Effects of NG-nitro-L-arginine and L-arginine on regional cerebral blood flow in the cat.

1. We studied the effects of NG-nitro-L-arginine (NOLA), a potent inhibitor of the L-arginine-nitric oxide pathway, and L-arginine, the precursor of nitric oxide, on regional cerebral blood flow, electrocortical activity and ex vivo cerebrovascular reactivity in the cat. Flow was measured via radiolabelled microspheres, and vascular responses were studied by measuring isometric tension of isolated middle cerebral arterial rings. 2. NOLA (30 mg kg-1 bolus followed by 1 mg kg-1 min-1 infusion) caused an approximately 40 mmHg elevation in the mean arterial blood pressure, a regionally heterogenous increase of the regional cerebrovascular resistance and a decrease in the regional cerebral blood flow 15 and 40 min after the start of its administration. In contrast L-arginine (30 mg kg-1 bolus followed by 10 mg kg-1 min-1 infusion) did not alter blood pressure, cerebrovascular resistance nor regional cerebral blood flow 15 min after the start of its administration. The NOLA-induced changes in tissue flow were the most pronounced in the cerebellum, pituitary and medulla oblongata, whereas there was no decrease in the flow of the cortex and white matter. 3. NOLA caused characteristic changes in total fronto-occipital EEG power and in power spectra which were unlikely to have been due to cerebral ischaemia. In addition, the ex vivo reactivity of the middle cerebral arteries showed signs of impaired endothelial nitric oxide synthesis: there were enhanced noradrenaline-induced contractions and N-ethoxycarbonyl-3-morpholino-sydnonimine (SIN-1)-induced relaxations and markedly attenuated acetylcholine- and ATP-induced relaxations after NOLA treatment. 4. The present data indicate that resting cerebral blood flow and cerebrovascular resistance are regulated by nitric oxide derived from L-arginine in a regionally heterogenous way and that exogenous L-arginine availability is not a limiting factor in this nitric oxide generation. Possibly, both the vascular endothelium and the neurons contribute to this basal nitric oxide release.


Effects of proteins on the permeability of monolayers of cultured bovine arterial endothelium.

1. Monolayers of arterial endothelium on porous membranes were exposed to a pressure of 15 cmH2O at 37 degrees C, or of 30 cmH2O at 0 degree C. At constant pressure, the rate of liquid flow per unit area (Jv/A) through each monolayer decreased with time, in the way previously described for cultured endothelium. This phenomenon has been called sealing. After Jv/A stabilized, the pressure was reduced and the hydraulic permeability (Lp) of the endothelium was calculated from the relationship between Jv/A and pressure. Endothelium was seen to be damaged after some experiments at 37 degrees C, but appeared undamaged after experiments at 0 degree C. 2. Bovine serum albumin (BSA) did not influence the Lp of cultured endothelium. At 37 degrees C, the mean (+/- S.E.M.) endothelial Lp was 47.2 +/- 7.3 x 10(-7) cm s-1 cmH2O-1 (n = 10) in the presence of BSA (5 g (100 ml)-1). This is not significantly different from the mean (+/- S.E.M.) Lp of 53.4 +/- 9.0 x 10(-7) cm s-1 cmH2O-1 (n = 9) in the absence of added protein (P greater than 0.10). At 0 degree C also, there was no significant difference between mean Lps in the presence of BSA (0.1 g (100 ml)-1) and in the absence of added protein. 3. Solutions of BSA (5 g (100 ml)-1 or of the neutral polymer Ficoll 70 (4 g (100 ml)-1) did not exert any effective osmotic pressure across endothelium at 37 or 0 degrees C, respectively. 4. BSA (0.1 g (100 ml)-1) did not enable solutions of Ficoll 70 (4 g (100 ml)-1) to exert an effective osmotic pressure across endothelium at 0 degree C. 5. The mean Lp of endothelium at 0 degree C was significantly lower in the presence of cationized ferritin (CF; 0.1 g (100 ml)-1) than in the absence of added protein (P less than 0.001). Native ferritin (NF; 0.1 g (100 ml)-1) had no effect on Lp. 6. In the presence of CF (0.1 g (100 ml)-1), solutions of Ficoll 70 (4 g (100 ml)-1) exerted a mean effective osmotic pressure of 27.7 cmH2O (n = 5) across endothelium at 0 degree C. The mean effective osmotic pressure exerted across endothelium by solutions of Ficoll 70 (4 g (100 ml)-1) plus NF (0.1 g (100 ml)-1) was 1.2 cmH2O (n = 4).


The pH dependence of chloride net flux in skeletal muscle fibres of Rana temporaria.

1. The pH- and voltage-dependent Cl- conductance in frog muscle may be related to the chloride equilibrium potential, VCl,eq, rather than to the absolute membrane potential, Vm. This hypothesis was tested in thin depolarized frog muscle fibre bundles by investigating the influence of pH on 36Cl- efflux during Cl- net efflux upon removal of external Cl-, which offers a state far from Cl- equilibrium by changing VCl,eq instead of Vm. 2. Upon the change from Cl- equilibrium at 20 mM-Clo- to Cl- net efflux at zero [Cl-]o, Vm changed only approximately 10 mV, but the 36Cl- efflux rate increased about three times at pH 5.5 and decreased to less than one tenth of the equilibrium efflux rate at pH 9.8. The switch between 'acid' and 'alkaline' responses occurs at a pH between 6.4 ('acid response') and 7.2 ('alkaline response'). 3. Changing pH between 5.5 and 9.8 during Cl- net efflux showed an increase of 36Cl- net flux rate upon acidification and a decrease upon alkalinization. The reactions are opposite to those seen by pH shifts at chloride equilibrium. 4. The changes of net flux rate coefficients upon changes of pH were transient, especially the activation at low pH that relaxed significantly during about 10 min. 5. The results are consistent with the notion that the state of the gCl mechanism in frog muscle is related to (Vm-VCl,eq) rather than to the absolute potential alone.


Respiratory modulation of the activity in sympathetic neurones supplying muscle, skin and pelvic organs in the cat.

1. The respiratory-related modulation of activity in neurones of the lumbar sympathetic outflow to skeletal muscle, skin and pelvic organs was investigated in anaesthetized, paralysed and artificially ventilated cats, using single- and multi-unit recordings. The activity of the neurones was analysed with respect to the phrenic nerve discharge under various experimental conditions. 2. Neurones tentatively classified as muscle vasoconstrictor and visceral vasoconstrictor neurones exhibited two activity peaks, one caused by baroreceptor unloading during the declining phase of the second order blood pressure waves and a respiratory drive-dependent peak in parallel with inspiration. The two peaks were separated by depressions of activity in early inspiration and post-inspiration. After cutting vagus and buffer nerves the activity peak during inspiration remained and was followed and sometimes preceded by a depression of activity. 3. The majority of the neurones tentatively classified as cutaneous vasoconstrictor neurones exhibited no respiratory modulation in their activity. Others exhibited an activity peak in expiration, an activity peak in inspiration, or a respiratory profile similar to that in muscle vasoconstrictor neurones. During increased respiratory drive (induced by hypercapnia) some neurones with unmodulated activity changed to an inspiratory or an expiratory pattern. Neurones discharging predominantly in inspiration projected preferentially to hairless skin. 4. Neurones which were tentatively classified as sudomotor neurones discharged predominantly in early expiration. 5. Some preganglionic neurones which were tentatively classified as motility-regulating neurones discharged during expiration. The majority of these neurones disclosed no respiratory modulation of their activity. 6. The study shows that different types of neurone of the lumbar sympathetic system exhibit distinct patterns of respiratory modulation in their activity. We conclude that the type and degree of central coupling between respiratory system and sympathetic nervous system may vary according to the destination of the sympathetic neurones.


Longitudinal chromatic aberration and emmetropization: results from the chicken eye.

1. Due to the chromatic dispersion of the ocular media, the focal length of the optics of the eye is about 3 diopters longer for red light than for blue light. Because emmetropization in the chicken (Gallus domesticus) does not require colour cues and operates properly in monochromatic light, one can, therefore, expect that chickens raised in red light become more myopic (with longer eyes) than chicks raised in short wavelength light. Prior to conducting this experiment, we matched the brightness of both light conditions by means of flicker electroretinograms such that equiluminance was obtained for the chickens. 2. Unexpectedly, refractive development was not different from controls in white light for either red or near-ultraviolet light. 3. We tested whether the visual mechanisms guiding refractive development were still sensitive to defocus under both illuminations by treating the chicks with spectacle lenses. 4. Similar to a previous experiment in white light, the growth of the eye in red light also changed such that it compensated for the imposed defocus. It failed to do so, however, in near-ultraviolet light. 5. A histological analysis of the sampling intervals for the ultraviolet receptor system revealed that its spatial resolving power was too low to detect the defocus imposed by the lenses, whereas the long wavelength receptors provided sufficiently good visual acuity. 6. The results show that, during emmetropization, the chicken eye elegantly bypasses the problem of multiple chromatic focal planes by having a low sensitivity to defocus in the blue end of the spectrum. Because the chromatic dispersion function is steep in the blue range but flat at the red end of the spectrum, the remaining chromatic defocus in the spectral range of high visual acuity is low and may match the depth of field of the eye.


Cholinergic activation of a non-selective cation current in canine gastric smooth muscle is associated with contraction.

1. The effects of acetylcholine on membrane electrical properties of single smooth muscle cells dissociated from the circular layer of the canine gastric corpus were investigated using the nystatin perforated patch technique. Cells retained their ability to contract during recording, making it possible to correlate changes in membrane potential and membrane currents with contractions. 2. Acetylcholine caused depolarization from -59 +/- 8 mV to -18 +/- 7 mV (means +/- S.D., n = 12) with no generation of action potentials. The depolarization was associated with a membrane conductance increase, consistent with acetylcholine activating an inward current. In addition, acetylcholine caused contraction of cells to 58% of initial length. Both depolarization and contraction were reversible and were antagonized by atropine. 3. Under voltage clamp, acetylcholine activated inward current associated with increased current noise. The current-voltage relationship of the acetylcholine-induced current was studied at steady-state voltages and with voltage ramp commands. The inward current was largest between -40 and -20 mV and reversed direction to outward close to 0 mV (reversal potential, Erev = +3 +/- 9 mV). Reduction of external Na+ concentration to 21 mM shifted Erev to -42 +/- 5 mV, as predicted for a non-selective cation current. The conductance activated by acetylcholine (gACh) increased sigmoidally with depolarization, with about 2.5 nS activated at 0 mV. 4. Cells consistently contracted upon stimulation with acetylcholine, even when studied under voltage clamp at potentials as negative as -100 mV. This was consistent with muscarinic receptor activation causing release of Ca2+ from internal stores. When cells were bathed in Ca(2+)-free solutions, the first application of acetylcholine elicited normal inward current and contraction. Thereafter, both inward current and contractions were greatly diminished or absent, suggesting that the stores of Ca2+ had been depleted. 5. Caffeine caused reversible contraction and activation of inward current similar to that elicited by acetylcholine. 6. It is concluded that muscarinic stimulation of canine gastric smooth muscle cells involves activation of a non-selective cation conductance and is consistently accompanied by contraction. The release of Ca2+ from internal stores may be a common trigger for both events.


