Cryopreservation of Adherent Human Pluripotent Stem Cells via a Novel Dish Culture Preservation Method.

BACKGROUND: Human induced pluripotent stem cells (hiPSCs) are valuable resources for cell therapy and drug discovery. Cryopreservation is a key technique used to realize these applications, which require a large number of cells. However, standard protocols for the preservation of the adherent cells involve multiple complicated steps, which can lead to technical difficulties. OBJECTIVE: To develop a more efficient method for cryopreservation of adherent cells using culture dishes. MATERIALS AND METHODS: Ice-seeding treatment was employed to avoid intercellular freezing, and rapid warming used to improve cell viability. RESULTS: The immediate survival rate after thawing was 48%. The recovery period of cells cryopreserved by the dish culture method was shortened upon subsequent passage culture, and the time for re-cultured cells to reach the appropriate confluency was reduced by two days. CONCLUSION: The hiPSCs can be successfully cryopreserved in culture dishes with improved viability and faster recovery. The optimization of the ice-seeding temperature and cooling rate increased the survival rate.

Expression and analysis of a cytolethal distending toxin (cdt) gene operon in Campylobacter lari.

The present study examines the expression of cytolethal distending toxin (cdt) gene encoding a cytotoxin in Campylobacter lari (n=6 urease-negative [UN] C. lari; n=4 urease-positive thermophilic Campylobacter [UPTC]). When reverse transcription polymerase chain reaction (RT-PCR) was carried out with 10 C. lari isolates using a primer pair to amplify the cdtB gene transcript segment, an approximate 260 bp RT-PCR amplicon was generated with all the isolates. In addition, cdtA, cdtB and cdtC gene operon was identified to be polycistronicly transcribed in the C. lari cells. The cdtB gene translation in the C. lari cells was also confirmed by Western blot analysis. Thus, the cdt gene operon in C. lari organisms, including UN C. lari and UPTC, was expressed at the transcriptional and translational levels in the cells. The present results suggest that all three cdt genes may be functional in the cells.

Exposure to clinical X-ray radiation does not alter antibiotic susceptibility or genotype profile in gram-negative and gram-positive clinical pathogens.

Inadvertent exposure of bacterial pathogens to X-ray radiation may be an environmental stress, where the bacterium may respond by increasing mutational events, thereby potentially resulting in increased antibiotic resistance and alteration to genotypic profile. In order to examine this, four clinical pathogens, including the Gram-negative organisms Escherichia coli O157:H7 NCTC12900 and Pseudomonas aeruginosa NCTC10662, as well as the Gram-positive organisms Staphylococcus aureus NCTC6571 and Enterococcus faecium were exposed to X-rays (35,495 cGy/cm2) over a seven-day period. Antibiotic susceptibility was assessed before, during and after exposure by examining susceptibility, as quantified by E-test with six antibiotics, as well as to a further 11 antibiotics by measurement of susceptibility zone sizes (mm). Additionally, the DNA profile of each organism was compared before, during and after exposure employing the enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC PCR). Results indicated that exposure of these organisms to this amount of X-ray radiation did not alter their antibiotic susceptibility, nor their genomic DNA profile. Overall, these data indicate that exposure of bacteria to X-ray radiation does not alter the test organisms' antibiotic susceptibility profiles, nor alter genomic DNA profiles of bacteria, which therefore does not compromise molecular epidemiological tracking of bacteria within healthcare environments in which patients have been exposed to X-ray radiation.

Comparative analysis of Campylobacter lari cytolethal distending toxin (CDT) effect on HeLa cells.

We aimed to clarify if Campylobacter lari exerts a cytolethal distending toxin (CDT) effect on HeLa cells. Campylobacter cell lysates (CCLys) from C. jejuni 81-176 and urease-positive thermophilic Campylobacter (UPTC) CF89-12 and UPTC NCTC12893 isolates were shown to exert a CDT effect on HeLa cells with morphological changes examined by Giemsa staining and microscopy. However, Campylobacter lari JCM2530(T) isolate showed no effect. In addition, Campylobacter cell culture supernatant wash gave low or absent toxic effects with both C. jejuni and C. lari organisms. When western blot analysis was carried out to clarify if there was a CDTB effect in the CCLys and soluble fractions from Campylobacter isolates, which had a CDT effect on HeLa cells or did not have any effect, anti-recombinant CjCDTB antibodies identified an immunoreactively positive signal at around approximately 25 kDa on all the C. lari isolates examined, as well as the C. jejuni 81116 strain. Thus, all the Campylobacter isolates including those without any CDT effect were shown to express CDTB at the translational level.

