In vitro system for differentiating pluripotent neural crest cells into smooth muscle cells

The change in vascular smooth muscle cells (SMC) from a differentiated to a dedifferentiated state is the critical phenotypic response that promotes occlusive arteriosclerotic disease. Despite its importance, research into molecular mechanisms regulating smooth muscle differentiation has been hindered by the lack of an in vitro cell differentiation system. We identified culture conditions that promote efficient differentiation of Monc-1 pluripotent neural crest cells into SMC. Exclusive Monc-1 to SMC differentiation was indicated by cellular morphology and time-dependent induction of the SMC markers smooth muscle alpha-actin, smooth muscle myosin heavy chain, calponin, SM22alpha, and APEG-1. The activity of the SM22alpha promoter was low in Monc-1 cells. Differentiation of these cells into SMC caused a 20-30-fold increase in the activity of the wild-type SM22alpha promoter and that of a hybrid promoter containing three copies of the CArG element. By gel mobility shift analysis, we identified new DNA-protein complexes in nuclear extracts prepared from differentiated Monc-1 cells. One of the new complexes contained serum response factor. This Monc-1 to SMC model should facilitate the identification of nodal regulators of smooth muscle development and differentiation.     

Expression of the mutant (1735T-DEL) tissue-nonspecific alkaline phosphatase gene from hypophosphatasia patients

Hypophosphatasia (HOPS) is an inherited disorder characterized by defects in skeletal mineralization due to the deficiency of tissue-nonspecific alkaline phosphatase (TNSALP). To date, various mutations in the TNSALP gene have been identified. Especially, a deletion of T at position 1735 (1735T-del) located in exon 12 has been detected in three genetically unrelated Japanese patients, which seems to be one of the hot spots among the causative mutations in Japanese HOPS patients. 1735T-del causes a frame shift downstream from codon 503 (Leu), and consequently the normal termination codon at 508 is eliminated. Since a new inframe termination codon appears at codon 588 in the mutant DNA, the resultant protein is expected to have 80 additional amino acids. Expression of the mutant TNSALP gene using COS-1 cells demonstrated that the protein translated from the mutant 1735T-del had undetectable ALP activity, and its molecule size was larger than normal, as expected. Interestingly, an immunoprecipitation study of patients' sera using antibody against TNSALP revealed an abnormal protein which corresponded in size to the mutated TNSALP expressed by COS-1 cells, suggesting that the abnormal TNSALP is made by HOPS patients. The detection of TNSALP in cells transfected with 1735T-del using an immunofluorescent method exhibited only a faint signal on the cell surface, but an intense intracellular fluorescence after permeabilization.     

Light responsiveness of the suprachiasmatic nucleus: long-term multiunit and single-unit recordings in freely moving rats

The suprachiasmatic nuclei (SCN) of the hypothalamus contain a pacemaker that generates circadian rhythms in many functions. Light is the most important stimulus that synchronizes the circadian pacemaker to the environmental cycle. In this paper we have characterized the baseline neuronal firing patterns of the SCN as well as their response to light in freely moving rats. Multiunit and single-unit recordings showed that SCN neurons increase discharge during daytime and decrease discharge at night. Discharge levels of individual neurons that were followed throughout the circadian cycle appeared in phase with the population and were characterized by low discharge rates (often below 1 Hz), with a twofold increase during the day. The effect of light on the multiunit response was dependent on the duration of light exposure and on light intensity, with light thresholds of approximately 0.1 lux. The light response level showed a strong dependency on time of day, with large responsiveness at night and low responsiveness during day. At both phases of the circadian cycle, the response level could be raised by an increase in light intensity. Single-unit measurements revealed that the time-dependent light response of SCN neurons was present also at the level of single units. The results show that the basic light response characteristics that were observed at the multiunit level result from an integrated response of similarly behaving single units. Research at the single-unit level is therefore a useful approach for investigating the basic principles of photic entrainment.     

