Calcineurin, a calcium/calmodulin-regulated protein phosphatase, in mammalian neuroendocrine cells and neoplasms. Calcineurin is a calcium/calmodulin-regulated protein phosphatase. By using enzyme-immunoassay and immunocytochemistry with an affinity-purified specific antibody to this protein, we have found that calcineurin is expressed in the central and peripheral neuroendocrine cells, also termed amine precursor uptake and decarboxylation cells. In addition, calcineurin immunoreactivity was found in the central neuroendocrine neoplasms such as pineocytoma, olfactory neuroblastoma and paraganglioma. The present findings indicate that the activity of phosphatase regulated by calcium and calmodulin is involved in neuroendocrine functions, and that the enzyme can be useful for the identification and characterization of neuroendocrine cell tumors.


Kindling and electrode effects on the benzodiazepine receptors density of olfactory bulb and hippocampus after olfactory bulb kindling. The olfactory bulb (OB) kindling is a model of limbic secondary generalized epilepsy. Ten days after the completion of OB kindling, we have studied the long term effects of both electrode insertion and kindling on the binding of [3H]diazepam to crude mitochondrial fractions. On the one hand, we have shown that electrode implantation in sham-operated controls induced an obvious increase in benzodiazepine (BZD) receptor density (Bmax) only at the site of the electrode in comparison to sham-unoperated rats. These results might indicate an additional mechanism extending earlier observations reported by others, who have shown that prolonged electrode implantation induced changes in sham-operated and kindled rats. On the other hand, the long lasting effect of OB kindling on the binding parameters of [3H]diazepam was examined in the focus and in the hippocampus. The results indicate a bilateral increase of BZD receptors in the OB and an ipsilateral increase in the hippocampus. These changes might be a regulation phenomenon in response to a hyperexcitability state and to focal stimulations.


A neurophysiological study of a lithium-sensitive phosphoinositide system in the hamster suprachiasmatic (SCN) biological clock in vitro. Lithium lengthens the period of free-running circadian rhythms in many species. In mammals the hypothalamic suprachiasmatic nucleus (SCN) has been identified as a biological clock which generates circadian rhythms. The effect of lithium-induced depletion of the intracellular pool of inositol, leading to decreased intracellular second messengers IP3 and DAG, was examined neurophysiologically. Extracellular recordings were obtained from spontaneously discharging SCN neurones maintained in vitro. Superfusion of slices with lithium-containing (0.1-30 mM) aCSF, but not rubidium-containing aCSF, suppressed neuronal firing in a dose-dependent manner. Lithium-induced suppressed firing was reversed by myo-inositol, but not by epi-inositol. These studies provide evidence for basal phosphoinositide turnover in neurones and implicate a lithium-sensitive phosphoinositide system in the maintenance of the spontaneous discharge activity of SCN neurones.


Bradykinin receptors in cultured astrocytes from neonatal rat brain are linked to physiological responses. Specific binding sites for bradykinin (BK) have recently been demonstrated on astrocytes of primary cultures from neonatal rat brain. In this study we demonstrate that BK induces membrane currents in concert with an elevation of [Ca2+]i. In 67% of astrocytes, BK induced an inward current as determined with the perforated patch-clamp technique in the whole-cell recording configuration. In a small population of astrocytes (20%), a BK-activated outward current was observed, while in the remainder of the cells (13%) no apparent current responses were detected. As recorded by fura-2 microfluorimetry, the peptide induced a transient rise of [Ca2+/bdi even when the extracellular calcium was removed. In the majority of astrocytes, the selective B1-agonist des-Arg9-BK elicited physiological responses with a much lower potency, indicating that the BK receptors are predominantly of the B2 subtype. A minor population of astrocytes was present which only responded to des-Arg9-BK.


The influence of basic fibroblast growth factor on acetylcholine receptors in cultured muscle cells. Acetylcholine receptors (AChRs) in Xenopus muscle cells undergo changes in channel kinetics during development in culture and these changes are somehow related to innervation. Recently we showed that basic fibroblast growth factor (bFGF), when locally applied, can mimic the effect of nerve in inducing a postsynaptic-type development. In this study, we examined whether bFGF can influence the developmental changes of AChRs. Patch clamp method was employed to record single AChR channel currents from cultured Xenopus myotomal muscle cells and the kinetics of low-conductance AChR channels were analyzed. In cultures treated with 1 microgram/ml bFGF at an early stage (stage 23), the burst duration of low-conductance AChR channels showed a 1.5-fold decrease between the first and second day in culture, while it underwent a remarkable 3-fold decrease during the same period in the control. Histogram analyses showed that the low-conductance channels were composed of a fast and a slow component and that the decrease in burst duration was due to a shift in the population from the slow to the fast. bFGF treatment appeared to slow down this shift by retaining the slow channels for a longer period of time. This effect is probably not due to channel modulation as the burst duration of short channel in older cells (stage 40) was not affected by bFGF. These data suggest that bFGF may enhance the metabolic stability of intrinsically short-lived AChRs. This effect seems to parallel the stabilization of junctional AChRs at the innervated endplate. Thus, bFGF, or a related polypeptide growth factor, may mediate this and other innervation-induced changes in the postsynaptic membrane.


Localization of melatonin binding sites in the pars tuberalis of the mink at three times during the seasonal testicular cycle. The strict photoperiodic dependence of gonadotropic function observed in mink provides an excellent physiological model for studying the activity of GnRH hypothalamic neurons. In mink, exposure to more than 10 h light inhibits the activity of this neurohormonal system. Melatonin plays an essential role in this type of regulation of gonadotropic function. In mink, contrary to results in many other photosensitive species, melatonin was observed to mediate the gonadostimulating effect of short days. However, the binding sites and action of this substance have not yet been defined. We thus attempted to identify melatonin binding sites in the mink brain. The study was carried out at three times during the seasonal testicular cycle in male minks maintained under natural environmental conditions. Coronal sections were taken from the whole brain and pituitary gland. Using 2-[125I]iodomelatonin, we observed a strong concentration of binding sites in the pars tuberalis and not in the part of the median eminence surrounded by the pars tuberalis. This concentrated localization of melatonin binding sites brings up the problem of defining the action of this substance in photoregulating the activity of the GnRH neurohormonal system.


Calcium channels in embryonic chick skeletal muscle cells after cultivation with calcium channel blocker. The effects of chronic treatment with calcium channel blockers were studied on the expression of voltage-dependent calcium channels (VDCCs) in chick skeletal muscle cells developing in culture. Myotubes were treated after 2 days in culture with either 20 microM D600 or 10 microM nifedipine, and measurements were made of the maximum rate of rise (M.R.R.) of the two components of action potential, operated by T- and L-type VDCCs, respectively. Treatment with either blocker reduced the M.R.R. of the action potential component operated by the L-type VDCC throughout the culture period examined. The M.R.R. of the T-type VDCC component, on the other hand, was unaffected by either treatment. The reduction in the M.R.R. of the L-type component in blocker-treated cells is thought to be due to the down-regulation of the expression of L-type VDCC. Thus, it appears that the expression of L-type VDCC in the chick skeletal muscle cells can be regulated by a function of L-type VDCC, which mediate the entry of Ca2+ into the cells. The physiological significance of the L-type VDCC, which expressed prominently early in the development of skeletal muscle cells, for the differentiation of excitability is discussed.


Sites of action of morphine on the ascending excitatory reflex in the guinea-pig small intestine. The effect of morphine on the ascending excitatory reflex of the circular muscle elicited by radial distension of the gut wall was studied in the isolated guinea-pig small intestine. A three compartment bath, in which an intermediate compartment divided the site of intraluminal stimulation (caudal compartment) from the site of reflex contraction recording (oral compartment), was used. Morphine (0.01-10 microM) applied independently to each compartment, caused a concentration-dependent depression (up to 90%) of the amplitude of distension-evoked reflex contractions. Concentration-response curves to morphine were shifted to the right by naloxone (30 nM) with an apparent pA2 value of about 8.5, which suggests an interaction with opioid mu-receptor subtypes. Our results indicate that morphine not only depressed transmission from excitatory motor neurons to the circular muscle but also neuro-neuronal transmission along the ascending excitatory reflex pathway.


Autoradiographic analysis of cyclic adenosine monophosphate phosphodiesterase using [3H]rolipram in the postischemic rat brain. Postischemic alteration of cyclic adenosine monophosphate phosphodiesterase was investigated in the rat brain, using [3H]rolipram in vitro autoradiography. After 90 min of middle cerebral artery (MCA) occlusion, [3H]rolipram binding sites decreased rapidly in the brain areas supplied by the occluded MCA. Moreover, 3 days after the ischemia, significant decreases of [3H]rolipram binding sites were observed in the thalamus and the substantia nigra, both areas had not been directly affected by the original ischemic insult. These results suggest that alteration of second messenger pathways may be involved in neuronal degeneration caused by transsynaptic process and that alteration of intracellular signal transduction may precede the neuronal damage in the exo-focal postischemic brain areas.


Local infusion of interleukin-6 attenuates the neurotoxic effects of NMDA on rat striatal cholinergic neurons. The potential neuroprotective effects of IL-6 against the excitotoxic neuronal loss induced by N-methyl-D-aspartate (NMDA) have been studied. Infusion into the rat striatum of excitotoxic amounts (250 nmol) of NMDA resulted in a 45% decrease in striatal choline acetyl transferase activity (ChAT; a marker of cholinergic neurons) and glutamate decarboxylase (GAD, a marker of GABAergic neurons) at 2 days post-injection. Co-infusion of 10 U of IL-6 reduced the loss of ChAT activity to 21% but failed to prevent the loss of GAD activity. IL-6 per se, up to the dose of 500 U, failed to affect ChAT or GAD activities. The in vivo effects of IL-6 are not mediated by a direct antagonism of NMDA toxicity, since IL-6 (up to a concentration of 500 and 5000 U/ml, respectively) did not antagonize either the increase in cyclic GMP levels resulting from NMDA receptor activation in cerebellar slices or the glutamate-induced release of lactate dehydrogenase, an index of neurotoxicity, by cultured cortical neurons. These results suggest that the increase in IL-6 levels observed in experimental brain lesions may play a role in the protection and regeneration of cholinergic neurons.


Mapping of GABAA receptor alpha 5 and alpha 6 subunit-like immunoreactivity in rat brain. The distribution of the alpha 5 and alpha 6 subunits of the GABAA receptor has been mapped in rat brain using affinity-purified antibodies generated against peptide sequences unique to the respective polypeptides. alpha 5 Subunit-like immunoreactivity was of low density but was distributed across several cell groups including cortical interneurones, hippocampal CA3 pyramidal neurones, the anterior thalamic reticular nucleus and cerebellar Purkinje neurones. alpha 6 Subunit-like immunoreactivity was observed in high density in cerebellar granule cells. These patterns are compatible with in situ hybridisation studies and provide a further anatomical substrate for GABAA receptor heterogeneity in the CNS.


