A 12-week placebo-controlled double-blind study of prazosin in the treatment of prostatic obstruction due to benign prostatic hyperplasia. A series of 93 normotensive patients with benign prostatic hyperplasia and maximum urinary flow rates < 15 ml/s, treated at 2 hospital centres using an identical protocol, was randomly assigned to receive a 12-week course of treatment with prazosin or placebo in a double-blind parallel group trial. A total of 75 patients completed the study and were suitable for the final analysis. Prazosin was administered orally in doses of 0.5 mg and then 1 mg twice daily for 4 days and 2 mg twice daily for the remainder of the trial. Patients on treatment with prazosin exhibited a significantly increased maximum urinary flow rate as compared with placebo, with a significant reduction in maximum voiding detrusor pressure. Prazosin therapy did not produce a significant effect on either frequency or standard parameters of detrusor instability. A double-blind overall assessment of drug efficacy and tolerance significantly favoured prazosin therapy. A total of 30 patients receiving prazosin and 28 receiving placebo reported varied adverse effects. Eighteen patients were excluded from the final analysis, 10 being withdrawn because of adverse effects, 7 on treatment with prazosin and 3 in the placebo group. In long-term usage oral prazosin was well tolerated and appeared to improve obstructed voiding in patients with benign prostatic hyperplasia.


Prostate specific antigen and bone scan correlation in the staging and monitoring of patients with prostatic cancer. The ability of serum prostate specific antigen (PSA) to predict bone metastases at initial presentation was determined in 146 patients, and in 66 patients during a 3-year period; 14.7% of patients with bone metastases at presentation had a PSA value less than 20 ng/ml. All patients who subsequently developed bone metastases had a PSA greater than 20 ng/ml and the rise in PSA often antedated the detection of bone metastases. Bone scans are still necessary in the initial staging but following diagnosis and treatment can be replaced by serum PSA measurement in monitoring patients with prostatic cancer.


Levels of prostate specific antigen that predict skeletal spread in prostate cancer. The ability of serum prostate specific antigen (PSA) and serum acid phosphatase (SAP) to identify skeletal spread was evaluated in untreated patients with prostatic cancer. Twenty patients with scintigraphic evidence of metastatic disease in bone (M1) at diagnosis were compared with 50 untreated patients in whom scans were repeatedly negative during long-term surveillance. Using the present laboratory upper limit of normal (ULN) of 3 iu/l, the sensitivity and specificity of SAP for M1 disease were 80 and 86% respectively. Stepwise discriminant analysis demonstrated that SAP was able to stage patients correctly (bone scan positive or negative) with 81% predictive accuracy at an optimum cut-off limit of 4.6 iu/l. By contrast, whilst PSA (Hybritech) was 100% sensitive for skeletal disease at 10 ng/ml--at the expense of poor (36%) specificity--analysis determined that an optimum cut-off limit of 58 ng/ml led to 79% predictive accuracy for disease in bone. It was concluded that PSA levels > 58 ng/ml are highly indicative of spread to the skeleton, even in the absence of radiological or scintigraphic evidence of metastases.


Cytokines in tumour therapy.

Cytokines in tumour therapy. Cytokines are low molecular weight proteins released by cells of the immune system that have therapeutic potential in cancer. They include the interleukins, the interferons, tumour necrosis factor and the colony-stimulating factors. Cytokines are capable of producing significant and sustained responses against a number of tumours. Clinically, the highest response rates to cytokine immunotherapy have been seen in melanoma and renal cell cancer. Current efforts aim to reduce treatment-related toxicity while maintaining the efficacy of cytokines. The therapeutic potential of these agents may be increased with genetic manipulation by introducing genes encoding cytokines into tumour-infiltrating lymphocytes and certain tumour cells. However, immunotherapy remains time consuming and expensive, and further developments are necessary before it can have a definitive role in tumour management.


The quantitative contribution of nitric oxide and sensory nerves to bradykinin-induced inflammation in rat skin microvasculature. Using a blister model in the rat hind footpad, the present study undertook to examine the relative contribution of sensory nerves and nitric oxide (NO) to the inflammatory response induced by bradykinin (BK). Using this model, combined with laser Doppler flowmetry, we were able to simultaneously monitor two parameters of the inflammatory response, namely vasodilatation (VD) and plasma extravasation (PE). Perfusion of BK (1, 10 or 100 microM) over the blister base elicited both VD and PE responses which were dose-dependent. The VD response was of rapid onset, sustained at the lowest concentration (1 microM), and showed tachyphylaxis at the highest two concentrations (10 and 100 microM). The PE response, however, was delayed in onset at the lower concentration but the response was maintained at all concentrations. The endothelium-independent vasodilator, sodium nitroprusside. (SNP, 100 microM), was used as an internal control and elicited a rapid maintained VD response. In rats pretreated as neonates with capsaicin to destroy primary sensory afferents, the inflammatory response to 10 microM BK was significantly smaller (50% and 64% decrease in VD and PE, respectively). The selective inhibitor of NO synthase, NG-nitro-L-arginine (L-NORAG) at 100 microM significantly attenuated the inflammatory response to BK in control rats (76% and 60% decrease in VD and PE, respectively) with a further decrease in the response in capsaicin pretreated rats. The inactive stereoisomer NG-nitro-D-arginine (D-NORAG) (100 microM) did not affect the inflammatory response to BK. The vasodilator response to SNP was intact in capsaicin pretreated rats and was not affected by either L-NORAG or D-NORAG.(ABSTRACT TRUNCATED AT 250 WORDS)


Expression of the Purkinje cell specific zebrin antigens in the cerebellum of the meander tail mutant mouse. The cerebellum of the meander tail mutant mouse is characterized by normal cytoarchitecture in the posterior lobe and agranular, abnormal cytoarchitecture in the anterior lobe. The Purkinje cells form a monolayer in the posterior lobe but are dispersed throughout the cortex of the anterior lobe. Examination of these cells with the zebrin antibodies demonstrates that in spite of the morphologic and laminar disorganization of these cells in the anterior lobe, they are organized into the appropriate number of correctly positioned immunopositive zebrin clusters.


Anticonvulsive effects of galanin administered into the central nervous system upon the picrotoxin-kindled seizure syndrome in rats. Galanin (2000, 1000 ng) administered into the lateral brain ventricle decreased the severity of picrotoxin-kindled convulsions in rats. The bilateral injection (200, 100 and 50 ng) of galanin into the hippocampus also evoked an anticonvulsive effect. When administered into the caudate nuclei or substantia nigra reticulata, galanin exerted anticonvulsive action only in a high dose (200 ng), whereas in the nucleus accumbens it did so in a low dose (50 ng). When administered into the ventral tegmental area in a dose of 50, 100 or 200 ng galanin failed to reduce the manifestations of picrotoxin-kindled seizures.