Experimental and Monte Carlo dosimetry of the Henschke applicator for high dose-rate 192Ir remote afterloading

We have performed extensive computational and experimental dosimetry of the Henschke applicator with respect to high dose-rate 192Ir brachytherapy using a GAMMAMED remote afterloader. Our goal was to generate clinically useful two- and three-dimensional look-up tables. Dose measurements of the Henschke applicator involved using TLD chips placed in a polystyrene phantom. Monte Carlo simulations were performed using the MCNP code. The computational models included the detailed geometry of 192Ir source, tandem tube, and shielded ovoid. The measured dose rates were corrected for the dependence of TLD sensitivity on the distance of measurement points from the source. Transit dose delivered during source extension to and retraction from a given dwell position was estimated by Monte Carlo simulations, and a correction was applied to the experimental values. For the applicator tandem, the ratio of dose rates obtained by MCNP to those measured by TLD chips ranges from 0.92 to 1.10 with an average of 0.98 and a standard deviation of 0.02. The measured and calculated dose rates at 1 cm on the transverse axis are 1.10 cGy U-1 h-1. For the shielded ovoid, the ratio ranges from 0.88 to 1.16 with an average of 1.00 and a standard deviation of 0.07. Causes of the discrepancy between the Monte Carlo and TLD results were identified. We found that the combined uncertainty of measured dose rates due to these causes is 5.6% for the applicator tandem and 8.4% for the shielded ovoid. Therefore, the results of the Monte Carlo simulation are considered to have been validated by the measurements within the uncertainty involved in the calculation and measurements.     

Nucleotide sequencing analysis of the 146-kilobase segment around the IkBL and MICA genes at the centromeric end of the HLA class I region

The biomechanics of medialization laryngoplasty are not well understood. An excised canine larynx model was used to test the effects of various sized silicon implants. The vocal fold length, position, and tension were measured. Medialization laryngoplasty did not affect vocal fold length. At the mid-membranous vocal fold, larger shims resulted in greater medialization and tension. Medialization laryngoplasty neither medialized nor stiffened the vocal process to resist lateralizing forces. We conclude that medialization laryngoplasty provides bulk and support for defects of the membranous region of the vocal fold, but does not appear to close a posterior glottal gap. The selection of a surgical procedure to treat glottal incompetence should take into account the unique biomechanical properties of the anterior (membranous vocal folds) and posterior (cartilaginous portion) glottis.     

Successful polymerase chain reaction-based diagnosis of fungal meningitis in a patient with chronic granulomatous disease

The structural and electronic properties of seventeen alkylxanthine derivatives were calculated using the MO program PM3 to elucidate the key features related to their inhibitory activity on phosphodiesterase (PDE) IV isoenzyme. Except for 7-alkylxanthine derivatives, a good correlation could be established between the distance between the tops of the two alkyl groups at the N1 and N3 positions of the xanthine skeleton (molecular length) and the PDE IV inhibitory activity (r=0.973, n=13). The same inhibitory activity could also be significantly correlated with the following electronic parameters of alkylxanthines: HOMO energy (r=0.850), absolute hardness (r=-0.806), and absolute electronegativity (r=-0.825). These results suggest that the electronic properties are partly responsible for PDE IV inhibition as far as the effects of structural properties associated with molecular length are concerned. Alkylxanthines may also act as electron donors in the charge-transfer interaction with the active sites on PDE IV isoenzyme.     

Comparison between Er:YAG laser and conventional technique for root caries treatment in vitro

Effective ablation of dental hard tissues by means of the erbium-doped:yttrium-aluminum garnet (Er:YAG) laser has been reported recently, and its application to caries removal and cavity preparation has been expected. However, few studies have investigated the capability of the Er:YAG laser to treat caries. In the present study, the effectiveness of caries removal by using an Er:YAG laser in vitro was compared with that of conventional mechanical treatment. Thirty-one extracted human teeth with root caries were used. Half of the caries in each tooth was treated with the Er:YAG laser, and the other was removed with a conventional bur or was left untreated as a control. Laser treatment was performed by means of a combination of contact and non-contact irradiation modes with cooling water spray, with a new fiber delivery and contact probe system. Conventional bur treatment was conducted by means of a low-speed micromotor. Measurements of the time required for caries removal, histopathological observations of decalcified serial sections, scanning electron microscope (SEM) observations, and hardness measurements of the treated cavity-floor dentin were performed for each treatment. Due to the careful irradiation technique, a longer treatment time was required for the complete removal of carious dentin by the Er:YAG laser. However, the Er:YAG laser ablated carious dentin effectively with minimal thermal damage to the surrounding intact dentin, and removed infected and softened carious dentin to the same degree as the bur treatment. In addition, a lower degree of vibration was noted with the Er:YAG laser treatment. The SEM examination revealed characteristic micro-irregularities of the lased dentin surface. Our results show that the Er:YAG laser system is promising as a new technical modality for caries treatment.     

