Shigenori Kitaoka

Two modes of human rotavirus entry into MA 104 cells.

SummaryEntry of the KUN strain of human rotavirus into MA 104 cells was studied by electron microscopy. Virus particles attached to the cell membrane appeared to be almost exclusively double-shelled virions. These attached virions followed two distinct pathways into the cytoplasm depending on pretreatment with trypsin. Using infectious rotavirus which had been pretreated with trypsin, the viral nucleoids passed directly into the cytoplasm within 5 minutes after inoculation, through dissolution of the viral capsid and cell membrane. Using non-infectious rotavirus that had not been pretreated with trypsin, phagocytosis or pinocytosis occurred in which virions were sequestered into lysosomes 20 minutes after virus attachment to the cell membrane. After being sequestered, uncoating of the rotavirus virions within lysosomes was seen, but it did not result in release of the genome.On the basis of these observations it was concluded that when virions were pretreated with trypsin, virus replication occurred following the direct passage of viral nucleoids into the cell cytoplasm. However, mere phagocytosis of virus particles into cell lysosomes, which occurred when trypsinuntreated virus was used, does not appear to be related to rotavirus replication.

Evidence for endocytosis-independent infection by human rotavirus.

SummaryThe effects of five lysosomotropic drugs (NH4Cl, chloroquine, methylamine, amantadine and dansylcadaverine) and cytochalasin B on human rotavirus (HRV KUN strain) and vesicular stomatitis virus (VSV Indiana strain) infection in monkey MA 104 cells were examined. These drugs had little effect on HRV yield but greatly reduced VSV yield. The results strongly suggest that HRV does not require endocytotic activity and intracellular acidic vesicles for the initial stage of infection and support our postulate that HRV enters the target cell by direct penetration of its nucleoid through the cell membrane.

Experimental study of afterload-reducing therapy: the effects of the reduction of systemic vascular resistance on cardiac output, aortic pressure and coronary circulation in isolated, ejecting canine hearts.

The relationship between cardiac output (CO) and peripheral resistance (Rp) was examined under the following conditions for coronary perfusion: constant coronary flow perfusion; perfusion with a pressure equal to mean aortic pressure (AoP perfusion); and perfusion with a pressure equal to the mean AoP - 30 mm Hg (AoP - 30 mm Hg perfusion). We also examined the coronary pressure-flow relationship. For these studies, we used paced, isolated, ejecting canine hearts, which were loaded by a hydraulic system that simulated the input impedance of a dogs systemic arterial tree.The CO in the constant coronary flow perfusion continued to increase with the reduction of Rp. The CO in the AoP perfusion became maximal at a slightly subphysiologic Rp, or at an average mean AoP of 65 mm Hg. This mean AoP was closely associated with the lower limit of the autoregulation of coronary blood flow. In the AoP - 30 mm Hg perfusion, the mean AoP at which CO became maximal was 72 mm Hg and the corresponding coronary perfusion pressure appeared to be lower than the lower limit of the perfusion pressure range for coronary flow autoregulation. The Rp value at that point was slightly higher than the physiologic range.We conclude that when coronary perfusion pressure changes with mean AoP, and when left ventricular enddiastolic pressure is fixed, there is a clear optimal Rp at which CO becomes maximal, and this optimal Rp is higher if coronary perfusion pressure is biased from mean AoP to a significant degree.

Further investigation on the mode of entry of human rotavirus into cells.

SummaryEntry of the KUN strain of human rotavirus into MA 104 cells was studied by electron microscopy. Double-shelled rotavirus particles attached to the cell membrane, and in the presence of trypsin their nucleic acids were expelled from the virus core into the cytoplasm through radial spaces between the capsomeres and the cell membrane pores formed after their attachment. This mechanism was considered to be analogous to those of phages.

The effect of graded coronary flow reduction in the left anterior descending and septal arteries on left ventricular function in the canine heart.

