Detection of flaviviruses by reverse-transcriptase polymerase chain reaction.

RNA sequences of five flaviviruses were detected by a modified polymerase chain reaction (PCR) that incorporated a reverse transcriptase and RNase inhibitor. Oligonucleotide primer pairs were synthesized to amplify sequences from St. Louis encephalitis (SLE), Japanese encephalitis (JBE), yellow fever (YF), dengue 2 (DEN-2), and dengue 4 (DEN-4) viruses. The amplified products were visualized as bands of appropriate size on ethidium bromide-stained agarose gels. The identity of these products was confirmed by restriction endonuclease cleavage to generate fragments of predicted lengths. The reverse-transcriptase PCR (RT-PCR) successfully amplified flavivirus sequences from cell cultures, frozen brain tissue, and formalin-fixed, paraffin-embedded brain tissue. The reactions were highly specific, and the method compared favorably to two conventional assays of viral infectivity. RT-PCR followed by PCR with nesting primers (N-PCR) was 1,000-fold more sensitive in detecting virus than classical infectivity titration by intracerebral inoculation of suckling mice and nearly 1,000-fold more sensitive than amplification of virus in cell culture followed by inoculation of mice.

Calcium transfer at the blood-nerve barrier of the frog sciatic nerve.

Calcium transfer across the blood-nerve barrier of the frog sciatic nerve was studied using an in situ perfusion technique and an in vivo i.v. bolus injection technique. The permeability-surface area product of 45Ca at the blood-nerve barrier, (PA)BNB, calculated from radioactivity in the desheathed nerve segment after 5 min of circulation of tracer, and corrected for the residual radioactivity in the blood space, equaled 4.4 +/- 0.4 (S.E.M.) X 10(-5) ml.s-1.g-1 wet wt. The (PA)BNB of 45Ca was independent of [Ca2+] in the perfusion medium between 0.18 and 18 mM. The permeability-surface area products of 45Ca across the perineurium [(PA)per] also was measured by an in situ incubation technique, and equaled 1.45 +/- 0.41 X 10(-5) ml.s-1.g-1 wet wt. (n = 8). The half time (t 1/2) for nerve calcium to equilibrate with plasma calcium was calculated to be 60 min. The low, passive permeability to calcium of the blood-nerve barrier probably limits marked calcium concentration changes in nerve endoneurium following transient changes of plasma calcium, but should not alter steady-state responses.

Calcium diffusion through perineurium of frog sciatic nerve.

Calcium and sucrose permeabilities (PCa or Psucrose) were calculated from the fluxes of 45Ca and [3H]sucrose across perfused everted and normal configurations of the perineurial cylinder isolated from the frog sciatic nerve and from fluxes into an intact nerve segment bathed in Ringer. Mean PCa for influx across the isolated perineurium equaled 10.2 +/- 0.6 X 10(-7) cm/s (n = 16) compared with Psucrose = 7.4 +/- 0.4 X 10(-7) cm/s. For efflux, PCa = 27.5 +/- 5.0 X 10(-7) cm/s and Psucrose = 23.2 +/- 4.7 X 10(-7) cm/s. The mean ratio of PCa for efflux to PCa for influx (2.7 +/- 0.5) was not significantly different from the flux ratio for sucrose (3.1 +/- 0.7). No effect on PCa or Psucrose was observed when the calcium concentration in the bath was varied from 0.5 to 20 mM, when Na-free Ringer was perfused, or when ouabain, La3+, or 2,4-dinitrophenol was applied. Asymmetrical fluxes across the perineurial cylinder were due presumably to bulk flow and resultant solvent drag out of the lumen caused by perfusion pressure. Calcium accumulated in the perineurial tissue in a saturable manner with a Km of 80 microM and a Bmax of 0.22 mumol/g wet wt. The half time for calcium exchange from the external medium to the nerve was calculated as 3 h. This long half time and the calcium-sequestering ability of the perineurium suggest that the perineurium can stabilize endoneurial calcium during transient changes in the calcium concentration of plasma.

Calcium content of frog sciatic nerve during chronic hypocalcemia and hypercalcemia.

