP8 plays an important role in cellular uptake of cadmium. Copyright © 2009 John Wiley & Sons, Ltd.     

Nafamostat is hydrolysed by human liver cytosolic long-chain acyl-CoA hydrolase

Although the authors recently reported that nafamostat, a clinically used serine protease inhibitor, was mainly hydrolysed by carboxylesterase in human liver microsomes, the involvement of human liver cytosol has not been elucidated. The current study examined the in vitro metabolism of nafamostat with human liver cytosols. Kinetic analysis indicated that the Vmax and Km values in the liver cytosols were 9.82 nmolmin(-1) mg(-1) protein and 197 microM for a liver sample HL-1, and 15.1 nmolmin(-1) mg(-1) protein and 157 microM for HL-2, respectively. The Vmax/Km values in both cytosols were at least threefold higher than those in the corresponding microsomes. The liver cytosolic activity for nafamostat hydrolysis was inhibited by phenylmethylsulfonyl fluoride (PMSF) (43% inhibition at 100 microM), whereas diisopropyl fluorophosphate (DFP) and bis(p-nitrophenyl)phosphate (BNPP) failed to inhibit the activity. Furthermore, the hydrolytic activity was also reduced by palmitoyl-CoA (67% inhibition at 100 microM) but not by acetyl-CoA. Effects of PMSF, DFP and BNPP on cytosolic palmitoyl-CoA hydrolytic activity were comparable with those of the cytosolic nafamostat hydrolytic activity. In addition, the palmitoyl-CoA hydrolytic activity was competitively inhibited by nafamostat with the apparent Ki value of 164 microM for the liver cytosol from HL-2. These results suggest that an isoform of long-chain acyl-CoA hydrolase may be responsible for the nafamostat hydrolysis in human liver cytosol.     

A case of antiphospholipid syndrome with cutaneous ulcer and intrauterine fetal growth retardation

A 28-year-old patient presented with severe intrauterine fetal growth retardation (IUGR) at 34 weeks' gestation. There was a prior history of a recurrent cutaneous ulcer on the left thigh. Serological tests for IgG anticardiolipin antibody were positive. A live premature male infant was delivered by an urgent cesarean section because of fetal distress. Histopathological examination revealed that the causes of the cutaneous ulcer and IUGR were thrombosis of the small blood vessels and placental infarction, respectively. Early diagnosis and proper treatment are important in the management of the antiphospholipid syndrome.     

Potential of embryonic stem cell-derived neurons for synapse formation with auditory hair cells

Recent studies have indicated that embryonic stem cells (ESCs) can be a source for the replacement of spiral ganglion neurons (SGNs), auditory primary neurons, and neurite projections from ESC-derived neurons to auditory sensory epithelia. However, the potential of ESC-derived neurons for synapse formation with auditory hair cells (HCs) has not been elucidated. The present study therefore aimed to examine the ability of ESC-derived neurons to form synaptic connections with HCs in vitro. Mouse ESC-derived neural progenitors expressing enhanced green fluorescence protein (EGFP) were cocultured with explants of cochlea sensory epithelia obtained from postnatal day 3 mice. After a 7-day culture, neurites of ESC-derived neurons predominantly elongated toward inner hair cells (IHCs), which play a crucial role in sound transmission to SGNs. Immunohistochemical analyses revealed the expression of synapsin 1 and synaptophysin in the nerve endings of ESC-derived neurons adjacent to IHCs, indicating the formation of synaptic connections. Transmission electron microscopy demonstrated synaptic contacts between nerve endings of ESC-derived neurons and IHCs. The present findings show that ESC-derived neurons can make synaptic connections with IHCs.