Mechanism of the galanin induced increase in acetylcholine release in vivo from striata of freely moving rats. Galanin (GAL) administered intracerebroventricularly (i.c.v.) induced a strong and long-lasting increase in the basal acetylcholine (ACh) release from striata of freely moving rats only when the excitatory corticostriatal input was removed, while its effect was transient in striata of sham-operated rats. This effect was dose-dependent (0.78, 1.56 and 3.12 nmol) and was completely prevented by the GAL receptor antagonist, galantide. GAL injected locally (3.12 nmol) in deafferented striata also induced a persistent increase in ACh release although to a lower extent. The impairment of monoaminergic neurotransmission caused by alpha-methylparatyrosine or p-chlorophenylalanine, respectively inhibitors of catecholamine and serotonin synthesis, completely prevented the rise in ACh output from deafferented striata while the muscarinic antagonist, scopolamine (0.5 mg/kg, s.c.), failed to do it. The data suggest that GAL in the deafferented striatum facilitates basal ACh release through an indirect mechanism. The effect seems to be at least partly mediated by an action of GAL on specific receptors in the striata.


Galanin-like immunoreactivity in autonomic regions of the rat lumbosacral spinal cord is sexually dimorphic and varies with the estrous cycle. These investigations show that there is a heterogeneous distribution of galanin-like immunoreactivity (GAL-LI) within laminae VII and X of the rat thoraco-sacral spinal cord. In either sex, GAL-LI fibers sparsely outline the position of male and female preganglionic sympathetic neurons in thoracic spinal segments; whereas in lumbosacral segments, far greater numbers of GAL-LI fibers surround autonomic preganglionic neurons. An unusual feature of the GAL-LI fibers in lumbosacral autonomic regions is their sexually dimorphic distribution with males containing greater numbers of GAL-LI fibers than all females examined. In this regard, although the number of GAL-LI fibers observed in males was consistent from animal to animal, the amount of GAL-LI in females fell into two qualitative categories: an 'average' and a 'heavy' amount. These data indicate that the difference in the amount of GAL-LI in the female rat lumbosacral spinal cord is related to the estrous cycle, such that heavy amounts of GAL-LI are observed during proestrus and estrus, while average amounts of GAL-LI are associated with metestrus and diestrus.


Physiological and pharmacological evidence for histamine as a neurotransmitter in the olfactory CNS of the spiny lobster. Perfusing histamine (HA, 0.1 microM-1 mM) into the brain of the spiny lobster reversibly altered the spontaneous activity in 24 (86%) of 28 morphologically unidentified, odor-responsive interneurons. The effects of HA were dose-dependent and could be selectively and reversibly antagonized by cimetidine, a vertebrate H2 antagonist, suggesting that the action of HA in the central nervous system (CNS) was mediated by a receptor pharmacologically similar to an HA receptor expressed by lobster olfactory receptor cells. Perfusing HA into the brain also reversibly altered the spontaneous and/or odor-evoked activity of 6 (67%) of 9 morphologically identified, odor responsive interneurons that arborized in the olfactory lobe (OL). These results extend previous evidence from our lab that the OL contains HA-immunoreactive interneurons and that OL tissue can synthesize HA from its precursor and further implicate HA as a putative neurotransmitter in the olfactory CNS of the spiny lobster.


Testosterone regulates substance P within neurons of the medial nucleus of the amygdala, the bed nucleus of the stria terminalis and the medial preoptic area of the male golden hamster. The medial nucleus of the amygdala, bed nucleus of the stria terminalis, and medial preoptic area appear to mediate steroidal regulation of mating behavior in male rodents. The mechanism of action has not been determined. One way testosterone could enhance neuronal function is by increasing neurotransmitter levels, thus altering neuronal transmission. To assess this hypothesis, we examined the effect of castration and testosterone treatment on substance P levels in the neurons of these three brain regions. Brains from male Syrian hamsters that were (1) gonadally intact, (2) castrated for 13 weeks, or (3) castrated for 9 weeks and treated with testosterone for 4 weeks, were processed for substance P, and the numbers of substance P immunoreactive neurons in the medial nucleus of the amygdala, bed nucleus of the stria terminalis, and medial preoptic area were determined. Castration reduced the number of substance P neurons in the bed nucleus of the stria terminalis and medial preoptic area relative to those in intact hamsters; the number of substance P neurons in these regions was restored by testosterone treatment. Castration did not reduce the number of substance P neurons in the medial nucleus of the amygdala; however, testosterone treatment increased the numbers of these neurons when compared to intacts. Thus, testosterone regulates substance P levels in areas that regulate mating behavior. As substance P enhances male copulatory behavior our results suggest that testosterone may regulate copulatory behavior by enhancing substance P levels in medial nucleus of the amygdala, bed nucleus of the stria terminalis and medial preoptic area.


WGA-HRP and choleragenoid-HRP as anterogradely transported tracers in vagal visceral afferents and binding of WGA and choleragenoid to nodose ganglion neurons in rodents. The axonal and terminal labelling pattern in the brain stem resulting from the injection of horseradish peroxidase (HRP) conjugate of wheat germ agglutinin (WGA) or choleragenoid into the nodose ganglion of guinea pigs was examined. In addition, the binding profiles of WGA and choleragenoid in the nodose ganglion of guinea pig and rat were examined. The results show that WGA-HRP and choleragenoid-HRP (B-HRP) produce almost identical distribution of axonal and terminal labelling, the difference being some contralateral fibre labelling present only with B-HRP. However, WGA-HRP shows the strongest labelling at short survival times, whereas B-HRP requires longer postoperative survival times to reach maximum labelling intensity. All nodose ganglion neurons appear to bind WGA as well as choleragenoid although to a varying degree. The results of this and previous studies support the view that visceral sensory ganglion cells and the large light subpopulation of somatic dorsal root ganglion cells both bind choleragenoid, whereas the small dark somatic cells show affinity for WGA but rarely for choleragenoid.