Molecular identification and characterization of clustered regularly interspaced short palindromic repeats (CRISPRs) in a urease-positive thermophilic Campylobacter sp. (UPTC).

Novel clustered regularly-interspaced short palindromic repeats (CRISPRs) locus [7,500 base pairs (bp) in length] occurred in the urease-positive thermophilic Campylobacter (UPTC) Japanese isolate, CF89-12. The 7,500 bp gene loci consisted of the 5'-methylaminomethyl-2-thiouridylate methyltransferase gene, putative (P) CRISPR associated (p-Cas), putative open reading frames, Cas1 and Cas2, leader sequence region (146 bp), 12 CRISPRs consensus sequence repeats (each 36 bp) separated by a non-repetitive unique spacer region of similar length (26-31 bp) and the phosphatidyl glycerophosphatase A gene. When the CRISPRs loci in the UPTC CF89-12 and five C. jejuni isolates were compared with one another, these six isolates contained p-Cas, Cas1 and Cas2 within the loci. Four to 12 CRISPRs consensus sequence repeats separated by a non-repetitive unique spacer region occurred in six isolates and the nucleotide sequences of those repeats gave approximately 92-100% similarity with each other. However, no sequence similarity occurred in the unique spacer regions among these isolates. The putative σ(70) transcriptional promoter and the hypothetical ρ-independent terminator structures for the CRISPRs and Cas were detected. No in vivo transcription of p-Cas, Cas1 and Cas2 was confirmed in the UPTC cells.

Comparison between in vitro qualities of platelets washed with commercially available additive solutions and those washed with M-sol.

BACKGROUND AND OBJECTIVES: We previously developed a novel additive solution (M-sol) with a high ability to preserve the in vitro qualities of platelets (PLTs) in washed PLTs Here, we compared the ability of M-sol with that of commercially available additive solutions (ASs) to preserve the in vitro qualities (pH, mean PLT volume, %disc, P-selectin, %hypotonic shock response and aggregation) of PLTs at a low plasma concentration. MATERIALS AND METHODS: The platelet concentrate was divided into two equal aliquots (control group and test group). After centrifugation of both groups and removal of as much supernatant as possible, the pellet of the control group was resuspended in M-sol and those of the test group were resuspended in other ASs, and subsequently stored in polyolefin bags with agitating at 20-24 degrees C. RESULTS: Compared with those stored in M-sol, the qualities of PLTs stored in PAS-B (alternative name; PAS-II or T-sol), PAS- C (alternative name; PAS-III or Intersol) or Plasma Lyte were degraded as early as 24 h after washing. The qualities of PLTs stored in PAS-D (alternative name; Composol PS) or PAS-E (alternative name; PAS-IIIM or SSP+) were comparable to that of those stored in M-sol 24 h after washing; however, the qualities had deteriorated 72 h after washing. CONCLUSIONS: At a low plasma concentration (5% or less), the M-sol showed a higher ability to preserve PLTs than the five ASs studied here. Although PAS-D and PAS-E are available as an AS for short-term storage of washed PLTs, M-sol is thought to be preferable for longer storage.

Accumulation of nuclear-encoded tRNA(Thr) (AGU) in mitochondria of the liverwort Marchantia polymorpha.

The mitochondrial genome of the liverwort Marchantia polymorpha does not encode the full complement of tRNAs for the threonine and isoleucine codon boxes. To find the missing tRNA genes specifically for tRNA(Thr) in mitochondria, we have searched the genomic library and identified two clones (pTT1 and pTT2), encoding the identical tRNA(Thr) (AGU) gene copy with different 5'- and 3'-flanking sequences. By northern analysis, we demonstrate considerable accumulation of the nuclear encoded tRNA(Thr) and moderate expression of native tRNA(Thr) (GGU) in mitochondria. Nonetheless, the imported and native tRNA(Thr) species together are not sufficient to translate all four threonine codons used in liverwort mitochondria, implicating mitochondrial import of at least one additional threonine isoacceptor tRNA.