Are Ca(2+)-permeable kainate/AMPA receptors more abundant in immature brain? The permeability properties of kainate/AMPA receptors are determined by subunit composition. The GluR1 and GluR3 subunits form Ca(2+)-permeable channels and exhibit inward rectification; heteromeric receptors containing the GluR2 subunit are Ca(2+)-impermeable and electrically linear. These observations raise the possibility of a developmental 'switch' in which turning on or off of GluR2 expression regulates the level of Ca2+ permeable kainate/AMPA receptors. We examined the ratio of GluR1 and GluR3 to GluR2 gene expression in developing and adult rat brain by in situ hybridization. A larger value of this ratio is likely to be associated with greater Ca2+ permeability. Our data suggest that in neocortex, striatum and cerebellum the number of Ca(2+)-permeable kainate/AMPA receptors is high at P4 and declines monotonically with age. In hippocampus, the number increases from P7 to P21, after which it declines. These findings provide evidence for a developmental 'switch' in which Ca2+ permeable glutamate receptors are turned off following early developmental events.


Calcium/calmodulin dependent protein kinase II mRNA in the gerbil brain after cerebral ischemia. The effect of transient cerebral ischemia on the expression of Ca2+/calmodulin dependent protein kinase II (CaM kinase II) mRNA in the gerbil brain was analyzed by Northern blots using cDNA clones for CaM kinase II. Ten minutes of bilateral carotid occlusion and 30 min of reperfusion resulted in reduced protein levels for alpha and beta subunits of the CaM kinase II, decreasing to 35% of control levels at 24 h. Recovery of immunoreactivity was detected in the cortex after 48 h. Eight to twelve hours after ischemia, the cortex showed a decrease in alpha and beta CaM kinase II mRNA levels. By 12-24 h of reperfusion the level of CaM kinase II mRNA was reduced to 26% of the control mRNA levels. CaM kinase II mRNA levels recovered by 48 h after ischemia, coinciding with the increase in CaM kinase II protein immunoreactivity. These results suggest that CaM kinase II is involved in neuronal survival through the reorganization of the neuroarchitecture and that the regulation of this role is controlled at the level of gene expression.


Organophosphate-induced alterations in muscarinic receptor binding and phosphoinositide hydrolysis in the human SK-N-SH cell line. Low level, chronic exposure (0.1 nM, 24 hrs) to the organophosphate paraoxon significantly inhibits N-[3H]methylscopolamine ([3H]NMS) binding to muscarinic receptors in a noncompetitive manner in the SK-N-SH neuroblastoma cell line. The effect of paraoxon on muscarinic receptor-mediated phosphoinositide hydrolysis was also investigated. At 0.1 nM, paraoxon caused a time-dependent increase in the accumulation of inositol phosphates, while the classical muscarinic receptor agonist carbachol was virtually ineffective at low concentrations. In contrast, carbachol-induced increases in phosphoinositide hydrolysis at higher concentrations (1 mM) were markedly larger (five-fold) than the increases induced by PX. Approximately 50% of the maximal increase in the accumulation of inositol phosphates due to paraoxon treatment was antagonized by saturating concentrations of muscarinic receptor antagonists, while the response to carbachol was completely antagonized. In addition, chronic treatment (24 hrs) of SK-N-SH cells with pertussis toxin blocked 75% of the stimulatory effects of carbachol, but inhibited only 38% of the paraoxon-induced response. Neomycin, a phospholipase C inhibitor, completely blocked both the paraoxon and carbachol-induced stimulation of phosphoinositide hydrolysis. The results suggest that paraoxon can modulate signal transduction by indirect activation of muscarinic receptors as well as by acting at a distal site along the pathway.


Seizures selectively impair agonist-stimulated phosphoinositide hydrolysis without affecting protein kinase C activity in rat brain. The influence of seizures on phosphoinositide hydrolysis and protein kinase C activity was measured in rat hippocampus and cerebral cortex, primarily using a model in which generalized convulsive status epilepticus was induced by administration of LiCl (3 mmole/kg) 20 hr prior to pilocarpine (30 mg/kg). A short (5 min) period of seizures reduced phosphoinositide hydrolysis in hippocampal slices stimulated by norepinephrine or ibotenate, but did not alter the responses to carbachol, 50 mM K+, or NaF. Induction of seizures with diisopropylfluorophosphate caused a similar reduction in the response to norepinephrine without altering carbachol-stimulated phosphoinositide hydrolysis. The inhibition of norepinephrine-stimulated phosphoinositide hydrolysis after seizures generated by lithium plus pilocarpine administration was apparently not due to inhibitory influences of quisqualate or activation of protein kinase C since both of these treatments caused similar inhibitions in slices from control and treated rats. Seizures induced by lithium plus pilocarpine or by kainate did not alter the activity of protein kinase C or the distribution of protein kinase C between membrane and cytosolic fractions. Thus, seizures cause a neurotransmitter-selective impairment of phosphoinositide hydrolysis, and this response may play a role in the severity or duration of seizure activity.


Dietary aluminum selectively decreases MAP-2 in brains of developing and adult rats. Administration of 0.3% aluminum in drinking water elevated serum aluminum concentrations 8-fold in rats. Further, chronic treatment with aluminum for 2-3 mon, in both developing and adult rats, significantly decreased the levels of MAP-2 in brain, as determined by quantitative immunoblot analysis. Aluminum treatment also decreased the level of brain spectrin, but only in the hippocampus of adult rats. These were selective effects, since the levels of tubulin, tau and the three proteins of the neurofilament triplet were unaltered. In the aluminum-treated adult rats MAP-2 levels were significantly decreased in the hippocampus and brainstem to 71% and 56% of control values, respectively. In developing rats, MAP-2 levels were significantly decreased in the cortex and brainstem (65 and 64% of control values, respectively) but not in the hippocampus. In support of these findings, immunohistochemical examination revealed that the intensity of hippocampal MAP-2 immunoreactivity was significantly decreased to 88% of control values with aluminum treatment in adult rats. To determine a possible mechanism by which MAP-2 levels are reduced, the effect of aluminum on calpain-induced proteolysis of MAP-2 was examined in vitro. At the aluminum concentrations tested, there was no apparent effect on calpain-induced proteolysis of MAP-2. In the developing rats, aluminum administration significantly increased the hippocampal cyclic AMP concentration, as reported previously in adult aluminum-treated rats, and decreased the inositol 1,4,5-trisphosphate concentration. These results demonstrate that chronic oral aluminum administration to rats selectively decreases the levels of MAP-2 in specific brain regions independent of calpain proteolysis. This decrease may be associated with increased cyclic AMP and protein phosphorylation, and the impairment of cognition previously observed in this model of aluminum intoxication.


Neurons in the nucleus of the solitary tract mediating inputs from emetic vagal afferents and the area postrema to the pattern generator for the emetic act in dogs. Roles of neurons in the nucleus of the solitary tract corresponding to the area subpostrema (mNST) for the retching reflex were investigated in decerebrate, paralyzed dogs. Retching was defined as rhythmic coactivation of the phrenic and abdominal muscle nerves. Retching which had been induced by stimulation of the left and right abdominal vagus nerves was impaired by cooling the left and right mNSTs, respectively. This result indicates that the mNST neurons mediate activities of emetic vagal afferents. All 40 non-respiratory neurons in the mNST, which had excitatory response to pulse train stimulation of the vagus nerve, were also activated by continuous stimulation of the vagus nerve to provoke retching. During provoked retching, however, these neurons did not exhibit any activities modulated in association with retching. The average latency of responses of these neurons to the pulse train stimulation (306.5 ms) was significantly shorter than that of the inspiratory neurons in the lateral NST and the adjacent reticular formation. Discharge frequencies of these neurons in the mNST gradually increased after administration of apomorphine (6/10) and glutamate (14/14) to the 4th ventricle. Antidromic responses to stimulation of the Bötzinger complex were observed in some (20/289) of the mNST neurons. These findings suggest that neurons in the mNST mediate the information from both the abdominal vagal afferents and the area postrema and drive the pattern generator for retching and vomiting, which is assumed to be located in the Bötzinger complex.


Non-respiratory neurons in the Bötzinger complex exhibiting appropriate firing patterns to generate the emetic act in dogs. This work was performed to prove the hypothesis that the pattern generator for the emetic act exists in the Bötzinger complex (BOT) and is driven by vagal afferents via the subpostrema portion of the nucleus of the solitary tract (mNST). Non-respiratory neurons (78) intermingling with BOT respiratory neurons in decerebrate dogs responded to pulse train stimulation of vagal afferents with a mean latency of 387 ms. During retching induced by vagal stimulation, one-half of the non-respiratory neurons exhibited high frequency burst firings synchronous with each retch (SH-firing, SH-neurons) and one-third of these neurons showed similar firings synchronous with the periods between retches (BH-firing, BH-neurons). Two-thirds of the SH-neurons and one-half of the BH-neurons fired with gradually augmenting frequencies (augmenting firing) during the period prior to retching, which may correspond to the period of prodromal signs of vomiting. Three SH-neurons were observed at fictive expulsion: all 3 exhibited burst firings concomitant with expulsion. During cooling block of transmission in the mNST, stimulation of the vagus nerve ipsilateral to the cooling failed to induce not only retching but also augmenting firing and SH-firing in all 11 BOT SH-neurons observed. In contrast, contralateral vagal stimulation induced retching and neuronal firings which had been observed before the cooling. These results support the hypothesis mentioned above. Respiratory firings were changed during retching in all BOT respiratory neurons observed. Respiratory firings were depressed during retching in the majority (15/25) of inspiratory (I) neurons and in a few expiratory (E) neurons (6/45). SH-firing was exhibited by 3 I- and 13 E-neurons. A few (2) I- and half (23) E-neurons showed BH-firing. These results indicate that all BOT respiratory neurons participate in central patterning of the emetic act.


Prominent expression of type 1 (gamma) protein kinase C in Purkinje cells located in the "central mass" of Reeler mutant mouse cerebellum as revealed by a computer image analysis technique. In Reeler mutant mice, cerebellar Purkinje cells exhibit abnormal synaptogenesis. In this study, Purkinje cells in the "central mass" with abnormal afferent connections were compared with those located in their normal position in the cortex in terms of immunoreactivity for type 1 (gamma) protein kinase C. A "computer image analysis technique" was developed for the purpose of this quantitative study, and it revealed that the immunoreactivity in the central mass was significantly higher than that in the cortex. The results suggest that type 1 (gamma) protein kinase C may be related to the abnormal synaptic formation of the Purkinje cells.