Effect of temperature on relaxation rate and Ca2+, Mg2+ dissociation rates from parvalbumin of frog muscle fibres.

1. Influence of temperature on relaxation rate as a function of isometric tetanus duration and on Ca2+ and Mg2+ dissociation rates from purified parvalbumin (PA) was examined to test the hypothesis that PA promotes relaxation in frog skeletal muscle. Single fibres and PA IVB from Rana temporaria skeletal muscle were utilized. 2. The magnitude of slowing of relaxation rate with increasing tetanus duration, relative to the final, steady value of relaxation rate, is 3-fold greater at O than at 10 degrees C. 3. In the 0-10 degrees C range, the Q10 for relaxation rate increases from 2.3 to 3.7 with increasing tetanus duration. 4. Dissociation of Ca2+ and Mg2+ from PA exhibited: (i) rate constants of 1.03 +/- 0.03 s-1 (mean +/- S.D., n = 5) and 3.42 +/- 0.14 s-1 (n = 5) at 20 degrees C and (ii) Q10 values of 2.3 and 1.9 in the 0-20 degrees C range, respectively. 5. Time courses of slowing of relaxation rate with increasing tetanus duration and recovery of relaxation rate with rest after a prolonged tetanus at 10 degrees C are similar to rates of dissociation of Mg2+ and Ca2+ from PA, respectively, as previously reported at 0 degree C. 6. Both the temperature dependence of the relative magnitude of slowing of relaxation rate and the increased Q10 of relaxation rate with increased tetanus duration can be explained if the Q10 for Ca2+ uptake by the sarcoplasmic reticulum is greater than the Q10 for Ca2+ sequestration by PA during relaxation. When Ca2+ and Mg2+ dissociation rates from PA at various temperatures are compared to other proposed indicators of PA function, it is concluded that PA facilitates relaxation of frog skeletal muscle in the 0-20 degrees C range.


Evidence that a long latency stretch reflex in humans is transcortical.

1. The hypothesis that the long latency reflex response to muscle stretch in humans uses a transcortical pathway was tested by looking for convergence onto cortical neurones in eleven normal subjects. 2. Postsynaptic events in single flexor pollicis longus (FPL) motoneurones were derived from changes in the firing probability of individual FPL motor units. 3. Extension of the terminal phalynx of the thumb resulted in both short latency and long latency facilitations of individual FPL motoneurones. These were not reproduced by electrical stimulation of afferents in the terminal phalynx. Magnetic stimulation over the contralateral motor cortex produced strong, short latency facilitation of FPL motoneurones. 4. When the facilitation produced by stimulation over the cortex was superimposed on the long latency facilitation produced by extension of the thumb, the facilitation produced by both stimuli was greater than the sum of the individual facilitations produced by either stimulus given alone. This was not the case when the superimposition occurred on the short latency response to stretch. 5. We conclude that afferent systems excited by the stretch of FPL converge onto cortical neurones which are known to facilitate motoneurones. Thus the cortex is likely to contribute to the long latency stretch reflex in humans.


Relationship between parasternal and external intercostal muscle length and load compensatory responses in dogs.

1. The effects of tracheal occlusion on peak parasternal (PA) and external intercostal (EI) (3rd interspace) EMG activities were examined at different end-expiratory lung volumes both above and below functional reserve capacity (FCR) in anaesthetized, vagotomized and spontaneously breathing dogs. 2. Parasternal (PA) and external intercostal (EI) muscle lengths were monitored in situ. The difference in peak EMG activity between free and occluded breaths (test breaths) was related to the coincident peak change in intercostal muscle length (delta L) for each muscle, respectively. 3. At FRC, tracheal occlusion resulted in compensatory augmentation of peak EI, but little change in peak PA EMG activities. At lung volumes below FRC, airway occlusion resulted in augmentation of both PA and EI activities. Responses to airway occlusion at lung volumes above FRC were variable. The magnitude and duration of these changes in EMG, however, could be linearly related to the value of delta L. With delta L = 0, there was no change in peak EI or PA EMG; for values of delta L less than 0, there was attenuation of EI and PA EMG; for delta L greater than 0, there was enhancement of EI and PA EMG activation. 4. The magnitude of the changes in EMG activity in response to tracheal occlusion was more prominent for the EI muscle compared to the PA, the latter of which are known to have much fewer muscle spindles than EI muscle. 5. Our results suggest that a difference in end-inspiratory muscle length between the control and occluded breaths is a stimulus for the intercostal response to applied loads implicating muscle spindles as the predominant receptor moderating these responses. We hypothesize that when delta L = 0, no change in EMG occurs since the spindles sense no change in muscle length. When delta L less than 0 (i.e. peak muscle length during the occluded breath is shorter than control) muscle spindles would be disengaged, resulting in a disfacilitation of EMG activity. Where delta L greater than 0 (i.e. peak muscle length during the occluded breath is longer than control), muscle spindles are stimulated, resulting in enhancement of EMG activity. 6. Additional doses of Nembutal (20 mg), which produced significant changes in breathing pattern, did not affect the magnitude of the load compensatory responses.


Response characteristics of the cells of cortical area 21a of the cat with special reference to orientation specificity.

1. Extracellular recording using tungsten-in-glass microelectrodes was conducted on 115 neurons in area 21a of fifteen anaesthetized cats. Quantitative analysis using computer-controlled display and collecting routines were used to investigate the excitatory and inhibitory regions of the receptive field and to see if interaction, within and between these regions, contributed to the response properties of the cells. 2. The responses of the cells in the sample appeared to arise from a single, homogeneous class. All cells had single discharge regions which responded with composite ON/OFF firing to a stationary flashing bar. The same region also responded to moving light and dark bars and edges. There was little evidence of inhibition as measured by the suppression of spontaneous or induced firing. Most cells had relatively small receptive fields (primary width: mean = 2.1 +/- 0.9 deg (S.D.); n = 108), all were binocular and were located within 15.0 deg of the visual axes. 3. All cells responded well to slowly moving stimuli but generally failed to respond to stimuli moving faster than 10.0 deg s-1. All responses were bi-directional and, although many showed evidence of length summation, there was no sign of linear summation. 4. Despite the absence of significant sideband inhibition many cells were acutely tuned for orientation (half-width at half-height: mean = 15.6 +/- 5.3 deg; n = 48). To investigate this property further, cells were analysed to assess the effect of changing the length of a moving bar stimulus on the acuteness of the orientation tuning curve. Short bars, of similar length to the width of the receptive field, had orientation tuning curves of equivalent sharpness to those obtained with longer bars. 5. The equivalence of orientation tuning for long and short bars stands in contrast to the results obtained for both simple (S) and complex (C) cells of the striate cortex where tuning for the longer bar is sharper than that for the shorter. The result from area 21a cells is consistent with the absence of sideband inhibition and can be related to an input from the striate cortex that passes through a threshold barrier. 6. The orientation tuning of cells of area 21a can be explained if it is assumed that they receive their major input from C or complex cells of the striate cortex in which firing must reach a threshold frequency to activate the recipient cell.


Neural regulation of acetylcholine receptors in rat neonatal muscle.

1. The neuronal regulation of the developmental decline in skeletal muscle acetylcholine (ACh) receptors was studied by comparing the effects of sciatic nerve section or of neuromuscular blockade with botulinum toxin (BoTX) on this decline in neonatal and adult rats, using 125I-alpha-bungarotoxin (125I-BTX) as a ligand for the receptor alpha-subunit. 2. The decline in 125I-BTX binding site concentration in neonatal rat triceps surae muscle homogenates towards low, adult levels followed a simple exponential with a time constant of 8 days. This decline occurred while the muscle is still rapidly growing, before the postnatal increase in numbers of sodium channels. It also preceded the decline in muscle ACh receptor alpha-subunit mRNA, reported in other studies, suggesting that subunit levels are not regulated only by mRNA availability. 3. Muscle denervation in the first two weeks of life prevented this developmental decline. Denervation increased the concentration of 125I-BTX binding sites but the magnitude of this increase became progressively smaller as the muscle matured, showing that removal of innervation during adult life does not revert the muscle, in toto, to its pre-innervation state. 4. Blockade of neuromuscular activity with BoTX increased 125I-BTX binding sites to a lesser extent than muscle denervation during neonatal life. This lesser effect of BoTX blockade contrasts with the equal effects of BoTX blockade and denervation in the adult.


Changes of intracellular pH due to repetitive stimulation of single fibres from mouse skeletal muscle.

1. The performance of skeletal muscle during repetitive stimulation may be limited by the development of an intracellular acidosis due to lactic acid accumulation. To study this, we have measured the intracellular pH (pHi) with the fluorescent indicator BCECF (2',7'-bis(carboxyethyl)-5(6)- carboxyfluorescein) during fatigue produced by repeated, short tetani in intact, single fibres isolated from the mouse flexor brevis muscle. 2. The pHi at rest was 7.33 +/- 0.02 (mean +/- S.E.M., n = 29, 22 degrees C). During fatiguing stimulation pHi initially went alkaline by about 0.03 units (maximum alkalinization after about ten tetani). Thereafter pHi declined slowly and at the end of fatiguing stimulation (tetanic tension reduced to 30% of the original; 0.3Po), pHi was only 0.063 +/- 0.011 units (n = 14) more acid than in control. 3. We considered three possible causes of acidosis being so small in fatigue: (i) a high oxidative capacity so that fatigue occurs without marked production of lactic acid; (ii) an effective transport of H+ or H+ equivalents out of the fibres; a high intracellular buffer power. 4. The oxidative metabolism was inhibited by 2 mM-cyanide in three fibres. After being exposed to cyanide for 5 min without stimulation, the tetanic tension was reduced to about 0.9 Po and pHi was alkaline by about 0.1 units. The fibres fatigued faster in cyanide and the pHi decline in fatigue was more than twice as large as that under control conditions. 5. Inhibition of Na(+)-H+ exchange with amiloride resulted in a slow acidification of rested fibres; resting pHi was not affected by either inhibition of HCO3(-)-Cl- exchange with DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) or inhibition of the lactate transporter with cinnamate. 6. Fibres fatigued in cinnamate displayed a markedly larger acidification (approximately 0.4 pH units) and tension fell more rapidly than under control conditions; inhibition of Na(+)-H+ and HCO3(-)-Cl- exchange did not have any significant effect on fatigue. 7. The intracellular buffer power, assessed by exposing fibres to the weak base trimethylamine, was about 15 mM (pH unit)-1 in a HEPES-buffered solution (non-CO2 or intrinsic buffer power) and about 33 mM (pH unit)-1 in a bicarbonate-buffered solution. Somewhat higher values of the intrinsic buffer power was obtained from changes of the partial pressure of CO2 (PCO2) of the bath solution. Application of lactate or butyrate frequently gave an infinite buffer power, which indicates that powerful pH-regulating mechanisms operate in these cases.(ABSTRACT TRUNCATED AT 400 WORDS)


Opioidergic inhibition of flexor and extensor reflexes in the rabbit.