Radionuclide angiographic assessment of left ventricular diastolic filling in amyloid heart disease: a study of patients with familial amyloid polyneuropathy.

To assess left ventricular diastolic filling in amyloid heart disease, 17 patients with familial amyloid polyneuropathy and 20 normal subjects were examined by radionuclide angiography. None of the patients showed clinical evidence of restrictive cardiomyopathy. All but two patients had normal left ventricular ejection fraction. Peak filling rate was significantly lower and time to peak filling rate was significantly greater in patients than in normal subjects (2.60 +/- 0.52 versus 3.10 +/- 0.44 EDV/s, p less than 0.001, and 215 +/- 53 versus 147 +/- 18 ms, p less than 0.001, respectively). The mean left ventricular filling volume during rapid diastolic filling and atrial systole in patients was 54.5 +/- 19.5% and 44.2 +/- 21.6% of the stroke volume, respectively, compared with 83.8 +/- 6.6% (p less than 0.001) and 20.0 +/- 6.0% (p less than 0.001), respectively, in normal subjects. Although 10 of the 14 patients without clinical evidence of overt heart disease had normal ventricular wall thickness as well as normal ejection fraction, 8 of the 10 showed abnormal diastolic filling. In patients with familial amyloid polyneuropathy, indexes of diastolic filling were significantly related to ventricular wall thickness alone. The incidence and magnitude of abnormalities in time to peak filling rate and contribution of rapid filling as well as atrial systole to ventricular filling increased with age and duration of illness. Thus, abnormal diastolic filling can be seen even in the early stage of familial amyloid polyneuropathy and may be related to myocardial amyloid deposition as well as to fibrosis. Careful consideration should be given to age and duration of illness when diastolic filling is assessed in this disorder.

A case of pulmonary arteriovenous malformation in a patient with brain abscess successfully treated with video-assisted thoracoscopic resection.

A 45-year-old women was admitted to the hospital with a brain abscess due to asymptomatic pulmonary arteriovenous malformation (PAVM). The brain abscess was removed by craniotomy and excision following antibiotic therapy. The stapled wedge excision of the lung with the PAVM was successful under video-assisted thoracoscopic surgery.

Comparison of the effects of different antiviral treatments on the antioxidant systems of stroma-free hemoglobin.

The effect of virus inactivation by 1,9-dimethylmethylene blue (DMMB) phototreatment, methylene blue (MB) phototreatment or heat on the activities of antioxidant systems of stroma-free hemoglobin (SFH) was studied. DMMB photoinactivated human immunodeficiency virus by > 3.69 log10 under conditions that inactivated 3.33 log10 of vesicular stomatitis virus (VSV). Under conditions which inactivated VSV by 6.10 log10 (1.37 J/cm2 irradiation and 2 microM DMMB), there was little change in the methemoglobin (Met-Hb) formation, concentration of reduced glutathione (GSH), or superoxide dismutase (SOD), catalase (CAT) or glutathione peroxidase (GPX) activities. However, the activity of glutathione reductase (GR) was decreased by 77%. Under conditions that inactivated VSV by 5.69 log10 (1.37 J/cm2 irradiation and 24 microM MB) there was little effect of MB phototreatment on SOD, CAT, GPX and GSH activities. However, GR activity was decreased by 74% and Met-Hb content reached 3.98%. Under conditions that inactivated VSV by more than 6.20 log10 (60 degrees C for 2 min), virucidal heat treatment resulted in 27% Met-Hb formation and decreased GPX activity by 43%. No significant decline in SOD, CAT or GR activities or GSH concentration was observed. These results suggest that, compared with heat treatment and MB phototreatment, virucidal DMMB treatment preserves not only the oxidative state of hemoglobin but also the antioxidant systems against superoxide and hydrogen peroxide, although the reduced GR activity may limit the quenching capacity of antioxidants in DMMB-treated SFH.

