Import of adenovirus DNA involves the nuclear pore complex receptor CAN/Nup214 and histone H1.

Adenovirus type 2 (Ad2) imports its DNA genome through the nuclear pore complex (NPC) of cells in interphase for viral production. Here we identify the NPC-filament protein CAN/Nup214 as a docking site for incoming Ad2 capsids. Binding to CAN is independent of cytosolic factors. Capsids disassemble at NPCs to free their DNA for import. This process requires binding of nuclear histone H1 to the stably docked capsids and involves H1-import factors, restricting this irreversible process to the proximity of the nucleus. Our results provide a molecular mechanism for disassembly of Ad2 and reveal an unexpected function of histone H1 in virus-mediated DNA import.

Intravenous crocetinate prolongs survival in a rat model of lethal hypoxemia.

OBJECTIVE: To examine whether a carotenoid, trans-sodium crocetinate, has beneficial effects on hemodynamic status and short-term outcome in a rat model of lethal hypoxemia. DESIGN: Randomized, placebo-controlled study. SETTING: Medical school laboratory. SUBJECTS: Eighteen spontaneously breathing, anesthetized Sprague-Dawley rats (six per group). INTERVENTIONS: Rats underwent instrumentation to measure blood pressure, aortic and renal blood flow, arterial blood gases, bladder epithelial oxygen tension (by an intraluminal Clark electrode), and hepatic microvascular oxygen tension (measured by porphyrin phosphorescence). After stabilization, the rats were subjected to breathing 10% inspired oxygen concentration. After 10 mins, they were administered 1.25 mL/kg intravenous boluses of either isotonic saline (control), normal strength crocetinate (40 microg/mL), or a concentrated crocetinate solution (60 microg/mL). These boluses were repeated at 30-min intervals until either death or 3 hrs had elapsed. MEASUREMENTS AND MAIN RESULTS: With the onset of hypoxemia, we observed a rapid reduction in blood pressure and renal blood flow, maintenance of aortic blood flow, an increase in arterial base deficit, and falls in oxygen tensions in arterial blood, bladder epithelium, and hepatic microvasculature. A progressive deterioration in the control rats was noted, with only two of the six animals surviving for 3 hrs. However, all 12 rats in the two crocetinate groups survived for 3 hrs, with hemodynamic stability until 150 mins and a slow decline thereafter. CONCLUSIONS: Trans-sodium crocetinate improved hemodynamic status and prolonged survival in this model of severe acute hypoxic hypoxia. To our knowledge, this is the first demonstration of an intravenous agent having such an effect.

The first step of adenovirus type 2 disassembly occurs at the cell surface, independently of endocytosis and escape to the cytosol.

Disassembly is a key event of virus entry into cells. Here, we have investigated cellular requirements for the first step of adenovirus type 2 (Ad2) disassembly, the release of the fibers. Although fiber release coincides temporally with virus uptake, fiber release is not required for Ad2 endocytosis. It is, however, inhibited by actin-disrupting agents or soluble RGD peptides, which interfere with integrin-dependent endocytosis of Ad2. Fiber release occurs at the cell surface. Actin stabilization with jasplakinolide blocks Ad2 entry at extended cell surface invaginations and efficiently promotes fiber release, indicating that fiber release and virus endocytosis are independent events. Fiber release is not sufficient for Ad2 escape from endosomes, since inhibition of protein kinase C (PKC) prevents Ad2 escape from endosomes but does not affect virus internalization or fiber release. PKC-inhibited cells accumulate Ad2 in small vesicles near the cell periphery, indicating that PKC is also required for membrane trafficking of virus. Taken together, our data show that fiber release from incoming Ad2 requires integrins and filamentous actin. Together with correct subcellular transport of Ad2-containing endosomes, fiber release is essential for efficient delivery of virus to the cytosol. We speculate that fiber release at the surface might extend the host range of Ad2 since it is associated with the separation of a small fraction of incoming virus from the target cells.

Microtubule-dependent plus- and minus end-directed motilities are competing processes for nuclear targeting of adenovirus.