1. Recordings were made from gastrocnemius medialis (GM), semitendinosus (ST) and tibialis anterior/extensor digitorum longus (TA/EDL) motor nerves during mechanical and electrical stimulation of the skin of the foot in decerebrated and spinalized rabbits. 2. GM motoneurones were excited from the heel and not from the toes, whereas TA/EDL responded to stimulation at the toes but not at the heel. ST also responded to electrical and mechanical stimulation at the toes, but there was a disparity between the effects of the two types of stimuli when they were applied at the heel: ST motoneurones fired in response to electrical stimulation of the heel but showed only an 'off' response to mechanical stimulation at this site. 3. The opioid antagonist naloxone caused a dose-dependent increase in all reflexes evoked by electrical stimulation of the skin. The heel-GM, toes-ST and toes-TA/EDL reflexes all increased to more than 3 times control levels with naloxone, which also caused significant decreases in the latencies of these reflex responses. On the other hand, the heel-ST response increased to just 1.4 times control levels and showed no decrease in latency with the opioid antagonist. 4. These data suggest that segmental withdrawal reflex pathways in the rabbit are suppressed by endogenous opioid peptides. This opioid-mediated inhibition seems to operate non-selectively on reflex pathways between cutaneous afferents and motoneurones.


The perceptions of force and of movement in a man without large myelinated sensory afferents below the neck.

1. Motor memory and the sense of effort have been investigated in a man with a complete large fibre sensory neuropathy for over 16 years. The perceptions of pain, heat, cold and muscular fatigue remained but he was without perceptions of light touch and proprioception below the neck. 2. The subject was able to discriminate weights held in the hand with an accuracy only slightly worse than control subjects (20 g in 200 g) when forearm movement and visual inspection were allowed. With eyes shut however he could only distinguish a weight of 200 g from 400 g. It is concluded that a crude sense of effort remains which may have a peripheral origin. 3. A limited motor memory was also present, which allowed him to maintain a posture or continue a simple repetitive movement. No novel movement was possible without visual feedback. 4. Differences in movement ability between this subject and others with similar if less pure sensory neuropathies are ascribed to rehabilitation.


The relationship between contraction and intracellular sodium in rat and guinea-pig ventricular myocytes.

1. The contraction, measured optically, and the intracellular Na+ activity (aNai), measured with the Na(+)-sensitive fluorescent dye SBFI, have been recorded simultaneously in rat and guinea-pig ventricular myocytes. 2. In rat and guinea-pig ventricular myocytes at rest, aNai was 7.8 +/- 0.3 mM (n = 4) and 5.1 +/- 0.3 mM (n = 16), respectively. 3. When both rat and guinea-pig ventricular myocytes were stimulated at 1 Hz after a rest there was usually a gradual increase in twitch shortening (referred to as a 'staircase') over several minutes accompanied by an increase in aNai over a similar time course. Twitch shortening increased by 21 +/- 3% (n = 6) and 20 +/- 4% (n = 16) (of steady-state twitch shortening during 1 Hz stimulation) per millimolar rise in aNai in rat and guinea-pig ventricular myocytes, respectively. 4. When rat and guinea-pig ventricular myocytes were exposed to strophanthidin to block the Na(+)-K+ pump, there were increases in twitch shortening and aNai over similar time courses. Twitch shortening increased by 24 +/- 4% (n = 5) and 20 +/- 3% (n = 10) (of control twitch shortening) per millimolar rise in aNai in rat and guinea-pig ventricular myocytes respectively. 5. The inotropic effect of cardiac glycosides, such as strophanthidin, is widely regarded to be principally the result of the rise in aNai. The similarity of the relation between twitch shortening and aNai during the staircase and on application of strophanthidin suggests that the progressive increase in the strength of contraction during the staircase was also linked to the rise in aNai. 6. In guinea-pig, but not rat, ventricular myocytes there was hysteresis in the relation between twitch shortening and aNai on application and wash-off of strophanthidin. This indicates that strophanthidin has another inotropic action in guinea-pig ventricular myocytes. 7. A computer model of excitation-contraction coupling has been developed to simulate the staircase and the action of cardiac glycoside and to account for the relation between contraction and intracellular Na+.


Developmental changes in troponin T isoform expression and tension production in chicken single skeletal muscle fibres.

1. The Ca2+ sensitivity of tension development was characterized in single skinned fibres from the slow anterior latissimus dorsi (ALD), fast posterior latissimus dorsi (PLD), and fast pectoralis major (PM) muscles of the chicken at adult and neonatal (2 weeks post-hatch) stages of development. In the adult, the PM was most sensitive, the ALD intermediate, and the PLD least sensitive to Ca2+. 2. PM and PLD fibres were less sensitive to Ca2+ at the neonatal stage of development than in the adult. However, ALD fibres exhibited no age-dependent changes in Ca2+ sensitivity. 3. Characterization of regulatory protein composition indicated that the PM and PLD fibres had identical fast isoforms of troponin C and troponin I at each developmental stage examined, but there were muscle-specific and age-dependent expressions of troponin T isoforms in these fibres. 4. In the ALD fibres, identical slow isoforms of troponin C, troponin I and tropomyosin were found at each stage. In addition, the troponin T isoform that was present did not change with age. 5. The results suggest a relationship between the specific troponin T isoform composition of individual muscle fibres and their calcium sensitivities of tension development.


Changes in synovial PO2 and blood flow in the rabbit knee joint due to stimulation of the posterior articular nerve.

1. Experiments were performed to measure the partial pressure of oxygen in the synovial fluid (Ps,O2) of the normal rabbit knee joint and assess the extent to which this varied with changes in knee joint blood flow. 2. With the hypodermic needle oxygen electrode sited just within the synovial cavity, Ps,O2 values ranged from 25 to 72 mmHg with a mean of 48.2 +/- 3.1 (S.E.M.; n = 18). 3. Ps,O2 was found to decrease with increasing depth of penetration of the oxygen electrode. Lowest values were observed close to the articular cartilage. 4. Electrical stimulation of the posterior articular nerve (PAN) of the knee resulted in vasoconstriction of knee joint blood vessels which was accompanied by a decrease in Ps,O2. The frequency-response and voltage-response profiles to electrical stimulation of PAN, although differing in magnitude, showed a high degree of correlation between blood flow and Ps,O2. 5. As judged by the conduction velocity, the vasoconstrictor response to nerve stimulation was mediated by unmyelinated nerve fibres, presumed to be sympathetic postganglionic fibres. 6. In view of the low Ps,O2 values occurring deep within the joint, avascular structures such as cartilage could be subject to injury if sustained reduction in synovial blood flow occurred. This could be a contributory factor in the pathogenesis of degenerative and inflammatory joint diseases.


Changes in presynaptic inhibition of afferents to propriospinal-like neurones in man during voluntary contractions.

1. The possibility was investigated that the facilitation of the transmission in the propriospinal-like system during voluntary contraction, documented in the companion paper (Burke, Gracies, Mazevet, Meunier & Pierrot-Deseilligny, 1992), is due to a decrease in presynaptic inhibition of afferents projecting to propriospinal-like neurones. 2. The radial nerve was stimulated to evoke presynaptic inhibition of the monosynaptic Ia projections to forearm flexor motoneurones (Berardelli, Day, Marsden & Rothwell, 1987) and, hopefully, of the afferent input to propriospinal-like neurones projecting to these motoneurones. 3. The propriospinal-like excitation of forearm motoneurones evoked from mixed afferent inputs was depressed by radial nerve stimulation, and this depression was long-lasting (200 ms). Despite the convergence of mixed nerve and cutaneous afferents onto common propriospinal-like neurones, the radial stimulation did not depress the cutaneous-induced excitation. This differential effect and the time course of the depression suggest that it results from presynaptic inhibition of mixed nerve afferents (presumably large muscle afferents) projecting to propriospinal-like neurones. 4. With voluntary contractions, phasic or tonic, the radial-induced depression of the propriospinal-like excitation evoked by mixed nerve afferents was much greater than at rest, but the cutaneous-evoked excitation was unchanged. Thus, with voluntary contractions, there was no evidence of decreased gating of the afferent input to propriospinal-like neurones whether the input was of muscle or cutaneous origin and it is concluded that changes in presynaptic inhibition cannot account for the facilitation of the transmission in the propriospinal-like system during voluntary contraction. 5. By contrast, presynaptic inhibition of the monosynaptic Ia projections to motoneurones was consistently reduced at the onset of contraction, and to a much lesser extent during a weak tonic contraction.


The role of nociceptors of cutaneous veins in the mediation of cold pain in man.

1. To test the hypothesis that nociceptors of cutaneous veins mediate cold pain, we studied in man the time course of pain intensity and skin sensibility in relation to both intracutaneous and vein wall temperature during cooling of the dorsum of the hand by ice water before and after perivenous and intravenous nerve block. 2. Upon exposure to cold, intracutaneous temperatures fell exponentially (half-life/45-75s) within 10 min to a median of 4 degrees C (range 2-9 degrees C) and returned to baseline with a similar time course during rewarming (half-life/40-85 s). 3. Skin sensitivity to pin prick disappeared and returned at almost the same intracutaneous temperatures (16-26 degrees C). Pain, however, occurred and eve increased when the skin was already numb. 4. Pain occurred during cooling and disappeared during rewarming at vein wall temperatures between 23 and 28 degrees C and its intensity increased to a maximum of 72-100% of visual analogue scale as vein wall temperature decreased to a minimum of 9 degrees C (range 7-10.5 degrees C). 5. the pain intensity-vein wall temperature relations derived from skin cooling with threshold temperature changes between -5.5 and -9 degrees C and slopes between 2.2 and 3.3 were congruent to those derived from intravenous cooling in a previous study to ours. 6. Perivenous and intravenous nerve block, which did not alter the sensitivity of skin and periosteum, relieved cold pain markedly (perivenous block) or completely (intravenous block). 7. These observations are consistent with the hypothesis that nociceptors of cutaneous veins mediate cold pain in humans.


Orofacial manifestations of the systemic mycoses.

