DNA formed an insoluble complex on mixing with chitosan (poly-D-glucosamine) in solution. The DNA content of the complex was about 50% and the DNA remained insoluble in aqueous media of pH 2-7%; e.g., on treatment of the DNA-chitosan complex with phosphate-buffered saline at pH 7 and 37 degrees C for 26 h, the DNA released into the aqueous phase was less than 0.05%. Obviously, DNA and chitosan formed a tight complex due to ionic interactions. The DNA can be solubilized by treatment with 0.1 N NaOH. RNA and other polynucleotides formed similar insoluble complexes with chitosan. The DNA attached to chitosan can be digested with a mixture of DNase I and phosphodiesterase. Cytosine residues in the DNA (denatured DNA) can be deaminated by treatment with sodium bisulfite, forming uracil DNA-chitosan. The uracil DNA-chitosan served as a substrate for uracil DNA glycosylase. Using polynucleotide-chitosan as an adsorbent, the affinities of reagents for polynucleotides can be determined directly. With this technique it was found that carcinogenic heterocyclic amines have an affinity for RNA as well as DNA. The results with homopolyribonucleotide-chitosans as adsorbents for 4 heterocyclic amines indicated that the binding occurs in a purine nucleotide-specific manner. These results suggest that the polynucleotides in the chitosan complex are accessible to enzymes and reagents. This new derivative may be useful in chemical and biological studies of polynucleotides and substances interacting with polynucleotides.

Chitin↗

PubMed1997

Effects of cAMP and cGMP on L-type calcium channel currents in rat mesenteric artery cells.

L-type Ca2+ channel currents in cultured rat mesenteric artery smooth muscle cells were recorded by the cell-attached patch-clamp technique. Depolarizing voltage steps from a holding potential of -40 mV elicited voltage-dependent inward Ba2+ currents. The inward currents were inhibited by nifedipine (10 microM) but enhanced by Bay K 8644 (5 microM), which suggests that the inward currents are carried almost exclusively by L-type Ca2+ channels. Application of dibutyryl cAMP (0.1-1 microM) and forskolin (0.01-1 microM) enhanced the activity of these Ca2+ channels. The dibutyryl cAMP induced enhancement of Ca2+ channels was antagonized by the serine/threonine kinase inhibitor H-8 (1 microM). Application of 8-bromo-cGMP (0.01-1 microM) and the cGMP inducer nitroglycerin (0.01-1 microM) inhibited the activity of these Ca2+ channels, and the inhibition of channel activity induced by 8-bromo-cGMP was antagonized by the serine/threonine kinase inhibitor H-8 (1 microM). These results suggest that in rat mesenteric artery cells, the L-type Ca2+ channel current is enhanced by a rise in intracellular cAMP levels and suppressed by a rise in intracellular cGMP levels. Furthermore, cGMP-induced Ca2+ channel inhibition may play a role in the expression of the nitric oxide-mediated vasodilating action of drugs such as nitroglycerin and atrial natriuretic peptide.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1997

Growth hormone increases serum 1,25-dihydroxyvitamin D levels and decreases 24,25-dihydroxyvitamin D levels in children with growth hormone deficiency.