Adenovirus (Ad) enters target cells by receptor-mediated endocytosis, escapes to the cytosol, and then delivers its DNA genome into the nucleus. Here we analyzed the trafficking of fluorophore-tagged viruses in HeLa and TC7 cells by time-lapse microscopy. Our results show that native or taxol-stabilized microtubules (MTs) support alternating minus- and plus end-directed movements of cytosolic virus with elementary speeds up to 2.6 micrometer/s. No directed movement was observed in nocodazole-treated cells. Switching between plus- and minus end-directed elementary speeds at frequencies up to 1 Hz was observed in the periphery and near the MT organizing center (MTOC) after recovery from nocodazole treatment. MT-dependent motilities allowed virus accumulation near the MTOC at population speeds of 1-10 micrometer/min, depending on the cell type. Overexpression of p50/dynamitin, which is known to affect dynein-dependent minus end-directed vesicular transport, significantly reduced the extent and the frequency of minus end-directed migration of cytosolic virus, and increased the frequency, but not the extent of plus end-directed motility. The data imply that a single cytosolic Ad particle engages with two types of MT-dependent motor activities, the minus end- directed cytoplasmic dynein and an unknown plus end- directed activity.

Alteration of fibronectin affinity during differentiation modulates the in vitro migration velocities of Hydra nematocytes.

In the fresh water Cnidarian Hydra nematocytes differentiate from stem cells in the body column of the polyps and are functional in the tentacles to where they migrate as single cells in an amoeboid fashion. The fluorescent vital stain TROMI (tetramethyl-rhodamine-5/6-maleimide) allows to easily discriminate between cells located in the body column and cells mounted in the tentacles. The two cell populations were found to have different in vitro migration properties. These differences appear to be due largely to a differential attachment to fibronectin. Nematocytes from the tentacles show significantly lower in vitro migration velocities on isolated pieces of the organisms extracellular matrix (the mesoglea) and attach more firmly to fibronectin-coated substrates than cells from the body column. Pretreatment of the mesogleae with antibodies against the cell binding domain of fibronectin or addition of RGD-peptides results in an increase of the average migration velocity of cells from the tentacles and a decreased velocity of the cells from the body column. These findings suggest that (1) modulation of the attachment to fibronectin is decisive for the observed differential migration properties of the two cell populations and (2) the in vitro migration of nematocytes is dependent on subtle and transient interactions of cell surface receptors (most probably integrins) and fibronectin.

Cardiorespiratory, tissue oxygen and hepatic NADH responses to graded hypoxia.

OBJECTIVE: To assess cardiorespiratory, tissue oxygen and hepatic nicotine adenine dinucleotide hydride (NADH) responses to graded hypoxia. DESIGN: Prospective, controlled, randomized study. SETTING: University laboratory. ANIMALS AND INTERVENTIONS: 18 anaesthetised Sprague-Dawley rats spontaneously breathing either 21% (controls), 12.5% or 10% inspired oxygen concentrations (6 rats per group). MEASUREMENTS AND RESULTS: All animals in the 21 and 12.5% O2 groups survived the 3-h study period, compared to only 1 in the 10% O2 group. In this latter group, mean arterial pressure and renal blood flow fell immediately with hypoxaemia, whereas aortic blood flow was maintained until the preterminal stages. Critical cellular hypoxia was suggested by an increasingly severe base deficit, an initial rise then a preterminal fall in hepatic NADH intensity and premature death in all but 1 animal. Hepatic NADH fluorescence intensity was unchanged in control animals but showed a progressive rise in the 12.5% O2 group, accompanied by a small though static increase in arterial base deficit. No significant differences were seen in arterial and tissue partial pressure of oxygen between the 12.5 and 10% O2 groups. CONCLUSIONS: This study demonstrates major differences in cardiorespiratory, hepatic NADH and outcome responses to small variations in the degree of hypoxic hypoxia. The fall in NADH fluorescence intensity presages impending death and is likely to reflect failure of cellular metabolic processes.

The role of the nuclear pore complex in adenovirus DNA entry.