Characterization of substances which promote or repel sympathetic fiber growth in vitro. To determine whether a recognition mechanism is involved in determination of sympathetic innervation patterns of various tissues, tissue-derived substances were applied to a restricted test surface region of dishes and the responses of cultured sympathetic neurites were examined. Sympathetic fibers exhibited a turning or ramifying response, resulting in a dense fiber growth on test regions coated with particulate (adheron) fractions of a conditioned-medium (CM) from expansor secundariorum, heart, peripheral blood vessel or abdominal aorta, whereas on test regions coated with those from lung, skeletal muscle or dorsal aorta the neurite growth was repelled and sparse fiber growth was observed. Particulate fractions of brain- or gizzard-CM had no effect. These patterns in vitro were in parallel with the dense sympathetic innervation in expansor secundariorum, heart, peripheral blood vessel and abdominal aorta, but little or no sympathetic innervation in lung, skeletal muscle and dorsal aorta in vivo. These results suggest that adheron particles may participate in determination of sympathetic innervation patterns. Activity which repels or promotes the sympathetic fiber growth was inactivated by pronase E or trypsin but not by DNase or neuraminidase. Repelling activity was lost after treatment with heparinase or heparitinase but not with chondroitinase ABC or hyaluronidase. Promoting activity was retained after treatment with these glycosidases. These results suggest that the factor(s) possessing a repellent effect is a heparan sulfate proteoglycan and one(s) possessing a promoting effect is a protein.


Neurotrophic effect of brain-derived neurotrophic factor on basal forebrain cholinergic neurons in culture from postnatal rats. We examined the effect of brain-derived neurotrophic factor (BDNF) on cholinergic neurons in culture from postnatal rat basal forebrain by assay of choline acetyltransferase (ChAT) activity and cytochemical staining for acetylcholinesterase (AChE). BDNF was found to increase the ChAT activities but failed to promote the survival of AChE-positive neurons in cultures from neonatal (P3) rats, suggesting that its main role is cholinergic differentiation. In contrast, an enhancement of the survival of AChE-positive neurons and of ChAT activity was observed in cultures from P15-16 rats, suggesting that BDNF's main action is the maintenance of cholinergic neurons. Our results indicate a similarity between BDNF and nerve growth factor effects on the responses of cholinergic neurons of postnatal rat basal forebrain in culture.


The molecular basis of the inhibition of collagenase by vitamin A. Retinoic acid inhibits the enzyme collagenase by forming an inactive complex between the liganded nuclear retinoic acid receptors and c-Jun, a protein that is itself an activator of the collagenase gene.


A ventilatory assistance device for use with Technegas. The demand for high-quality ventilation imaging of the lungs has resulted in the advent of Technegas, which has proven its superiority to the more conventional agents in a number of clinical trials. Images of similar diagnostic quality have been difficult to obtain in patients who are uncooperative, intubated or very young. With this problem as the impetus, a ventilatory assistance device (VAD) was designed to exploit the superior imaging qualities of Technegas. The Technegas generator was interposed between the patient and the VAD, allowing the controlled movement of Technegas into the patient from the generator. Utilizing this device, the problems of increased dead space, turbulent gas flow and possible contamination were circumvented. Twenty patients, comprising nine children and eleven adults, were studied for various indications including assessment of pulmonary parenchymal disease, suspected pulmonary embolism and the lung clearance of modified Technegas. In all cases, images of diagnostic quality were obtained with ease, facilitating relevant clinical decision making. We conclude from this preliminary appraisal that the VAD is a simple, safe addition to the Technegas generator for imaging a group of patients in whom images of diagnostic quality have traditionally proven difficult to obtain.


Hairy leukoplakia in an HIV-negative, nonimmunosuppressed patient. Oral hairy leukoplakia is an Epstein-Barr virus-associated lesion that is considered to be a marker of immunosuppression. We report a case of oral hairy leukoplakia in a healthy nonimmunosuppressed elderly patient with no evidence of HIV infection on repeated investigation. This report has important implications on our understanding of the significance of this lesion.


Human papillomavirus type 16 in an oral squamous carcinoma and its metastasis. DNA was extracted from fresh frozen tissues of eight patients with primary oral squamous carcinoma. Samples of normal oral mucosa were available in seven cases and metastatic tumor in two cases. The samples were probed for human papillomavirus types 16 and 18 by Southern hybridization. In one case of squamous carcinoma of the floor of the mouth, human papillomavirus type 16 was identified in the primary tumor and a lymph node metastasis, but it was not detectable in normal oral mucosa from this patient. Human papillomavirus DNA was not detected in any other sample of primary tumor, metastasis, or normal oral mucosa. Restriction enzyme digests of the human papillomavirus positive primary tumor and its metastasis revealed that the viral DNA was identical to the prototype human papillomavirus type 16 and present at 50 to 100 copies per cell in an episomal state with no evidence of integration into the host DNA. Compared to the human papillomavirus DNA in the primary tumor, the viral DNA in the metastasis was of the same type, in the same physical state, and at the approximately the same copy number. The consistent maintenance of human papillomavirus DNA in metastases from human papillomavirus positive primary tumors supports the hypothesis that human papillomaviruses are cofactors in the pathogenesis of some carcinomas.


[Malignant fibrous hystiocytomas of unusual localizations]. 25 cases of malignant fibrous histiocytomas of different localisation were diagnosed in the St. John's Hospital (Budapest, Hungary) in a 10-year period from 1982 to 1992. In the present study ten patients are reported with rare localisation of this tumor caused abdominal complications. Special attention is payed to diagnostic difficulties. The importance of morphological diagnosis is emphasized because modern therapeutic methods favourably influence the prognosis of this tumor.


Role of Ras in signal transduction from the nerve growth factor receptor: relationship to protein kinase C, calcium and cyclic AMP. A dominant inhibitory ras mutant (Ha-ras Asn-17) has been used to investigate the role of Ras in nerve growth factor (NGF)-mediated signal transduction in PC12 cells. Expression of Ha-Ras Asn-17 blocks neuronal differentiation of these cells in response to NGF treatment. The Ha-Ras Asn-17 block was bypassed by treatment with NGF plus dibutyryl cAMP or NGF plus the Ca2+ ionophore ionomycin, but not by NGF plus 12-O-tetradecanoyl phorbol acetate (TPA). Direct stimulation of the cAMP or Ca2+ pathways thus appeared to act synergistically with a Ras-independent NGF signaling pathway. This Ras-independent pathway was also distinct from protein kinase C, since its activity was not affected by protein kinase C down-regulation. It thus appears that NGF stimulation generates a Ras-independent intracellular signal that contributes to neuronal differentiation independently of the cAMP, Ca2+ or protein kinase C second messenger systems. Since TPA did not bypass the Ha-Ras Asn-17 block to differentiation, protein kinase C also did not appear to be sufficient for Ras-dependent pathways mediating NGF-induced differentiation. Down-regulation experiments further indicated that protein kinase C was not required for NGF induction of early response genes via either Ras-dependent or Ras-independent pathways. Moreover, the formation of inositol phosphates and mobilization of intracellular calcium in response to NGF was not inhibited in PC12 cells expressing the Ha-Ras Asn-17 protein. Therefore, although calcium was able to bypass the Ha-Ras Asn-17 block to PC12 differentiation, Ras activity was not required for activation of phospholipase C in response to NGF. It thus appears that both Ras-dependent and Ras-independent signaling pathways contribute to NGF-induced PC12 cell differentiation independently of the cAMP, calcium and protein kinase C second messenger systems.


Cloning and characterization of the latent membrane protein (LMP) of a specific Epstein-Barr virus variant derived from the nasopharyngeal carcinoma in the Taiwanese population. A DNA fragment containing Epstein-Barr virus (EBV) terminal fragment sequence was obtained from a genomic library of nasopharyngeal carcinoma (NPC). One of the clones (clone 1510) contained the gene encoding latent membrane protein (LMP). Sequence analysis revealed that this gene had 95% homology with the LMP sequence of the B95-8 strain. Among the sequence variations, there was a change from G to T at nucleotide position 169,426, resulting in the loss of an XhoI site in exon 1 of the LMP gene. A pair of primers bracketing the XhoI site were designed to synthesize the EBV DNA fragment from nucleotides 169,081-169,577 by using the polymerase chain reaction (PCR) method. The PCR products were then subject to XhoI digestion and to DNA sequencing analysis. This restriction enzyme site polymorphism along with the sequence variations were also observed in 50 biopsy tissues as well as in the throat washings of 6 out of 20 healthy individuals that we examined, indicating that the EBV strain predominantly existing in these biopsy tissues was different from strains of B95-8, Jijoye or nude mouse passaged cells (C15) with an African origin, but closely resembled other nude mouse passaged CAO cells which were originally derived from China. Balb/c 3T3 cells carrying this NPC-LMP gene showed a transformed cell morphology and were tumorigenic in nude mice. The relationship between this unique type of EBV and NPC has yet to be established.


Identification of mutations in the WT1 gene in tumours from patients with the WAGR syndrome. Individuals with constitutional, heterozygous deletions of chromosome region 11p13 are predisposed to the development of Wilms' tumour, indicating the site of the tumour predisposition gene. The WT1 gene is a candidate for this cancer predisposition gene. If this gene is truly involved in tumorigenesis it would be expected to be mutant in tumour tissue from patients with 11p13 deletions. We have used single-stranded conformation polymorphism (SSCP) and polymerase chain reaction sequencing to test this hypothesis in an exon-by-exon analysis of the gene. Four tumours were analysed, two of which were from unilaterally affected individuals and two from a bilaterally affected patient. SSCP analysis identified mutations in the two unilateral tumours which, on sequencing, were shown to involve a 10-bp insertion in exon 7 and a single base pair change in exon 8. Both mutations result in the generation of premature stop codons and the predicted proteins would lack part of the zinc finger motif. Despite complete sequencing of the WT1 gene in both of the bilateral tumours, no mutations were identified. These results possibly suggest that WT1 may not be involved in tumorigenesis in all tumours. All four tumours retained heterozygosity in the 11p15 region, making it unlikely that a second recessive oncogene in this region was involved in tumorigenicity.


Met and hepatocyte growth factor/scatter factor signal transduction in normal melanocytes and melanoma cells. The proto-oncogene c-MET encodes a transmembrane tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF). HGF/SF stimulates the proliferation and motility of various cell types. Because HGF/SF is also a melanocyte mitogen, we investigated the biological role of HGF/SF, including c-Met expression, activation and signal transduction, in normal and malignant human melanocytes. We show that HGF/SF is mitogenic in the presence of synergistic factors, such as basic fibroblast growth factor (bFGF) and mast cell growth factor (MGF) and that, by itself, it stimulates the motility of normal human melanocytes. The ligand also maintained high levels of tyrosinase activity and melanin content in theses cells. Signal transduction by HGF/SF included phosphorylation of tyrosyl residues on c-Met, a cascade of tyrosine phosphorylations on several other proteins and activation of microtubule-associated protein kinase/extracellular signal-regulated kinase. Met expression and activity are normal in human melanomas, and constitutive activity of HGF/SF in retrovirally infected autonomously proliferative mouse melanocytes is insufficient to confer the malignant phenotype. Our findings suggest that activation of Met in response to HGF/SF may contribute to malignant progression synergistically with the aberrant expression of bFGF in malignant melanocytes and that, in addition, the peptide may promote dispersion of factor-dependent melanocytes from early stages of primary melanomas to ectopic sites.