Aspergillosis, cryptococcosis and zygomycosis (mucormycosis) are overall the most common systemic mycoses but histoplasmosis is particularly endemic in parts of central USA and other areas worldwide. Orofacial lesions caused by systemic mycoses have rarely been reported in the past though they have been recorded particularly in outdoor workers from geographic areas with a high prevalence of infection and occasionally in immunocompromised individuals. Increasing world-wide travel, and the dramatic increase in numbers of immunocompromised persons, especially those with human immunodeficiency virus (HIV) disease, have been responsible for an increase in reports and other studies of orofacial disease in systemic mycoses and new opportunists are now being recognized. Those in Oral Medicine and Pathology must now be aware of the possibility of a systemic mycosis as the cause of chronic oral ulceration, chronic maxillary sinus infection, or bizarre mouth lesions, especially in patients with HIV disease, lymphoproliferative disorders, or diabetes mellitus, or in those who have been in endemic areas. Diagnosis and management should be undertaken in consultation with a physician with appropriate expertise, as pulmonary and other systemic infection may well be present. This paper reviews the eight main systemic mycoses.


The effect of the stimulation pattern on the fatigue of single motor units in adult cats.

1. The main purpose of this study was to examine the effects of two subtly different stimulus patterns on the force developed by fast-twitch, fatiguable motor units in a cat hindlimb muscle during control (pre-fatigue) and fatiguing contractions. 2. The peak force and the force-time integral responses of nineteen high fatigue (FF) and three intermediate fatigue (FI) motor units of the tibialis posterior muscle in five deeply anaesthetized adult cats were measured at selected times during the course of a 360-s fatigue test. 3. The fatigue test involved a pseudo-random alternation of two patterns of stimulation. One pattern (regular) was composed of a train of stimuli with constant interpulse intervals, set at 1.8 x the twitch contraction time of each unit (interval range, 27-51 ms), and delivered for 500 (or 400) ms. For the total (FF + FI) motor-unit sample, the mean (+/- S.D.) stimulation frequency was 26 +/- 4 Hz (range, 19-37 Hz). The other stimulus pattern (optimized) consisted of three initial stimuli with short (10 ms) interpulse intervals, followed by a constant interpulse-interval train that was adjusted (interval range, 29-62 ms; frequency, 23 +/- 5 Hz; frequency range, 16-36 Hz) such that the total train had the same number of pulses, and the same average frequency and duration as the regular train. 4. The stimulus trains were delivered at 1 s-1 for 360 s, using three-train sequences of each pattern, randomly alternating with one another. The response of the third train in each sequence was selected for the force measurements. The force profile obtained from the fatigue test was subsequently decomposed into two profiles: one attributable to regular and one to optimized stimulation. 5. During the initial responses to the fatigue test, the optimized stimulus pattern produced significantly more force than the regular stimulus pattern. For FF units, the mean increase in peak force (141%) was significantly greater than the increase in the force-time integral (59%). 6. All motor units exhibited an initial potentiation of peak force with the regular stimulation pattern, whereas peak force declined monotonically with the optimized pattern. In contrast, the force-time integral potentiated in the first 30 s for both regular and optimized stimulus patterns. 7. Each motor unit maintained an increased force response to optimized stimulation during the fatigue test, with the greatest relative increase occurring about 120 s into the test, well after the potentiation effect had subsided.(ABSTRACT TRUNCATED AT 400 WORDS)


The role of saliva in aggregation and adherence of Pseudomonas aeruginosa in patients with cystic fibrosis.

The aggregation and adherence activity of P. aeruginosa, mediated by whole saliva from cystic fibrosis (CF) patients and non-CF subjects, was investigated. CF saliva-mediated aggregation of P. aeruginosa was stronger than the activity of non-CF saliva. Likewise, P. aeruginosa adherence to buccal epithelial cells (BEC) of CF patients was stronger than to BEC of non-CF subjects. Adherence of non-mucoid P. aeruginosa to BEC of CF patients was increased by saliva, whereas the mucoid variant was not. CF patients colonized with P. aeruginosa showed higher adherence of the non-mucoid variant than non-colonized CF patients. CF patients with high saliva-mediated adherence of non-mucoid P. aeruginosa also had high salivary aggregation activity. Increased CF saliva-mediated aggregation activity may be linked to the increased non-mucoid P. aeruginosa adherence to BEC of CF patients.


Oral carriage of yeasts, coliforms and staphylococci in patients with advanced malignant disease.

Many patients with advanced cancer have oral problems, some of which may have a microbiologic basis. The oral flora in such patients has not, however, been characterized. This study has assessed the prevalence of yeasts, coliforms and coagulase positive staphylococci in the oral flora of 197 patients with advanced cancer. Both imprint cultures (n = 197) and oral rinses (n = 161) were collected. Yeasts were isolated from the mouths of 83% of the patients, coliforms from 49.1% and coagulase-positive staphylococci from 28%. All these percentages are considerably in excess of reported levels for healthy individuals. The results indicate a loss of colonization resistance of the oral mucosa in terminal cancer, with potential implications for the development of mouth care regimes for such patients.


Gingival cyst in adults.

A retrospective study is reported of 14 cases of gingival cyst of adults. Clinical data were pooled with well-documented cases in the literature. Most cases occurred in the 5th and 6th decades and in the premolar-canine-incisor area. Microscopically, the epithelial lining ranged from a thin, flat to cuboidal layer, 1-3 cells thick, resembling reduced enamel epithelium, to a nonkeratinized stratified squamous epithelium without rete ridges, of about 5-6 layers. Epithelial cells were frequently atrophic and this could involve the full thickness of the epithelium leaving an amorphous band of eosinophilic material. Plaque-like epithelial thickenings are sometimes present. Multicystic or "botryoid" forms may occur. The cysts are of developmental origin, arising either from basal cell extensions of overlying epithelium; or from postfunctional odontogenic epithelial residues of the dental lamina or rests of Malassez; or from junctional epithelium in the gingival crevice. The latter theory of origin is favored.


Epidermal growth factor receptor in odontogenic cysts and tumors.

The expression of epidermal growth factor receptor (EGFR) was investigated in 67 cases of odontogenic cysts and 35 cases of odontogenic tumors using monoclonal antibody to EGFR (Biomarker, Israel) to determine the presence and significance of this transmembrane growth factor receptor. The cystic epithelial cells of odontogenic cystic lesions (keratocyst 60%; primordial cyst 75%; radicular cyst 35%; and follicular cyst 47.4%) were positive to EGFR staining. Cytochemical characterization of EGFR in those cystic epithelium was cell membrane positive type as in the normal epithelium. No expression of EGFR was found in the odontogenic tumors. This diversity of EGFR represents no binding activity of EGF, or loss of EGFR in the tumor cell upon EGFR mediated growth in odontogenic tumors was suggested a different tumor cell growth factor status or microenvironment in cell proliferation mechanism at the cellular level in cysts and tumors of odontogenic origin.


Primary malignant lymphoma of the salivary gland: a tumor of mucosa-associated lymphoid tissue.

The clinical, morphologic and immunohistochemical features of 10 cases of the low-grade mucosa-associated lymphoid tissue (MALT) lymphoma of salivary glands are described. Although the initial histologic diagnosis in nine of these cases was myoepithelial sialadenitis, the diagnosis of primary salivary gland MALT lymphoma was based on the demonstration of light chain restriction and on morphologic characteristics. Histologic study showed a characteristic cytology, which included centrocytoid cells (composed of small centrocytes and monocytoid B cells) and a varying degree of plasma cell differentiation; the occurrence of epithelial or acinar invasion by neoplastic centrocytoid cells; and the presence of reactive lymph follicles among the neoplastic cells. Furthermore, multinucleate giant cells resembling Warthin-Finkeldey cells were detected in seven cases. In the light of these findings, cases previously diagnosed as myoepithelial sialadenitis require careful assessment and nine out of 32 cases are, in reality, examples of primary salivary gland MALT lymphomas. Immunohistochemical analysis of paraffin sections revealed the following characteristic immunophenotype of MALT lymphoma: L26, KiB3 and LN2 positive, and a monotypic immunoglobulin pattern (predominantly IgM/kappa). It was of interest that salivary gland parenchyma, infiltrated by neoplastic centrocytoid cells, reacted with LN3 for cells expressing human leukocyte antigen-DR (HLA-DR) antigens. Whereas salivary gland epithelia devoid of a neoplastic invasion were invariably negative for LN3. This suggests a lymphocyte-mediated role in salivary epithelial HLA-DR expression. It appears that HLA-DR expression is an inducible phenomenon in MALT lymphomas of salivary gland.


Possible premalignant character of oral lichen planus. The Amsterdam experience.

In the present paper the 21-yr experience of the Department of Oral and Maxillofacial Surgery and Oral Pathology, and the Department of Otorhinolaryngology of the Free University Hospital, Amsterdam, is reported with regard to the possible premalignant character of oral lichen planus. The study consists of two parts: 1) the follow-up of 113 patients with histopathologically proven oral lichen planus, and 2) a search for lichen planus in 727 patients who have been admitted for oral cancer. Three patients with histopathologically proven lichen planus developed a squamous cell carcinoma in an average follow-up period of 7 yr. In the 727 patients with oral cancer, two additional patients with the simultaneous occurrence of oral lichen planus were observed. Our experience gives some but not very strong support to the hypothesis that oral lichen planus is a premalignant condition.


A chemostat study on proline uptake and metabolism of Leishmania donovani.

Leishmania donovani grew in the chemostat on proline as its sole carbon and energy source at a maximum growth rate of 1.39 divisions per day. The efficiency of proline metabolism decreased with increasing external proline concentration. The internal concentration of proline and its intracellular metabolites was low when proline was the growth rate limiting substrate and high when proline was available in excess. In time-course experiments proline uptake leveled off after 30 min, independent of the culture conditions prior to the experiment. Proline uptake depended on the external proline concentration in a manner that is best described as the combination of an enzymatic and a diffusion component. Adaptation to different proline concentrations did not occur and no evidence was found that proline is actively transported by L. donovani.


Isoenzyme comparison of axenic Giardia lamblia strains.

We obtained isoenzyme patterns by polyacrylamide gradient gel electrophoresis (PGGE) of water-soluble protein fractions prepared from trophozoites of 11 axenic G. lamblia strains. The strains were isolated from animals and humans (both symptomatic and asymptomatic) from various geographic locations. Isoenzymes were also separated by isoelectric focusing. Of 12 enzymes attempted, eight exhibited well-defined and reproducible isoenzyme patterns by PGGE, based on which the strains were grouped into four zymodemes. Although the 11 strains were grouped into four zymodemes based on PGGE, no correlation between zymodeme and the known characteristics of the strains existed. Thus, a high degree of characteristic sharing appears to occur among genetically different G. lamblia strains.