We examined the calcium contents of desheathed peripheral nerve, perineurial sheath, and whole sciatic nerve in the frog as a function of the steady-state plasma concentration of ionized calcium. Chronic hypocalcemia was induced by parathyroidectomy and by bathing frogs in a phosphate medium. Chronic hypercalcemia was induced by administering vitamin D3 and by bathing frogs for up to 2 wk in medium containing 50 mM CaCl2. Calcium was measured with a calcium-sensitive electrode and by atomic absorption spectroscopy. The calcium contents (mmol/kg wet wt) in whole nerve, desheathed nerve, and the perineurial sheath varied linearly with slopes of 0.72, 0.71, and 1.72, respectively, with plasma concentration (mM) of ionized calcium, which ranged from 0.3 to 8.0 mM. In the same animals the calcium content in the cerebrum was independent of plasma calcium between 0.5 and 1.5 mM but rose at higher plasma concentrations. Our results indicate that net calcium concentration in the frog peripheral nerve is not regulated during chronic hypocalcemia and hypercalcemia, whereas brain calcium is regulated at plasma calcium concentrations less than 1.5 mM. The lack of calcium regulation in the nerve is attributed to the lack of calcium regulation in the endoneurial compartment.

Fertilization of rabbit ova and the role of ovum investments in the block to polyspermy.

The rabbit ovum seldom becomes polyspermic despite the presence of supernumerary sperm with easy access to the ovum plasma membrane. The purpose of this study was to characterize the role of ovum investments in blocking polyspermy. Ova were inseminated with capacitated sperm in vitro and were fertilized. Early stages of development were normal. The incidence of polyspermy was determined by cytological examination of fixed ova. The incidence of polyspermy increased following removal of both the zona pellucida and corona radiata but not following removal of only the corona radiata. These results suggest that the failure of supernumerary sperm to penetrate the ovum plasma membrane is at least in part due to the presence of the zona pellucida.

Rabbit oocyte maturation: changes of membrane resistance, capacitance, and the frequency of spontaneous transient depolarizations.

Immature oocytes from rabbits were examined with electrophysiological techniques to determine if their membrane properties change during maturation. The input resistance increased and input capacitance decreased during maturation, although no significant change in membrane potential was observed. The changes observed were consistent with a decrease of corona radiata-oocyte electrical coupling accompanying maturation. Spontaneous transient depolarizations were recorded from immature oocytes surrounded by corona radiata, but not from mature ova. Each event consisted of a rapid depolarization, sustained for 1-100 sec, and a slow repolarization to the resting potential. Spontaneous inward currents with a time course similar to the spontaneous transient depolarizations occurred when the oocyte's membrane potential was held constant by voltage clamp. The frequency with which spontaneous transient depolarizations occurred decreased during maturation. These findings are consistent with a model in which spontaneous depolarizations originate in corona radiata cells and are detected in the oocyte via electrical coupling.

Peripheral nerve as an osmometer: role of endoneurial capillaries in frog sciatic nerve.

The sciatic nerve of the frog was perfused in vivo with isotonic Ringer solution followed by Ringer made hypertonic by addition of sucrose or of NaCl. Nerve diameter and endoneurial hydrostatic pressure fell during hypertonic Ringer perfusion. Using a model that describes the elastic and osmotic properties of the nerve, sigma sLp, the product of the osmotic reflection coefficient at endoneurial capillaries for s equals sucrose or NaCl (which approximates 1), and of capillary hydraulic conductivity, was found to equal 73 X 10(-13) cm3 X s-1 X dyn-1. The nerve is elastic. It has a compliance K of 3.7 X 10(-5) cm2 X mmHg-1, corresponding to a modulus of elasticity E of the perineurium equal to 1.2 X 10(6) dyn X cm-2. The results indicate that the nerve behaves as an osmometer during vascular perfusion, due to the low permeability of endoneurial capillaries to small solutes such as NaCl and sucrose. A low capillary hydraulic conductivity limits bulk water flow between blood and nerve, and a low compliance limits nerve swelling and edema.

Conduction velocity changes along lumbar primary afferent fibers in cats.

Though peripheral conduction velocity is widely used to characterize afferent fibers according to somatosensory modality, disagreement exists as to whether or not conduction velocity varies along such an axon's length. Therefore, in this experiment, conduction velocities were measured over very short axonal segments (7.5 to 15 mm) within the posterior tibial nerve, sciatic nerve, and L7 dorsal root, using the method of spike-triggered averaging of neurograms recorded from tripolar electrodes. The conduction velocities for several units were also determined using electrical stimulation, so that the accuracy of the two techniques could be compared. For most units, dorsal root and sciatic nerve conduction velocities were not significantly different; however, they were not tightly correlated. Tibial nerve conduction velocity averaged 86% of that within the sciatic nerve. Variations in sciatic nerve conduction velocity within adjacent axonal segments (9 mm in length) rarely exceeded experimental error. It appears that spike-triggered averaging of signals from tripolar electrodes separated 15 mm or more apart is a more accurate method for measuring conduction velocity than electrical stimulation, which was subject to several large errors.