The various domains of v-myb and v-ets oncogenes of E26 retrovirus contribute differently, but cooperatively, in transformation of hematopoietic lineages. The genome of the avian leukemia virus E26 is a unique example of association between two transcription factors which appear as a fused composite nuclear oncoprotein, P135gag-myb-ets. Previous studies with E26 have shown that v-myb and v-ets must cooperate to fully transform both erythrocytic and myelomonocytic precursor cells in vivo and in vitro. To analyse further the contribution of the individual domains involved in the transformation of various hematopoietic lineages, we have constructed several mutant viruses expressing a fusion protein with deletions in either v-myb or v-ets. We show here that integrity of the v-ets oncogene is necessary for transformation of the erythrocytic cells but that neither the DNA-binding domain nor the trans-activating domain of v-myb is required for this transformation. The DNA-binding domain of v-ets is necessary to transform myelomonocytic cells. Furthermore, we show that E26 onco-protein also transforms granulocytic cells. The v-ets DNA-binding domain is not necessary to transform them, whereas deleting the v-myb DNA-binding domain strongly reduces transformation of these cells. These data show that the v-myb and v-ets DNA-binding domains provide quite different contributions to the transformation of various hematopoietic lineages by E26.


Activation of Jun/AP-1 by protein kinase A. The product of the c-jun proto-oncogene is the major component of the 12-O-tetradecanoyl phorbol 13-acetate (TPA)-inducible transcription factor AP-1. Jun binds to the TPA-responsive elements (TREs) present in a large number of TPA-inducible genes, thereby regulating their expression in response to activation of protein kinase C. Previously we have shown that Jun/AP-1 can also activate cAMP-responsive elements (CREs), indicating the existence of cross-talk in signal transduction at the transcriptional level. Here we show that Jun/AP-1 is activated by the cAMP-dependent protein kinase A (PKA). In transient transfection experiments, TRE activation by Jun is strongly enhanced by co-transfection of the catalytic subunit of PKA or forskolin treatment, although not in all cell types studied. Jun activity can be significantly inhibited by co-transfection of the regulatory subunit of PKA. Furthermore, we show a cell-specific increase in AP-1 binding in response to forskolin treatment. However, since direct phosphorylation of Jun by PKA does not occur, we suggest an indirect activation mechanism.


Head and neck synovial cell sarcoma.

Head and neck synovial cell sarcoma. Our experience with 14 patients treated for synovial cell sarcoma during the past 30 years is described. These tumors were manifested in young people whose ages ranged from 12 to 43 years. The diagnosis of synovial cell sarcoma proved difficult because approximately one third of the patients initially received incorrect pathologic diagnoses. A painless neck mass was the most common presenting symptom. Initial treatment included surgical excision in five patients and surgical excision and radiation therapy in nine. Recurrent or metastatic tumor occurred from 4 months to 62 months later; thus, long-term followup is important. Four of the nine patients who were followed more than 5 years died of their disease. Favorable prognostic findings included early diagnosis and the performance of wide surgical removal.


Endocrine control of steroidogenesis in granulosa cells. This review offers a summary of an increasingly complex literature which exists on the specific endocrine control of granulosa cell steroidogenesis. Further developments in cell biology will ultimately aid our greater understanding of pathophysiological regulation of the ovarian events underlying the human menstrual cycle. While we are beginning to unravel many of the important operational facets of this integrated process, our knowledge is still limited. Important questions remain to many clinical syndromes involving altered ovarian function, for example primary ovarian failure, polycystic ovary disease, resistant ovary syndrome, and so on. However, with the rapid continuing expansion of cellular and molecular technologies gaps in our understanding may be bridged in the near future, and thus enable physicians to evaluate better and treat disorders of ovarian function.


A kinetoplast DNA probe diagnostic for Leishmania infantum. A 196 bp fragment of cloned kinetoplast (k) DNA of Leishmania infantum has been sequenced and shown to be diagnostic for this species. The DNA from 10(4)-10(5) promastigotes was routinely and specifically detected in crude dot blots of whole cells lysed in situ, and there was no cross-hybridization with 10(6) cells of L. major, L. tropica, L. killicki, L. aethiopica, L. braziliensis and L. mexicana. A low degree of hybridization was found with L. donovani and L. chagasi. This probe will be of particular use in Mediterranean countries, such as Italy, in which dermotropic zymodemes of L. infantum can be difficult to isolate and grow in numbers sufficient for isoenzyme typing.


Schistosoma mansoni: praziquantel-induced tegumental lesion exposes actin of surface spines and allows binding of actin depolymerizing factor, gelsolin. Praziquantel is widely used for treatment of schistosomiasis. Elimination of intravascular worms involves a combination of drug-induced changes at the worm surface and a host immune response against exposed parasite components. Loss of spines is a typical feature of schistosomes obtained from praziquantel-treated infected animals. The mechanism for this is unknown. We have considered the possibility that praziquantel treatment involves exposure of surface spines consisting of 'paracrystalline' actin and a subsequent host response against actin through mechanisms established in the host independently from schistosomal infection. Drug-induced exposure of actin was demonstrated by fluorescence microscopy using anti-actin antibodies and phallacidin, an actin-binding mushroom toxin. Actin spines remained intact at the schistosome surface after in vitro exposure, but spine morphology was lost after in vivo exposure to praziquantel. Disintegration of spines in vivo was associated with binding of host antibodies. In vitro spine destruction could be seen in the presence of normal human serum. The effect was linked to calcium-dependent binding of actin depolymerizing factor, gelsolin.


Management of chronic asthma.

Management of chronic asthma. The treatment of asthma is changing to reflect the importance of inflammation in the disease pathogenesis. Medicines that alter the inflammatory response are the cornerstone of therapy for patients with persistent symptoms. Bronchodilators are important in acute care, but in chronic illness they are adjuvant therapy. Patient education is essential for successful outcome.


Color Doppler sonography in pyelonephritis. Renal sonography detects abnormality in only 40% of pediatric pyelonephritis. In eight patients shown to have acute pyelonephritis by 99mTc DMSA renal cortical scintigraphy, five were found to have focal abnormalities of renal perfusion by color Doppler sonography in the same sites as the scintigraphic defects (sensitivity-63%). Two of the five patients had normal plain sonograms. False positive studies occurred in patients with documented chronic renal scarring. The specificity of vascular asymmetry was 70%. This preliminary report suggests that, particularly in the patient without pre-existing renal scarring, color Doppler evaluation of the renal vasculature may increase the sensitivity of sonography in the diagnosis of pyelonephritis in children.


[Evaluation of prognostic parameters in colorectal carcinoma. I. Histopathological variability]. A careful histopathological analysis was performed on a series of 244 unselected surgically removed primary colonic and rectal cancers. Tumours were staged according to the TNM system. Tumour type (adenocarcinoma or mucinous carcinoma), grade of differentiation, character of invasive margin, degree of peritumoural lymphocytic infiltration, venous and neural invasion were found to be correlated with the clinico-pathological stage and in some ways interrelated. As a whole our results seem to suggest that histological evaluation of the variables examined may provide information of clinical relevance in the management of patients with large bowel cancer.


Progressive hearing loss in infants with asymptomatic congenital cytomegalovirus infection. Congenital cytomegalovirus (CMV) infection is a major public health problem because 30,000 to 40,000 neonates with the infection are born each year in the United States. Although 90% of the congenitally infected infants are asymptomatic at birth, evidence is accumulating that these infants are at risk for audiologic, neurologic, and developmental sequelae. The current study describes the audiologic outcome of 59 infants with asymptomatic congenital CMV infection compared with 26 control infants. Eight of 59 infected infants had congenital sensorineural hearing loss (SNHL) but none of the control subjects did. Longitudinal audiologic assessments revealed that 5 of the 8 infants had further deterioration of their SNHL; a ninth infant with initially normal hearing experienced a unilateral SNHL during the first year of life, with further deterioration subsequently. The frequency of SNHL was similar for infected infants born to mothers with recurrent CMV infections during pregnancy (2 of 9) and for those born to mothers who experienced primary CMV infections (5 of 26). There was a significant difference between the occurrence of hearing loss in infected infants with normal computed tomographic scans (2 of 40) compared with those with either periventricular radiolucencies (4 of 13) or calcifications (1 of 3). Children with SNHL often have no identified cause of the loss; thus, it is likely that many of these children had asymptomatic congenital CMV infection. Given the progressive nature of SNHL associated with asymptomatic congenital CMV infection, longitudinal audiologic assessments are mandatory.


[Prenatal cardiology: can we treat the fetal heart?]. Antenatal diagnosis of heart disease has become very efficient with the advances in ultrasound technology. Accordingly, antenatal treatment of some heart diseases is possible and this is mainly represented by fetal tachycardia and brachycardia. In fetal tachycardia, medical treatment leads to improvement or total curve in approximately 85% of cases. In contrast, there is no efficient medical treatment for fetal bradycardias. In any case, fetal therapy must fulfill several conditions, namely: transplacental transfer of the drug, innocuity for the mother and the fetus, absence of teratogenic effect and low extracardiac tissular accumulation.


[Fetal surgery: for what purpose?].

[Fetal surgery: for what purpose?]. Widespread use of prenatal ultrasonography allows accurate diagnosis of many fetal malformations. The natural history and pathophysiology of some fetal diseases will undoubtedly induce lethal issue. Prenatal ultrasounds allow to determine prognosis during the second trimester of pregnancy. Experimental model and clinical practice have shown that prenatal surgery could be proposed for some correctable malformations: congenital diaphragmatic hernia, cystic adenomatoid lung disease, sacrococcygeal teratoma, lower urinary tract obstruction. To purpose this fetal therapy, great attention has to be paid to maternal risk and her future fertility, keeping in mind the aim of such fetal surgery: a new hope for improved management of the fetus with life-threatening defect.


[Prevention of sexual ambiguity in children with 21-hydroxylase deficiency by treatment in utero]. In the classical form of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency, female fetuses are virilized by the excessive production of adrenal androgens. Sexual ambiguity is a major complication. Prenatal treatment has been proposed with the aim of reducing the adrenal androgen overproduction and hence of preventing the in utero virilization of CAH affected females. As there is good placental transfer of natural or synthetic glucocorticoids, dexamethasone (Dex) in particular, efficient treatment can be administered to the fetus via the mother. In early protocols the mothers were given 0.5 mg of Dex either 8- or 12 hourly. Analysis of the results of a French multicentric study has shown that the first consideration is to start treatment as early as possible, and no later than the 7th week of gestation. Also, the daily dose should be adjusted to maternal size. A dose of 20-25 micrograms/kg body weight is suggested. As this condition has to be treated early it is not possible to make a prenatal diagnosis before instituting therapy, which is not necessary in 7 out of 8 fetuses. It is thus important to establish as soon as possible a reliable diagnosis of sex and CAH. The generalization of villus chorionic biopsies and the recent progress in molecular genetics--restriction length fragment polymorphism and polymerase chain reaction (PCR)--now permit a direct analysis of the DNA during the first trimester. In particular, the determination of point mutations using PCR amplification of specific alleles in the proband an the parents, simplifies the procedure and also increases the reliability of prenatal diagnosis. On the whole, prenatal treatment with an adequate protocol has been fully or partially successful in almost all cases. No teratogenic or major adverse effects in the mother or child have been reported.