Adenovirus targets its genome to the cell nucleus by a multistep process involving endocytosis, membrane penetration and cytoplasmic transport, and finally imports its DNA into the nucleus. Using an immunochemical and biochemical approach combined with inhibitors of nuclear import, we demonstrate that incoming viral DNA and DNA-associated protein VII enter the nucleus via nuclear pore complexes (NPCs). Depletion of calcium from nuclear envelope and endoplasmic reticulum cisternae by ionophores or thapsigargin blocked DNA and protein VII import into the nucleus, but had no effect on virus targeting to NPCs. Calcium-depleted cells were capable of disassembling incoming virus. In contrast, inhibitors of cytosolic O-linked glycoproteins of the NPC blocked virus attachment to the nuclear envelope, capsid disassembly and also nuclear import of protein VII. The data indicate that NPCs have multiple roles in adenovirus entry into cells: they contain a virus-binding and/or dissociation activity and provide a gateway for the incoming DNA genome into the nucleus.

Cardiorespiratory and tissue oxygen dose response to rat endotoxemia.

The dose response to endotoxin (Escherichia coli serotype 127:B8) was assessed in a spontaneously breathing, halothane-anesthetized, Sprague-Dawley rat model monitoring blood pressure, aortic and renal blood flows, blood gases, and bladder epithelial PO2, a marker of organ perfusion. The animals received either saline or endotoxin at doses of 1, 10 and 100 mg/kg body wt. Blood pressure changed significantly in all three endotoxin groups, though only the 100 mg/kg group showed significant changes in arterial PCO2, arterial PO2, and body temperature compared with controls. Whereas aortic and renal blood flow rose significantly in the two lower-dose groups, an approximate one-third fall occurred in the 100 mg/kg group (P < 0.001). Notwithstanding these macrocirculatory hemodynamic changes, both bladder epithelial PO2 and arterial base deficit rose significantly in all groups, though only the base deficit showed a progressive dose response. This model illustrates that responses to endotoxin are dose dependent but with changing patterns for different variables. The consistent finding of an elevated tissue PO2 in endotoxemia, regardless of dose, is suggestive of defective cellular oxygen metabolism.

Oxygen tension in the bladder epithelium rises in both high and low cardiac output endotoxemic sepsis.

The effect of endotoxin on tissue oxygen tension measured at the bladder epithelium was assessed in spontaneously breathing Sprague-Dawley rats anesthetized with halothane. Hyperdynamic (high cardiac output, group A, n = 6) and hypodynamic (low cardiac output, group B, n = 6) circulatory responses were achieved by intravenous administration of Escherichia coli lipopolysaccharide, 10 mg/kg over 30 min or 20 mg/kg over 1 min, respectively. Comparison was made against sham-operated control rats (group C, n = 6). Aortic and renal blood flows increased in group A and fell in group B (P < 0.001). However, in both groups, bladder epithelial oxygen tension rose significantly compared with control (P < 0.01), despite an increasing metabolic acidosis. This is in contradistinction to previous studies of nonseptic insults where bladder epithelial oxygen tension fell in line with an increasing arterial base deficit. If a raised tissue oxygen tension could be demonstrated in other organ beds, this would suggest that decreased utilization of oxygen rather than reduced tissue oxygen availability is responsible for the apparent anaerobic respiration seen in sepsis.

The effect of norepinephrine and dobutamine on bladder epithelial oxygen tension.

STUDY OBJECTIVES: To assess the effects of two contrasting vasoactive agents (dobutamine [DOB] and norepinephrine [NE]) on (1) global and regional cardiorespiratory variables, (2) acid base status, and (3) bladder epithelial oxygen tension (BEOT), a putative marker of organ perfusion. DESIGN: Measurement of aortic blood flow (ABF) and renal blood flow (RBF), mean arterial blood pressure, arterial blood gases, and BEOT were made during infusion of placebo and varying doses of DOB and NE. SETTING: Medical school laboratory. SUBJECTS: Eighteen anesthetized, spontaneously breathing, male Sprague-Dawley rats divided into three groups. INTERVENTIONS: Two groups were allocated to receive escalating doses of DOB (to 40 micrograms/kg/min) or NE (to achieve a 50% change in any hemodynamic variable). The drug therapy was then discontinued for 15 min and restarted at the previous maximum dose. A third group received 0.9% saline solution at the same infusion rate (16 mL/kg/h). MEASUREMENTS AND RESULTS: There was a dose-related increase in mean blood pressure with NE and fall with DOB. Compared with control values, NE had no effect on ABF but decreased RBF significantly whereas DOB significantly increased ABF but had no effect on RBF. Base excess and BEOT decreased significantly and in parallel with both agents, more so with NE. CONCLUSIONS: Despite their different macrocirculatory effects, DOB and NE both produced a significant but reversible fall in BEOT and a metabolic acidosis. BEOT shows potential as a monitor of the effectiveness of organ perfusion.

