PARVB overexpression increases cell migration capability and defines high risk for endophytic growth and metastasis in tongue squamous cell carcinoma.

BACKGROUND: Tongue squamous cell carcinoma (TSCC) is highly diverse, even in its early stages. This cancer is classified into three subtypes (superficial, exophytic, and endophytic) based on macroscopic appearance. Of these subtypes, the endophytic tumours have the worst prognosis because of their invasiveness and higher frequency of metastasis. METHODS: To understand the molecular mechanism underlying the endophytic subtype and to identify biomarkers, we performed a comprehensive gene expression microarray analysis of clinical biopsy samples and also confirmed the clinical relevance of differential gene expression. RESULTS: Expression of the parvin-beta (PARVB) gene and its encoded protein was significantly upregulated in endophytic-type TSCC. PARVB is known to play a critical role in actin reorganization and focal adhesions. Knockdown of PARVB expression in vitro caused apparent decreases in cell migration and wound healing, implying that PARVB has a crucial role in cell motility. Moreover, metastasis-free survival was significantly lower in patients with higher tumour expression of PARVB. CONCLUSIONS: These findings suggest that PARVB overexpression is a candidate biomarker for endophytic tumours and metastasis. This protein may be a clinically useful target for adjuvant TSCC therapy.

Lateral dominance of stretch-shortening cycle performance in unilateral and bilateral athletes.

AIM: Few studies have been conducted on the lateral dominance of stretch-shortening cycle (SSC) performance. This study aimed to make an index for evaluation of the ability to use SSC for powerful elbow flexion and to examine the lateral dominance of SSC performance in unilateral and bilateral athletes. METHODS: Thirty-three male athletes (19.9+/-1 years) participated in this study. Each subject pulled a submaximal load (25% of maximum voluntary contraction [MVC]) in a single burst via elbow flexion of the dominant and non-dominant upper limb from the following two preliminary conditions: a static relaxed muscle state (SR condition) and using a counter-movement (SSC condition). Muscle power was measured accurately with a power instrument containing a rotary encoder, and the SSC index was calculated in order to evaluate the ability to use SSC. RESULTS: For the SSC index, the non-dominant upper limb showed higher values than the dominant upper limb. The unilateral athletes showed high values of power output when using only SSC in the dominant upper limb compared with bilateral athletes. The correlation in the SSC index between dominant and non-dominant limbs was found to be low. CONCLUSION: The non-dominant limb is superior to the dominant limb in the ability to use SSC in the upper limbs and there is the possibility of left side dominance. Unilateral athletes were superior to bilateral athletes in their ability to use SSC with their dominant limb, but a transfer of learning to the non-dominant limbs was not apparent.

Effects of diltiazem on left ventricular diastolic behavior in patients with hypertrophic cardiomyopathy: evaluation with exercise pulsed Doppler echocardiography.

To estimate the effects of diltiazem on the left ventricular diastolic abnormalities in patients with hypertrophic cardiomyopathy, transmitral flow velocity during diastole was studied before and immediately after dynamic leg exercise with the pulsed Doppler technique combined with two-dimensional echocardiography. Seventeen patients with hypertrophic cardiomyopathy and 24 apparently healthy men performed bicycle ergometer exercise in the supine position with the target heart rate set at 120 beats/min. The patients with cardiomyopathy were directed to perform the exercise at the same intensity after receiving 30 to 60 mg of diltiazem, three times daily, for 1 or 2 weeks. The pattern of transmitral flow velocity in diastole had two components, one corresponding to the rapid filling phase in early diastole and the other to the atrial contraction phase in late diastole. To assess left ventricular diastolic behavior, the following variables were analyzed: peak velocity in the rapid filling and atrial contraction phases, the ratio of peak velocity in the atrial contraction phase to that in the rapid filling phase, and pressure half-time. The changes in peak velocity in the atrial contraction phase, pressure half-time and the ratio of peak velocity in the atrial contraction phase to that in the rapid filling phase with exercise differed significantly between patients with hypertrophic cardiomyopathy with no medication and control subjects. After diltiazem, the response of these variables to exercise was almost identical in the two groups. These results suggest that diltiazem can lessen the left ventricular diastolic abnormality in patients with hypertrophic cardiomyopathy on dynamic exercise of mild intensity.

Cytoplasmic architecture of the axon terminal: filamentous strands specifically associated with synaptic vesicles.