[Treatment of malaria in children in France]. Currently, most of the subjects presenting with Plasmodium falciparum malaria in France come from areas where chloroquine resistance has already been reported. Treatment of uncomplicated malaria should consist of oral administration of either quinine or mefloquine or halofantrine. In children, halofantrine seems to be the treatment of choice at any age. The prognosis of cerebral malaria depends on how fast the diagnosis is made and the treatment is undertaken. The detection of clinical and biological risk factors is crucial. The treatment of cerebral malaria is based on quinine perfusion administered according to pharmacokinetic data.


[Contribution of Doppler in fetal prognosis]. Uteroplacental and cerebral artery velocimetry is a predictive factor in the outcome of many high-risk pregnancies. Positive predictive value is good for intrauterine growth retardation (complications due to preeclampsia, diabetes or twin pregnancy). Velocimetry is also a predictor of adverse outcome in small for gestational age fetuses and for oligohydramnios but not for anemic fetuses of isoimmune pregnancies or post-term pregnancies.


[Utero-vaginal malformations].

[Utero-vaginal malformations]. This review deals with the various anatomo-clinical features and the embryogenesis of utero-vaginal malformations. Three anatomo-clinical groups are distinguished from the vaginal malformations: vaginal aplasia, vaginal septa and hydrometrocolpos. Similarly, there are 3 types of uterine malformations, each corresponding to a chronologically different embryopathological anomaly: mullerian aplasia, most often responsible for utero-vaginal aplasia, mullerian fusion defects leading to bicornuate uterus, and resorption defects of the mullerian septum resulting in uterine septum. Only the utero-vaginal malformations with a vaginal component, mainly neonatal hydrometrocolpos, can really be detected during infancy and childhood, provided that the examination of the external genitalia is systematically and carefully performed. Due to their usual clinical latency during childhood, most malformations are only discovered by the end of puberty or after menarche, the main clinical signs being primary amenorrhea, cryptomenorrhea, unilateral dysmenorrhea or isolated dyspareunia. After the clinical gynecological examination, ultrasound diagnosis is very useful to explore the internal genitalia and to search for a frequently associated anomaly of the urinary tract.


[Mid-term and long-term outcome in newborn infants with periventricular leukomalacia (53 cases)]. During the period 1983-1987, 53 neonates (30 boys, 23 girls, mean birth weight 1,438 +/- 317 g, mean gestational age 30 +/- 2 weeks) were found to have cystic periventricular leukomalacias (CPVL) detected by brain ultrasonography. The neuro-developmental follow-up was 3-7 years. Seventeen CPVL were isolated and CPVL were associated with minor peri-intraventricular hemorrhage (0-1 to II-II) in 36 cases. CPVL included 16 minor forms (11 normal children; 2 minor, 3 moderate sequelae), 29 moderate forms (15 normal children; 4 minor, 7 moderate, 3 major sequelae) and 8 major forms (7 major sequelae, one moderate mental retardation without motor deficit). According to reverse analysis, a normal evolution (26 cases) followed CPVL which were sometimes widely, but thinly spread over the ventricles (thickness 1/5-1/3 of the cerebral mantle); minor sequelae (6 cases) were associated with a 1/4-1/3 thickness, moderate sequelae (11 cases) with a 1/3-1/2 thickness, major sequelae (10 cases) with a 1/2-2/3 thickness of CPVL. The thickness of the lesions appeared to be more predictive of sequelae than the sagittal extent, in particular that of the posterior CPVL which played a major role in the severity of sequelae, and mainly in the presence of motor deficits.


[Retinoblastoma].

[Retinoblastoma]. Retinoblastoma (RB) is the most frequent ocular tumor in childhood. Due to recent advances in molecular biology, RB has become a study model for cancer suppressor genes, and antenatal diagnosis has now become feasible. The goals of therapy include an improved survival rate, decrease in iatrogenic sequelae (especially enucleation), and avoidance of radio-induced neoplasias. This review examines recent data from the literature.


["Cyrano" angiomas].

["Cyrano" angiomas]. Hemangioma of the nasal tip (Cyrano nose) is a cavernous or capillary cavernous form. Its development is related to immaturity. It occurs in infants a few days after birth, and increases with regularity. The natural outcome is favorable, and the angioma decreases in most cases after 5 or 6 years of age. Due to the location and the aesthetic prejudice involved, the parents often ask for early surgical treatment. A study of 4 cases and a literature review indicate the surgical intervention should take place later, ie one should wait and only operate on the sequelae resulting from the development of the hemangioma. Late surgical treatment led to resection of the remaining lesion and to repair of the nasal tip.


[Abnormalities of the nervous system in sudden infant death syndrome]. Anomalies seem to be present in the brain of infants who died of sudden infant death syndrome. They imply dendritic synapses, myelinisation, some neurotransmitters, some peptides, their enzymes and receptors. More precisely, catecholaminergic systems seem to be abnormal. The changes were found mainly in brain stem, in vegetative central areas. These anomalies are probably maturational in nature. They could represent a fragility of vegetative control systems, especially cardiorespiratory and swallowing control systems. They probably do not explain, per se, the occurrence of death; they probably would need to be potentiated by additional etiological factors (environmental, viral, etc). These anomalies have to be considered as provisional and, in our opinion, need confirmation. Apart from the brain stem, other areas are under investigation, particularly cerebral structures implied in temperature control, sleep-wakefulness control and hemispheric structures.


[Cardiac failure by major arterial hypertension secondary to nephroblastoma]. The authors report on the case of a 7 week-old boy, in whom a renal mass was discovered after general symptoms were observed. Within 48 h, cardiac failure secondary to systemic arterial hypertension occurred, requiring intensive care. After a few days of mechanical ventilation and alternating elevated and low blood pressure, improvement was obtained with captopril and frusemide enabling further investigations to be carried out which lead to the diagnosis of Wilms tumor. During left-sided nephrectomy, elevated renin from the left renal vein was found. The post surgical course was excellent. Several authors have reported on the association between arterial hypertension and nephroblastoma as being the result of hyperreninism due to hilar compression; however severe hypertension was uncommon. Renin activity determination from the tumoral tissue had led to a different interpretation, ie primary hyperreninism: in the case of mesoblastic nephroma, only the non tumoral but compressed tissue contains a large quantity of renin; in the case of nephroblastomas, only the tumoral tissue contains renin. The question now is whether all or only certain nephroblastomas secrete renin.


[Neonatal herpes: recurrence after treatment with acyclovir]. A case of cutaneous herpes relapse with meningitis is reported in a 1.5 month-old infant treated during the first three weeks of life with acyclovir (ACV) for a neonatal herpes infection. Such a relapse has previously been described in older children as well as in adults. In this case report, there was immunological response to herpes virus infection, 2.5 months after the onset of the infection. The relapse is discussed taking into account the mechanism of action of ACV, the age of the patient and the immunological response profile. Because of the high risk of neurological involvement, we suggest that the relapse should be treated with ACV for a period of time longer than actually recommended.


[Cerebrovascular accidents and sickle cell disease]. A 7 year-old girl, with homozygous sickle cell disease experienced two cerebrovascular strokes over an 8-month period. The diagnostic investigations were performed with highly sensitive imaging methods (MRI, angiography). Such a complication raises problems in curative and prophylactic treatment. New diagnostic methods are being evaluated.


[Unusual cause of neonatal hemorrhage: congenital cutaneous aplasia of the vertex]. We report on a case of familial congenital scalp defect with fetal bleeding during labor. The authors emphasize the frequency of familial forms and the potential risk of such abnormalities during labor's monitoring. Treatment and classification are reviewed. The prognosis is good, as shown by the mother's case 27 years later.


[Is pericerebral hemorrhage a cause of severe malaise in infants?]. The authors report 7 cases of infants presenting with an apparent life-threatening event associated with acute pericerebral haemorrhage (subarachnoid haemorrhage and/or subdural hematoma) without evidence of traumatism, abuse, or shaking. Clinical characteristics were the same in all cases, including limpness, severe dysautonomic disorders, and pallor; all infants had retinal and pre-retinal haemorrhages. Two infants died; the five survivors have severe neurologic sequelae. The symptoms revealing an infant's pericerebral haemorrhage are usually axial hypotonia and pallor. Traumatism remains the most common aetiology and must be searched for. Non-traumatic aetiologies are unusual and were excluded in these reported cases. The 'shaken baby' syndrome is not the sole aetiology of an apparent spontaneous pericerebral haemorrhage: a slight bump associated with predisposing vascular factors particular to infancy could be involved. When confronted with an apparent life-threatening event associating limpness and pallor, one must consider the diagnosis of pericerebral haemorrhage.


[Congenital toxoplasmosis in the Alpes-Maritimes region. Apropos of 26 cases observed between 1984 and 1990]. Twenty-six cases of congenital toxoplasmosis observed in the department of Alpes Maritimes (Chief Town: Nice) between 1984 and 1990 are reported. All affected children were treated by pyrimethamine and sulfonamide as soon as the diagnosis was established. None of them exhibited serious sequellae. In most cases (58%) there were no clinical manifestations in agreement with other reports. Benign manifestations were observed in 10 cases (38%): 4 chorioretinitis; 4 intracranial calcifications: 1 febrile seizure; 7 hyperproteinorachias: 3 isolated and 4 associated with other signs. In 3 cases, secondary lesions appeared during the treatment period. This confirms the need for active therapeutic research in order to decrease the risk of late ocular complication which is the major problem of the disease.


[Cirrhosis and cirrhogenic diseases in Tunisian children. Multicenter study of 65 cases]. Over a period of 10 years, 65 cases of hepatic cirrhosis and cirrhogenic disease have been observed in five Pediatric Centers in mid Tunisia. The age of the patients ranged from 30 days to 14 years. The main etiology was biliary cirrhosis (24 cases) followed by post-hepatic cirrhosis (15 cases). Eight cases had a metabolic origin, which was Wilson's diseases in five cases. Three children had cirrhosis of a pre-hepatic origin. In 15 children, the liver biopsy showed the presence of cirrhosis but the etiology could not be found. Preventive measures are needed in order to reduce the frequency of cirrhosis among Tunisian children: 1) early recognition of biliary atresia, 2) vaccination against hepatitis B virus of at risk neonates and children, 3) Genetic counselling and search for familial cases when cirrhosis of metabolic origin is identified, particularly Wilson's disease.