Ciliate protozoa in the rumen of Kafue lechwe, Kobus leche kafuensis, in Zambia, with the description of four new species.

The composition of the rumen ciliate fauna in 76 Kafue lechwe inhabiting a limited area in Zambia was surveyed and five genera containing 24 species with 16 formae belonging to the family Ophryoscolecidae were identified. Four new species belonging to Diplodiniinae were recognized and described as Diplodinium lochinvarense n. sp., Diplodinium leche n. sp., Diplodinium zambiense n. sp., and Metadinium ossiculi n. sp. In addition, Ostracodinium gracile form fissilaminatum Dogiel, 1932 was found for the second time and described as Metadinium fissilaminatum n. comb. The species composition was fairly unusual. Seven of the species have been found only in African wild antelopes and these species were found more frequently than cosmopolitan species. There was no evidence of isotrichid species. The average density of ciliates per 1 ml of the rumen fluid was 25.7 x 10(4), and the number of ciliate species per head of host was 10.8.


Acanthamoeba healyi n. sp. and the isoenzyme and immunoblot profiles of Acanthamoeba spp., groups 1 and 3.

Two strains of Acanthamoeba isolated from human brain tissue and a strain of Acanthamoeba isolated from a fish were compared with 10 species of Acanthamoeba belonging to groups 1, 2 and 3 based on their isoenzyme profiles and antigenic characteristics. A total of 12 enzymes were studied. The isoenzymes and antigens were electrophoretically separated on polyacrylamide gradient gels, and the patterns obtained were compared after appropriate staining for particular enzymes and reactivities with homologous and heterologous rabbit anti-Acanthamoeba antisera. One of the human strains (CDC:1283:V013) was identified as A. healyi n. sp. because of its unique isoenzyme profiles for 11 of the 12 enzymes tested. The other human isolate was reidentified as A. culbertsoni because its isoenzyme profiles for 10 of 12 enzymes resembled those of A. culbertsoni, Lilly A-1 strain. Since the isoenzyme profiles and the antigenic patterns of the fish isolate as well were remarkably similar to those of A. royreba, it was considered as a strain of A. royreba. Polyacrylamide gradient gel electrophoresis appears to be a powerful technique for the study of isoenzymes and antigens of Acanthamoeba.


Production and characterization of three polyclonal antibodies raised against cyst wall proteins of a hypotrichous ciliate.

Three polyclonal antibodies raised against Paraurostyla sp. cyst wall polypeptides of molecular weight 110,000 (p110), 66,000 (p66) and 52,000 (p52) have been obtained. The specificity of the antisera was tested by immunoblotting. Anti-p110 antibody detected five bands of 300, 170, 135, 110 and 40 kDa, respectively. Antiserum obtained against p66 recognized only this protein. Anti-p52 antiserum showed reaction for two different bands of 52 and 44 kDa, respectively. The precise localization of these proteins in the cyst wall was assessed by light microscope immunocytochemistry. Anti-p110 antiserum produced a strong positive reaction in both the ectocyst and endocyst. Both anti-p66 and anti-p52 antibodies recognized the ectocyst.


Amphotericin B-induced carbohydrate changes on the Trypanosoma cruzi surface membrane.

Changes in the cell surface carbohydrates of Trypanosoma cruzi epimastigotes induced by Amphotericin B (AmB) were assessed by chemical methods and by agglutination assay employing a panel of highly purified lectins of various sugar specificities. Escherichia coli K12 with mannose-sensitive fimbriae was also used as an agglutination probe. Amphotericin B caused a decrease in the total carbohydrate content of all glycoconjugate fractions isolated. Exposure to AmB strongly affected the mannose/galactose ratio (1:5) in the CHCl3/methanol/H2O soluble fraction. These sugars in 1.4:1 ratio were the major hexose components of control cells. The decrease in the mannose content (48 to 15%) after AmB treatment agrees with the marked decrease in the T. cruzi cell surface receptors for fimbriated E. coli K12. Also, an increase in the galactose content (74%) as compared with control cells (34%) is in agreement with the peanut agglutinin and Euonymus europaeus lectins agglutination results. Differences in the cell surface carbohydrates induced by AmB could be associated with alterations in the membrane structure and organization.


Arginine catabolism by Leishmania donovani promastigotes.

Leishmania donovani promastigotes were grown to late log phase, washed and resuspended in iso-osmotic buffer containing L-arginine, and the rate of urea formation was then measured under various conditions. Addition of glucose or mannose activated urea formation, whereas 2-deoxyglucose inhibited and 6-deoxyglucose had no effect. Addition of alanine or of alpha-aminoisobutyrate inhibited urea formation, alanine causing a greater inhibition than alpha-aminoisobutyrate. Addition of leucine, proline, glycine, or lysine had no effect on urea formation. The presence of glutamate also increased the rate of urea formation from arginine, but to a lesser extent than did glucose. The presence of both glucose and alanine caused no net change in urea formation, whereas the inhibitory effect of alanine exceeded the activating effect of glutamate, so that a small inhibition in the rate of urea formation occurred in the presence of both alanine and glutamate. Cells grown to 3-day stationary phase had a markedly reduced rate of arginine catabolism to urea, but the activating effect of glucose and the inhibitory effect of alanine were qualitatively similar to their effects on late log phase cells. Addition of water to cells suspended in buffer also inhibited urea formation, but this appeared to be due primarily to the release of alanine caused by the hypo-osmotic stress. Addition of mannitol to cells suspended in buffer caused a small inhibition of arginine catabolism. Addition of dibutyrylcyclic AMP, 3',5'-cyclic GMP, phorbol myristic acid, or A23187 had no effect on the rate of urea formation from arginine.(ABSTRACT TRUNCATED AT 250 WORDS)


Trypanosoma brucei brucei: in vitro production of metacyclic forms.

An in vitro method has been established to obtain metacyclic form populations of Trypanosoma brucei brucei. Trypanosome populations containing more than 98% of metacyclic forms were obtained from cultures which were: 1) initiated with bloodstream forms in primary cultures in the presence of Microtus montanus embryonic fibroblast-like cells (feeder cell layers); 2) maintained in glucose-free Eagle's minimum essential medium supplemented with 10 mM L-proline, 2 mM L-glutamine and 20% (v/v) fetal bovine serum at 27 degrees C without medium change for five days; 3) subcultured in the absence of the feeder cell layers but in the presence of Cytodex 3 beads; 4) maintained for an additional nine days with medium changes on days 5, 8 and 11; and 5) harvested on day 14 by means of diethylaminoethyl cellulose column chromatography prior to the appearance of other infective forms. Most of the trypanosomes obtained under these conditions were morphologically similar to metacyclic forms derived from tsetse fly vectors, coated with variable surface glycoprotein and were infective for mice. In the primary cultures procyclic forms, epimastigotes and metacyclic forms appeared by day 8. When the duration of the subculture was prolonged to 17 days or more at 27 degrees C, the metacyclic forms decreased in number while short trypomastigotes, long slender epimastigotes, and long slender trypomastigotes increased in number. These forms in such long-term cultures also appeared in diethylaminoethyl cellulose-isolated populations along with metacyclic forms.


Plasmodium falciparum: differential sensitivity in vitro to E-64 (cysteine protease inhibitor) and Pepstatin A (aspartyl protease inhibitor).

We investigated the effect of a cysteine proteinase inhibitor (E-64) and an aspartyl proteinase inhibitor (Pepstatin A) on asexual erythrocytic stages of Plasmodium falciparum in culture. These two protease inhibitors showed different patterns of activity. E-64 acted preferentially against trophozoite and schizont stages. After 48 h incubation at high concentrations of E-64 (28, 140, 280 microM), growth was totally abolished and the parasites presented characteristic enlarged food vacuoles. Morphological alterations were also seen after shorter incubation periods (6 h at 28 microM) or 12 h at the inhibitory concentration 50% (12 microM), but an additional culture period (24 h) in inhibitor-free medium allowed normal parasite development, demonstrating a parasitostatic effect. E-64 acts on parasite multiplication; the normal merozoite maturation was altered and the normal reinvasion process partially impaired. Pepstatin A used at the inhibitory concentration 50% (4 microM) killed the parasites before trophozoite development and had a major effect on schizonts maturation. No altered parasite development occurred during an additional culture period without Pepstatin A, demonstrating a parasiticidal effect. E-64 and Pepstatin A used in combination inhibit the parasite growth with a strong synergistic effect.


Continuous cultivation and drug susceptibility testing of Plasmodium falciparum in a malaria endemic area.

Isolates (UCH-23 and OM) and cloned strains of Plasmodium falciparum (Clones W-2 and D-6) were maintained in continuous culture for 28 to 150 days using culture media supplemented with 10% (v/v) heat inactivated semi-immune human plasma. Microscopic appearance and growth rates (R) of the parasites in media supplemented with semi-immune human plasma [R = 1.13 (W-2), 0.92 (D-6), 0.75 (OM) and 0.84 (UCH-23)] were comparable to those of parallel cultures maintained in media supplemented with 10% (v/v) heat inactivated non-immune human plasma [R = 1.42 (W-2), 0.83 (D-6), 0.66 (OM) and 0.89 (UCH-23)]. In addition, IC50 for chloroquine and mefloquine against the two cloned strains of P. falciparum maintained in culture media supplemented with either non-immune human plasma or semi-immune human plasma were identical. Although growth rates of new isolates (UCH-23 and OM) fluctuated over time, they stabilized between the 12th and 19th day of adaptation to culture. This fluctuation in growth rates of the new isolates underscores the influence of population dynamics during adaptation of P. falciparum to continuous culture. Sixty-eight percent of the primary isolates (170 of 250) obtained from patients in Ibadan were successfully adapted and maintained in continuous culture using semi-immune human plasma. The results of these studies indicate that semi-immune human plasma is a suitable supplement for continuous cultivation and drug susceptibility testing of P. falciparum.(ABSTRACT TRUNCATED AT 250 WORDS)


Exceptions to mutual exclusion among cell surface i-antigens of Tetrahymena thermophila during salt stress and stationary phase.

In ciliates, only one of the alternative forms of the immunodominant membrane glycoprotein usually coats the external surface of the cell. Such mutual exclusion is regulated at the pretranslational level by mechanisms that result in the expression of a single protein gene. In the holotrich Tetrahymena thermophila five alternative cell surface immobilization proteins (i-antigens) are expressed under different conditions of temperature (L, H, T) and culture media (I, S). Using polyclonal and monoclonal antibodies to these proteins and a cDNA probe derived from the SerH3 gene, we have reinvestigated expression of i-antigens in media supplemented with 0.2 M NaCl. We find that in addition to S, the H and L antigens are also present on the cell surface. While all three i-antigens may be simultaneously present on the cell surface, the combinations S/L and S/H are more frequent. Compared to cells expressing H and L singly, the level of H3 mRNA is diminished, and a subset of the L family of polypeptides is variably expressed. The expression of S begins within 30 min after transfer to NaCl-supplemented medium, while the expression of L begins three days to several weeks after transfer. When cells are transferred out of NaCl-supplemented medium, S is turned off within 24 h, and L is expressed for at least 1 wk prior to the return of full H expression. Although these differences in kinetics suggest differences in control mechanism(s), the absence of I and T on the surface of NaCl-grown cells suggests that there is also a common regulatory link among H, S and L.(ABSTRACT TRUNCATED AT 250 WORDS)


Inter- and intrasyngenic variations of the hemoglobin components in Paramecium caudatum.