Species diversity of neuronectin and cytotactin expression patterns in the vertebrate central nervous system. Two extracellular matrix proteins of brain tissue, neuronectin (NEC1) and cytotactin (CT), are disulfide-bonded multimers of M(r) 180,000-250,000 subunits. The previously known distribution of these molecules is, however, very different. Human NEC1 is found throughout the white matter of rostral segments of the adult central nervous system (CNS) but not in rostral gray matter or in caudal CNS segments, including the cerebellum. In contrast, CT is absent or expressed at a low level in the adult chicken cerebrum but highly expressed in the cerebellum. Despite these differences in distribution, results obtained with antibodies that recognize NEC1 and CT in several vertebrate species indicate that these molecules are identical or at least closely related: (1) alpha NEC1 antibodies recognize proteins affinity-purified with CT-binding proteoglycan; (2) proteins recognized by alpha NEC1 and alpha CT antibodies in cells constitutively expressing the molecules, cells in which expression is induced by growth factors and phorbol ester and cells treated with tunicamycin (to block glycosylation) are identical in subunit composition and mobility on SDS gels; (3) the removal of NEC1 from culture supernatants by immunoprecipitation removes all molecules reactive with alpha CT antibodies and vice versa; (4) immunoblots of brain extracts with alpha NEC1 and alpha CT antibodies yield identical results. Having demonstrated the structural similarity between NEC1 and CT, we reexamined their distribution in the CNS. Surprisingly, the temporal and spatial distribution pattern of NEC1/CT varied greatly among species. Immunohistochemical and immunoblot experiments with adult human CNS tissues revealed significant levels of NEC1/CT in rostral but not caudal segments. In contrast, in cows and pigs the molecule is found throughout the CNS. Adult rat and mouse brains show regionally restricted expression of NEC1/CT in several areas of the cerebrum--distinct from those showing NEC1/CT in the human--and in the molecular layer of the cerebellum. Tests with fetal and newborn tissues revealed that CNS development in humans, cows and pigs is not accompanied by the marked decline in NEC1/CT levels or the changes in subunit composition found in the chicken CNS. The marked species diversity in temporospatial expression patterns suggests that intrinsic and/or extrinsic elements controlling the expression of NEC1/CT have diverged during vertebrate evolution.


Photoperiodic regulation of substance P immunoreactivity in the mating behavior pathway of the male golden hamster. Mating behavior in the male golden hamster is regulated by both gonadal steroids and photoperiod. Gonadal steroids may regulate mating behavior by actions on the medial nucleus of the amygdala, bed nucleus of the stria terminalis, and medial preoptic area. Neurons in these areas actively accumulate gonadal steroids and lesions of these nuclei disrupt mating behavior in male hamsters. Photoperiodic regulation of mating behavior is regulated, at least in part, by decreased responsiveness to gonadal steroids. Therefore, we sought to determine if the changes induced by changes in gonadal steroids would mimic those induced by changes in photoperiod. The number of substance P-containing neurons in these areas decrease following castration and are restored with testosterone treatment suggesting that this peptide may mediate steroidal regulation of male mating behavior. To determine the effect of photoperiod on substance P, peptide containing neurons were counted in (1) enucleates (n = 6), (2) enucleated castrates treated with testosterone (n = 6), (3) castrates treated with testosterone (n = 4), and (4) intact controls (n = 6). Bilateral enucleation caused a decrease in the number of substance P neurons in the medial nucleus, bed nucleus of the stria terminalis, and medial preoptic area (P less than 0.05). Testosterone treatment prevented this decrease (P less than 0.05). Thus, a decrease in daylength causes a decrease in substance P in the medial nucleus of the amygdala, the medial bed nucleus of the stria terminalis and the medial preoptic area that is mediated by changes in testosterone levels.


Prenatal ontogeny of substance P-like immunoreactivity in the parabrachial nucleus of the human fetus--an immunocytochemical study. Using an immunocytochemical method, the prenatal ontogeny of substance P-like immunoreactivity (SP-LI) was demonstrated in the parabrachial nucleus (PB) of human fetus, fetal age (menstruation age) 11.5 to 40 weeks. The time of initial appearance of SP-LI in the human brainstem PB was between fetal age 11.5 weeks and 16 weeks. While the fetus grew, the density of SP-LI fibers and terminals in the human fetus brainstem PB increased constantly from fetal age 16 weeks to 40 weeks. These findings indicate that substance P (SP) might play an important role in the human parabrachial nucleus development and in its functional establishment during the prenatal period.


Effects of benzylpiperazine derivatives on the neurotoxicity of 3,4-methylenedioxymethamphetamine in rat brain. The neurotoxicity of 3,4-methylenedioxymethamphetamine (MDMA) in rat brain was attenuated significantly by coadministration of several benzylpiperazines (p-nitrobenzylpiperazine, p-chlorobenzylpiperazine and 1-piperonylpiperazine), which were weak inhibitors for [3H]6-nitroquipazine binding to the 5-hydroxytryptamine (5-HT) transporter in rat brain. These results suggest that these benzylpiperazines may inhibit the MDMA-induced neurotoxicity by a novel neuropharmacological effect other than 5-HT uptake inhibition.


Trigeminal and dorsal column nuclei projections to the anterior pretectal nucleus in the rat. The projections of the trigeminal (V) sensory nuclei (VSN) and the dorsal column nuclei (DCN) to the anterior pretectal nucleus (APT) of the rat were investigated by the use of anterograde and retrograde transport of wheat-germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). Injections of WGA-HRP into the APT retrogradely labeled neurons in the contralateral VSN and DCN. The labeled neurons in the VSN were most concentrated in the rostral V subnucleus interpolaris (Vi), but were also found in caudal V subnucleus oralis (Vo). No labeled neurons were seen in V subnucleus caudalis. In the DCN, retrogradely labeled neurons were observed in rostral portions of both the cuneate (Cu) and gracile (Gr) nuclei. Injections of WGA-HRP into the rostral Vi or caudal Vo resulted in dense anterograde terminal labeling in the ventral two-thirds of the APT; the labeling was maximal in the ventromedial part of the caudal half of the APT and did not extend into its most rostral portion. Labeling resulting from injections of tracer into Cu or Gr was located primarily in the ventral half of the APT, was maximal in the mid-levels of the nucleus and extended into its rostral portions. These results indicate the existence of prominent somatosensory projections to the APT and are consistent with recent findings suggesting a role for the APT in sensorimotor integration.


Reduction of blood PO2 decrease and PCO2 increase during asphyxia by paramedian reticular nucleus in cats. Effects of activation of paramedian reticular nucleus (PRN) on the systemic arterial blood pressure (SAP), heart rate, renal nerve activity (RNA), and changes of the partial pressure of the arterial blood oxygen (PO2) and carbon dioxide (PCO2) during asphyxia were studied in cats anesthetized with chloralose (40 mg/kg) and urethane (400 mg/kg). During a 35-s period of asphyxial anoxia, SAP and RNA increased while heart rate decreased significantly. The arterial blood PO2 decreased by 64.6 +/- 4.7% while the PCO2 increased by 54.6 +/- 6.3%. Electrical stimulation of PRN produced a mild to moderate decrease of the SAP, heart rate, and RNA, but arterial PO2 and PCO2 did not change significantly. When PRN was stimulated simultaneously with asphyxia, increases of SAP and RNA and changes of blood gases subsequent to asphyxia reduced significantly. Arterial PO2 decreased only 54.0 +/- 4.9% while the PCO2 increased 39.4 +/- 10.5% (p < 0.01). Similar effects were observed in the venous blood from inferior vena cava. In addition, when the arteriovenous difference of PO2 and PCO2 was compared, simultaneous PRN stimulation during asphyxia produced a higher PO2 reserve (66.3%) and less PCO2 production (-7%) than without PRN stimulation; PO2 54.2%, PCO2 (-2.9%). The results suggest that PRN is a structure that can exert inhibition over a wide spectrum of body functions; not only autonomic system but probably also metabolism.