Aortic pH oscillations in conscious humans and anaesthetised cats and rabbits.

Respiratory oscillations in arterial blood gas composition influence breathing in cats and dogs. Their role in the control of breathing in humans in less certain. To determine whether oscillations are very small or absent in mammals who are large or breathe fast, aortic pH oscillations, recorded with a tridodecylamine based hydrogen-ion selective electrode, were compared in humans (n = 13), cats (n = 7) and rabbits (n = 4) over a wide range of ventilation. For comparison, data were analysed in terms of the ratio of tidal volume to functional residual capacity (VT/FRC). During spontaneous breathing in rabbits, cats and humans (mean respiratory frequency fR = 61, 20.4 and and 17.5 min-1), mean VT/FRC were 1.35, 0.63 and 0.36 respectively. Corresponding pH amplitudes (pHamp) of 0.009 (0.004), 0.016 and 0.013 (0.005) pH units (mean +/- 1SD) were not significantly different. The pHamp decreased exponentially with increasing FR in each species and pHamp increased linearly with increasing VT in the 3 cats in which this was studied. The study confirms the dependence of pHamp on FR and VT and its comparability among species despite differences in body size. It also demonstrates that oscillations can be recorded in humans at FR in excess of 20 min-1.

The attachment of nematocytes from the primitive invertebrate Hydra to fibronectin is specific and RGD-dependent.

The transient attachment of cells to components of the extracellular matrix is an important step in the complex molecular mechanisms involved in amoeboid cell locomotion. We have analyzed the attachment of nematocytes from the freshwater cnidarian Hydra to fibronectin which is a constituent of the mesoglea, the extracellular matrix, of the polyps. The percentage of attaching cells increased gradually in a concentration-dependent manner and reached a plateau value at a fibronectin concentration of 50 micrograms/ml. Attachment was inhibited by exposure of the fibronectin-coated surfaces to antibodies against the cell binding domain of fibronectin or by incubating the cells with peptides containing the recognition sequence Arg-Gly-Asp (RGD) known from vertebrate cells. This, together with data obtained by affinity chromatography, indicates that RGD-dependent binding to fibronectin, mediated by a receptor which possibly belongs to the integrin family, already occurs in Hydra, a member of an evolutionary low invertebrate phylum.

The contributions of microtubules and F-actin to the in vitro migratory mechanisms of Hydra nematocytes as determined by drug interference experiments.

In order to investigate the contributions of microtubules and of F-actin to the in vitro migration mechanisms of Hydra nematocytes we have studied the effects of agents directed against cytoskeletal structures. Disassembly of microtubules by treatment with the drug nocodazole in moving nematocytes resulted in the loss of all locomotory activity within 20 min after the onset of treatment and in the detachment from the substratum after about 30 min. Depolymerization of microtubules by exposure to low temperatures had the same effect but was reversible in this case. Locomoting cells treated with cytochalasin D, which disrupts the actin filaments, stopped movement 2 min after drug administration and detached from the substratum after 15 min. The pattern of F-actin, alpha-tubulin, and tyrosinated tubulin in drug- or cold-treated cells was determined by immunocytochemical techniques and confocal laser scanning microscopy. These patterns and the reactions of the cells to the various drug treatments suggest that both actin filaments and microtubules play a crucial role in nematocyte locomotion. Analysis of the cytoskeletal pattern in drug-treated cells shows that the microtubules which are involved in locomotion are mostly tyrosinated. Furthermore it is suggested that microtubules and actin filaments interact with each other during the locomotion of nematocytes.

Carotid chemoreceptor response to increases in CO2 output.