The ciliated protozoan Paramecium contains hemoglobin in heterogeneous monomeric forms. In particular, Paramecium caudatum is characterized by the presence of a major component called Hb10 and a basic component named bHb. We found that in P. caudatum both of these hemoglobin components show some variation according to stock. The types and distributions of these hemoglobin components were examined on 16 stocks in five different syngens and one stock in an unidentified syngen using high performance liquid chromatography. The results indicate that in a variety of stocks the major component, Hb10, was divided into three types, A, B or A + B, and that the basic hemoglobin component was composed of a combination of two or three variants out of four possible, i.e. bHb 1, bHb 2, bHb 3 and bHb 4. Neither the Hb10 types nor the bHb variants, however, could be used to distinguish syngen in P. caudatum, since all of the Hb10 types and bHb variants were widely distributed over syngens and identical profiles appeared to some stocks in different syngens.


Codon usage in Giardia lamblia.

A codon usage table for the intestinal parasite Giardia lamblia was generated by analysis of the nucleotide sequences of eight genes comprising 3,135 codons. Codon usage revealed a biased use of synonymous codons with a preference for NNC codons (42.1%). The codon usage of G. lamblia more closely resembles that of the prokaryote Halobacterium halobium (correlation coefficient r = 0.73) rather than that of other eukaryotic protozoans, i.e. Trypanosoma brucei (r = 0.434) and Plasmodium falciparum (r = -0.31). These observations are consistent with the view that G. lamblia represents the first line of descent from the ancestral cells that first took on eukaryotic features.


Eimerians in harvest mice, Reithrodontomys spp., from Mexico, California and New Mexico, and phenotypic plasticity in oocysts of Eimeria arizonensis.

Between May 1979 and August 1991, 48.7% (57/117) of the harvest mice (Reithrodontomys spp.) examined from 10 localities in Mexico, California and New Mexico had coccidian oocysts in their feces. A total of 46.7% (49/105) of the Reithrodontomys megalotis examined were positive for coccidian oocysts; this included samples from five states in Mexico (47.1%, 8/17), three counties in California (66.7%, 4/6) and two counties in New Mexico (45.1%, 37/82); 66.7% (8/12) of the Reithrodontomys montanus from one county in New Mexico also were infected. Only two coccidian species, Eimeria arizonensis and Eimeria langebarteli, were found in these hosts. Oocysts of E. langebarteli were found only in R. megalotis: in all three infected mice from Madera County, California, in the only mouse from San Bernardino County, California, and in 63% (5/8) of the infected mice from four states in Mexico. Oocysts of E. arizonensis were found in R. megalotis in Mexico, California, and New Mexico and in R. montanus from New Mexico. Sporulated oocysts of E. langebarteli differed slightly from those in previously published reports by having wider oocysts and larger sporocysts. Sporulated oocysts of E. arizonensis were variable in size, with those recovered from R. montanus significantly larger in length and width and sporocyst width than those from R. megalotis. The structure of the oocyst residuum was polymorphic, both within and between host species, and within the same mouse; it could appear as one large globule, two globules, several to many smaller globules, or as a compact mass of many small granules. Oocysts with a variable residuum were larger than those with one globule in all oocyst/sporocyst dimensions.(ABSTRACT TRUNCATED AT 250 WORDS)


Unusual selective immunoglobulin deficiency in an Arabian foal.

A 10-month-old Arabian foal was evaluated for a suspected immunoglobulin (Ig) M deficiency. Decreased to nondetectable concentrations of IgM, IgA, and IgG (T), and a normal concentration of IgG, were present. Results of in vitro testing of the blood lymphocyte blastogenesis showed a weak response to the B-cell mitogen, lipopolysaccharide (LPS), but normal responses to T-cell mitogens. Results of postmortem examination showed synovitis of the left tibiotarsal and both scapulohumeral joints. Atrophy and edema of the lymph nodes and lymphocyte depletion in the thymus and spleen were seen. A subacute inflammatory infiltrate was observed in the kidney, synovium, liver, and brain. Etiologic agents were not identified. This case represents a previously unreported form of immunodeficiency disease in the horse.


Immunosuppressive therapy in the treatment of immune-mediated disease.

Immune-mediated diseases represent some of the most frustrating types of disorders that are diagnosed and treated in veterinary medicine. Drug-induced immunosuppression is an attempt to control the aberrant immune response against self antigens but the immunosuppression can result in sepsis or other unacceptable adverse effects. If the pathophysiology of immune-mediated and autoimmune disease is considered, the immune response can be divided into several components and attempts can be made to selectively deal with each component separately. The components of the immune response that can be manipulated by therapy include antibodies, effector cells, the mononuclear phagocytic system, and the peripheral manifestations of disease. This article reviews the therapy of immune-mediated and autoimmune diseases based on a pathophysiologic approach and discusses conventional as well as current therapies in the treatment of these devastating diseases.


Effect of breed and body weight on echocardiographic values in four breeds of dogs of differing somatotype.

Eighty normal dogs of four morphologically disparate breeds (Pembroke Welsh Corgi, Miniature Poodle, Afghan Hound, Golden Retriever) (twenty of each breed), were studied by echocardiography to determine the importance of breed and weight in establishing normal echocardiographic reference ranges. Echocardiographic measurements included left-ventricular chamber dimension at systole and end-diastole, right-ventricular chamber dimension at end-diastole, interventricular septal thickness at systole and end-diastole, left-ventricular free wall thickness at systole and end-diastole, E-point septal separation, aortic root dimension at end-diastole, left atrial dimension, and fractional shortening. Analyses of covariance indicated that for all measurements except right-ventricular chamber dimension, the means were significantly different among breeds, after the differences in weight were taken into account. Echocardiographic measurements are variable even within the same breed. Breed must be considered in establishing echocardiographic measurement reference ranges. Echocardiographic values for each breed are presented.


Differentiation of chronic lymphocytic leukemia in the horse. A report of two cases.

Chronic lymphocytic leukemia (CLL) was diagnosed in two horses: an 18-year-old Quarter Horse gelding that was examined because of edema of the prepuce and ventral abdomen; and a 20-year-old mixed breed gelding that was referred because of lymphocytosis, ventral edema, and weight loss. The first horse had enlarged peripheral lymph nodes and cool nonpainful pitting edema of the ventral abdomen and prepuce. The second horse had enlarged peripheral lymph nodes, cool nonpainful pitting edema of the ventral thorax and cranial ventral abdomen, and a 3/5 holosystolic heart murmur. The diagnosis of CLL was based on increased blood lymphocyte counts and infiltration of marrow and other tissues by lymphocytes. In horse 1, the lymphocytosis persisted for 2 months between initial examination and death. The results of flow cytometric analysis on blood lymphocytes using anti-lymphocyte antibodies suggested that horse 1 had T-cell CLL, and horse 2 had B-cell CLL. In addition, the second horse had a monoclonal gammopathy (IgG), with light-chain proteinuria.


Ontogeny of immunosuppressive activity, MHC antigens and leukocytes in the rat testis.

The levels of immunosuppressive activity and the presence of MHC antigens and leukocytes were studied in the immature and the sexually mature rat testis. The immunosuppressive activities were measured from high-molecular weight (greater than 5 kDa) fractions of testis extracts using the protectin bioassay. The presence of MHC antigens and leucocytes was studied using the indirect immunoperoxidase method. In the immature rats, clusters of class I MHC antigen positive cells and a few cells expressing class II MHC antigen were present in the testicular interstitium. In the sexually mature rats, all the cells were MHC I+, and MHC II+ cells were numerous in the testicular interstitium. The seminiferous epithelium was MHC-negative in both the immature and the sexually mature testis. W3/25+ leukocytes were present in the interstitium and the tubular wall in both the immature and the sexually mature rat testis, but not in the seminiferous epithelium at any age. At 20-30 days of age, the testicular extracts were neutral or slightly stimulated 3H-TdR incorporation into peripheral blood lymphocytes, but at 44-60 days of age they inhibited lymphocyte proliferation significantly. In gel filtration, a peak of immunosuppressive activity was observed at approximately 400 kDa (protectin A) in both 20- and 60-days-old rat testes. A smaller peak was present at approximately 200 kDa in both age groups. This study shows that the testicular immunoregulatory microenvironment is different in the immature and the sexually mature rats. This may be important in such age-dependent human diseases as mumps orchitis and the testicular relapses of acute lymphoblastic leukemia.


Evaluation of L-asparaginase: polyethylene glycol conjugate versus native L-asparaginase combined with chemotherapy. A randomized double-blind study in canine lymphoma.

L-asparaginase is an enzyme that inhibits protein synthesis by the depletion of sources of L-asparagine, which is necessary for transformed lymphoid cells to proliferate. L-asparaginase is used in the treatment of childhood acute lymphoblastic leukemia. A problem with L-asparaginase therapy is the immunogenicity of the enzyme and the development of anaphylactic reactions. Canine lymphoma is a predominantly B-cell tumor with widespread disease; without treatment, dogs with lymphoma usually survive 1-2 months. Canine lymphoma will respond to L-asparaginase therapy. A randomized double-blind study evaluated a polyethylene glycol (PEG) conjugate L-asparaginase combined with chemotherapy (vincristine, cyclophosphamide, doxorubicin, and prednisone). Thirty-five dogs were randomized to the PEG L-asparaginase group, and 34 dogs were randomized to the native L-asparaginase group. Thirty dogs (85.7%) achieved a complete remission (CR) with a median time to relapse of 217 days, and 32 (94.1%) dogs in the native L-asparaginase group achieved a CR with a median time to relapse of 214 days (P greater than 0.05). The asparaginase was well tolerated in both groups. Two dogs in the native L-asparaginase group had severe allergic reactions, and one dog in the PEG asparaginase group had a generalized urticarial reaction after repeated injections. This study indicates that PEG L-asparaginase has equal therapeutic efficacy to native L-asparaginase.


Isolation of a phylogenetically conserved and testis-specific gene using a monoclonal antibody against the serological H-Y antigen.