[Two-dimensional and Doppler echocardiography in the non-invasive diagnosis of coronary cardiac fistula]. Two asymptomatic children with an atypical continuous murmur and a clinical diagnosis of coronary artery fistula are reported. Cross-sectional and Doppler echocardiography led to the diagnosis of a right coronary artery-right ventricule fistula in both cases, which was confirmed by coronary angiography. Surgical closure of the fistula was realized without complications.


[Acquired peripheral facial palsy in children. Current data illustrated by 66 recent personal cases]. The authors observed 66 cases of peripheral facial palsy (PFP) in children during a 5-year period (1986-1990). Bell's palsy (idiopathic facial paralysis) occurred in 26 children (39.3%), 1 month to 14.5 years old, with a complete recovery in 95% of the cases; a surgical decompression was carried out in 2 cases. The PFP was related to otitis in 16 cases (24.2%): acute otitis media (6), mastitis (4), serous otitis (5), cholesteatoma (1); the treatment was medical and surgical in all cases with complete recovery in 15 cases. In 15 cases the PFP was secondary to trauma (13) or surgery (2); complete spontaneous recovery occurred in 11 cases, and partial recovery following surgical treatment in 2 cases. A viral origin was retained in 6 cases: herpes zoster (3), mumps (1), echovirus (1), herpes (1); the recovery was complete in 4 cases, partial in 2 cases. In 3 cases the PFP was related to a rare cause: lymphoma, metabolic acidosis, Melkerson-Rosenthal syndrome. Bell's palsy remains the main cause of PFP in childhood; other etiologies can be ruled out by the case history, a careful physical examination, and a limited number of laboratory and/or X-ray studies; medical treatment, in particular prednisone, does not seem to have an effect upon the rate of recovery which is spontaneously high; similarly there is no evidence that surgical decompression really modifies the rate of recovery so that the authors suggest that it should be reserved to the complete forms with no clinical and electrical evidence of recovery after 3 weeks.


[Value of approach monitoring of infusion pressure in neonatology]. The consequences of catheter occlusions are particularly dangerous in neonates. For early detection and to efficiently prevent such occlusions, the monitoring of infusion pressure by a variable pressure pump is reported. Two ways of monitoring can be recommended: easy viewing of infusion pressure and programming a pressure limit. Infusion pressure is recorded at around +/- 30 cm water pressure of the mean day pressure. Consequently, a rise above this gap must be considered as suspect. The regular viewing of this measurement is a way of predicting such problems as occlusions. The optimum pressure limit is programmed at 30 cm water pressure above the infusion pressure. This alarm may identify the beginning of occlusions or reduce the extent of tissue damage in early detecting of an infiltration.


Effect of calcitonin on the regulation of intracellular pH in primary cultures of rabbit early distal tubule. To examine the intracellular pH (pHi) regulation in primary cultures of rabbit distal convoluted tubules (DCTb) we used the pH-sensitive dye 2,7-bis-carboxyethyl-5(6)-carboxyfluorescein (BCECF/AM) and a video-microscopy technique. DCTb segments were microdissected from rabbit kidney cortex and cultured in a hormonally defined medium. The culture epithelia were grown on semi-transparent permeable supports. Before pHi measurement, DCTb primary cultures were maintained for 48-96 h in growth-factor-free medium to obtain quiescent cells. We had previously shown that two mechanisms are involved in the regulation of intracellular pH: a basolateral Na+/H+ exchanger and an apical Cl-/HCO3- exchanger. The pHi of DCTb cells was significantly decreased by the addition of 60 nM human calcitonin (from 7.30 +/- 0.04 to 7.08 +/- 0.04). This response to calcitonin was dose-dependent and mimicked by both forskolin and permeant cyclic AMP derivatives. An initial acidification (of 0.25 pH unit in 7-8 min) was observed after the addition of basolateral amiloride (1 mM). The persistence of the effect induced by human calcitonin in these conditions, suggests that the Na+/H+ exchanger is not involved in the response. However, the acidification response was blocked in both the absence of chloride at the apical side and by the apical addition of 0.1 mM 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS). These experiments suggest that the target for the human calcitonin effect on pHi is the Cl-/HCO3- exchanger. This study confirms the importance of this transporter in pHi regulation within the physiological pHi range and the influence of calcitonin in the regulation of DCTb cell function.


Roles of inositol trisphosphate and protein kinase C in the spontaneous outward current modulated by calcium release in rabbit portal vein. We examined the effects of heparin, guanosine nucleotides, protein kinase C (PKC) modulators, such as phorbol 12,13-dibutyrate (PDBu) and H-7 on Ca(2+)-dependent K+ currents in smooth muscle cells of the rabbit portal vein using the whole-cell patch-clamp technique, to explore the effects of PKC on the oscillatory outward current (Ioo). Neomycin (30 microM), an inhibitor of phospholipase C, and intracellular applications of heparin (10 micrograms/ml) and guanosine 5'-O-(2-thiodiphosphate) (GDP[beta S]; 1 mM) partly but consistently inhibited the generation of Ioo, whereas a higher concentration of heparin (100 micrograms/ml) transiently enhanced then suppressed the generation of Ioo. Inhibition of Ioo generation by heparin was more powerful at the holding potential of +20 mV than at -20 mV. Inositol 1,4,5-trisphosphate (InsP3; 30 microM) continuously generated Ioo at holding potentials more positive than -60 mV. Noradrenaline (10 microM) and caffeine (3-20 mM) transiently augmented, then reduced the generation of Ioo. Heparin (10 micrograms/ml) completely inhibited responses induced by InsP3 and noradrenaline, but not those induced by caffeine. Intracellular application of guanosine 5'-triphosphate (GTP; 200 microM) or low concentrations of guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma S]; < or = 3 microM) continuously augmented the generation of Ioo. High concentrations of GTP[gamma S] (> or = 10 microM) transiently augmented, then inhibited Ioo. Neither GTP[gamma S] nor noradrenaline induced the transient augmentation or the subsequent inhibition of Ioo when applied in the presence of GDP[beta S] (1 mM), neomycin (30 microM) or heparin (10 micrograms/ml). PDBu (0.1 microM) reduced the generation of Ioo but failed to produce an outward current following application of caffeine (3-5 mM). This action of PDBu was inhibited by pretreatment with H-7 (20 microM). In the presence of H-7, GTP[gamma S] continuously enhanced the generation of Ioo. The suppression of the generation of Ioo during application of noradrenaline (10 microM) was reduced by pretreatment with H-7. Thus both InsP3 and protein kinase C contribute to the generation of Ioo in smooth muscle cells of the rabbit portal vein and heparin is not a specific InsP3 antagonist on the InsP3-induced Ca(2+)-release channel (PIRC). InsP3 opens PIRC and protein kinase C may deplete the stored Ca2+ by either inhibiting the reuptake of Ca2+ or by enhancement of the releasing actions of InsP3.


The acute paralysis in Guillain-Barré syndrome is related to a Na+ channel blocking factor in the cerebrospinal fluid. The effect of cerebrospinal fluid (CSF) from patients with severe polyradiculoneuritis (Guillain-Barré syndrome, GBS) on voltage-dependent Na+ channels of myoballs was studied. The transient Na+ currents, elicited by repetitive stimulation at 1 Hz, were inhibited by the CSF from most of the GBS patients to 10%-40% the control value. The inhibition was complete in about 5 s and was fully reversible. Such inhibition was never seen with control CSF. The blocking property of the CSF from GBS patients was lost after the number of cells and the protein content had been lowered by means of a clinical filtration technique for cerebrospinal fluid. The results demonstrate that in Guillain-Barré syndrome blocking factors of Na+ channels are present in the CSF, impairing neuron impulse conduction, and thereby causing muscular weakness and sensory disturbances in the affected patient.


Tetrandrine blocks a slow, large-conductance, Ca(2+)-activated potassium channel besides inhibiting a non-inactivating Ca2+ current in isolated nerve terminals of the rat neurohypophysis. The effects of tetrandrine, a bis-benzyl-isoquinoline alkaloid, on voltage-gated Ca2+ currents (ICa) and on Ca(2+)-activated K+ current (IK(Ca)) and channels in isolated nerve terminals of the rat neurohypophysis were investigated using patch-clamp techniques. The non-inactivating component of ICa was inhibited by external tetrandrine in a voltage- and dose-dependent manner, with an IC50 = 10.1 microM. IK(Ca) was elicited by depolarizations when approximately 10 microM Ca2+ was present on the cytoplasmic side. Only externally applied tetrandrine, at 1 microM, decreased the amplitude of IK(Ca), whereas the fast inward Na+ current and transient outward K+ current were not affected. Tetrandrine, applied to the extracellular side of outside-out patches excised from the nerve terminals, induced frequent and short closures of single type II, maxi-Ca(2+)-activated K+ channels. Tetrandrine decreased the channel-open probability, within bursts, with an IC50 = 0.21 microM. Kinetic analysis of the channel activity showed that the open-time constant decreased linearly with increasing tetrandrine concentrations (0.01-3 microM), giving an association rate constant of 8.8 x 10(8) M-1 s-1, whereas the arithmetic mean closed time did not change, giving a dissociation rate constant of 136.6 s-1. These results show that tetrandrine is a high-affinity blocker of the type II, maxi-Ca(2+)-activated K+ channel of the rat neurohypophysial terminals.


The colon carcinoma cell line Caco-2 contains an H+/K(+)-ATPase that contributes to intracellular pH regulation. The presence of an H+/K(+)-ATPase and its contribution to the regulation of intracellular pH (pHi) was investigated in Caco-2 cells. The H+/K(+)-ATPase was detected immunologically using the monoclonal antibody 5-B6, which was raised against hog gastric H+/K(+)-ATPase. Cell pH was determined using the pH-sensitive dye 2',7'-bis(carboxyethyl)-carboxyfluorescein. Control pHi, measured in HCO(3-)-free medium, was 7.62 +/- 0.03 (n = 27) when cells were cultured for 14 days and decreased to 7.40 +/- 0.03 (n = 18) after 35 days in culture. Recovery of pHi following a NH+4/NH3 pulse could be reduced by either 100 microM SCH 28080 or 1 mM amiloride, or by removing extracellular Na+. The inhibitory effects of SCH 28080 and amiloride were additive, demonstrating the involvement of a gastric-like H+/K(+)-ATPase and a Na+/H+ exchanger in regulating pHi. Recovery rates at pHi 6.8 were not significantly different in cells cultured for up to 21 days, but were significantly lower in cells cultured for 28 and 35 days. This decrease in recovery rate was due to a decrease in the SCH-28080-insensitive recovery, indicating a reduction of the relative importance of Na+/H+ exchange to the recovery. Recovery of pHi was also inhibited by 1 mM N-ethylmaleimide. However, it is unlikely that N-ethyl-maleimide inhibited a vacuolar type of H+-ATPase, since bafilomycin A1 had no effect on pHi recovery. In conclusion, Caco-2 cells contain a SCH-28080-sensitive mechanism for regulating pHi, which is most conveniently studied after 28 days in culture, when the relative contribution of a Na+/H+ exchanger to pHi regulation is decreased.