Shape of the chromophore binding site in pharaonis phoborhodopsin from a study using retinal analogs.

To investigate the shape of the chromophore binding site of pharaonis phoborhodopsin (ppR), ppR-opsin was incubated with five ring-modified retinal analogs: an acyclic retinal, phenylretinal, alpha-retinal, cyclohexylretinal and 5-isopropyl-alpha-retinal. The experimental results were compared with those obtained from bacteriorhodopsin-opsin (bR-opsin) and the same retinal analogs. It was suggested that ring chain conformation is important in affecting the spectral shoulder unique for the absorption spectrum of ppR. The rate of pigment formation depended greatly on the analogs used with the planar analogs showing rapid formation. Thus, we concluded that the space of the retinal binding site of ppR is restricted to the plane of the cyclohexenyl ring of the chromophore, whereas that of bR is less restricted.

Photoinactivation of virus infectivity by hypocrellin A.

We investigated the photoinactivation of virus infectivity by hypocrellin A and its mechanism. The titers of vesicular stomatitis virus (VSV) and human immunodeficiency virus type 1 (HIV-1), both of which are enveloped viruses, were reduced upon illumination with hypocrellin A in a concentration-dependent manner, whereas canine parvovirus, a nonenveloped virus, was not killed. The removal of oxygen or addition of sodium azide or beta-carotene both inhibited VSV inactivation. Mannitol and superoxide dismutase had no effect on VSV inactivation. These results indicate that singlet oxygen was involved in the process of VSV inactivation. Of the three major VSV membrane proteins, peripheral membrane protein M was most damaged by the hypocrellin A phototreatment.

Virus photoinactivation in stroma-free hemoglobin with methylene blue or 1,9-dimethylmethylene blue.

Photoinactivation of vesicular stomatitis virus (VSV) in stroma-free hemoglobin (SFH) was carried out using methylene blue (MB) or 1,9-dimethylmethylene blue (DMMB). The VSV was more sensitive to inactivation by 660 nm light with 1 microM DMMB than with the same concentration of MB. Under conditions that inactivated 6 log10 of VSV, the methemoglobin content (Met-Hb[%]) and P50 of hemoglobin were changed by 1 microM MB phototreatment but were not changed by 1 microM DMMB phototreatment. The migration of hemoglobin during electrophoresis and the activity of superoxide dismutase were not changed by MB or DMMB phototreatment. In contrast to the results obtained with DMMB at 660 nm, 580 nm irradiation of SFH with DMMB resulted in a significant increase of Met-Hb(%) under conditions that only inactivated 1.19 log10 VSV. The 580 nm irradiation primarily activates the dimer and higher-order aggregates of the dyes, while 660 nm irradiation primarily activates the monomer. These results indicate that the monomer form of DMMB can effectively inactivate viruses without damage to SFH.

Islet amyloid polypeptide (IAPP/amylin) causes insulin resistance in perfused rat hindlimb muscle.

It has been reported that islet amyloid polypeptide (IAPP) has insulin antagonistic effects in vivo and in vitro. To determine whether IAPP affects glucose metabolism in skeletal muscle, we performed in situ rat hindlimb perfusion which is a near-physiological system. Forty min after the beginning of insulin infusion at 1000 microU/ml, the synthesized rat amide form of IAPP was infused at 1 nM or 10 nM for 50 min and glucose concentration in the effluent was measured to calculate glucose uptake (GU). The GU did not change during the 1 nM IAPP infusion, but significantly decreased during 10 nM IAPP infusion (554 +/- 24 to 445 +/- 29 nmol/g/min, P less than 0.01). Rat calcitonin gene-related peptide (CGRP), which has sequence homology with IAPP and has been reported to inhibit insulin action, was also administered. Similar to the effect of IAPP, the GU did not change during 1 nM CGRP infusion but significantly decreased during 10 nM CGRP infusion (507 +/- 7 to 323 +/- 15 nmol/g/min, P less than 0.01). In the experiments without insulin infusion, the GU was not changed even by 10 nM IAPP infusion. Therefore, IAPP directly reduced only the insulin-mediated GU in the skeletal muscle, and this effect of IAPP occurred at the same dose as that of CGRP. These data suggest that both IAPP and CGRP may cause insulin resistance in skeletal muscle not through a CGRP receptor but a yet unknown receptor, which has similar binding affinity for both IAPP and CGRP.

