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.

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.

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.

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.

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.

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.

Left ventricular function in asthmatic children chronically treated with theophylline evaluated by exercise Doppler echocardiography.

To evaluate the chronic effects of theophylline on cardiac function, M-mode and pulsed Doppler derived variables were measured at rest and the suprasternal continuous wave Doppler measurement of ascending aortic flow was used during treadmill exercise testing. Subjects consisted of 13 children with stable asthma (mean 11.7 +/- 2.2 years) who were treated with theophylline for at least one year and 16 age-matched, untreated normal volunteers. In the resting state, the chronic administration of theophylline seemed to produce a slight increase in percent fractional shortening, outflow peak velocity and atrial contribution to ventricular filling in the asthmatic children as compared to normals, but these changes were not statistically significant. The asthmatic children showed significantly lower values than the controls in exercise induced changes in the peak velocity, stroke index and cardiac index, but not in the heart rate. Therefore, chronic administration of theophylline appears to have a minimal effect on resting cardiac function, but a possibly deleterious effect on the cardiac response to exercise testing.

Discrepancy between systolic and diastolic dysfunction of the left ventricle in patients with Duchenne muscular dystrophy.

To assess the systolic and diastolic dysfunction of the left ventricle (LV) in relation to age and the severity of impairment in Duchenne muscular dystrophy (DMD), we performed M-mode, two-dimensional and pulsed-wave Doppler echocardiography in 45 male subjects with DMD aged 8 to 25 years and in 40 age-matched healthy controls. Systolic dysfunction started in the first decade of life, with some patients showing severe systolic dysfunction in their early teens. This dysfunction, however, did not always depend on the severity of the skeletal muscle disease. No patients with DMD showed an increase in peak atrial velocity and time-velocity integrals of the atrial contraction velocity curve, findings frequently reported to precede the abnormalities in many cardiac diseases; it was thought therefore that these patients had no increase in left atrial compensation. Diastolic dysfunction may not routinely precede or accompany the systolic dysfunction in DMD, in contrast with what is reported in patients with ischaemic or hypertensive heart disease. DMD patients usually show a predominant systolic dysfunction.

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.

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.

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.

Determinants of left ventricular filling dynamics: alteration in the Doppler-derived transmitral filling profile with progressive impairment of cardiac function in a dog preparation.

To clarify the factors determining transmitral filling, left ventricular and atrial pressures (LVP and LAP) and Doppler-derived diastolic indices were analysed in six anaesthetized dogs at various right atrial pacing rates during dextran infusion. The relationship of the late to early diastolic peak velocity ratio (A/E ratio) to end-diastolic LVP (LVEDP) showed a quadratic curve concave to the LVEDP axis in five animals (r2 = 0.320-0.588). An elevation in LVEDP up to 25 mmHg accompanied an increase in A/E ratio (ascending limb), and further LVEDP elevation caused its inverse decline (descending limb). Multiple regression analysis indicated that A/E ratio correlated positively with maximal LVP, a-wave LAP and heart rate, and negatively with v-wave LAP in both limbs. The time constant of isovolumic LVP decline, which was prolonged as LVEDP was elevated, was a positive correlate of A/E ratio in the ascending limb, but lost its influence on A/E ratio in the descending limb. An elevation in v-wave LAP must have masked the expected effect of left ventricular relaxation abnormality on A/E ratio in this limb. Thus, the transmitral filling profile did not alter unidirectionally, but returned to that seen before volume loading, with a simultaneous progressive impairment of cardiac function.

Metabolism of pravastatin sodium in isolated rat hepatocytes. I. Glutathione conjugate formation reaction.

1. The metabolic fate of pravastatin sodium (sodium (+)-(3R,5R)-3,5-dihydroxy-7-((1'S,2'S,6'S,8'S,8'aR)-6'-hydroxy-2'methyl- 8'-[(S)-2"-methylbutyryloxy]-1',2',6',7',8', 8'a-hexahydro-1'-naphthyl) heptanoate) was studied in isolated rat hepatocytes. 2. Two polar metabolites were isolated and identified as a glutathione conjugate and a dihydrodiol. 3. Both metabolites were formed via an epoxide which has been identified as the 4'a beta,5' beta-epoxide on the decalin moiety. 4. Formation of the glutathione conjugate was enzymic, while the dihydrodiol was formed by non-enzymic hydrolysis of the epoxide accompanied by the intramolecular migration of the double bond.

Role of left atrial booster pump function in a worsening course of congestive heart failure.

We evaluated changes in left ventricular (LV) preload and the Doppler-derived transmitral late to early diastolic peak velocity ratio (A/E ratio) during the exercise in 27 patients with ischemic heart disease. After the exercise, A/E ratio decreased in 16 patients with a remarkable elevation in LV preload, and increased in 11 with a mild elevation. Further, Doppler transmitral flow in conjunction with pulmonary venous flow and hemodynamic parameters were analyzed in 11 dogs during a worsening course of heart failure induced by dextran infusion. The relationship of A/E ratio to LV end-diastolic pressure showed a quadratic curve concave to the pressure axis. A/E ratio, an index expressing left atrial (LA) contribution to LV filling, returned to that seen before volume loading under the condition of cardiac dysfunction. Pulmonary venous reflux fraction determined as the ratio of peak velocity of pulmonary venous reflux during LA systole to the sum of systolic and diastolic peak velocities of pulmonary venous antegrade flow, did not increase here. In this situation, blood could not be ejected from the left atrium into the left ventricle and even into the pulmonary veins during LA contraction. Finally, LV filling was not compensated by the left atrium, and LA booster pump function itself was deteriorated.

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.

Metabolism of pravastatin sodium in isolated rat hepatocytes. II. Structure elucidation of the metabolites by n.m.r. spectroscopy.

1. The structures, including stereochemistry, of the two major metabolites of pravastatin sodium in an isolated rat hepatocyte system, i.e. the 4'a alpha-glutathione conjugate (CM-1) and the 3',5'-dihydrodiol (CM-2), were determined by one- and two-dimensional n.m.r. spectroscopy. 2. The structures of two synthetic pravastatin epoxides, possible precursors of the metabolites, were also established. 3. One of the synthetic epoxides, 4'a beta, 5' beta-epoxide was converted to the pravastatin metabolite, 4'a alpha-glutathione conjugate (CM-1) by a rat liver cytosol system and is proposed as the common metabolic intermediate between pravastatin sodium and the metabolites, CM-1 and CM-2.