Characteristics and modulation by thyrotropin-releasing hormone of an inwardly rectifying K+ current in patch-perforated GH3 anterior pituitary cells. Hyperpolarization of patch-perforated GH3 rat anterior pituitary cells in high-K+ Ca(2+)-free medium reveals an inwardly rectifying K+ current. This current showed potential-dependent activation and inactivation kinetics, complete inactivation during strong hyperpolarization and rectification at depolarized potentials. The current was blocked by millimolar concentrations of external Cs+, Ba2+, Cd2+ and Co2+, but it was almost insensitive to tetraethylammonium, 4-aminopyridine and two dihydropyridines, nisoldipine and nitrendipine. Verapamil and methoxyverapamil produced a strong and reversible inhibition of the current. In the presence of 100 nM thyrotropin-releasing hormone (TRH), the current was reduced. This reduction was increased by holding the cell at more negative potentials and was accompanied by a shift in steady-state voltage dependence of inactivation towards more positive voltages. Furthermore, the current slowly returned to the initial levels upon washout. Treatment of the cell with the protein phosphatase inhibitor okadaic acid increased the magnitude of the inhibition caused by TRH. Moreover, the current did not return towards the control level during a 30-min washout period. It is concluded that protein phosphatases participate in modulation of the GH3 cell inwardly rectifying K+ channels by TRH. Furthermore, these data indicate that either a protein phosphatase or a factor necessary for its activation is lost under whole-cell mode, which could account for the permanent reduction of the current in response to TRH.


Voltage-dependent noradrenergic modulation of omega-conotoxin-sensitive Ca2+ channels in human neuroblastoma IMR32 cells. High-threshold (HVA) Ca2+ channels of human neuroblastoma IMR32 cells were effectively inhibited by noradrenaline. At potentials between -20 mV and +10 mV, micromolar concentrations of noradrenaline induced a 50%-70% depression of HVA Ba2+ currents and a prolongation of their activation kinetics. Both effects were relieved at more positive voltages or by applying strong conditioning pre-pulses (facilitation). Facilitation restored the rapid activation of HVA channels and recruited about 80% of the noradrenaline-inhibited channels at rest. Re-inhibition of Ca2+ channels after facilitation was slow (tau r 36-45 ms) and voltage-independent between -30 mV and -90 mV. The inhibitory action of noradrenaline was dose-dependent (IC50 = 84 nM), mediated by alpha 2-adrenergic receptors and selective for omega-conotoxin-sensitive Ca2+ channels, which represent the majority of HVA channels expressed by IMR32 cells. The action of noradrenaline was mimicked by intracellular applications of GTP[gamma S] and prevented by GDP[beta S] or by pre-incubation with pertussis toxin. The time course of noradrenaline inhibition measured during fast application (onset) and wash-out (offset) of the drug were independent of saturating agonist concentrations (10-50 microM) and developed with mean time constants of 0.56 s (tau on) and 3.6 s (tau off) respectively. The data could be simulated by a kinetic model in which a G protein is assumed to modify directly the voltage-dependent gating of Ca2+ channels. Noradrenaline-modified channels are mostly inhibited at rest and can be recruited in a steep voltage-dependent manner with increasing voltages.


Membrane capacitance increases induced by histamine and cyclic AMP in single gastric acid-secreting cells of the guinea pig. During acid secretion, gastric parietal cells undergo profound morphological changes including formation of the apical secretory membrane. To examine the mechanism of histamine-induced increases in the apical membrane area at the single cell level, we monitored the membrane capacitance by applying a time-resolved phase-sensitive detection method to singly isolated parietal cells of guinea pig. A real-time increase in the membrane capacitance was detected within several min after stimulation with histamine. An H2-blocker (cimetidine), but not an H1-blocker (pyrilamine), inhibited the histamine response. Dibutyryl cyclic AMP mimicked the histamine effect. The capacitance response to histamine was sensitive to cytosolic Ca2+, temperature and N-ethylmaleimide. The histamine response was inhibited by intracellular application of a non-hydrolyzable ATP analog (AMP-PNP) and an isoquinolinesulfonamide derivative that works as an inhibitor of protein kinase A (H-8). These results indicate that in parietal cells, elevation of intracellular cyclic AMP induces exocytotic insertion of intracellular membranes into the plasma membrane, presumably by activating protein kinase A.


Aldosterone actions on basolateral Na+/H+ exchange in Madin-Darby canine kidney cells. In recent studies, there has been a re-evaluation of the polarity of Na+/H+ exchange in Madin-Darby canine kidney (MDCK) cells. This study was designed to examine aldosterone actions on basolaterally located Na+/H+ exchange of MDCK cell monolayers grown on permeant filter supports; pHi was analysed in the absence of bicarbonate by using the pH-sensitive fluorescent probe 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. Pre-exposure of MDCK cells to aldosterone led within 10-20 min to an alkalization of pHi (approximately 0.3 pH unit); this effect is prevented by an addition of dimethylamiloride to the basolateral superfusate. Addition of aldosterone led to stimulation of the basolaterally located Na+/H+ exchange activity (Na(+)-dependent recovery from an acid load); this effect required preincubation (more then 3 min) and was observed at 0.1 nM aldosterone. Pre-exposure (15 min) of MDCK monolayers to phorbol 12-myristate 13-acetate also led to an activation of Na+/H+ exchange; pre-exposure to 8-bromo-cAMP led to inhibition of Na+/H+ exchange activity. An inhibitory effect of aldosterone was observed if Na+/H+ exchange activity was analysed in the presence of aldosterone; the highest inhibitory effects (20%-30%) occurred at concentrations of 5 nM and higher. Aldosterone-dependent inhibition does not require preincubation and is fully reversible; it was only observed at low (20 mM) but not at high Na+ concentrations (130 mM). The data suggest that aldosterone has an instantaneous inhibitory effect on basolaterally located Na+/H+ exchange activity under conditions of low Na+, but stimulates the rate of transport activity upon preincubation under conditions of physiological Na+ concentrations.


Molecular basis for pharmacological differences between brain and cardiac sodium channels. Sodium channels from brain and heart, whose primary structures are known, differ in their sensitivity to block by the guadinium toxins tetrodotoxin and saxitoxin and to block by external Zn2+ and Cd2+. Studies using site-directed mutagenesis have identified the SS2 and adjacent regions of all four repeats as critical determinants for toxin sensitivity. Within and in the immediate vicinities of the SS2 segments, there are only two amino-acid differences between rat brain sodium channel II and rat heart I sodium channel, both located in repeat I. Here we show that replacement of phenylalanine 385 of brain sodium channel by cysteine that is present at the equivalent position in heart channel (F385C) not only reduces sensitivity to the guadinium toxins but also increases sensitivity to Zn2+ and Cd2+, thus conferring properties of heart sodium channel on brain sodium channel. Replacement of asparagine at the second non-conserved position by arginine (N388R) only marginally affects sensitivity to the toxins, Zn2+ or Cd2+, but this mutation markedly reduces sensitivity to block by Ca2+ and Co2+. The double mutant channel (F385C.N388R) shows combined properties of the two mutant channels. These results give a structural insight into the different properties of the two channel proteins.


Ser/Thr-specific protein phosphatases are required for both catalytic steps of pre-mRNA splicing. We have used a combination of highly specific protein phosphatase inhibitors and purified mammalian protein phosphatases to show that at least two separate Ser/Thr protein phosphatase activities are required for pre-mRNA splicing, but not for spliceosome assembly. Okadaic acid, tautomycin, and microcystin-LR, which are potent and specific inhibitors of PP1 and PP2A, two of the four major types of Ser/Thr-specific phosphatase catalytic subunits, block both catalytic steps of the pre-mRNA splicing mechanism in HeLa nuclear extracts. Inhibition of PP2A inhibits the second step of splicing predominantly while inhibition of both PP1 and PP2A blocks both steps, indicating a differential contribution of PP1 and PP2A activities to the two separate catalytic steps of splicing. Splicing activity is restored to toxin-inhibited extracts by the addition of highly purified mammalian PP1 or PP2A. Protein phosphatase activity was not required for efficient assembly of splicing complexes containing each of the U1, U2, U4/U6 and U5 snRNPs. The data indicate that reversible protein phosphorylation may play an important role in regulating the pre-mRNA splicing mechanism.


Characterization of cDNA encoding the mouse DNA topoisomerase II that can complement the budding yeast top2 mutation. Several cDNA clones encoding mouse DNA topoisomerase II were obtained from a mouse spermatocyte cDNA library and the entire coding sequence of the gene was determined. The mouse DNA topoisomerase II consists of 1528 amino acids with a molecular weight of 173 kDa. It shares significant homologies with the other eucaryotic enzymes, although species-specific sequences are observed in their highly charged C-terminal regions. The complete mouse TOP2 cDNA was put under yeast GAL1 promoter and examined for complementation of top2ts mutation in S.cerevisiae. We found that the cloned mouse gene could rescue the temperature-sensitive top2ts mutation, depending on its induction by galactose. The functional expression of the mouse DNA topoisomerase II in yeast was further confirmed by enzymatic assays and by immunological methods with antibodies specific for the mouse enzyme.


Trans-dominant inhibition of transcription activator LFB1. Liver-enriched factor LFB1 (also named HNF1) is a dimeric transcription activator which is essential for the expression of many hepatocyte-specific genes. Here we demonstrate that LFB1 mutants in the POU A-like or in the homeo domains inhibit wild-type DNA binding by forming inactive heterodimeric complexes. Co-transfection of one of these mutants with wild-type LFB1 in HeLa cells eliminated LFB1 DNA binding and transcriptional activities through a trans-dominant mechanism. Expression of the same dominant negative mutant in human hepatoma HepG2 cells only partially inhibited endogenous LFB1 activity, due to stabilization of LFB1 dimers in these cells. Dimer stabilization in hepatoma cells is mediated by a heat-labile association with an 11kD polypeptide, analogous to the DCoH cofactor identified in rat liver by Mendel et al. (1). The property of stabilizing LFB1 dimers is also shared by HeLa cells which produce a HeLa homolog of DCoH. These results demonstrate that LFB1 dimer stabilization as well as the synthesis of 'stabilizing factors' are not restricted to cells expressing LFB1 or other members of its family.


DNA helicase III from HeLa cells: an enzyme that acts preferentially on partially unwound DNA duplexes. Human DNA helicase III, a novel DNA unwinding enzyme, has been purified to apparent homogeneity from nuclear extracts of HeLa cells and characterized. The activity was measured by using a strand displacement assay with a 32P labeled oligonucleotide annealed to M13 ssDNA. From 305 grams of cultured cells 0.26 mg of pure protein was isolated which was free of DNA topoisomerase, ligase, nicking and nuclease activities. The apparent molecular weight is 46 kDa on SDS polyacrylamide gel electrophoresis. The enzyme shows also DNA dependent ATPase activity and moves unidirectionally along the bound strand in 3' to 5' direction. It prefers ATP to dATP as a cofactor and requires a divalent cation (Mg2+ > Mn2+). Helicase III cannot unwind either blunt-ended duplex DNA or DNA-RNA hybrids and requires more than 84 bases of ssDNA in order to exert its unwinding activity. This enzyme is unique among human helicases as it requires a fork-like structure on the substrate for maximum activity, contrary to the previously described human DNA helicases I and IV, (Tuteja et al. Nucleic Acids Res. 18, 6785-6792, 1990; Tuteja et al. Nucleic Acids Res. 19, 3613-3618, 1991).


