Evolution of human endogenous retroviral sequences: a conceptual account.

Endogenous retroviruses (ERVs) most likely are remnants of ancient retroviral infections. ERVs preserve functions of exogenous retroviruses to a varying extent, and can be parasites, symbionts or more or less neutral genetic 'junk'.Their evolution has two facets, pre- and post-endogenization. Although the two are not clearly separated, the first pertains to retroviral evolution in general and the second to the fate of repetitive DNA and the evolution of the host organism and its genome. The study of ERVs provides much material for the understanding of retroviral evolution. This sequence archive reflects the history of successes and shortcomings of antiviral resistance, but also of strategic evolutionary decisions regarding genome organization and new gene acquisition. This review discusses retroviral evolution illustrated through HERVs, bioinformatic prerequisites for ERV studies, the endogenization process and HERV evolution post-endogenization, including relation to disease. (Part of a multi-author review).

Analysis of mean retinal transit time from fluorescein angiography in human eyes: normal values and reproducibility.

OBJECTIVE: To evaluate three different techniques to quantify retinal blood flow transit times in normal human eyes from fluorescein angiograms. SUBJECTS AND METHODS: Fluorescein angiograms were recorded on two different occasions in 18 normal individuals with a scanning laser ophthalmoscope. The angiograms were digitized (5 frames per second) and the images were aligned. Mean transit times (MTT) were analysed with a newly developed technique based on an impulse-response analysis (MTTIR) and again with the conventional technique (MTTSLOPE). Arterio-venous passage times (AVP) were also calculated. RESULTS: At the first determination, mean values (SD) for MTTIR, MTTSLOPE, and AVP were 3.22 (0.78), 4.88 (1.86), and 1.46 (0.57) seconds, respectively. Detection of an increase of 25% with a power of 80% requires groups of 12, 86 and 17 individuals for the three techniques, respectively. CONCLUSIONS: Mean transit time is a well-defined physiological parameter. The technique based on impulse-response analysis allows for analysis of even badly defined dye curves. We found this technique to be superior to the conventional technique in terms of reproducibility.

Reconstitution of neurotransmission by determining communication between differentiated PC12 pheochromocytoma and HEL 92.1.7 erythroleukemia cells.

Neurotransmitter release was monitored using fura-2-loaded HEL 92.1.7 cells dispersed among differentiated PC12 cells (loaded with another Ca2+ indicator fluo-3) and immobilised using transparent polycarbonate membrane filters with uniform pore size. Depolarisation with K+ caused a rapid rise in Ca2+ concentration in the PC12 cells, followed by a delayed secondary Ca2+ response in simultaneously monitored nearby HEL cells. There was a lag period of about 20 s between the responses of the two cell types. Voltage-gated Ca2+ channels in PC12 cells were inhibited by the P/Q-type (omega-conotoxin MVIIC, omega-agatoxin IVA), N-type (omega-conotoxin GVIA) and L-type channel blockers (nifedipine) as determined using fura-2 or whole-cell patch-clamp recordings. The communication between the cell types on the other hand was sensitive to P/Q- and N-type but not to L-type channel blockers. This suggests that, as in neurons, P/Q- and N-type Ca2+ channels mediate the release of neurotransmitters acting on HEL cells. Theoretically, the procedure employed should be sensitive enough to detect single exocytotic events. Our results demonstrate that a random distribution between effector and target cells is sufficient to allow communication between cells in a manner similar to extrasynaptic transmission.

Comparison of retinal transit times and retinal blood flow: a study in monkeys.

PURPOSE: To determine the correlation between transit times of retinal blood flow calculated from fluorescein angiograms and retinal blood flow determined by the microsphere method. METHODS: Two fluorescein angiograms were obtained in each eye of six monkeys, followed by determination of retinal blood flow with labeled microspheres. Angiograms in 10 eyes were analyzed for mean transit time (MTT) and arteriovenous passage time (AVP). MTT was determined in two ways: from dye curves reconstructed by extrapolation of semilogarithmic plots of the recorded curves (MTT(slope)) and by a new technique based on an impulse-response analysis (MTT(ir)). RESULTS: Mean values (+/-SD) for retinal blood flow in 10 eyes were 23.2 +/- 6.9 mg/min. Corresponding values for MTT(ir), MTT(slope), and AVP were 2.22 +/- 0.38, 4.89 +/- 5.89, and 1.08 +/- 0.14 seconds. There was a weak, but not statistically significant, correlation between retinal blood flow and MTT(ir) (r = -0.60, P = 0.06) but no useful correlation between retinal blood flow and either MTT(slope) or AVP. CONCLUSIONS: The relationship between retinal blood flow and transit times determined from fluorescein angiograms is weak. Of the three transit parameters tested, MTT(ir), determined with the recently developed impulse-response technique, had the best correlation with retinal blood flow. Further studies are needed to determine the ability of these transit parameters to detect a change in retinal blood flow and the possibility that transit times may provide useful clinical information unrelated to absolute values of retinal blood flow.