The effect on carotid chemoreceptor activity of alterations in PaCO2 oscillations produced by venous CO2 loading via the small intestines was studied in anaesthetised cats, which were paralysed and ventilated at constant frequency. Changes in PaCO2 oscillations were assessed from continuously recorded oscillations of arterial pH. Chemoreceptor activity was averaged over 20-50 pH cycles to determine the amplitude (csnd ampl) and mean level (csnd mean) of the respiratory fluctuation in discharge frequency during control and CO2 loaded periods. Two groups of 5 cats were studied. In both groups, the amplitude of the PaCO2 oscillations increased by an overall average of 74.5% (standard error, SE = 9.2) with minimal change in mean PaCO2 (average increase 0.7 mm Hg), in response to an increase in VCO2 of 77.5% (SE = 7.4). When isoxia was maintained (Group 1) csnd ampl increased by 51.3% (SE = 11.1) and csnd mean by 17.3% (SE = 7.8). In Group 2, PaO2 rose by 16.7 mm Hg (SE = 1.5) during CO2 loading and both csnd ampl and csnd mean decreased by 7.4% (SE = 9.3) and 2.3% (SE = 13.3) respectively. The results demonstrate a sensitivity of chemoreceptors to changes in VCO2 which is influenced by small changes in PaO2.

A single layer of microtubules is part of a complex cytoskeleton in mature nematocytes of hydra.

The architecture of microtubules in mature nematocytes (stinging cells) of Hydra attenuata was investigated in detail by an indirect immunofluorescence study and by scanning and transmission electron microscopy in order to comprehend the function of the cytoskeleton in this extremely complex cell type. Microtubules were detected in all types of nematocytes in the tentacles and were found to be arranged in parallel arrays forming a highly organized basket-like structure around the nematocysts.

Is hypercapnia necessary for the ventilatory response to exercise in man?

1. Continuous recordings of arterial pH, ventilation, airway CO2 and heart rate were made during rest and during 3-4 min periods of rhythmic leg exercise in four renal patients with arteriovenous shunts. 2. The patients were anaemic (haemoglobin 6.5-9.0 g/dl) but had a normal ventilatory response to exercise as judged by the ratio of the change in ventilation to the change in CO2 production. 3. Breath-by-breath oscillations in arterial pH disappeared for the majority of the exercise period in each patient. 4. Changes in mean arterial pH and end-tidal CO2 tension with exercise were inconsistent between subjects but consistent within a given subject. On average, mean arterial pH rose by 0.011 pH unit. Changes in end-tidal CO2 tension reflected changes in mean pHa by falling on average by 1 mmHg (0.13 kPa). 5. Hypercapnia and acidaemia were not found to be necessary for the ventilatory response to moderate exercise.

Derivation of CO2 output from oscillations in arterial pH.

Theory predicts that the rate of rise of the oscillation in arterial CO2 partial pressure (PaCO2) is linearly dependent on CO2 flux from venous blood to alveolar gas. We have measured, in the anesthetized cat, CO2 output (VCO2) and oscillations in arterial pH. The pH signal was differentiated to give the maximum rate of fall of pH on the downstroke of the oscillation (dpH/dt decreases max). Since oscillations in pH are due to oscillations in arterial PCO2, dpH/dt decreases max was considered to be equivalent to the maximum rate of rise of the PCO2 oscillation. VCO2 was increased by ventilating the intestines with CO2 and by the intra-arterial infusion of 2,4-dinitrophenol. VCO2 was decreased by filling the intestines with isotonic tris(hydroxymethyl)methylamine buffer. The maximum range of VCO2 covered was 7.8-51 ml/min, and the mean range was from 13.6 +/- 1.3 to 29.7 +/- 1.6 (SE) ml/min. Although CO2 loading produced a small rise and CO2 unloading a small fall in mean PaCO2, the changes were not statistically significant, so that overall the response was close to isocapnia. Over the limited range of VCO2 studied there was a highly significant linear association between dpH/dt decreases max and VCO2 which supports the contention that the slope of the upstroke of the PaCO2 oscillation is determined by the CO2 flux from mixed venous blood to alveolar gas. As such this slope is a potential chemical signal linking ventilation to CO2 production.

The effect of small changes in arterial carbon dioxide tension on carotid chemoreceptor activity in the cat.