Cytoplasmic architecture of axon terminals in rat central nervous tissue was examined by quick-freeze deep-etch method to determine how synaptic vesicles and their associated cytoplasmic environment are organized in the terminal and to know how these structures participate in the mechanism for neurotransmitter release. The axoplasm is divisible into two domains: one occupied by mitochondria in the middle of the terminal, called the mitochondrial domain, the other situated in the periphery and exclusively filled with spherical synaptic vesicles, 50-60 nm in diameter, the synaptic vesicle domain. The most characteristic feature of the mitochondrial domain was the appearance of many microtubules connected with mitochondria by filamentous strands. Large vesicles, 80-100 nm in diameter, were preferentially associated with the mitochondrial domain, and linked with microtubules wherever they appeared. The cytoplasmic matrix of the synaptic vesicle domain showed a more fibrillar texture than that of the mitochondrial domain because of the distribution of filamentous strands associated with synaptic vesicles. These strands were significantly thicker and longer (mean 11.7 nm thick and 42.7 nm long) than those linking membrane-bound organelles to microtubules (mean 8.3 nm thick and 23.0 nm long), and connected vesicles to one another or to the plasma membrane, making a complicated network around the vesicles. Further, both strands were significantly different in dimension from actin filaments (mean 9.9 nm thick and 73.5 nm long) showing 5-nm axial periodicity. These strands, especially synaptic vesicle-associated ones including their network, were readily broken down in the most part by detergent treatment or chemical fixation, indicating that they are very delicate in nature. Granular materials, which are spherical and vary in size (6-20 nm in diameter), are also more conspicuous in the synaptic vesicle domain than in the mitochondrial domain. More fibrillar and granular cytoplasmic structure of the synaptic vesicle domain may be crucial for synaptic vesicles to perform an essential role in releasing the transmitter.

Evaluation by exercise Doppler echocardiography of maintenance of cardiac output during ventricular pacing with or without chronotropic response.

To examine the effectiveness of activity-initiated rate-responsive pacing, this study assessed the increases in stroke volume and cardiac output during randomized treadmill exercise in rate-responsive and fixed-rate ventricular (VVI) pacing in 10 patients. Stroke volume index and cardiac index were determined by suprasternal Doppler measurements. Compared with the findings during VVI pacing, the rate-responsive pacing was associated with (1) prolongation of exercise duration (8.0 +/- 4.0 vs 7.3 +/- 3.6 minutes, p less than 0.05); (2) greater exercise-induced positive chronotropic response (mean maximal heart rate 127 +/- 12 vs 78 +/- 15 beats/min, p less than 0.001); (3) smaller increase in stroke volume index (38 +/- 10 vs 50 +/- 11 ml/m2, p less than 0.001), and (4) greater increase in cardiac index (4.7 +/- 1.1 vs 3.9 +/- 1.0 liters/min/m2, p less than 0.001). A significant correlation was observed between age and percent increase in stroke volume index during VVI pacing (p less than 0.05). These findings indicate that VVI pacing increased stroke volume more than did rate-responsive pacing, especially in younger patients, but the increase in cardiac output was less than that seen with rate-responsive pacing due to the absence of chronotropic response. Accordingly, an activity-sensing, rate-responsive pacemaker can effectively increase the heart rate, significantly augment cardiac output and extend the duration of exercise.

Neuron-targeted gene transfer by adenovirus carrying neural-restrictive silencer element.

Adenovirus transfers genes to a wide range of cell types, but its application to neurons has been hampered by its reduced efficiency of infection as compared with that for glia. To achieve neuron-targeted gene transfer, we have produced an adenovirus carrying the reporter lacZ gene driven by the SCG10 minimum promoter containing the neural-restrictive silencer element (NRSE), which element selectively represses the transcription of genes in non-neuronal cells. When rat hippocampal slice cultures were infected with NRSE-bearing adenovirus, beta-galactosidase-positive cells were mostly pyramidal and granular neurons, whereas infection with virus carrying a mutated NRSE resulted in beta-galactosidase expression in both neurons and glia. The results suggest that the adenovirus carrying NRSE to be a useful tool for neurontargeted gene transfer.

Gene transfer into hippocampal slice cultures with an adenovirus vector driven by cytomegalovirus promoter: stable co-expression of green fluorescent protein and lacZ genes.