Effects of sequence and length on imino proton exchange and base pair opening kinetics in DNA oligonucleotide duplexes. The base catalysed imino proton exchange in DNA oligonucleotides of different sequences and lengths was studied by 1H-NMR saturation recovery experiments. The self-complementary sequences studied were GCGCGAATTCGCGC (I), CGCGAATTCGCG (II), GCGAATTCGC (III), and CGCGATCGCG (IV). The evaluation of base pair lifetimes was made after correction for the measured 'absence of added catalyst' effect which was found to be characterized by recovery times of 400-500 ms for the AT base pairs and 250-300 ms for the GC base pairs at 15 degrees C. End effects with rapid exchange is noticeable up to 3 base pairs from either end of the duplexes. The inner hexamer cores GAATTC of sequences I-II show similar base pair lifetime patterns, around 30 ms for the innermost AT, 5-10 ms for the outer AT and 20-50 ms for the GC base pairs at 15 degrees C. The shorter sequences III and particularly IV show much shorter lifetimes in their central AT base pairs (11 ms and 1 ms, respectively).


High levels of reactive oxygen metabolites in colon cancer tissue: analysis by chemiluminescence probe. Reactive oxygen metabolites (ROM) have been postulated to contribute to the development of various carcinomas, including colon cancer. Indeed, the effects of ROM scavengers are being tested for chemoprevention of adenocarcinoma of the colon. However, there has been no evidence to indicate that high levels of ROM are indeed present in cancerous tissue. In this study, we used a chemiluminescence probe to estimate ROM levels in cancerous and neighboring noncancerous colonic tissues from seven patients with colon cancer. Cancerous tissues contained significantly (p less than 0.05) more luminol-enhanced chemiluminescence (4,808 +/- 2,282 counts/min/mg protein) than neighboring noncancerous tissues (2,175 +/- 1,111). The addition of an ROM scavenger, catalase (2, 4, and 8 micrograms/ml), to the tissue suspension inhibited chemiluminescence produced by both noncancerous (-74%, -85%, and -71%) and cancerous (-11%, -61%, and -53%) tissues. This study shows that colonic cancerous tissue contains high levels of ROM, which may play an important role in the pathogenesis of colon cancer.


Effect of a dietary fiber (beet fiber) on dimethylhydrazine-induced colon cancer in Wistar rats. The modifying effect of a dietary fiber, Fibeta (beet fiber), on experimentally induced colorectal cancer was studied in Wistar rats. The rats were fed a powdered semisynthetic casein-based diet in which the carbohydrate pool was substituted with Fibeta as the sole source of fiber. Dimethylhydrazine dihydrochloride (DMH-2HCl) was used as initiator in a dose of 20 mg/kg body wt and given by gavage once a week for 10 weeks. Throughout the experiment the rats were offered the diets with different levels of fiber in a preinitiation period of 8 weeks, during the initiation, or in a 30-week postinitiation period. The study was terminated after one year. A protective effect of the fiber was not found at any stage of the colorectal carcinogenic process. Even though differences (not statistically significant) in tumor incidences were seen, these did not reflect any effect of the high or low fiber intake during the study. Analysis for volatile fatty acids in cecal content showed that continuous feeding with a fiber-rich diet resulted in significant increase in most of the volatile fatty acids. The relative change was highest for butyric acid. These findings do not support the hypothesis that butyric acid has a protective effect on colorectal cancer. The tumor yield in the present study was low compared with that reported in the literature, and possible causes for this are discussed.


Comparative assessment of the effect of lomefloxacin, ciprofloxacin, and placebo on cerebral blood flow, and glucose and oxygen metabolism in healthy subjects by positron emission tomography. The mechanism by which the fluorinated quinolones produce central nervous system (CNS) effects is currently unknown. We measured the effect of lomefloxacin on cerebral blood flow and metabolism using positron emission tomography. Eighteen healthy, nonsmoking volunteers were randomized to receive lomefloxacin 400 mg, ciprofloxacin 750 mg, or placebo given in a single-blind fashion every 12 hours for 72 hours, the time window for maximum lomefloxacin CNS effects. Subjects receiving lomefloxacin had a mean (+/- SEM) cerebral blood flow (CBF) of 46 (2.9) ml/min/100 g, glucose metabolism (FDG) 4.7 (0.4) mg/min/100 g, oxygen metabolism (OM) 3.3 (0.3) ml/min/100 g, and oxygen extraction (%OM) 0.4 (0.04). Posttreatment values were 43 (3.6) ml/min/100 g, 4.2 (0.4) mg/min/100 g, 2.6 (0.3) ml/min/100 g, and 0.4 (0.03), respectively. Values for subjects receiving ciprofloxacin were CBF 44.8 (1.6) ml/min/100 g, FDG 4.9 (0.7) mg/min/100 g, OM 4.1 (0.4) ml/min/100 g, and %OM 0.6 (0.03) at baseline, and 40.3 (3.5), 4.5 (0.6), 3.4 (0.4), and 0.5 (0.09), respectively, after treatment. For placebo-treated subjects, baseline values were CBF 41.4 (1.9) ml/min/100 g, FDG 4.9 (0.5) mg/min/100 g, OM 3.3 (0.4) ml/min/100 g, and %OM 0.5 (0.07), and respective posttreatment values were 42.1 (2.3), 5.0 (0.6), 3.5 (0.3), and 0.5 (0.02). No effect was observed on visual (qualitative), blinded reading of the scans. No significant effect on cerebral blood flow or metabolism was detected. We conclude that short-term administration of lomefloxacin or ciprofloxacin to healthy volunteers does not have a significant effect on cerebral blood flow, or on oxygen or glucose metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)


In vivo and in vitro beta 2-adrenergic receptor responsiveness in young and elderly asthmatics. This pilot study was undertaken to characterize age-related alterations in airway and metabolic beta 2-adrenergic receptor response using terbutaline as a probe. A single dose of terbutaline 0.007 mg/kg was administered subcutaneously to 10 young (range 20-35 yrs) and 10 elderly (range 60-73 yrs) men and women with asthma. beta-Receptor function was assessed by serial pulmonary function tests, heart rate, blood pressure, plasma potassium, glucose, insulin, and catecholamine levels, and in vitro 3', 5'-cyclic adenosine monophosphate (cAMP) production of pure T lymphocytes. The trend was for lung beta 2-receptor response (bronchodilation) and T lymphocyte cAMP production to be lower in elderly than in young asthmatics, but differences did not reach statistical significance. Elderly subjects' beta 1-adrenergic receptor responsiveness (systolic blood pressure) was also dampened. These data suggest that T lymphocyte stimulation does reflect pulmonary beta 2-receptor response. Whether elderly asthmatics require and tolerate higher dosages of parenteral terbutaline than younger asthmatics deserves further study.


Clinical implications of antibiotic resistance. Great advances have been made in the last two decades in understanding mechanisms of antibiotic resistance among pathogenic bacteria. From the clinician's perspective, the value of this scientific information at the bedside is not always obvious. Its importance can be emphasized by exploring two separate avenues: anecdotal reports illustrating the clinical cases in which one often sees resistance, and reports to the Centers for Disease Control (CDC) and some relevant findings from the results of a recent multicenter study of Enterobacter bacteremia.


Seizures associated with ganciclovir therapy. A 32-year-old man diagnosed with acquired immunodeficiency syndrome and a disseminated cytomegalovirus infection experienced seizures associated with the administration of ganciclovir. Seizures began 1 month after initiation of therapy and worsened with increasing dosages. Despite phenytoin administration, the seizure-like activity subsided only after discontinuing ganciclovir. After rechallenge with ganciclovir the seizures recurred. Although this case was confounded by numerous patient and disease factors, the Naranjo algorithm produced a score of 7, indicating a probable association between ganciclovir and seizure activity.


A study of the effect of the irreversible delta receptor antagonist [D-Ala2,Leu5,Cys6]-enkephalin on delta cx and delta ncx opioid binding sites in vitro and in vivo. Several lines of data support the existence of two classes of delta receptors: the delta cx binding site, which is the delta binding site of the mu-delta opioid receptor complex, and the delta ncx, which is the noncomplexed delta receptor. [D-Ala2,Leu5,Cys6]Enkephalin (DALCE) is an extended analog of [Leu5]enkephalin, which has been shown to bind irreversibly to delta receptors via the terminal cysteine by formation of a disulfide bond with the receptor. In vivo studies have shown that DALCE produces short-lived antinociceptive actions, followed by long-term antagonism of delta receptor-mediated antinociception. The major goal of the present study was to examine the effect of DALCE on the delta cx and delta ncx binding sites in vitro and in vivo. Intracerebroventricular administration of 40 micrograms DALCE failed to decrease [3H][D-Ala2,D-Leu5]enkephalin binding to the delta cx and delta ncx binding sites. Pretreatment of membranes with DALCE in vitro greatly reduced the Bmax of the delta ncx binding site, without significantly altering the Bmax of the delta cx binding site. These findings suggest that when administered in vivo, DALCE fails to distribute uniformly throughout the brain, and that it therefore binds covalently to opioid receptors mostly in the periventricular regions. Viewed collectively, these data support the hypothesis that DALCE acts as a selective delta ncx antagonist, and that the delta ncx binding site, which is sensitive to DALCE, is most likely synonymous with the recently described delta 1 receptor.


Purification and characterization of joining peptide and N-terminal peptide of proopiomelanocortin from the pars distalis of the bullfrog pituitary. The joining peptide (JP) and the N-terminal peptide of proopiomelanocortin (NPP) were isolated from an acid-acetone extract of the distal lobe of the pituitary of the bullfrog, Rana catesbeiana, and purified by gel filtration and reverse-phase high performance liquid chromatography. The amino acid sequence of the bullfrog JP resembled the sequences of the JPs of Rana ridibunda (86% similarity) and Xenopus laevis (54% similarity), as deduced from the nucleotide sequences of their cDNAs. The amino acid sequence of bullfrog NPP showed 100%, 85%, and 50% similarity with those of Rana ridibunda, Xenopus laevis, and human NPPs, respectively. Administration of bullfrog NPP (0.05-5 micrograms/ml) to perifused Rana ridibunda interrenal slices induced a dose-dependent stimulation of corticosterone and aldosterone release. The present results indicate that the primary structure of NPP has been highly conserved during evolution. These data also reveal that NPP, which has no sequence homology with ACTH, exhibits a substantial corticotropic activity.