A new, simple myelin stain.

A new, simple myelin stain. A new and convenient myelin stain is described. Paraformaldehyde fixed tissue is serially immersed in a nitroblue tetrazolium solution and then in a diaminobenzidine solution. The result is distinct blue staining of myelinated fiber tracts. This technique has advantages over presently used myelin stains.


[Congenital indifference and congenital insensitivity to pain]. Congenital indifference to pain is often mistaken for congenital insensitivity. It is characterized by the occurrence since childhood of lesions, mainly cutaneous and osteoarticular secondary to strictly painless traumas. However, despite the lack of pain, the patient is able to discriminate a painful stimulus. Autopsy shows no abnormality of the nervous system. A dysfunction of the central endomorphinic systems has been suggested. Congenital analgesia is associated with anhidrosis in Swanson's syndrome (in which Lissauer the tractus is absent in the spinal cord) and with dysautonomia in Riley-Days's disease (in which there is a lack of amyelicinic fibres). On account of these data, some authors refuse the autonomy of congenital indifference and classify it in the group of the various autonomic and sensory neuropathies. However it seems justified to acknowledge the congenital analgesia with two varieties: congenital indifference in which there is no sensation of pain but normal sensory pathway and tonic function of endomorphinic system, congenital insensitivity in which the painful stimulus is not transmitted to the central nervous system.


Alpha-fetoprotein synthesis in human hepatocellular carcinoma: correlation with hepatitis B surface antigen expression. We analyzed the pattern of alpha-fetoprotein (AFP) synthesis in 40 consecutive human hepatocarcinomas (HCC) in relation to hepatitis B viral (HBV) infection. In addition, histopathological characteristics of liver parenchyma and the tumor itself were examined. Elevated AFP (> 20 ng/ml) were found in 90% of HCC patients and in none of the controls. In 35% of HCC cases, serum AFP was above 100,000 ng/ml. AFP levels were significantly higher in patients seropositive for hepatitis B surface antigen (HBsAg) compared with their negative counterparts (mean log[AFP]: 4.28 +/- 1.67 vs. 3.28 +/- 1.96, respectively; geometric mean (GM): 19,322.6 ng/ml and 1939.5 ng/ml, respectively; p < 0.05). Furthermore, serum AFP levels were higher in HCC patients with liver cirrhosis than in those without (log[AFP]: 4.43 +/- 1.58 vs. 3.23 +/- 1.98, respectively; p < 0.05). However, the relationship of cirrhosis with AFP was confounded by the high prevalence of HBsAg in cirrhotic HCC patients. There was no correlation of AFP with either liver necrosis (abnormal AFP in 45% of cases; mean log[AFP]: 3.99 +/- 1.91 vs. 3.75 +/- 1.85 for HCC with and without necrosis, respectively; 0.05 < p < 0.68, not significant (NS)), or inflammation (abnormal AFP in 25%; mean log[AFP]: 4.33 +/- 1.62 vs. 3.70 +/- 1.93 for HCC with and without inflammation, respectively; 0.05 < p < 0.39, NS). A vast majority of HCC (75%) were moderately (grade 2-3) or poorly differentiated tumors (grade 3, grade 4, or combined grade 3-4). Serum AFP did not correlate with tumor grade. Immunohistochemical analysis of HBsAg and AFP confirmed the serological findings, and confirmed earlier observations of elevated AFP in HBsAg-positive patients. These results may reflect pathogenic and biological differences between HBsAG-secreting and nonsecreting HCC.


[Total chemical synthesis of natural transfer RNA]. New improvements in the chemical synthesis of oligoribonucleotides are reported and they are applied to the first total chemical synthesis of a natural RNA. This E. coli K12 alanine tRNA contains in its sequence dihydrouridine, ribothymidine and pseudo-uridine. The synthetic tRNA was fully sequenced and showed a 42% aminoacyl acceptance activity. When tRNA was used as a template, reverse transcriptase directed the incorporation of adenine opposite dihydrouridine, ribothymidine and pseudouridine.


Relaxant effects on iloprost in canine cerebral artery. Iloprost caused relaxation of rings of canine cerebral arteries precontracted with prostaglandin F2 alpha or the thromboxane A2 analogue U46619, but it was without effect on arteries precontracted with potassium chloride. Pretreatment with iloprost did not significantly affect the concentration-response curve to any agent. Contractile responses to oxyhemoglobin were completed relaxed by iloprost. In arteries from animals with moderate cerebrovascular spasm, the response to prostaglandin F2 alpha was also reduced by iloprost. The observation that iloprost relaxes the response to oxyhemoglobin to prostaglandin F2 alpha in spastic arteries may be of interest in the management of cerebral vasospasm.


Renorenal reflexes: interaction between efferent and afferent renal nerve activity. In rats, stimulation of renal mechanoreceptors by increasing ureteral pressure results in a contralateral inhibitory renorenal reflex response consisting of increases in ipsilateral afferent renal nerve activity, decreases in contralateral efferent renal nerve activity, and increases in contralateral urine flow rate and urinary sodium excretion. Mean arterial pressure is unchanged. To study possible functional central interaction among the afferent renal nerves and the aortic and carotid sinus nerves, the responses to renal mechanoreceptor stimulation were compared in sinoaortic denervated rats and sham-denervated rats before and after vagotomy. In contrast to sham-denervated rats, there was an increase in mean arterial pressure in response to renal mechanoreceptor stimulation in sinoaortic-denervated rats. However, there were no differences in the renorenal reflex responses among the groups. Thus, our data failed to support a functional central interaction among the renal, carotid sinus, and aortic afferent nerves in the renorenal reflex response to renal mechanoreceptor stimulation. Studies to examine peripheral interaction between efferent and afferent renal nerves showed that marked reduction in efferent renal nerve activity produced by spinal cord section at T6, ganglionic blockade, volume expansion, or stretch of the junction of superior vena cava and right atrium abolished the responses in afferent renal nerve activity and contralateral renal function to renal mechanoreceptor stimulation. Conversely, increases in efferent renal nerve activity caused by thermal cutaneous stimulation increased basal afferent renal nerve activity and its responses to renal mechanoreceptor stimulation. These data suggest a facilitatory role of efferent renal nerves on renal sensory receptors.