Virus-mediated gene transfer into identified neurons of organotypic hippocampal slice cultures offers a great potential for studying the cellular and molecular mechanisms of synaptic plasticity. We describe here a new adenovirus vector Ad-GFP-lacZ carrying an early cytomegalovirus (CMV) gene promoter that efficiently co-transferred the beta-galactosidase (lacZ) and green fluorescent protein (GFP) genes in rat organotypic hippocampal slice cultures. Monitoring of GFP fluorescence and immuno-histochemical staining for beta-galactosidase showed that the expression of the transferred genes was widespread in the glial cells and neurons of CA1, CA3/4, and dentate gyrus regions. Immunoblot analyses showed that the expression of gamma-galactosidase and GFP was maximal about 48 h after infection of hippocampal slices with the adenovirus vector and the expression levels were maintained for several weeks. Also, immunoblot analyses showed no significant differences in the MAP-2 and glial fibrillary acidic protein levels in the adenovirus vector infected and uninfected hippocampal slices. In addition, we found that the infection of hippocampal slices with the adenovirus vector caused no significant increase in the induction of heat shock protein (HSP)-70 and showed no change in their electrophysiological properties as measured by stable field synaptic potentials in CA1 region and its reactivity to high frequency stimulation. Our data suggest that this adenovirus vector can be exploited to transfer multiple genes into neurons and may have implications for developing strategies for gene therapy.

Effects of vasodilators on venous tone in vivo in dogs.

The authors investigated, in vivo, the effects of four vasodilators on venous tone in dogs. Baseline venous tone was determined from the pressure: diameter relationships in the inferior vena cava (VSIVC) and femoral vein (VSFV) as measured during several seconds of occlusion of the proximal inferior vena cava. All of the slopes were nearly linear. All vasodilators were administered in dosages sufficient to lower blood pressure by approximately 20%; these dosages also decreased systemic vascular resistance by 15% to 30%. Isosorbide dinitrate reduced VSIVC from 7.17 +/- 0.81 to 5.81 +/- 0.73 mmHg/mm and VSIVC from 59.4 +/- 13.5 to 37.2 +/- 6.6 mmHg/mm. Neither nifedipine nor nisoldipine altered VSIVC or VSFV. However, prazosin decreased VSIVC from 13.2 +/- 3.3 to 10.7 +/- 2.7 mmHg/mm and VSFV from 43.5 +/- 11.3 to 29.9 +/- 8.8 mmHg/mm. These results suggest that isosorbide dinitrate and prazosin decrease venous tone in vivo, whereas nifedipine and nisoldipine do not.

[Hemodynamic study of negative pressure ventilation using diaphragm pacing].

A negative pressure ventilation (NPV) by unilateral or bilateral diaphragm pacing (DP) was prepared for canine experiments. A shift from positive pressure ventilation (PPV) to NPV resulted in elevation of mean aortic pressure, increase in stroke volume and depression of mean pulmonary arterial pressure. Examination of the interaction between respiratory cycle and cardiac function during PPV, disclosed a reduction of right ventricular stroke volume and elevation of mean aortic and right ventricular end-diastolic pressure at end inspiration, compared to those at end expiration. During NPV with DP, left ventricular stroke volume, heart rate and mean aortic pressure were increased immediately after DP (immediately after inspiration) compared to those at end expiration. The experimental model of DP, in which respiratory condition could be easily altered, was considered to be useful to evaluate the effect of NPV on cardiac function.

Ultrastructure of the zonula adherens revealed by rapid-freeze deep-etching.

The zonula adherens (ZA) in adult chicken retinal pigment epithelium was examined with cryo-electron microscopic methods. Deep-etching of the cross-fractured ZA showed globules in the intercellular space. These globules apparently correspond to the electron-dense structure seen in thin sections. Deep-etching of obliquely fractured ZA further revealed rod-like structures extending from the extracellular surface into the intercellular space. These rods (mean approximately 9 nm thick, approximately 20 nm long) were straight and sometimes divided into two or three segments. The rods typically canted at approximately 60 degrees with respect to the plasma membrane, and they were often connected to the intercellular globules at their distal ends. When the rods are compared with the isolated cadherins reported previously, it is suggested that a combination of a rod and a globule may represent an extracellular part of cadherin. Membrane particles were observed on the P-face of the ZA plasma membrane, and their distribution density was approximately seven times that of the rods. The freeze-etching also revealed a characteristic particle complex on the ZA cytoplasmic surface, which may represent the cytosolic proteins linking cadherins to actin bundles.