We quantitatively analyzed the effect of graded left anterior descending and septal coronary flow (LAD + septal flow) reduction on left ventricular function with a left ventricular end-diastolic pressure (LVEDP) of 6 mm Hg and 12 mm Hg. We used an isolated, ejecting, canine heart preparation (n = 8), the coronary flow of which could be controlled independently of the aortic pressure. We kept the other hemodynamic variables — heart rate, left circumflex coronary flow, right coronary flow and aortic input impedance — constant within their normal physiologic range. We considered this reduction in LAD + septal flow to be analogous to that of the most frequent lesion in ischemic heart disease. There was no plateau in the left ventricular work caused by this reduction of the regional coronary flow. Therefore, the plateau commonly reported in previous studies may be partially a result of the compensatory elevation of LVEDP, which is necessary to maintain the left ventricular work.

Patterns of polypeptide synthesis in human rotavirus infected cells

SummaryPolypeptide analysis of three strains of human rotavirus (KUN, Wa and MO) were conducted using a hypertonic culture which suppressed host protein synthesis and unmasked rotavirus specific protein synthesis. As a result, eleven human rotavirus specific polypeptides (Vp 1–Vp 11) were detected by pulselabeling infected cells with [14C]-leucine. Among the 11 polypeptides, three polypeptides (Vp 7, Vp 10 and Vp 11) underwent post-translational processing, and two (Vp 7 and Vp 10) were glycosylated. Six polypeptides (Vp 1, 2, 3, 4, 6 and 7) were identified as viral structural proteins. Comparisons of three strains of different serotypes revealed that their polypeptide profiles differed from each other in electrophoretic mobility; in particular, profiles of the glycosylated polypeptide, Vp 7, were distinct among the three strains.

A comparison of ST segment deviation and calculated solid angle during acute regional ischemia in the isolated canine heart at precordial, epicardial and intramyocardial lead surfaces

Although solid angle analysis has been considered to be reasonable for explaining the distribution of ST segment deviation following ischemia, it has not been tested fully, especially for ST segment changes in various sites at different lead surfaces. Thus, we investigated the applicability of solid angle theory to the mechanism of ischemic ST segment deviation at intramyocardial, epicardial and precordial leads. We used seven isolated, coronary perfused, isovolumic contracting canine hearts in a homogeneous cylindrical volume conductor. ST segment potentials from 246 electrodes were continuously measured during left circumflex coronary artery occlusion for five minutes. The ischemic boundary was obtained from a postmortem angiography, and the solid angle subtended by the ischemic boundary was calculated at every electrode site. Despite the difference between epicardial and precordial ST segment potential distributions, there was a high correlation between measured ST segment potential and calculated solid angle at epicardial (r = 0.86 +/- 0.05, 0.77-0.93), precordial (r = 0.93 +/- 0.05, 0.84-0.99), and intramyocardial leads (r = 0.95 +/- 0.03, 0.91-0.99). We conclude that solid angle analysis can be used to approximate the distribution of ischemic ST segment deviation at different lead surfaces in acute ischemia.

Effect of trypsin and chymotrypsin on polypeptides of human rotavirus KUN strain.

In view of the fact that trypsin enhances the infectivity of human rotavirus and decreases its hemagglutination, the trypsin-mediated structural modification of the viral polypeptides was analysed, using the KUN strain, a cultivable human rotavirus isolate, grown in the absence of trypsin. A major polypeptide sensitive to trypsin treatment was Vp4 with a molecular weight of 80,000, being cleaved into three polypeptides with molecular weights of 54,000 (P54), 30,000 (P30), and 24, 000 (P24). Vp4 was also sensitive to chymotrypsin treatment, generating cleavage products different from those obtained with trypsin.

Effect of hypertonic conditions on protein synthesis in MA104 cells infected with human rotavirus

When a high NaCl concentration was used to decrease selectively the synthesis of cell proteins, the synthesis of most cellular polypeptides was greatly diminished relative to human rotavirus proteins. Thus, in the presence of 150 mM excess NaCl, 11 viral polypeptides were clearly identified. However, hypertonic conditions also reduced viral protein synthesis to a different extent with individual proteins. No significant changes in viral protein synthesis occurred during incubation under the hypertonic condition for up to 6 h, and infectious virus yields of MA104 cells incubated in the hypertonic medium did not differ from the yields of untreated MA 104 cells. These results indicate that hypertonic conditions provide a useful tool for qualitative studies of viral protein synthesis in human rotavirus infected cells.