Several cDNA clones of a gene termed male-enhanced antigen-2 (Mea-2), have been isolated from a mouse testicular expression cDNA library using a monoclonal histocompatability Y (H-Ys) antibody which detects specific protein(s) present in the mouse testis but not the ovary. The Mea-2 gene is phylogenetically conserved among various mammalian species examined, and is expressed at high levels in adult mouse testis. The expression pattern of Mea-2 is very similar to that of another gene, the male-enhanced antigen-1 (Mea-1), previously isolated using a polyclonal H-Ys antibody. Northern blotting and RT-PCR analyses demonstrated that Mea-2 is also expressed in other adult and fetal mouse organs at low levels. The testis-enhanced expression of this gene is associated with germ cell development at mid- to late-meiotic stages of spermatogenesis. Analysis of an intersubspecies mouse backcross has assigned this gene to chromosome 5, between the loci Gus and Hnf-1.


Canine leptospirosis. A retrospective study of 17 cases.

Seventeen dogs were diagnosed with leptospirosis on the basis of clinical findings, laboratory abnormalities, and serology. This article summarizes and characterizes the historical and physical findings, laboratory data, serology, treatment, and outcome of these dogs. All of the dogs had serologic evidence of infection with interrogans serovars pomona and grippotyphosa. These findings are compared with previous reports of canine infection with Leptospira interrogans serovars icteroaemorrhagiae and canicola. The clinical presentation of these dogs did not correspond to the classic description of the disease in dogs in which concurrent renal and hepatic diseases are present. This may be due to infection with different serovars than those previously reported. In addition, this article suggests that canine leptospirosis should be considered in the differential diagnosis of dogs with acute or subacute renal failure.


Proacrosin binding protein: immunocomparative studies in boar, bovine, hamster, human and ram.

The cross reactivities of acidic extracts from boar, bovine, hamster, human and ram spermatozoa to a polyclonal antibody of the boar proacrosin binding protein has been investigated. The pH 3.0 extracts of the washed spermatozoa from each species were subjected to Western blot analysis using a polyclonal antibody developed against the 28 kDa boar proacrosin binding protein. The boar sperm extracts had a major 28 kD and a minor 29 kDa proacrosin binding protein. A similar protein of 29 kDa was present in the bovine and ram samples, while the hamster had bands at 22 and 25 kDa. Extracts of human sperm yielded a diffuse but distinct area of cross reactivity of about 25-30 kDa. These results demonstrate that proacrosin binding proteins are present in sperm of different mammalian species and they have similar molecular weights.


Phase I evaluation of doxorubicin and whole-body hyperthermia in dogs with lymphoma.

Fifteen previously untreated dogs with histologically confirmed, high-grade multicentric lymphoma were entered into a phase I study to evaluate combined doxorubicin and whole-body hyperthermia (DOX/WBH). Groups of three, four, and eight dogs were treated with whole-body hyperthermia and concurrent doxorubicin at 12 mg/m2, 24 mg/m2 and 30 mg/m2, respectively, after one doxorubicin induction dose at 30 mg/m2. Plateau temperature (42 +/- 0.1 degree C) was maintained for 90 minutes using a radiant heating device. A total of five DOX/WBH treatments per dog were planned, and these were given every 21 days. Treatment-related toxicity was not seen in the 12-mg/m2 doxorubicin dose group. Tumor progression prohibited administration of more than three DOX/WBH treatments to any dog in the 12-mg/m2 group. Premature ventricular contractions developed after the fifth treatment in one of the four dogs treated with 24 mg/m2 of doxorubicin. Two dogs (25%) in the 30-mg/m2 dose group had treatment-related toxicity. One dog experienced acute serious myelosuppression 1 week after the third treatment. This dog received all planned DOX/WBH treatments. Asymptomatic cardiac toxicosis consisting of decreased ejection fraction and fractional shortening developed in the second dog. This dog received only two DOX/WBH treatments. The three dogs treated at 12 mg/m2 had partial responses of short duration (60-83 days). Four dogs treated at 24 mg/m2 had complete responses for 150, 164, 186, and 200 days. Eight dogs treated at 30 mg/m2 had complete responses with a mean and median duration of 241 and 190 days, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


Monoclonal antiphospholipid antibody reactivity against human placental trophoblast.

Naturally occurring antibodies against the negatively charged phospholipids cardiolipin (CL) and phosphatidylserine (PS) have been associated with recurrent pregnancy loss. One prevalent hypothesis proposes that antiphospholipid antibody (aPL) mediated pathophysiology is through increased placental thrombosis. In this study we investigated the reactivity of three mouse monoclonal aPLs with term and 26 week human placental preparations. Each monoclonal antibody reacted differently with CL and PS; 3SB9b reacted with PS (CL-/PS+), D11A4 reacted with CL (CL+/PS-) and BA3B5C4 reacted with both CL and PS (CL+/PS+). 3SB9b reacted strongly with the syncytiotrophoblastic layer of both formalin fixed and frozen placental tissue. Sporadic reactivity was observed against the cytotrophoblastic layer. BA3B5C4 reacted strongly and specifically with cytotrophoblastic cells. D11A4 had only weak reactivity in the subtrophoblastic stromal region of the placenta in frozen sections. aPL staining was also observed against extravillous cytotrophoblast. BA3B5C4 stained cytoplasmic structures, whereas 3SB9b stained the plasma membrane region with little cytoplasmic staining. These data suggest that the trophoblastic layer is reactive with aPLs and may potentially be directly damaged through mechanisms unrelated to thrombosis. In addition, the trophoblastic layer directly in contact with the maternal circulation is most reactive with aPLs that are PS+ rather than CL+. The differential reactivity of 3SB9b and BA3B5C4 suggests that the antigenic conformation involving PS on the cytotrophoblast is altered concurrent with fusion into the syncytium.


Autoantibodies in women with primary recurrent spontaneous abortion of unknown etiology.

A group of 153 women with 3 or more recurrent spontaneous abortions (RSAs) with unknown etiology and 90 normal multigravida controls were evaluated for antibodies to phospholipids and nuclear antigens. We demonstrate that women with recurrent spontaneous abortions showed significantly higher incidence of antibodies to phospholipids than normal multigravida controls. In contrast, the incidence of antibodies to polynucleotides and histones was not different between these two groups. These findings suggest that antiphospholipid antibodies are either epiphenomena or causally related to recurrent spontaneous abortions.


Effect of sperm-associated antibodies on the dynamics of sperm movement and on the acrosome reaction of human spermatozoa.

Anti-sperm antibodies were eluted from the sperm cell fraction of autoimmune human ejaculates and transferred onto normal motile spermatozoa. The movement and the acrosomal status of these antibody-coated spermatozoa were evaluated after incubation in a capacitating medium. The amplitude of lateral head displacement (ALH) and the straight line velocity (VSL) were analyzed using an HTM automated motility analyser. Acrosomal loss was monitored by an FITC-conjugated lectin binding technique. During the 6-h incubation in BWW-BSA medium, antibody-free and antibody-coated spermatozoa exhibited significant changes of ALH and VSL distribution that evolved differently in the two populations. The dynamics of sperm movement in control spermatozoa were apparently modified by the presence of antibodies on the sperm membrane. The low percentage of spontaneous acrosomal loss obtained in control populations, even after 20 h of incubation, was not modified by the presence of antibodies on spermatozoa. However, the same antibodies decreased the acrosomal loss induced by a calcium ionophore after 3 h of incubation in capacitating conditions. These results suggest that sperm capacitation and acrosome reaction, considered as essential for successful fertilization, can be altered by antisperm antibodies present on human ejaculated spermatozoa.


Secretory immune system of the male reproductive tract: effects of dihydrotestosterone and estradiol on IgA and secretory component levels.

Immunoglobulin A (IgA) is present at mucosal sites of the body which are exposed to the external environment. In this study we evaluated the levels of IgA and its transport protein secretory component (SC) in organs of the male reproductive tract of both intact and castrate-hormone-treated rats. Our goals were to determine whether these proteins are present in the male reproductive tract and whether sex hormones can influence the amounts of IgA and SC in selected organs. We found that in intact animals, IgA was present in the prostate, epididymis, vas deferens and testis and that SC levels in the prostate were 22-fold greater than in these same organs. Dihydrotestosterone (DHT) and/or estradiol, when administered to castrate animals, dramatically increased the levels of prostatic SC. In contrast, the levels of IgA were only minimally affected. DHT administration also resulted in a significant increase in SC found in the seminal vesicles. These studies demonstrate that IgA and SC are present in the male reproductive tract of the rat. Further, they show that androgens and estrogens act at selected sites in the male reproductive tract to play an important role in maintaining SC levels and thereby suggest that these hormones influence the movement of IgA from tissues into secretions.


Biological actions of monoclonal antibodies to bovine lutropin receptor.

Monoclonal antibodies (Mabs) were produced against bovine Lutropin receptor (LH-R). Antibodies were detected by an enzyme linked immunosorbant assay (ELISA). Hybridomas were subcloned to achieve monoclonality. Ascites were developed in Balb/c mice. Hybridoma supernatants were purified by ammonium sulfate precipitation and chromatography on hydroxylapatite columns. LH-R antibodies showed upto 50% inhibition of 125I-labeled hCG binding to bovine luteal cell membranes and up to 80% inhibition of testosterone production by hCG stimulated mouse Leydig cells. LH-R antibodies were predominantly IgM isotype. Purified antibodies showed a 78-kDa band, in SDS-PAGE, as the heavy chain of the immunoglobulin. LH-R antibodies were localized specifically in the thecal and luteal cells of the rat ovaries as well as in the Leydig cell of mouse testes. Injections of the LH-R antibody caused a constant estrus in normal rats. One month after the cessation of the injections the animals returned to normal estrus cycle and fertility. Pregnant mice injected with LH-R antibodies produced only 3 viable pregnancies and 10 pups, as compared to 8 pregnancies and 45 pups born to normal controls. LH-R antibodies also caused, approximately, a 50% reduction in testosterone production in normal male rats. These observations indicate a high degree of specificity of the Mab to LH-R and their potential in studies on gonadal function.


Tissue reconstruction with nonpenetrating arcuate-legged clips. Potential endoscopic applications.

A new system for tissue approximation consisting of a nonpenetrating arcuate-legged clip applied to everted tissue edges to form an elastomeric flanged joint is described. The flanged joint has unusual physical and morphologic properties. Novel systems for tissue eversion, clip application and clip removal have been tested at the micro scale in blood vessels and the rat vas deferens (vasovasostomy). Human applications have been successful (cerebrovascular reconstruction, free-flap transfer, skin grafting, A-V access). The system is biologically and technically equivalent to or superior to the needle-and-suture technique. Avoidance of intimal or mucosal penetration or intraluminal foreign body is associated with prompt wound healing and the reconstitution of tubular integrity. The system is readily adaptable for endoscopic surgical reconstructions, providing the surgeon with enhanced reconstructive abilities.