Ultrastructure of intermediate filaments of nestin- and vimentin-immunoreactive astrocytes in organotypic slice cultures of hippocampus.

Glial cells in rat hippocampal slices cultured for 4 weeks were examined with immunocytochemical and cryoelectron microscopical methods. Astrocytes possessing long processes were similarly stained with antibodies against nestin, vimentin, and glial fibrillary acidic protein as seen by confocal microscopy. The three antibodies also labeled intermediate filaments in these astrocytes. In order to examine the fine structure of these intermediate filaments, slices were rapid-frozen for freeze-substitution and freeze-etching. By freeze-substitution the processes of the astrocytes were packed with large hundles of intermediate filaments. In rapid-freeze deep-etched slices, these filaments were often interconnected with filamentous cross-bridges. These cross-bridges were rather uniform in size and shape (mean 2.9 nm thick and 14.8 nm long). These results suggest that the filament network with these cross-linkers is important for shaping the long processes of nestin- and vimentin-immunoreactive astrocytes in slice cultures.

Dendritic spine density and LTP induction in cultured hippocampal slices.

Transverse hippocampal slices were cut from 8- to 9-day-old rats and maintained in an interface chamber for periods of 1-4 wk, in tissue culture conditions. Neurons in the slice preserved their spatial organization and connectivity. Dendritic spine density in CA1 neurons was very low at 1 wk in culture, and long, filopodia-like structures were abundant. Spine density increased in these neurons nearly threefold during the course of 3 wk in vitro, to approach values of those of the normal, in vivo hippocampus. The magnitude of long-term potentiation (LTP) of reactivity of Ca1 to stimulation of CA3 neurons also increased during weeks in culture in parallel with the change in spine density. Chronic exposure of slices to drugs that interact with synaptic activity caused changes in their dendritic spine density. Blockade of the N-methyl-D-aspartate (NMDA) receptors with the receptor antagonist 2-aminophosphonovalerate (D-APV) or blockade of action potential discharges with tetrodotoxin (TTX) prevented dendritic spine development in immature cultures. Enhancing synaptic activity by blockade of GABAergic inhibition with picrotoxin did not affect spine density to a significant degree. D-APV-treated slices expressed larger LTP than controls. TTX-treated slices expressed smaller LTP than controls. Picrotoxin treated slices did not express LTP. It is proposed that LTP and dendritic spine density are correlated strongly during development, whereas they are not correlated in the more mature slice/culture of the hippocampus where spine density can be modulated by chronic exposure to blockers of synaptic activity, which will not affect LTP in a similar manner.

Evidence for the transport of opsin in the connecting cilium and basal rod outer segment in rat retina: rapid-freeze, deep-etch and horseradish peroxidase labelling studies.

In order to clarify the pathway of opsin transport in the connecting cilium and basal rod outer segment, we examined rat rod cells by a rapid-freeze and deep-etch technique and also examined the uptake of horseradish peroxidase into isolated retina. The distribution of intramembrane particles on the P-face of the cilium indicated that the ciliary plasma membrane has similar opsin content to the basal rod outer segment plasma membrane. Dilated cisternae were detected below the stack of disk membranes at the basal rod outer segment in fresh retina. The fine structure of the P-face and true surface of these cisternae was identical to that of the disk membrane. Uptake of horseradish peroxidase was detected in the cisternae or in both cisternae and most basal disk, indicating that the cisternae are formed prior to the disk membrane. In the distal part of connecting cilium, we found axially oriented infoldings on the P-face of the plasma membrane, and subplasmalemmal tubules or cisternae adjacent and parallel to them. Such subplasmalemmal membranes were labeled by exogenous horseradish peroxidase, suggesting that the infoldings are invaginating plasma membrane. These results may indicate that opsin molecules are conveyed on the ciliary plasma membrane, and that this opsin-rich plasma membrane is internalized in the distal connecting cilium to form dilated cisternae, which subsequently change to the disk membranes.

Direct visualization of a transmembrane channel associated with ribosomes.