The influence of growth hormone (GH) on calcium-phosphorus metabolism and modulation of vitamin D metabolism has been demonstrated, but the mechanism remains unclear. We investigated the effect of a 6-month course of GH therapy on vitamin D and mineral metabolism in twelve GH-deficient children. Before GH therapy, levels of vitamin D metabolites and other biochemistry data were within normal ranges. All patients responded to GH therapy with increased growth velocity. 1,25-Dihydroxyvitamin D levels increased after 1 month of treatment and remained at these higher levels, with a significant increase found at 3 months (P < 0.05), whereas 24,25-dihydroxyvitamin D levels were decreased at 1 and 3 months, the latter being a significant decrease (P < 0.05), and then returned to the baseline levels at 6 months. 25-Hydroxyvitamin D levels did not change significantly. A significant increase in serum insulin-like growth factor-I (IGF-I) levels occurred during the 6 months of treatment (1 month, P < 0.01; 3 and 6 months, P < 0.001). Serum parathyroid hormone (PTH) levels decreased significantly at 3 and 6 months (3 months, P < 0.01; 6 months, P < 0.05). Serum calcium and phosphorus levels did not change significantly. Significant increases were found in the urinary calcium/urinary creatinine ratio (3 and 6 months, P < 0.05) and the percent tubular reabsorption of phosphorus levels (1 and 3 months, P < 0.05). The results of this study confirmed the actions of GH on renal tubules with increases in calcium excretion and phosphorus reabsorption, and indicate that the action of GH on modulating vitamin D metabolism may be IGF-I mediated, not PTH mediated.

24,25-Dihydroxyvitamin D 3↗

PubMed1997

3D analysis of human locomotion before and after caloric stimulation.

Human locomotion was analysed in the sagittal and coronal planes using a position detector system composed of 2 infrared video cameras and a data processor. Normal healthy volunteers with 8 marker points on the body were asked to walk in place (WIP) and on a treadmill (WOT). Vertical and medial/lateral (M/L) translational movements were measured. Head angular movements in the sagittal (pitch) and coronal (roll) planes were also analysed. Pitch movements counteracted the vertical head movements. Head movement was remarkably attenuated in the vertical axis compared with that of the trunk. However, head M/L movement showed no difference with that of the lower part of the body. Ice water caloric stimulation was introduced to cause acute unilateral vestibular deficit. The stride length and step cycle became small after caloric stimulation in WOT, but not in WIP. The characteristic change in locomotive pattern was a large lateral sway of the hip joint occurring to the side away from calorization (right) during one foot standing (right foot) (t-test, p = 0.057). Measurement of M/L sway amplitude showed an increase only at the hip joint. As the changes in head and neck movements were not significant after caloric stimulation, it appears that the vestibulo-spinal reflex contributes little to maintaining the dynamic balance of the upper body. The importance of the hip joint for locomotion (hip strategy) was confirmed from the present data.

Adult↗

PubMed1997

Localization of heat shock protein in osteoarthritic cartilage.

In osteoarthritic (OA) chondrocytes, expression of 70 kD heat shock protein (HSP70) is enhanced under stress. The HSP70 level is also related to OA severity in humans. In the present study, specimens of OA cartilage were obtained from 14 surgically treated female patients. Their disease severity was evaluated according to Mankin's histological/histochemical grading scores. The ratio of HSP70-positive cells was examined by using immunohistochemistry. As OA became more severe, the ratio of HSP70-positive cells increased in deeper areas from the surface, and there was a positive correlation between the grade and the ratio. Distribution of cells expressing HSP70 mRNA detected by in situ hybridization was similar to the distribution of positive cells in the immunohistochemistry. It was presumed that HSP70 plays a role in cell protection from stress, and that mechanical and biological factors which are related to the enhancement of HSP70 expression, have a considerable influence on OA progressions.

Aged↗

PubMed1997

Excitatory amino acid receptors in the paraventricular hypothalamic nucleus mediate pressor response induced by carotid body chemoreceptor stimulation in rats.

In urethane-anesthetized rats with spinal transection, antagonists of excitatory amino acid receptors, P2 purinoceptors and adrenoceptors were microinjected into the paraventricular hypothalamic nucleus (PVN) and their effects on the pressor response evoked by carotid body chemoreceptor stimulation were examined. Microinjections of the non-selective excitatory amino acid antagonist kynurenate, the non-NMDA receptor antagonist CNQX and the NMDA antagonist 2-amino-5-phosphonovalerate (AP5) into the PVN inhibited the chemoreceptor reflex-induced pressor response. The excitatory amino acid agonist L-glutamate injected into the PVN produced an increase in blood pressure. The P2 purinoceptor antagonist suramin did not affect the pressor response and ATP did not affect basal blood pressure. The alpha adrenoceptor antagonist phentolamine, prazosin and yohimbine also inhibited the chemoreceptor-induced pressor response, while the beta antagonist propranolol did not affect it. These findings indicate that excitatory amino acid receptors and alpha adrenoceptors in the PVN are involved in mediating the pressor response induced by carotid body chemoreceptor stimulation in rats.