The indirect negative inotropic effects of the P1-receptor agonist, L-phenylisopropyladenosine, in guinea-pig isolated cardiac preparations: comparison with cromakalim. The possible mechanisms of the indirect negative inotropic responses to the P1-receptor agonist, L-phenylisopropyladenosine (L-PIA) were evaluated in electrically paced (2 Hz, 5 ms pulse width, voltage 50% above threshold) left atria and papillary muscles of guinea pigs. The responses were compared in naive tissues (direct effects) or after prestimulation with submaximal concentrations of either cAMP-dependent positive inotropes (isoprenaline or forskolin) or the cAMP-independent inotrope Bay K 8644. Cumulative concentration-response curves were obtained in naive or prestimulated preparations for L-PIA or the potassium channel activator, cromakalim, for comparison. L-PIA and cromakalim exerted negative inotropy in naive atrial tissues, whereas only cromakalim was active in naive papillary muscles. In atria prestimulated with isoprenaline (31 nM) or forskolin (1.4 microM), the negative inotropy of L-PIA was enhanced compared with naive tissues. In contrast, prestimulation with Bay K 8644 (1 microM) exerted a significant functional antagonism of the response to L-PIA. In the case of cromakalim, prestimulation with isoprenaline exerted a functional antagonistic effect. In papillary muscles, an indirect negative inotropic effect of L-PIA was only seen in tissues prestimulated with the cAMP-dependent inotropes isoprenaline (31 nM) or forskolin (2.4 microM), and not in naive tissues or those prestimulated by Bay K 8644 (333 nM). As with atria, prestimulation with isoprenaline exerted a functional antagonistic effect on the response to cromakalim. These results suggest that the P1-receptor agonist, L-PIA, exerts its indirect negative inotropic effects in left atria by two mechanisms.2+ with cAMP-dependent positive inotropes.(ABSTRACT TRUNCATED AT 250 WORDS)


Neurological findings in HIV-infected children: a review of 49 cases. Many HIV-infected children have neurological involvement. We present our observations in 49 cases, 58% of which had some form of clinical neurological impairment. Most of the patients affected (71%) presented with progressive encephalopathy, characterized by developmental delay with loss of acquisitions and cognitive decline, an impaired growth curve, microcephaly and corticospinal dysfunction. CT-scan imaging shows cerebral atrophy in all cases and basal ganglia calcifications in 29%. Non-specific abnormalities are found on the EEG in two-thirds of cases and in the CSF in slightly less than half the cases. Pathological studies sometime revealed HIV encephalitis or lateral corticospinal tracts degeneration. Neurological impairment secondary to vascular events, neoplasms or opportunistic infections were rare, especially when compared with the adult HIV population.


Immunophenotyping of subtypes of B-chronic (mature) lymphoid leukemia. A study of 242 cases. The French-American-British group's proposal for the classification of chronic lymphoid leukemias is unique at this time. Testing, expanding, and adding to the theory by immunophenotyping will help to additionally characterize this group of diseases. Peripheral blood samples from 242 patients with chronic lymphoid leukemias were analyzed for immunologic evaluation of the following subtypes: typical chronic lymphocytic leukemia (CLL), 189; CLL with pleomorphic lymphocytes (CLL-pleo), 19; CLL of mixed cell type (CLL/PL), 20; prolymphocytic leukemia (PLL), 22; hairy cell leukemia (HCL), 10; HCL-variant, 1; and splenic lymphoma with villous lymphocytes, 1. The phenotype of CLL and CLL-pleo was weak surface immunoglobulin (SIg) with positive results of mouse rosettes (MR+), CD5+, and CD22-. Of PLL and HCL, it was strong SIg, MR-, CD5-, and CD22+. By analyzing the four markers and accepting the relevant results of two or more as sufficient for diagnosis, all cases (100%) of CLL, CLL-pleo, PLL, and HCL were diagnosed. CLL/PL showed the phenotype of CLL in 66.67% and of PLL in 33.33% of patients. The frequency of cases with weak fluorescence in decreasing order was CLL, CLL-pleo, CLL/PL, and PLL and HCL. The same sequence applied to the mean percentage of mouse rosette-forming cells and CD5 cells, but the sequence was reversed for CD22 cells. SIg intensity, MR, CD5, and CD22 constitute the minimum number of immune markers for the differential diagnosis of the subtypes of chronic lymphoid leukemia. The frequency of the four markers among the subtypes suggested that CLL and CLL-pleo have identical phenotypes and that the five subtypes follow a continuous range of B-cell differentiation from early mature (CLL and CLL-pleo) to late mature pre-plasma cell stages (PLL followed by HCL), with CLL/PL of intermediate maturity.


Expression of developmentally relevant proteins by rodent embryo CNS cells in vivo and in vitro: proto-oncogene pp60c-src and high molecular weight neurofilament protein. The expression of proteins that play a role in neuronal differentiation was examined in central nervous system (CNS) micromass embryo cell cultures and compared to expression at comparable developmental stages in vivo. The protein product of the src proto-oncogene (pp60c-src) has been postulated to have a specific role in development because, although it is expressed in many tissues, marked increases in amount and activity of pp60c-src occur in neurons at the time of differentiation. Another protein of interest, high molecular weight neurofilament (NF) protein, is found in differentiated neurons. In the present study, changes over time in the expression of these two proteins in vitro and in vivo were examined. In the micromass cell cultures, primary cells from day 12 rat embryo CNS are plated at high density and differentiate into neurons during five days in culture. Tissues from embryos grown in vivo were assessed at 12 and 17 days post-coitum. Proteins were quantified by PAGE separation of equal amounts of total protein followed by transfer to membranes, immunoblotting, and densitometric scanning of blots. Increases in the amount of both proteins with neuronal differentiation was shown. Protein kinase activity of immunoprecipitated pp60c-src also increased in cell cultures and in embryos. Similarity in patterns of expression between in vitro and in vivo tissue samples provides further evidence that the cultures closely simulate in vivo differentiation and are a useful system for examining expression of developmental genes in vitro.


Comparison of psychosocial adaptation and sexual function of survivors of advanced Hodgkin disease treated by MOPP, ABVD, or MOPP alternating with ABVD. Survivors of advanced Hodgkin disease, who were assigned randomly to treatment by mechlorethamine, vincristine, procarbazine, and prednisone (MOPP); doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD); or MOPP alternating with ABVD in a clinical trial of the Cancer and Leukemia Group B (protocol 8251), were compared in terms of their psychosocial adaptation and psychosexual function an average of 2.2 years after completion of treatment (range, 1-5 years). The study was undertaken to determine if there were differences among treatments in these functional areas as a consequence of differential long-term gonadal damage in the three regimens. Ninety-three disease-free survivors of advanced Hodgkin disease (56 men and 37 women) were studied (a minimum of 1 year after completion of treatment) by an interview conducted over the telephone. Standardized measures were used to assess their psychologic, sexual, family, and vocational functioning, including the following tests: the Psychosocial Adjustment to Illness Scale--Self Report, the Brief Symptom Inventory, the Profile of Mood States, and the Impact of Event Scale. Contrary to expectation, no statistically significant differences in survivors' psychosocial adaptation or psychosexual function were found by treatment arm. Infertility (based on survivors' reports of medical test results and perceptions) and lower income 1 year before the diagnosis of cancer were significant predictors of poorer adjustment. Most survivors reported a range of problems that they attributed to having had cancer: 35%, proven or perceived infertility; 24%, sexual problems; 31%, health and life insurance problems; 26%, a negative socioeconomic effect; and 51%, conditioned nausea, associated with visual or olfactory reminders of chemotherapy. No significant long-term advantage in psychosocial adaptation or psychosexual function was found for survivors of Hodgkin disease treated by the less gonadally toxic ABVD regimen 1 to 5 years after completion of treatment.