Brain uptake of a food dye, erythrosin B, prevented by plasma protein binding.

Although food colors have been held responsible for several behavioral disorders and do affect neuronal function when directly applied, there is no information on whether significant quantities of the dyes appear in the brain after consumption or parenteral administration. [14C]erythrosin B was administered directly into the circulation of mature rats and radioactivity was measured thereafter in brain regions at several times. Although insignificant parenchymal radioactivity was detected in brains perfused with dye in whole blood, significant concentrations of [14C]erythrosin B were detected in all brain regions when perfused with protein-free Ringers, as predicted from the octanol-water partition coefficient of the dye. Thus, significant brain uptake of intravascular dye is normally prevented by its binding to plasma protein (greater than 99% bound) and by the blood-brain barrier impermeability to the dye-protein complex. Sensitivity to food dyes such as erythrosin B in some individuals may reflect altered plasma protein binding capacity, which can vary with age and disease.

Vicissitudes of the suicidal impulse in dreams.

The author presents several dreams reported by psychosomatic patients which contain an overt or disguised act of suicide. In the latter instances various transformations of the act of suicide had been brought about by the defensive functions of the dreaming ego. Paradoxically, in several of these instances it was the very efforts of the dreaming ego to defend itself which allowed the suicidal impulse to reach its consummation.

Dreams which culminate in migraine headaches.

23 dreams culminating in attacks of migraine were collected. Despite problems of overlap it was possible to separate out the dreams into six categories on the basis of their manifest content. All but one of the categories were characterized by the presence of powerful negative affect. By far the largest category was made up of dreams of terror.

Insemination of rabbit eggs is associated with slow depolarization and repetitive diphasic membrane potentials.

The plasma membrane of the rabbit egg allows only one sperm to enter the egg during fertilization, but the mechanism of this block to polyspermy is unknown. Electrophysiology and in vitro fertilization techniques were employed in this study to investigate the possibility that a voltage block to polyspermy exists in rabbit eggs. Ovulated zona-intact eggs had a mean membrane potential of -71 +/- 2.1 mV (interior negative). A stereotypic response occurred 12-135 min following in vitro insemination in 19 of 40 eggs. Association of this stereotypic response with the appearance of pronuclei suggested that the electrical response was related to some interaction of gametes. This response consisted of a slow transient 8 +/- 1.5 mV depolarization upon which were superimposed up to 36 repetitive diphasic insemination potentials. Each potential consisted of a brief 2.0 +/- 0.44 mV hyperpolarization followed by a slow 2.5 +/- 0.45 mV depolarization. The small amplitude of the stereotypic response when compared with the large variation of resting potentials suggested that the response was insufficient to block polyspermy by a mechanism dependent upon the magnitude of the rabbit egg membrane potential.

Suicidal trends in patients with asthma and hypertension. A chart study.

A chart study revealed a significantly higher percentage of suicidal trends in patients with asthma and hypertension as compared to patients in two control groups. The theoretical significance of this finding is briefly discussed.

Neurotransmitter release and nerve terminal morphology at the frog neuromuscular junction affected by the dye Erythrosin B.

1. The quantal release of neurotransmitter and the fine structure of frog neuromuscular junctions has been examined in the presence of the xanthene dye Erythrosin B.2. At concentrations of 10 muM or greater, Erythrosin B produced time- and dose-dependent increases in transmitter release from presynaptic nerve terminals.3. Miniature end-plate potential (m.e.p.p.) frequency increased in an exponential manner during continuous exposure to the dye. The rate constant for this exponential was dose-dependent, increasing with concentrations from 10 muM to 1 mM.4. The amplitude of evoked end-plate potentials (e.p.p.s) also increased exponentially during dye treatment, primarily due to an increase in quantal content. Rate constants for this effect were also dose-dependent, and were approximately 1/5 as large as those for m.e.p.p.s.5. While the frequency of m.e.p.p.s was increasing, their amplitude distribution did not qualitatively change. Thus the dye has little effect on the size of individual quanta.6. The presynaptic effects of Erythrosin B were irreversible under these experimental conditions. Brief exposure to the dye caused increases in m.e.p.p. frequency and e.p.p. amplitude which were maintained at steady levels during extensive rinsing with dye-free Ringer solution.7. Prolonged exposure to the dye caused an eventual decrease in m.e.p.p. frequency and abolition of e.p.p.s. Coincident with this decline ;giant' m.e.p.p.s as large as 40 mV were observed.8. At dye concentrations greater than approximately 200 muM, Erythrosin B rapidly and reversibly increased the membrane potential and input resistance of muscle fibres. This post-synaptic effect was small and variable in normal saline, but was pronounced in low potassium solutions.9. During the period that release was enhanced by Erythrosin B, presynaptic nerve terminals contained the normal complement of synaptic vesicles and other organelles. Mitochondria were swollen in this condition.10. After m.e.p.p. frequency declined below normal levels and ;giant' m.e.p.p.s appeared, the number of synaptic vesicles within nerve terminals declined and dilated cisternae were present. Mitochondria were swollen further.11. These results do not reveal any mechanism to explain the ability of Erythrosin B to increase transmitter release, but the decline in release may be caused by partial depletion of synaptic vesicles. The ;giant' m.e.p.p.s could be due to the discharge of acetylcholine from cisternae.