2-Amino-5-phosphonovalerate↗

PubMed1997

[Clinical features and utricular dysfunction in patients with benign paroxysmal positional vertigo].

We report the clinical features in patients with benign paroxysmal positional vertigo (BPPV), who met the following criteria; (1) a history of brief episodes of positional vertigo, and (2) a characteristic torsional paroxysmal positional nystagmus that was observed visually. The mean age of onset was 55 years. There was no sex predominance. In most patients, positional vertigo disappeared within 2 months. We then used the eccentric rotatory test to examine the patients otolithic function. In the eccentric rotatory test, a patient is rotated around an axis located behind his head (eccentric rotation, ECR). We have demonstrated that the enhancement of vestibulo-ocular reflex (VOR) gain in ECR can be used as an index of the utricular function. Enhancement of VOR gain was observed in ECR to the lower ear in positional vertigo, but not to the uppermost ear. The findings suggest an imbalance in utricular function in patients with BPPV. Otoconia dislodged from the deranged utricle may be a source of canalolithiasis/cupulolithiasis.

Adult↗

PubMed1997

Diagnosis of fetal anomalies by three-dimensional imaging using helical computed tomography.

We present the first report on the use of helical computed tomography (CT), a new, non-invasive diagnostic technique that produces three-dimensional (3-D) images, in prenatal diagnosis. This technique was used to construct 3-D images in the prenatal diagnosis of two anomalous fetuses. The 3-D images provided clear information about the anomalies: trisomy 18 in one case and cystic hygroma in the other. In one case, rapid intervention after a planned Caesarean section prevented respiratory distress. Surgery to correct the anomaly was performed 2 days postnatally; the infant recovered uneventfully.

Adult↗

PubMed1997

[Shearing injuries of parasagittal white matter, corpus callosum and basal ganglia: possible radiological evidences of hemiplegia in diffuse axonal injury].

The relationship between spastic hemiplegia in diffuse axonal injury (DAI) and neuroradiological findings was studied in 100 cases. These cases were prospectively collected from the files of Automobile Insurance Rating Organization in Japan between 1993 from to 1996. Requirements for entry to this study were as follows: presence of initial unconsciousness after head injury without any lucid interval. Existence of CT scan or MRI film obtained within 12 hours of injury showing no significant mass effects, as well as follow-up CT scan or MRI film obtained more than 3 months after the injury. Psychosocial outcome was described both by the medical professional and the caregiver. The hemiplegia was rated severe, mild, or none. The outcome and diffuse ventriculomegaly were classified as reported by the authors previously. Spastic hemiplegia or quadriplegia was documented in the chronic stage in 63 cases, including 53 severe cases with difficulty in walking and 10 mild cases with only pyramidal signs detected. Chi-square analysis showed significant correlation between hemiplegia and the DAI outcome level or ventriculomegaly rating. Focal brain contusion was noticed in 33 cases, but did not correlate with the hemiplegia at all. Radiological findings included 25 cases of parasagittal white matter injury (gliding contusion), 20 cases of callosal injury, 19 cases of basal ganglionic region injury, 5 cases of brain-stem injury, and 3 cases of cerebellar injury. Chi-square analyses of hemiplegia and contralateral presence of these injuries were significant in the former three types of injury. Presence of at least one of these 3 lesions was defined as GCB injury. There were altogether 46 GCB injury cases which were significantly correlated with contralateral hemiplegia by chi-square analysis and by Spearman rank analysis. Partial correlation analysis with hemiplegia as the target variable indicated highly significant correlation only with GCB injury and outcome level. In conclusion, spastic hemiplegia in DAI is a manifestation of primary shear injury. Neuroradiological findings of GCB injury were statistically able to be significantly correlated with the presence of hemiplegia, and suggested pyramidal tract injury either at the corona radiata or the internal capsule level.