Specific expression of the ras-related rab3A gene in human normal and malignant neuroendocrine cells. The authors originally demonstrated the tissue-specific expression of the rab3A gene in the mouse brain. In the current study, they analyze the activity of this gene in fresh human tumors associated with neuronal phenotype compared with normal and malignant cells from other origins. The authors studied the transcription levels of the rab3A gene by Northern blot in 81 fresh tumors. A high rab3A gene expression was observed in tumor samples derived from the neural tube (i.e., neuroblastomas, ganglioneuroblastomas, and adult nervous system neoplasms). In addition, this tissue-specific expression extended to neuroendocrine tumors of the gut, small cell lung cancers, and pheochromocytomas. These results suggest a specific restriction pattern to human cells derived from the neural tube and neural crests. The GTP/GDP-binding rab3A protein may be a useful differentiation marker of neuro-endocrine cells in the characterization of undifferentiated neoplasms.


Treatment of childhood germ cell tumors. Review of the St. Jude experience from 1979 to 1988. The outlook for patients with germ cell tumors was poor before the advent of effective chemotherapy. The authors assessed the outcome of treatment with multiagent chemotherapy (with or without radiation therapy) in children treated for germ cell tumors at St. Jude Children's Research Hospital (SJCRH). Sixty children with germ cell tumors were treated between January 1979 and June 1988. Postsurgical treatment was based on tumor site, stage, and histology. Most patients received chemotherapy with vincristine, actinomycin-D, and cyclophosphamide (VAC), or a modified Einhorn regimen (cisplatin, bleomycin, and vinblastine [PVB]); in the absence of response to initial therapy, patients received alternating courses of VAC and PVB (VAC/PVB regimen). Exceptions were patients with Stage I testicular tumors (observation only) and ovarian germinomas (Stage I tumors measuring less than 10 cm, observation only; tumors larger than 10 cm or Stage II-III disease, radiation only; and Stage IV disease, VAC plus radiation). The estimated 5-year survival is 100% for patients with Stage I disease (n = 18), 87% for patients with Stage II (n = 8), 72% for Stage III (n = 25), and 56% for Stage IV (n = 9). Patients with testicular tumors of any stage or with Stage I-II ovarian tumors had 100% 5-year survival. Extragonadal tumors responded poorly to VAC alone with recurrent or progressive disease in eight of nine patients. Treatment for those tumors was changed to alternating courses of VAC and PVB, which failed in only one of seven patients. Nine of 19 patients with advanced ovarian tumors had disease recurrence with VAC; these patients then received PVB, which was effective in four cases. For patients with advanced germ cell cancers, intensification of therapy or the development of new approaches is necessary. In contrast, future trials in children with limited stage should focus on reducing acute and long-term toxicities.


Acute arterial thrombosis after escalated-dose methotrexate, vinblastine, doxorubicin, and cisplatin chemotherapy with recombinant granulocyte colony-stimulating factor. A possible new recombinant granulocyte colony-stimulating factor toxicity. Combination chemotherapy with a regimen of methotrexate, vinblastine, doxorubicin, and cisplatin (M-VAC) has produced long-term survival times in a significant proportion of patients with advanced nodal and metastatic urothelial tumors. The availability of the colony-stimulating factors has spurred interest in studies evaluating escalated dosing schedules of M-VAC in an attempt to improve major response rates and overall survival. More frequent use of the colony-stimulating factors, however, in this and other settings may be associated with unrecognized side effects. The authors report a case of arterial thrombosis in a 69-year-old man receiving recombinant granulocyte colony-stimulating factor (rhG-CSF) and escalated-dose M-VAC for treatment of a transitional cell carcinoma of the bladder. Incidents of venous thrombosis have been reported previously with the use of colony-stimulating factors, but, to the knowledge of the authors, this case represents the first report of an arterial thrombosis occurring in a patient receiving rhG-CSF.


Estradiol- and estriol-binding in human benign prostatic hyperplasia. Binding of the natural estrogens, estradiol and estriol, was investigated, in 34 samples of human benign prostatic hypertrophy (BPH) tissue, using Scatchard analysis and agar gel electrophoresis. Saturation binding analysis using a wide range of concentrations of both ligands resulted in curvilinear Scatchard plots. This confirmed the presence of two binding forms for estradiol: a true estrogen receptor, and a protein with lower affinity and higher capacity. Both binding species were also demonstrated and quantified with estriol. The electrophoretic process, after incubation at low and high ligand concentrations also resulted in separation, for both estrogens, of two binding peaks. They are probably two distinct forms of the low affinity, high capacity binding measured by Scatchard. The procedure used in our laboratory was not able to provide accurate determination of the concentrations of these binding forms. Possible modifications to alleviate these drawbacks are discussed.


Expression of c-kit receptor in normal and transformed human nonlymphoid tissues. The protooncogene c-kit encodes a tyrosine kinase with a molecular weight of 145,000, highly related to the platelet derived growth factor/colony stimulating factor receptors. Mutations of the murine gene result in impairment of hematopoiesis, gametogenesis, and of the melanocyte cell lineage. In order to elucidate c-kit functions in development and oncogenesis we have analyzed immunohistochemically its expression in human normal and transformed nonlymphoid tissues. The receptor has been detected in spermatogonia, melanocytes, and unexpectedly, in astrocytes, renal tubules, parotid cells, thyrocytes, and breast epithelium. While the gene product is expressed in seminoma, lung tumors, and melanoma of low invasiveness, no detectable levels have been detected in thyroid and breast carcinomas, astrocytomas, and invasive melanomas. In breast tumors these findings were confirmed by paired, Northern blot analysis of RNA preparations from normal and transformed tissue. The present results demonstrate that the c-kit receptor plays a role in the development of a larger spectrum of cell lineages. Furthermore, on the basis of the transformation associated changes, we speculate that, while in some cell types, c-kit expression positively regulates mitogenesis and is selected for in neoplastic transformation, in other tissues the c-kit pathway is involved in morphogenesis and differentiation and is, therefore, negatively selected in the course of tumor progression.