Effects of microemboli on local blood flow in the rabbit brain.

This work describes changes in relative blood flow caused by discrete emboli reaching the brain of conscious rabbits. With [14C]iodoantipyrine autoradiography, small ischemic foci were observed scattered throughout the brain. After correction for tracer diffusion from the surroundings, the ischemic regions located in the deeper parts of the brain were found to have a lower blood flow than the foci in the cortex. Further, the cortical foci were significantly smaller than those in the basal ganglia (the area where the average size of the foci was found to be largest). The results and conclusions of this work should aid in interpretation of future experiments with potential treatments. Microinfarctions may well need different treatments depending on their localization.

In situ freezing of the rabbit and rat brain.

We have measured the effectiveness of different standard cryogenic liquids (liquid nitrogen, chilled isopentane, chilled hexane, and chilled ethanol) and applied a mathematical model for calculating the temperature changes at different depths in rabbit and rat heads cooled in liquid nitrogen. We have also evaluated the extent to which the freezing time is shortened when the skin is removed prior to immersion. We conclude that: (i) of the coolants used, liquid nitrogen gives the most rapid freezing. (ii) dry ice-chilled ethanol is as effective as dry ice-chilled hexane. Liquid nitrogen and ethanol are safer for the environment than isopentane and hexane and should satisfy most needs. (iii) By removing the skin prior to immersion, the freezing time can be reduced by about 20 s for the rabbit cortex and rat hypothalamus, 30 s for the rat cortex, and 40 s for the rabbit hypothalamus.

A diffusion model of cerebral microischemia.

We have recently published an experimental method useful for causing discrete cerebral microischemic foci in rabbits. In that study it was shown that on reaching the brain, plastic beads (with an average diameter of 80 or 110 microns) give rise to foci of increased [14C]2-deoxyglucose accumulation, indicating anaerobic glycolysis. Further, it was suggested that the relatively low accumulation of [14C]2-deoxyglucose, as observed in the center of some of the ischemic foci located in areas which are thought to have a poor collateral blood supply, was an indication of glucose depletion. This work is intended to be a useful approach for the study of diffusion-limited consumption of glucose and oxygen in microischemic regions, located in endarterial regions. It is shown that a mathematical model with reasonable parameters can be fitted to a measured [14C]2-deoxyglucose profile. The results of this study are compatible with the view that the glucose concentration in the ischemic center is very low and limits the rate of energy turnover. Together with animal experiments, this mathematical model may impart new information of importance for evaluating the treatment of subcortical infarctions in humans.

Retinal mean transit time determined with an impulse-response analysis from video fluorescein angiograms.

PURPOSE: To evaluate a new technique for determining the Mean Transit Time (MTT) of retinal circulation, based on an impulse-response analysis. The results obtained were compared to those obtained with a conventional method based on semilogarithmic extrapolation. METHODS: 128 frames at a rate of 5 frames per second were digitized from video fluorescein angiograms and processed with a specially designed image analysis system. The MTT was determined from 25 curve pairs from 12 eyes in 8 young, healthy volunteers by the conventional technique (MTT[SLOPE]) and by our newly developed Impulse-Response analysis (MTT[IR]). RESULTS: MTT(SLOPE) and MTT(IR) calculated for the 25 curve pairs were 3.54 +/- 1.00 and 2.67 +/- 0.88 seconds, respectively (Mean +/- SD). There was a good correlation between the two techniques (r2 = 0.81, p < 0.001). Unlike MTT(SLOPE), MTT(IR) could be calculated also from curves with a badly defined bolus. CONCLUSION: We conclude that MTT(IR)-analysis of the retinal circulation may be used for clinical studies on retinal blood flow from angiograms obtained in patients.

An experimental model of cerebral microischemia in rabbits.