Adolescent↗

PubMed1997

The proportion of fetal nucleated red blood cells in maternal blood: estimation by FACS analysis.

The purpose of this study was to determine the proportion of fetal nucleated red blood cells (NRBCs) among enriched NRBCs and to evaluate the effectiveness of enriching NRBCs in maternal blood using fluorescence-activated cell sorting (FACS) to separate NRBCs. The origin of enriched NRBCs was determined using fluorescence in situ hybridization (FISH) methods. Y-specific signals were observed in 4.6 +/- 1.5 per cent of the enriched cells from 14 of 16 (87.5 per cent) pregnant women who gave birth to boys. In this series, the specificity of the fetal sex diagnosis was 100 per cent, the sensitivity 88 per cent, and the negative predictive value 86 per cent. Fetal NRBCs are present in maternal blood and FACS has the potential to enrich fetal NRBCs. Fetal cells were estimated to be enriched more than 10,000-fold in the first trimester and more than 100-fold in the third trimester. Average frequencies of fetal cells in maternal blood were 8.1 x 10(-5) and 1.6 x 10(-5) in the first trimester and the second/third trimesters. However, most of the NRBCs in maternal blood are maternal in origin.

Antigens, CD↗

PubMed1997

Excitatory synaptic transmission in the rat medical vestibular nucleus.

In vitro brainstem slice preparations have provided the opportunity to obtain accurate information about the neuropharmacology of the vestibular system. By stimulating the root of the eighth cranial nerve, excitatory synaptic transmission in the rat medial vestibular nucleus has been observed and analyzed with extra- and intra-cellular recordings. Recently, tight-seal, whole-cell recordings, perforated whole-cell recordings and microfluorometry of Ca2+ were applied to in vitro brainstem slice preparations to obtain detailed information. Excitatory synaptic transmission to MVN neurons is reviewed.

Animals↗

PubMed1997

Transformation of rice mediated by Agrobacterium tumefaciens.

Agrobacterium tumefaciens has been routinely utilized in gene transfer to dicotyledonous plants, but monocotyledonous plants including important cereals were thought to be recalcitrant to this technology as they were outside the host range of crown gall. Various challenges to infect monocotyledons including rice with Agrobacterium had been made in many laboratories, but the results were not conclusive until recently. Efficient transformation protocols mediated by Agrobacterium were reported for rice in 1994 and 1996. A key point in the protocols was the fact that tissues consisting of actively dividing, embryonic cells, such as immature embryos and calli induced from scutella, were co-cultivated with Agrobacterium in the presence of acetosyringonc, which is a potent inducer of the virulence genes. It is now clear that Agrobacterium is capable of transferring DNA to monocotyledons if tissues containing 'competent' cells are infected. The studies of transformation of rice suggested that numerous factors including genotype of plants, types and ages of tissues inoculated, kind of vectors, strains of Agrobacterium, selection marker genes and selective agents, and various conditions of tissue culture, are of critical importance. Advantages of the Agrobacterium-mediated transformation in rice, like on dicotyledons, include the transfer of pieces of DNA with defined ends with minimal rearrangements, the transfer of relatively large segments of DNA, the integration of small numbers of copies of genes into plant chromosomes, and high quality and fertility of transgenic plants. Delivery of foreign DNA to rice plants via A. tumefaciens is a routine technique in a growing number of laboratories. This technique will allow the genetic improvement of diverse varieties of rice, as well as studies of many aspects of the molecular biology of rice.

Agrobacterium tumefaciens↗