Retinoid status and the control of keratin expression and adhesion during the histogenesis of squamous metaplasia of tracheal epithelium. We induced vitamin A depletion to define early and late changes during the histogenesis of squamous metaplasia of hamster tracheal epithelium. An early change is the "minimal morphological change" (MMC), in which the mucociliary epithelium is separated from the basement membrane by a continuous layer of basal cells. Immunohistochemistry showed an exclusive localization of the keratins K5 and K14 in basal cells of normal and MMC epithelia. At the MMC stage no staining was observed above the basal layer with antibodies to K5, but upon progression of the lesion to a squamous focus all cells from basal to terminally differentiated were positive for K5 and K14. In contrast, when we used antibodies to the keratins K6 or K13 all cells were negative in the normal epithelium and in the MMC epithelium. Successive layers of suprabasal squamous cells found in squamous metaplasia failed to express normal epidermal differentiation marker keratins K1 and K10 but expressed the proliferation marker keratin K6 and the internal stratified epithelium keratin K13, not normally found in the epidermis or in the trachea. Hamster tracheal epithelial cells could be maintained in culture in serum-free medium for at least 4 weeks in the presence of retinoic acid (RA). In non-RA-containing medium, cells from vitamin A-deficient hamsters showed markedly reduced growth and an increase in the expression of keratins K5, K6, K13, and K14. Since our previous work had implicated retinoids in the control of cell adhesiveness, we were interested to find out whether changes in cell adhesion occur in vitamin A-deficient hamster tracheal epithelial cells, compared to normal cells. Functional assays demonstrated that hamster tracheal epithelial cells, obtained from non-RA-treated tracheas or maintained in culture, displayed reduced attachment to laminin, compared to RA-treated cells. Immunofluorescence studies did not show a decrease either in the alpha 6 integrin subunit, which was localized in the basal aspect of basal cells, or in basement membrane laminin. However, the expression of laminin-binding protein 37 decreased as the epithelium changed from pseudostratified to stratified. Therefore, a coordinated pattern of changes in keratin gene expression, as well as in the expression of laminin-binding protein 37, the precursor to the cell surface laminin receptor 67LR, and in adhesive properties takes place in tracheal epithelium when its phenotype changes from mucociliary to the preneoplastic stage of squamous metaplasia.


Complementary DNA cloning for galactosyltransferase associated with tumor and determination of antigenic epitopes recognized by specific monoclonal antibodies. The galactosyltransferase associated with tumor (GAT) was the name given to the isoenzyme that tends to polymerize resulting in slower moving in a nondenaturing polyacrylamide gel electrophoresis than normal (beta 1-4)galactosyltransferase (normal GalT). A complementary DNA (cDNA) library was constructed from a human ovarian cancer cell line, RMG-I, which secreted an amount of GAT into the culture supernatant and screened with monoclonal antibodies (MAbs) against GAT and normal GalT. One of six cDNA clones, UG86-1, encoded an epitope recognized by a GAT-specific MAb, 8513. Recombinant proteins expressed by UG86-1 in Escherichia coli also had antigenic epitopes recognized by the other MAbs against normal GalT. The 229-base pair nucleotide sequence encoded by UG86-1 was identical to the stem region sequence of HGT832 which encodes a full-length cDNA of human GalT. Using recombinant proteins directed by deletion mutant cDNAs, the antigenic epitopes recognized by each MAb were determined. The epitope of MAb8628, which reacts to both the GAT and normal GalT, was localized to the COOH-terminal side of proteolytic cleavage site where the membrane-bound form enzyme is cleaved to be converted to soluble forms, while MAb8513 epitope was at the NH2-terminal side from this cleavage site between the COOH-terminal end of the membrane-binding domain and the cleavage site. These results demonstrate that GAT is produced by aberrant proteolytic cleavage at the different site, closer to the membrane-binding domain, from the normal GalT.


Colocalization of basal and luminal cell-type cytokeratins in human prostate cancer. In the epithelium of secretory acini of the prostate two different cell types can be discriminated on the basis of localization, morphology, and degree of differentiation, the luminal and basal cells. The possibility of a developmental relationship between basal and luminal cells has been a subject of interest in several studies. According to the stem cell model at least three cell types, i.e., stem, amplifying, and transit cells, can be discriminated in the epithelium of prostate secretory acini. We previously reported that in the process of degeneration and regeneration in normal rat prostate a population of cells could be identified as candidates for the amplifying cells. These cells showed a keratin expression profile intermediate between those of basal and luminal cells. We now show, by using keratin antibodies, that also in normal human prostate at least three subpopulations of cells can be identified, one of them putatively representing amplifying cells as defined in the stem cell model. Furthermore, these antibodies were used to obtain a better insight into the different cell types involved in the etiology and progression of prostatic carcinoma. Both primary and hormone-independent prostatic tumors were investigated. Our results indicated that the candidate stem cell population was absent in prostatic carcinoma. Unlike earlier reports on the unique presence of cells with luminal characteristics in prostatic carcinoma, we identified also a population of cells coexpressing basal and luminal cell-type cytokeratins in primary and hormone-independent prostatic carcinoma. Since amplifying cells are defined in the stem cell model as precursors of transit (luminal) cells in the hierarchical pathway of prostatic epithelium differentiation, we postulate that on the basis of the keratin expression profile this subpopulation is most likely the target for neoplastic transformation.


Prolonged survival of thymoma-bearing mice after vaccination with a soluble protein antigen entrapped in liposomes: a model study. EG7-OVA cells are mouse thymoma EL4 cells stably transfected with the complementary DNA of chicken ovalbumin (OVA) and thus express OVA epitopes as a unique antigen. Cytotoxic T lymphocytes specific to OVA can be elicited by immunization of mice with OVA osmotically loaded into syngeneic splenocytes or entrapped in liposomes. Cytotoxic T lymphocytes thus induced can specifically cytolyse the EG7-OVA cells in vitro in an antigen-specific and major histocompatibility complex-restricted manner. In the present study, we have examined in this model system whether immunization with liposomal OVA can protect mice against tumors induced by EG7-OVA cells. Vaccination with OVA either entrapped in liposomes or osmotically loaded in the syngeneic splenocytes prolonged the survival of mice which had been challenged with EG7-OVA cells, but not those mice challenged with the parent EL4 cells. The antitumor effect was attributed to the induced OVA-specific cytotoxic T lymphocyte activity, since other forms of acquired immunity such as interaction of tumor cells with specific antibody could not be detected. Our results demonstrate that immunization with antigen incorporated in liposomes could be a useful means of inducing a protective antitumor response.