Multiinfarct dementia is the second most common form of dementia in the elderly. An animal model of microischemia may provide information about the pathophysiology relevant when searching for prevention or treatment of microinfarctions in humans. The purpose of the present study was to develop an experimental model useful for studying discrete microischemic foci. In order to achieve single cerebral microischemic foci plastic beads with diameters of about 100 microns were injected into the left heart ventricle of anesthetized rabbits. 2-Deoxy-[14C]glucose (2-DG) and autoradiography were used to detect regions with disturbed metabolism. The tissue sections were inspected for impacted beads. Foci with markedly increased 2-DG accumulation and with diameters of about 1 mm were detected in all parts of the brain, indicating hypoxic regions with enhanced glycolysis. In some foci, located mainly in the basal ganglia, a central dip in the 2-DG profile was seen, suggesting poor glucose supply to the central ischemic region. The ratio foci/beads was about 1 in the brain stem (diencephalon included) and about 0.5 in the cortex. Twenty-four hours after embolization, infarctions, mainly in the deeper brain regions, were seen. There were still foci with increased 2-DG uptake, which were mainly located in the cortex. The results suggest that microemboli reaching the deeper brain regions give rise to metabolic disturbances more often than emboli reaching the cortex and that the ischemic foci in deeper brain regions are more prone to develop further into infarctions.

Measurements of magnetic field variations in the human brain using a 3D-FT multiple gradient echo technique.

A magnetic resonance 3DFT multiple gradient-echo technique was used for measurements of the proton spectrum for each voxel in the measured slice. Water, fat, magnetic field and T2 distributions in the head of a normal volunteer and a patient with intracerebral hematoma were computed. Magnetic field variations caused by the head were calculated after correction for the static magnetic field inhomogeneity. Large local magnetic field variations up to 3 ppm were found in the human brain near interfaces between air or bone and brain tissues and 0.5 ppm between hematoma and brain tissue. Information about magnetic field variations could be useful for shimming procedures in vivo and for correcting artifacts in imaging and spectroscopy.

Effects of high intraocular pressure on the glucose metabolism in the retina and optic nerve in old atherosclerotic monkeys.

BACKGROUND: There are reasons to suspect that in patients with high intraocular pressure and glaucoma, there is underperfusion of the intraocular tissues and optic nerve head, leading to tissue hypoxia and neuronal damage. Studies in young, healthy monkeys have demonstrated that there is efficient autoregulation of the blood flow, and essentially normal glucose consumption, even at very high intraocular pressures that reduce the perfusion pressure to levels around 30 mm Hg. It seemed likely that the conditions might be different in old monkeys that had been on atherogenic diet for long periods of time and that such monkeys were a better model for glaucomatous patients. METHODS: The perfusion pressure in one eye was reduced to 30-35 mmHg in four old rhesus monkeys that had been on atherogenic diet for 12.5 years, and the glucose consumption in the eyes and optic nerves was studied with the 14C-2-deoxyglucose (2DG) method of Sokoloff et al. RESULTS: There was enhanced uptake of 2DG in the inner as well as outer parts of the retina and in the optic nerve head in all four monkeys studied, indicating compromised supply of oxygen resulting in anaerobic glycolysis. CONCLUSION: Old monkeys that have been on atherogenic diet seem more susceptible to elevation of the intraocular pressure than young, healthy monkeys, thereby suggesting defective autoregulation in them. Such differences in susceptibility may play a role also in the development of ischemic disorders of the optic nerve head and glaucomatous optic neuropathy.

Measurement of magnetic susceptibility and MR contrast agent concentration.

This paper describes an MR imaging method for determining magnetic susceptibility constants of solutions containing paramagnetic contrast agents. The method's validity is demonstrated on Gd(DTPA) and Dy(DTPA) water solutions. The method can be used for measurement of the volume magnetic susceptibility or concentration of contrast agents in biological tissues.

Properties of the phase-alternating phase-shift (PHAPS) multiple spin-echo protocol in MRI: a study of the effects of imperfect RF pulses.