Electrical stimulation of the rat lumbar spine induces reflex action potentials in the nerves to the lower abdomen.

STUDY DESIGN: The distribution of the nerve action potentials reflexively elicited by electrical stimulation of the lumbar spine was investigated in rats. OBJECTIVES: To elucidate the relation between the lumbar spine and other body regions that compose the spinal reflex. SUMMARY OF BACKGROUND DATA: The hypothesis was that the ventral portion of the L5-L6 disc spatially corresponds to the groin. METHODS: In Experiments 1 and 2, wire electrodes were placed 1) in the ventral and dorsal portions of the disc, facet joint, and muscle fascia at L5-L6, and 2) in the ventral portions of L3-L4, L4-L5, L5-L6, and L6-S discs. A needle electrode was inserted in the L5-L6 disc by 0.4-mm increments, and action potentials were serially recorded from the genitofemoral nerve. RESULTS: Experiments 1 and 2: Reflex action potentials were elicited in the iliohypogastric (T13 and L1), ilioinguinal (L1), and genitofemoral (L2) nerves. Experiment 1: Stimulation of the disc induced reflex discharges significantly more frequently than stimulation of the facet joint and muscle fascia. Experiment 2: The more cranial the disc stimulated, the more frequently the reflex discharge was induced in the iliohypogastric nerve. Experiment 3: The depth of stimulation did not influence the size of the reflex action potential. CONCLUSIONS: Electrical stimulation of the lumbar disc and facet joint induced reflex discharges in the nerves to the lower abdominal regions. It was postulated that the reflex discharges are related to muscle contraction resulting in referred pain in the loin and groin.

Sensory innervation of the dorsal portion of the lumbar intervertebral disc in rats.

STUDY DESIGN: The vertebral levels of dorsal root ganglia innervating the dorsal portion of the L5-L6 intervertebral disc were investigated in rats using a retrograde transport method. The pathways and functions of nerve fibers supplying the dorsal portion of the disc were determined by denervation and immunohistochemistry. OBJECTIVES: The dorsal portion of the lumbar intervertebral disc has been reported to be innervated segmentally, but anesthetic block of the paravertebral sympathetic trunks and the L2 spinal nerve can relieve discogenic low back pain. In the current study, the sensory innervation of the dorsal portion of the L5-L6 intervertebral disc was investigated, because the disc anatomically corresponds to the L4-L5 disc in humans, and the dorsal portion of the human L4-L5 disc is frequently subject to injury that causes low back pain. METHODS: A retrograde transport of Fluoro-Gold (F-G; Fluorochrome, Denver, CO) was used. Subjects included nontreated control (n = 32) and sympathectomized rats in which paravertebral sympathetic trunks were removed from L2 to L3 (n = 9). In a ventral approach, Fluoro-Gold crystals were placed on the dorsal portion of the L5-L6 disc, and labeled neurons in the bilateral dorsal root ganglia from T10 to L6 were counted. RESULTS: Fluoro-Gold crystals did not leak from the dorsal portion of the L5-L6 disc in 14 of the 32 nontreated rats and in 5 of the 9 sympathectomized rats. These rats were used for analysis. Fluro-Gold-labeled neurons were found in dorsal root ganglia from T13 to L6 in the 14 control rats but only from L2 to L6 in the 5 sympathectomized rats. CONCLUSION: The dorsal portion of the L5-L6 disc of rats was shown to be multisegmentally innervated by the T13 to L6 dorsal root ganglia. The sensory fibers from T13, L1, and L2 dorsal root ganglia were shown to innervate the dorsal portion of the L5-L6 disc through the paravertebral sympathetic trunks. In contrast, those from the L3-L6 dorsal root ganglia may innervate the dorsal portion of the L5-L6 disc through the sinuvertebral nerves.