The hypothesis that the fluctuations in carotid chemoreceptor discharge represent more than a simple proportional response to respiratory oscillations of Pa, CO2 has been examined. Simultaneous recordings of the activity in few- or multi-fibre chemoreceptor preparations of the cut carotid sinus nerve and oscillations of carotid arterial pH have been made in four anaesthetized cats which were paralysed and artificially ventilated at constant frequency. Chemoreceptor activity was averaged over a minimum of twenty consecutive pH cycles and the amplitude of the fluctuations in discharge frequency determined. Using the mean Pa, CO2 obtained from blood-gas analysis and the slope of the relationship between log Pa, CO2 and pH obtained in each cat, the amplitude of the pH oscillations was converted to Pa, CO2 amplitude. This amplitude relative to the mean Pa, CO2 has been compared with the amplitude of the corresponding fluctuation in the chemoreceptor discharge frequency recorded from the carotid sinus nerve (e.s.n.d. amplitude) relative to its mean level. Whereas the Pa, CO2 amplitude was always less than 8% of the mean Pa, CO2, the c.s.n.d. amplitude ranged from 40 to 186% of the mean discharge frequency. C.s.n.d. amplitude was divided by the corresponding Pa, CO2 amplitude to give an index of the sensitivity of the chemoreceptors to Pa, CO2. This sensitivity has been compared with that determined from the mean discharge frequency produced in response to changes in mean Pa, CO2 comparable in magnitude to the Pa, CO2 amplitude. The former sensitivity was usually at least 3 times greater than the latter. Examination of the fluctuations in chemoreceptor discharge frequency in relation to the corresponding pH oscillations revealed that whereas the minimum discharge frequency coincided with the alkaline peak of the pH oscillation (trough of the Pa, CO2 oscillation), the maximum discharge frequency did not invariably coincide with the acid trough of the pH oscillation (peak of the Pa, CO2 oscillation). On 30% of occasions, the maximum discharge frequency was associated with the region of maximum rate of fall of pH (maximum rate of rise of Pa, CO2). It was concluded that fluctuations in the discharge of carotid chemoreceptors cannot be accounted for on the basis of a simple proportional relationship to Pa, CO2. They contain a large rate of change component.

Regulation of intestinal brush border microvillus length during development by the G- to F-actin ratio.

We examined ultrastructural changes in developing chicken intestinal microvilli and correlated these with changes in the G- to F-actin ratio and the amount of actin per milligram cell protein. Three discrete morphological and temporal changes occur during late microvillus morphogenesis: an increase in microvillus number associated with microvilli becoming hexagonally packed on the cell surface; an increase in core actin filament number; and an increase in the length of microvilli. Dramatic rises in the amount of cell actin occur at the time of the first two morphological changes. Changes in the G- to F-actin ratio suggest that increases in the level of monomeric actin drive the elongation phase of microvillus growth since immediately prior to growth the G- to F-actin ratio shifts from its embryonic and adult 3:7 ratio to a 1:1. Our results also indicate, but do not prove, that an increase in the amount of G-actin precedes the rise in level of F-actin and growth of microvilli by 1 day, implying that an increase in the content of G-actin stimulates actin polymerization. Our findings also suggest that the G- to F-actin ratio and their absolute amounts, perhaps in combination with cytoskeletal protein turnover and/or the pool size of actin binding proteins, plays a role in restricting the mature constant length of microvilli.

The brush border cytoskeleton is not static: in vivo turnover of proteins.

The shape and stability of intestinal epithelial cell microvilli are maintained by a cytoskeletal core composed of a bundle of actin filaments with several associated proteins. The core filaments are intimately associated with the overlying plasma membrane, in which there occur rapid turnover of proteins and constant incorporation of new membrane. Previous work has shown that starvation or inhibition of protein synthesis results in modulation of microvillar length, which indicates that there may be cytoskeletal protein turnover. We demonstrate herein, by means of in vivo pulse labeling with radioactive amino acids, that turnover of brush border cytoskeletal proteins occurs in mature absorptive cells. Turnover of cytoskeletal proteins appears to be quite slow relative to membrane protein turnover, which suggests that the turnover of these two microvillar compartments is not coupled. We thus conclude that cytoskeletal protein turnover may be a factor used to maintain normal length and stability of microvilli and that the cytoskeleton cannot be considered a static structure.