The effects of imperfect radiofrequency (RF) pulses on the echo amplitudes from the Carr-Purcell (CP), Carr-Purcell-Meiboom-Gill (CPMG), and the PHase-Alternating Phase-Shift (PHAPS; combination of CP and CPMG) multiple spin-echo schemes were studied. Properties of the PHAPS scheme for transverse relaxation time measurements was emphasized. Numerical simulations on non-relaxing spin systems were performed to assess the properties of selective (damped sinc shaped) and nonselective refocusing pulses in terms of effective spatial selectivity and generation of secondary echo signal. Analytical solutions of the Bloch equations were applied to study the generation and propagation of stimulated echo signal caused by nonideal 180 degrees phase reversals, and the results were used to analyse the numerical simulations in terms of primary and stimulated echo components. Finally, the simulated echo train patterns from the different MSE schemes were compared with MR imaging measurements. It was found that the underestimation of T2 values by the PHAPS protocol with selective refocusing pulses is mainly an effect of an "artificial" echo amplitude decay in the CP scheme, while the CPMG scheme produces a typical even-odd echo pattern (different from corresponding echo patterns in conventional high resolution NMR). Both effects are related to the flip angle error and phase dispersion along the slice selection direction from selective RF pulses, and are not significantly influenced by stimulated echo interference for nonrelaxing spin systems. However, the presence of stimulated echoes at the time of the primary echoes implies a dependence on T1 of the PHAPS echo amplitudes. In the CPMG protocol, different gradient schemes have been implemented to defocus stimulated echoes. However, the results indicate that there exists stimulated components that will not be affected by such gradients, and that the optimization of the RF refocusing pulses then remain the main objective.

Dependence on T1 of the echo amplitudes from multiple spin-echo sequences with equidistant echoes: simulation studies.

Transverse relaxation times were estimated from numerical simulations on spin systems using multi-echo spin-echo MRI protocols. The influence of T1 on the echo amplitudes via stimulated echo components was studied. The resulting effects on T2 estimates from the Carr-Purcell (CP), Carr-Purcell-Meiboom-Gill (CPMG), and Phase-Alternating-Phase-Shift (PHAPS; combination of CP and CPMG), multiple echo schemes were examined. Protocols with either spatially selective or nonselective refocusing pulses were studied. An intravoxel static field inhomogeneity of 0.1, 1, and 10 ppm was stimulated. The dependence on T1 of the T2 estimates was notable for T1 values below approximately 800 msec for all protocols. The PHAPS scheme provided rather accurate, but underestimated, T2 values when selective refocusing was used. With nonselective refocusing, PHAPS T2 values were overestimated and demonstrated a pronounced dependence on magnetic field inhomogeneity. In general, long T2 values were erroneous with the PHAPS protocol. The results indicate that a CPMG protocol structure provides a more robust method for T2 estimations than the PHAPS protocol.

MRI measurements of the dependence on T1 of the echo amplitudes using a multiple spin-echo scheme.

Analytical calculations using the Bloch formalism were performed to assess the dependence on T1 of the echo amplitudes for the Phase-Alternating Phase-Shift (PHAPS) multiple spin-echo protocol. Measurements in a 0.5 T MR imaging unit were performed to ratify the analytical results. Especially for low T2 values, the echo amplitudes were erroneous, with an increasing contribution from stimulated echo components with increasing T1. Apart from affecting T2 estimates, stimulated echoes generated a non-monoexponential signal decay of the echo trains. The results confirmed previous simulation studies as regards the dependence on T1 of T2 estimates from PHAPS.

Quantification of inhomogeneities in malignancy grading of non-Hodgkin lymphoma with MR imaging.

In a previous study of 50 patients with non-Hodgkin lymphoma (NHL) it was shown that the inhomogeneous appearance of a tumor at MR imaging strongly indicated a high malignancy grade. In this study of 33 patients with NHL, the administration of an i.v. contrast medium, Gadolinium-DTPA, improved the subjective detectability of the inhomogeneities. A method of quantifying the degree of inhomogeneity in the tumors (inhomogeneity index, IH-index) was developed and tested. The mean value of IH-index in the T2-weighted image before contrast medium administration, and of the T1-weighted image after contrast medium administration, as well as the IH-index value in the T2-weighted image before contrast medium administration alone, was able to discriminate well between low- and high-grade NHL. This method of quantifying the degree of inhomogeneity in tumors improved sensitivity in detecting high-grade NHL.

Computer-assisted quantification and image processing of whole-body autoradiograms.

A computerized image-processing system especially adapted for analysis of whole-body autoradiograms has been developed. It consists of commercially available standard components, including a black-and-white video camera, a microcomputer, and graphics equipment. The lower performance of the hardware has been compensated for by more flexible software. When the system was calibrated, special attention was paid to local variations in the measuring system in different parts of the picture. Utility programs for the manipulation of contrast, pseudocoloring, and image enhancement, etc., are available. Some programs have been especially designed to comply with specific problems and demands related to different autoradiographic applications. A program displaying the density histogram for an area of interest is particularly useful for the quantitation of whole-body autoradiograms. It allows the operator to select interactively a range of densities. Image elements (pixels) corresponding to the densities in this range are shown in red on the monitor, and their average true density is calculated. This procedure permits the marking and analysis of delicate structures on autoradiograms. Other programs allow a picture, stored in memory, to be rotated or translated, and two pictures to be superimposed for comparison. Various applications of using image analyses in whole-body autoradiography are presented and illustrated.