Presynaptic effect of Erythrosin B at the frog neuromuscular junction: ion and photon sensitivity.

1. The anionic dye Erythrosin B increases quantal transmitter release from frog neuromuscular synapses. Experiments were performed to determine the role of ions and light in this presynaptic effect. 2. In calcium-free saline containing 1 mM-EGTA, Erythrosin B increased miniature end-plate potential (m.e.p.p.) frequency at a more rapid rate than in normal saline. 3. The dye's effect was influenced by extracellular calcium ions in a complex manner. Dye-induced release was minimal in Ringer solution containing 0.1 mM-calcium, and higher in calcium concentrations above or below 0.1 mM. 4. Erythrosin B-induced spontaneous release also occurred in saline which contained 1 mM-EDTA and was free of both calcium and magnesium ions. 5. Temporary removal of external sodium ions did not alter the progressive increase in m.e.p.p. frequency produced by the dye. 6. Elevation of the potassium concentration of the external medium (from 2 to 20 mM), which presumably depolarized nerve terminals and increased their calcium permeability, did not change the rate of increase of dye-induced release when preparations were in a reversed (outward) electrochemical gradient for calcium ions. 7. A reduction in light intensity of at least six orders of magnitude reduced the effect of Erythrosin B by 50%, suggesting that photoactivation is not the primary basis for the dye's action. 8. These results indicate that Erythrosin B is not acting solely by altering the ionic permeability of the presynaptic nerve terminal to calcium, magnesium, or sodium ions, or by altering the calcium metabolism of the terminal. The enhanced effect of the dye in calcium-free saline suggests that it may be competing with calcium at a common site, while the enhancement of its effect in elevated external calcium suggests that the dye may also increase the permeability of the nerve terminal to calcium ions.

Peripheral nerve as an osmometer: role of the perineurium in frog sciatic nerve.

Measurements of volume and hydrostatic pressure in the frog sciatic nerve in vitro demonstrate that the nerve acts as an osmometer, in large part because the perineurium is a semipermeable membrane for water flow. Endoneurial hydrostatic pressure in nerves in isotonic Ringer exceeds bath pressure by about 7 mmHg. In Ringer made hypertonic by addition of sucrose, nerve volume and endoneurial pressure fall linearly in relation to 1/osmolality. The slope of the plot of pressure against volume provides a value for nerve compliance equal to 0.006 mm2/mmHg. Calculations based on the model of the nerve as an osmometer indicate that the nerve has an osmotically "inactive" volume equal to 0.19 mm3/mm, which is about 75% of the total volume of a nerve segment of unit length in normal Ringer. Perineurial hydraulic conductivity (Lp) equals 7.5 x 10(-13) cm3.s-1.dyn-1, a value characteristic of nonleaky epithelia. The perineurium is an elastic tissue with a constant modulus of elasticity equal to 3 x 10(6) dyn/cm2 when not markedly stretched and may limit nerve swelling under pathological conditions of nerve edema.

Explicit incestuous motifs in psychosomatic patients.

In this study, which is the first ever to deal with the issue of incestuous motifs in psychosomatic patients, the author reviewed the charts of all his psychosomatic patients, seen in at least 40 interviews between 1975 and 1980, and the charts of all his non-psychosomatic patients, seen in at least 40 interviews between 1970 and 1975, in order to determine the frequency of explicit incestuous motifs in each group. It was found that the motifs had been reported by more psychosomatic patients than non-psychosomatic patients. Also, it was found that the explicit incestuous motifs which had been reported by the psychosomatic patients were more intense and pervasive than those which had been reported by the non-psychosomatic patients. These findings are discussed with reference to the issue of failure of the ego's defense mechanisms in psychosomatic patients.