Examination of directly frozen rough endoplasmic reticulum (ER) of retinal pigment epithelial cells by freeze-fracture and freeze-substitution revealed distinct paired transmembrane proteins associated with membrane ribosomes. Ribosomal subunits on intact ER membrane are directly visualized for the first time, providing a global view of the structure of the ribosome and the corresponding structures on the ER membrane. The ribosomal intersubunit cleft appears to be continuous with a cleft between paired transmembrane proteins that extends into the lumen of the ER. This continuous cleft may be the path taken by nascent polypeptides.

Dependency of the pulsed Doppler-derived transmitral filling profile on the sampling site.

In previous reports that evaluated pulsed Doppler transmitral filling, the sampling site has varied; we examined the effect of the sample volume location on Doppler measurements. Studied were 97 patients: 58 with normal echocardiograms, 20 with mitral regurgitation, and 19 with miscellaneous cardiac diseases. Transmitral filling was recorded at the mitral anulus and at the left atrial and left ventricular (LV) sides of the mitral tips. As the sample volume was moved from the mitral anulus to the LV side, the peak velocity and time-velocity integral of early diastole increased (40.6 +/- 13.8 versus 59.0 +/- 19.0 cm/sec, 5.26 +/- 1.65 versus 8.35 +/- 2.37 cm; p less than 0.001) as did those of late diastole (48.7 +/- 11.5 versus 57.5 +/- 17.0 cm/sec, 3.48 +/- 0.97 versus 4.59 +/- 1.39 cm; p less than 0.001). The late-to-early diastolic peak velocity and time-velocity integral ratios and the late-to-total diastolic time-velocity integral ratio decreased (1.33 +/- 0.51 versus 1.06 +/- 0.41, 0.71 +/- 0.24 versus 0.58 +/- 0.19, 0.40 +/- 0.09 versus 0.36 +/- 0.08; p less than 0.001). The dependency of these indices on the sampling site was in the same degree in all three groups. However, the peak filling rate normalized to mitral stroke volume (4.71 +/- 1.43 versus 4.63 +/- 1.32 l/sec; p = NS) was not influenced by the sample volume location; thus this parameter may be more reliable for assessing LV filling.

Characteristic findings on the standard 12-lead ECG in patients with the fasciculoventricular Mahaim fiber.

Standard 12-lead electrocardiograms with a Q wave in lead V1 were obtained from 32 subjects without organic cardiac disease and analyzed for features that might characterize an abnormal atrioventricular conduction through the fasciculoventricular Mahaim fiber. Following an infusion of ajmaline, the Q wave in V1 vanished abruptly and changed to an rS pattern in the 12 ajmaline responders. Discriminant analysis was performed to distinguish the ajmaline responders from the others. The explanatory variables were number of precordial leads with the abnormal Q wave, existence of the septal q waves, existence of the slurring of the Q wave in V1, existence of clockwise rotation, and existence of high voltage (RV5 + SV1 > 3.5 mV). Three variables, the absence of the septal q waves, the presence of the slurring, and the absence of clockwise rotation, were found to predict a positive response to ajmaline (discriminant probability = 77%). These findings associated with the Q wave in V1 suggest that the fasciculoventricular fiber may be present.

Topography of opsin within disk and plasma membranes revealed by a rapid-freeze deep-etch technique.

Rod outer segments in fresh rat retinas were examined by a rapid-freeze, deep-etch technique to explore how membrane proteins are organized at the macromolecular level. Cross-fractures revealed that intradiscal membranes are adherent to each other except at the rim. When an isolated fresh retina was incubated in a hypotonic solution for a few minutes, the interdiscal space was expanded and the cytoplasmic surface of the disk membrane was found to be covered with protrusions except at the rim. A few particles were scattered among the protrusions and were attached to the cytoplasmic surface. Since the distribution density of the cytoplasmic surface protrusions was similar to that of the P-face particles, which are known to reflect opsins, the protrusions were considered to be portions of opsins extending into the cytoplasm. The intradiscal surfaces in chemically-fixed retinas were rather smooth and were labelled with anti-opsin antibodies and wheat germ agglutinin. The true surfaces of the plasma membrane were found to be similar in fine structure to those of the disk. A model of the macromolecular organization of rod outer segments is proposed on the basis of these observations. The model shows apposed opsins within a disk membrane adhering to one another except at the rim. These opsins, as well as those in the plasma membrane, are minimally exposed to the extracellular surface, but protrude deeply into the cytoplasm.