Control of retinal and choroidal blood flow.

Earlier studies on the control of retinal and choroidal blood flow are reviewed and some recent observations on the effects of light on retinal metabolism and retinal and choroidal blood flow in monkeys (Macaca fascicularis) are reported in preliminary form. The retina is nourished by the retinal blood vessels, where blood flow is autoregulated and the choroidal blood vessels where autoregulation is absent. Studies with the deoxyglucose method of Sokoloff indicate that flickering light tends to increase the metabolism of the inner retina, while constant light reduces the metabolism in the outer retina. Retinal blood flow in flickering light, 8 Hz, is higher than in constant light. The sympathetic nerves of the choroid are probably involved in a protective mechanism, preventing overperfusion in fight and flight situations with acute increments in blood pressure. The facial nerve contains parasympathetic vasodilator fibres to the choroid; the physiological significance of these fibres is unknown. The neuropeptides NPY, VIP and PHI are likely to be involved in autonomic reflexes in the eye.

Aspects of oxygen and glucose consumption in the retina: effects of high intraocular pressure and light.

Studies on retinal oxygen and glucose consumption in cats and pigs are reviewed and recent experiments with the deoxyglucose method in monkeys are described. In all three species, the retina is supplied with nutrients by both the retinal and the choroidal blood vessels. Studies on regional blood flow and differences in arteriovenous concentration in cats have indicated that under conditions of general anesthesia and exposure to laboratory light, the oxygen supply from the choroid is about 5.6 microliters/min and that from the retinal vessels, 1.3 microliters/min. In pigs the corresponding figures were about 4 and 2.9 microliters/min, respectively. Moderate reductions in perfusion pressure caused by increments in intraocular pressure or reductions in arterial blood pressure resulted in little change in oxygen tension in the inner retina, due to efficient autoregulation of retinal blood flow. Reduced choroidal blood flow was to a large extent compensated by increased differences in arteriovenous concentration. Studies using the deoxyglucose method in monkeys indicated that pentobarbital anesthesia and constant illumination tend to reduce the metabolism of the retina. In darkness, glucose consumption in the photoreceptors was higher than that recorded under conditions of constant illumination with white light. Flickering light at 4 Hz enhanced glucose consumption in the inner retina. At very high intraocular pressures glucose consumption in the retina was enhanced, probably as a result of partial ischemia, with a shift to more anaerobic glucose metabolism.

The effect of prostaglandin F2 alpha-1-isopropylester (PGF2 alpha-IE) on uveoscleral outflow.

These studies assessed the acute effects of a single dose of prostaglandin F2 alpha-1-isopropylester (PGF2 alpha-IE) on the IOP and AHF, as well as on conventional and uveoscleral outflow, in cynomolgus monkeys. The AHF was determined by a dilution method, using radioactive albumin as a marker. Arterial blood samples were collected and analyzed for radioactivity to determine the flow of aqueous humor to blood, corresponding to the conventional outflow. The uveoscleral outflow was calculated as the difference between the measured AHF and the conventional outflow. Topical application of PGF2 alpha-IE (1 microgram free acid equivalents) to eyes of cynomolgus monkeys caused a small initial increase in IOP followed by a gradual decrease, which was greatest (2.9 +/- 0.6 mmHg below the control eye) at about three hours after the PG application. The mean AHF during 4 hours was slightly higher in the experimental eyes than in the control eyes. The mean uveoscleral outflow during 4 hours was significantly higher in the experimental eyes (0.98 +/- 0.12 microliters.min-1) than in the control eyes (0.61 +/- 0.10 microliters.min-1), while the conventional outflow was significantly lower. The ability of PGF2 alpha-IE to increase the uveoscleral outflow was also demonstrated by autoradiography. These results suggest that PGs exert at least part of their ocular hypotensive effect by increasing the uveoscleral outflow. It remains to be established whether this effect is caused by relaxation of the ciliary muscle or whether other mechanisms, such as structural and metabolic changes, are involved.