Endometrial ablation. Improvement in PMS related to the decrease in bleeding.

Of 108 patients evaluated before and after ablation of the endometrium since July 1986, for menorrhagia and symptoms associated with the menstrual period, 57 met criteria for premenstrual syndrome (PMS). An additional 35 patients had symptoms associated with their menstrual period but did not qualify for a diagnosis of PMS. Following endometrial ablation most patients in both groups reported either amenorrhea or spotting and decreased severity of symptoms. The degree of improvement in both PMS and perimenstrual symptoms seemed to be related to the degree to which bleeding was reduced.


Analysis of tissue margins of cone biopsy specimens obtained with "cold knife," CO2 and Nd:YAG lasers and a radiofrequency surgical unit.

Analysis of the tissue margins of cone biopsy specimens obtained from 40 patients showed varying degrees of thermal and mechanical artifact at the tissue margins. The least artifact was seen in the tissue margins of specimens obtained with the scalpel ("cold knife"). The amount of thermal damage to biopsies obtained via lasers and the radiofrequency unit varied with the instrument employed. However, the quality of the tissue margins of specimens obtained using a radiofrequency surgical unit equipped with a needle electrode on a "pure cut" setting approached the quality of those obtained with the cold knife in their lack of thermal and mechanical artifact.


Ectopic pregnancy following the treatment of tubal infertility.

To evaluate the prognosis for the patient who becomes pregnant after infertility treatment, we analyzed the occurrence of ectopic pregnancy following reconstructive surgery and in vitro fertilization/embryo transfer (IVF/ET) for tubal infertility. The results of 474 microsurgical operations and the results of 2,119 stimulated IVF/ET cycles for tubal infertility in the Reproduction Unit of Ljubljana University Department of Obstetrics and Gynecology are presented. The ratio of patients who subsequently had only ectopic pregnancies to the number of operations was 12%. Ectopic pregnancies represented 28% of all pregnancies after surgery. In IVF/ET cycles for tubal infertility, ectopic pregnancy represented 2.8% of all pregnancies and 3 permiles of all transfers. There was one (0.5%) heterotopic pregnancy. The likelihood of live births (30%, one or more times) after surgery compensates the high risk for ectopic pregnancy. While the risk for ectopic pregnancy after IVF/ET is much lower than the risk after tubal surgery, it is still rather high compared with the risk in the normal population. In the cases with severe tubal lesions IVF/ET is preferable to tubal surgery. The results show the importance of considering ectopics when deciding upon treatment and in patients who become pregnant after treatment for tubal infertility.


Correlation of focal pelvic tenderness with implant dimension and stage of endometriosis.

Reported symptoms in patients with endometriosis have shown poor correlation with the extent of disease at surgery. Recent data have suggested that signs and symptoms may correlate with depth and volume of individual implants. In this study preoperative focal tenderness was correlated with the presence or absence of endometriosis, depth and volume of the endometrial implants and the presence of other pathology. The findings suggest that focal tenderness has limited value in predicting the stage of endometriosis, but is strongly associated with the presence of disease in the cul de sac and uterosacral ligaments. There was also significant association of tenderness with deeper and larger volumes of implants, a finding that may be of clinical significance in the surgical management of endometriosis-associated symptoms and tenderness.


Endometrial ablation repeat procedures. Case studies.

Of 143 women who underwent endometrial ablation from May 1986 through August 1991, 16 requested repeat endometrial ablation and 7 underwent hysterectomy. Only two of the hysterectomies were performed for bleeding, and no hysterectomy was needed for any woman who had a repeat endometrial ablation. For patients undergoing one ablation, the results were amenorrhea in 55 women (38%), staining in 32 (22%), light flow in 33 (23%), 7 hysterectomies (5%) and 16 patients requesting repeat endometrial ablation (11%). Repeat endometrial ablation resulted in amenorrhea in 10 women (63%), staining in 3 (19%) and light flow in 3 (19%). Repeat endometrial ablation can eliminate the need for hysterectomy in women who continue to have bleeding problems after one endometrial ablation. Gynecologists should not hesitate to offer repeat ablation since the results will usually be excellent.


Fatal fetal abnormalities. Route of delivery and effect of the development of antepartum diagnostics in the last 13 years.

The appropriateness of cesarean section as the route of delivery in pregnancies complicated by fatal fetal abnormalities was evaluated in an obstetric population of 47,924 deliveries during the years 1978-90. The incidence of fatal fetal abnormalities was 0.50% and did not change during the study period. The cesarean section rate was twice that in the whole obstetric population, and the procedure was performed mainly for inappropriate fetal indications. One-third of the sections were caused by antepartum or intrapartum fetal distress. After the advent of sophisticated real-time ultrasound equipment, the obstetric management of those cases seemed to increase, but later the incidence of cesarean sections for fetal indications decreased, from increased experience with assessing the prognosis of malformed fetuses and increased opportunities to examine the fetal karyotype. Some conservatism seems justified when choosing obstetric management of a malformed fetus before large, controlled, clinical studies become available.


Identifying the obstetric patient at high risk of multiple-unit blood transfusions.

We sought to characterize the obstetric patient who required more than the 1 or 2 units of blood products typically available by autologous donation. Medical records from 1988 were reviewed retrospectively. During this period, 16,462 deliveries were performed. Twenty-seven patients (0.16%) received more than 2 units of blood products during their pregnancies. The most common diagnoses associated with a transfusion were placenta previa (10), uterine atony (8) and abruption (5). Platelets, fresh frozen plasma and cryoprecipitate were administered to 7, 12 and 4 patients, respectively. An antepartum condition associated with hemorrhage was identified in only 11 of the 27 patients (41%) who received greater than or equal to 3 units of blood products. The remainder of the cases were not diagnosed during the antepartum period, when autologous donation is an option.


Snake bite poisoning in pregnancy. A review of the literature.

With our 2 cases, there are 30 reported cases in English of snake bite poisoning in pregnancy with some details presented and 20 cases without details, for a total of 50 cases. We reviewed the maternal and fetal outcomes in the 30 cases that had such information. Poisoning by members of the Crotalidae family (rattlesnakes, cotton-mouths [water moccasins] and copperheads) during pregnancy carries with it a fetal wastage rate of 43% and a maternal mortality rate of 10%. Pit viper bites cause a bleeding diathesis from the fact that the venom of these snakes contains a procoagulant that defibrinates the blood and leads to bleeding throughout the body.


Oral methotrexate for persistent ectopic pregnancy. A case report.

Persistent ectopic pregnancy complicates a significant percentage of conservative tubal pregnancy procedures. The optimal treatment of persistent trophoblast has yet to be determined. Most reported cases have been treated by reoperation and removal of the affected tube. Use of methotrexate may avoid the need for reoperation and permit tubal salvage. We report on a case of persistent ectopic pregnancy treated successfully using an oral methotrexate regimen originally described for treating nonmetastatic gestational trophoblastic neoplasia. Oral methotrexate provides a simple, convenient, outpatient treatment option for cases of persistent ectopic pregnancy diagnosed by a rise or plateau in human chorionic gonadotropin levels following conservative surgery.


Structure of tobraviral particles: a model suggested from sequence conservation in tobraviral and tobamoviral coat proteins.

Comparisons of the coat protein sequences of four tobraviruses with those of seven tobamoviruses indicate that these proteins share a common evolutionary origin. Numerous amino acids for which specific functions have been identified in the molecular structure of the tobacco mosaic virus vulgare protein have identical or closely similar counterparts among the tobraviral proteins. These include those with roles in the hydrophobic core of the protein, those that contribute to the RNA binding site and those involved in the control of virus assembly. We suggest a model for the structure of the tobraviral particle that not only offers an explanation for the greater diameter of the tobraviral particle but also confirms an early suggestion for RNA placement within this particle.


G+C-rich tract in 5' end of human introns.

Analysis of an artificial neural network trained to classify DNA as coding or non-coding revealed compositional differences between sequence parts translated into protein and those that were not. The 5' end of human introns was found to have a base composition that was non-random to an extent matching the non-randomness in the 3' end that contains the polypyrimidine tract. The prevailing nucleotides in the initial 50 nucleotides of human introns are guanine and cytosine, the trinucleotide GGG was found to occur almost four times as frequently as it would in sequences with a uniform distribution of the nucleotides. The initial part of terminal exons and their associated terminal introns were shown to have a very special base composition deviating strongly from the normal picture in other exons and introns.


Structural organization of the genes encoding the small nuclear RNAs U1 to U6 of Tetrahymena thermophila is very similar to that of plant small nuclear RNA genes.

We report the sequences of the genes encoding the small nuclear RNAs (snRNAs) U1 to U6 of the ciliate Tetrahymena thermophila. The genes of the individual snRNAs exist in two to six slightly different copies per haploid genome. Sequence analyses of the gene-flanking regions indicate that there are two classes of snRNA genes. Both classes are characterized by several conserved sequence elements, some of which are unique to each class and some of which are found in both classes. Comparison of the promoter structure of the snRNA genes of T. thermophila with the promoter structures of snRNA genes of other organisms revealed several similarities to plant snRNA genes. These similarities include the overall promoter architecture as well as specific sequence elements. The structural organization of the 3' flanking region of some of the T. thermophila snRNA genes is not observed in other organisms. This finding is discussed in relation to a possible role in snRNA 3'-end formation.


Crystal structures of two mutant neuraminidase-antibody complexes with amino acid substitutions in the interface.

The site on influenza virus N9 neuraminidase recognized by NC41 monoclonal antibody comprises 19 amino acid residues that are in direct contact with 17 residues on the antibody. Single sequence changes in some of the neuraminidase residues in the site markedly reduce antibody binding. However, two mutants have been found within the site, Ile368 to Arg and Asn329 to Asp selected by antibodies other than NC41, and these mutants bind NC41 antibody with only slightly reduced affinity. The three-dimensional structures of the two mutant N9-NC41 antibody complexes as derived from the wild-type complex are presented. Both structures show that some amino acid substitutions can be accommodated within an antigen-antibody interface by local structural rearrangements around the mutation site. In the Ile368 to Arg mutant complex, the side-chain of Arg368 is shifted by 2.9 A from its position in the uncomplexed mutant and a shift of 1.3 A in the position of the light chain residue HisL55 with respect to the wild-type complex is also observed. In the other mutant, the side-chain of Asp329 appears rotated by 150 degrees around C alpha-C beta with respect to the uncomplexed mutant, so that the carboxylate group is moved to the periphery of the antigen-antibody interface. The results provide a basis for understanding some of the potential structural effects of somatic hypermutation on antigen-antibody binding in those cases where the mutation in the antibody occurs at antigen-contacting residues, and demonstrate again the importance of structural context in evaluating the effect of amino acid substitutions on protein structure and function.