Prognostic factors and rational approach in the treatment of submucosal cancer of the stomach

Metallothionein (MT) mRNA levels were analyzed following exposure of neonatal rat primary astrocyte cultures to physiologic pH (7.4), acidosis (pH 6.5 and 6.0), and dimethyl sulfoxide (DMSO). Treatments were carried out both in the presence and absence of the bioflavonoid, quercetin. Total RNA was probed on northern blots with [alpha32P]dCTP-labeled synthetic cDNA probes specific for rat MT isoform mRNAs. MT-I and MT-II mRNA levels in astrocytes exposed to pH 6.5 or pH 6.0 were increased compared to controls (pH 7.4). Treatment with DMSO in the presence and absence of acidosis, also increased MT-I and MT-II mRNA levels compared to controls (pH 7.4). The DMSO-induced increase in MT mRNA expression was reversed by treatment of astrocytes with quercetin, such that MT-I and MT-II mRNA levels in DMSO plus quercetin-treated astrocytes were indistinguishable from mRNA levels in their respective controls at pH 7.4, pH 6.5, and pH 6.0. These findings suggest that both acidosis and DMSO exposure are associated with increased astrocytic MT synthesis at the mRNA level, and that quercetin, effectively blocks MT mRNA induction by DMSO.     

Chitosanase activity of the enzyme previously reported as beta-1,3-1,4-glucanase from Bacillus circulans WL-12

Chitosanases 33 kDa and 40 kDa in size were detected in the culture supernatant of Bacillus circulans WL-12. One of the two chitosanases, chitosanse 40 (40-kDa chitosanase) has been shown to be identical to the enzyme which has been reported previously as a beta-1,3-1,4-glucanase by Bueno et al. The enzyme has been classified into family 8 glycosyl hydrolases together with the enzymes formally known as cellulase family D. This enzyme named chitosanase 40/beta-1,3-1,4-glucanase hydrolyzed both chitosan and beta-1,3-1,4-glucan with similar efficiency. However, the production of the enzyme was induced with chitosan but not by beta-1,3-1,4-glucan. Therefore, it seems possible that the major substrate of this enzyme is chitosan rather than beta-1,3-1,4-glucan. Analysis of degradation products generated from partially N-acetylated chitosan showed that chitosanase 40/beta-1,3-1,4-glucanse hydrolyzes GlcN-GlcN and GlcN-GlcNAc linkages but not GlcNAc-GlcNAc nor GlcNAc-GlcN. The specificity for hydrolyzing linkages of this enzyme is similar to that of the chitosanase from S. griseus HUT6037.     

Inter-isoformal regulation of nitric oxide synthase through heteromeric dimerization

A 130-kD protein that coimmunoprecipitates with the tight junction protein ZO-1 was bulk purified from Madin-Darby canine kidney (MDCK) cells and subjected to partial endopeptidase digestion and amino acid sequencing. A resulting 19-amino acid sequence provided the basis for screening canine cDNA libraries. Five overlapping clones contained a single open reading frame of 2,694 bp coding for a protein of 898 amino acids with a predicted molecular mass of 98,414 daltons. Sequence analysis showed that this protein contains three PSD-95/SAP90, discs-large, ZO-1 (PDZ) domains, a src homology (SH3) domain, and a region similar to guanylate kinase, making it homologous to ZO-1, ZO-2, the discs large tumor suppressor gene product of Drosophila, and other members of the MAGUK family of proteins. Like ZO-1 and ZO-2, the novel protein contains a COOH-terminal acidic domain and a basic region between the first and second PDZ domains. Unlike ZO-1 and ZO-2, this protein displays a proline-rich region between PDZ2 and PDZ3 and apparently contains no alternatively spliced domain. MDCK cells stably transfected with an epitope-tagged construct expressed the exogenous polypeptide at an apparent molecular mass of approximately 130 kD. Moreover, this protein colocalized with ZO-1 at tight junctions by immunofluorescence and immunoelectron microscopy. In vitro affinity analyses demonstrated that recombinant 130-kD protein directly interacts with ZO-1 and the cytoplasmic domain of occludin, but not with ZO-2. We propose that this protein be named ZO-3.