Identification of a novel CD56- lymphokine-activated killer cell precursor in cancer patients receiving recombinant interleukin 2. Circulating lymphokine-activated killer (LAK) cell activity in cancer patients receiving recombinant interleukin 2 (rIL-2) therapy is confined to cells expressing the CD56- surface marker. However, CD56- cells from these patients but not normal individuals have been reported to exhibit LAK cytotoxicity only following in vitro activation with rIL-2. Studies were performed to document the existence of CD56- LAK precursor cells and to phenotypically characterize this population in patients receiving rIL-2 therapy using fluorescence-activated cell sorter-purified CD56- cell subsets. Initial studies confirmed that CD56- cells exhibit NK activity [20 +/- 7 (SE) LU/10(6) cells] but not LAK activity (0 +/- 0 LU/10(6) cells) when evaluated directly from peripheral blood of patients receiving rIL-2. CD56- cells from patients but not normal individuals developed significant LAK cytolytic activity against NK-resistant COLO 205 targets (16 +/- 3 LU/10(6) cells) when cultured for 3 days with 1500 units/ml rIL-2. The CD56- LAK precursor activity was confined to cells expressing a CD56-CD16+ phenotype and a large granular lymphocyte morphology; little or no NK or LAK precursor activity was detectable in CD56-CD5+ T-cells from patients. Phenotypic characterization of CD16+CD56- cells revealed that this population is uniformly CD11a+,CD18+, and CD38+ and is heterogeneous in its expression of CD11b, CD11c, and CD16/Leu 11c. These results indicate that rIL-2 administration induces enhanced LAK precursor activity in a novel population of CD5-CD16+CD56- cells.


Effects of differentiating agents on cell surface expression of the breast carcinoma-associated DF3-P epitope. The DF3 antigen is a member of a family of high-molecular-weight glycoproteins aberrantly expressed in human breast carcinomas. Recent work has described the generation of a monoclonal antibody (MAb), designated DF3-P, that reacts with immature, underglycosylated precursors of DF3 antigen. Immunoperoxidase staining studies have demonstrated that MAb DF3-P exhibits selective reactivity with malignant mammary epithelium. Using flow cytometry and live cell radioimmunoassays, the present studies demonstrate that the epitope recognized by MAb DF3-P is expressed on the surface of MCF-7 and other human breast carcinoma cell lines. We also demonstrate that treatment of MCF-7 cells with 12-O-tetradecanoylphorbol-13-acetate, an agent known to induce a more differentiated mammary cell phenotype, is associated with increased expression of the DF3-P epitope. Similar findings were obtained with sodium butyrate. The results indicate that these agents increase both cell surface DF3-P antigen density and the percentage of DF3-P-positive cells. Immunofluorescence studies performed on chamber slides further demonstrate that MAb DF3-P-reactive cells are detectable in small clones or clusters. Similar studies with 12-O-tetradecanoylphorbol-13-acetate- and butyrate-treated cells demonstrate increases in the size and number of these clusters. Taken together, these results indicate that the DF3-P epitope is expressed on the surface of human breast carcinoma cell lines and that the heterogeneity of this expression is related to the presence of differentiating signals.


Intracellular localization of human DNA repair enzyme methylguanine-DNA methyltransferase by antibodies and its importance. The human DNA repair enzyme, methylguanine-DNA methyltransferase (MGMT, M(r) 21,000), which protects cells against the mutagenic effect of alkylating carcinogens, was found to be localized in the cell nucleus (except the nucleolus) by immunofluorescence staining using polyclonal and monoclonal antibodies. The supporting experiments came from differential staining of the MGMT-deficient (mer-) and -proficient (mer+) cells, Western blotting analysis, and specific antibody depletion studies with the immobilized fusion protein, GSTMGMT-glutathione-Sepharose. Its localization in the nucleus agrees with its biological function and possibly explains the ineffective protection of mammalian cells (mer-) transfected with the Escherichia coli MGMT genes from bifunctional alkylating agents.


Monoclonal antibodies to the myogenic regulatory protein MyoD1: epitope mapping and diagnostic utility. Monoclonal antibodies (MoAbs) were developed against recombinant wild-type murine MyoD1 protein. Each of 4 MoAbs was immunologically reactive with recombinant MyoD1 protein by enzyme-linked immunosorbent assay, and each specifically stained the nuclei of myogenic cells. Epitopes were mapped using fusion protein constructs with specific deletions of defined regions of the MyoD1 molecule. MoAb 5.2F recognized an epitope in the amino terminal region between amino acid residues (AAR) 3 and 56, whereas epitopes for MoAbs 1.1A, 5.4G, and 5.8A were in the carboxyl terminus (AAR 167-318) of the MyoD1 protein. The epitope for MoAb 5.8A was further delineated to AAR 170-209 by Western analysis and immunoprecipitation of in vivo transcribed and translated MyoD1 protein having specific deletions in the carboxyl terminus. The 5.8A epitope was ultimately localized to the region between AAR 180 and 189 of the protein by enzyme-linked immunosorbent assay using 10-amino acid residue synthetic peptides. This sequence is apparently unique to MyoD1 and has little homology to other myogenic regulatory proteins (myogenin, Myf5, Myf6, and MRF4). Transfection of cDNA for murine MyoD1 into a nonmuscle cell line conferred 5.8A reactivity, confirming the specificity of this reagent. MoAb 5.8A was then used to examine the expression of MyoD1 in normal and malignant human tissues. MyoD1 was not detected in any normal adult tissue but was detected in 25 of 25 histologically confirmed rhabdomyosarcomas. Staining was localized to the nucleus and showed marked heterogeneity between cells as well as differential staining within nuclei. Specific subcellular localization of 5.8A was further determined by immunoelectron microscopy, where antibody was found to localize to electron-dense areas, more frequently associated with the nuclear submembranous region. In addition to rhabdomyosarcomas, MoAb 5.8A stained 2 of 5 Wilms' tumors and one ectomesenchymoma, neoplasms known to contain myogenic elements. The 5.8A reagent was also of value in the accurate histopathological classification of 2 of 4 tumors previously diagnosed as extraosseous Ewing's sarcoma and 2 of 3 tumors diagnosed as undifferentiated sarcomas.


Antitumor effect of interferon plus cyclosporine A following chemotherapy for disseminated melanoma. Interferon (IFN) increases the expression of major histocompatibility (MHC) antigens on the surface of tumor cells. Cyclosporine A (CsA) administration following myeloablative therapy and syngeneic bone marrow transplantation results in the generation of cells with autoreactive and antitumor effects that are related to the expression of MHC antigens. We used these two agents following conventional chemotherapy in a non-bone marrow transplantation setting for a melanoma in a murine model. Treatment with IFN alone or CsA alone was ineffective in controlling the dissemination of melanoma. A combination therapy with both these agents resulted in a significant control in the dissemination of the tumor, prolonged the survival of the tumor-bearing mice over that with chemotherapy alone, and generated cells with potent MHC-unrestricted cytotoxic potential in vitro. Adoptive transfer of these cells to secondary tumor bearers treated with chemotherapy showed potent antitumor effect; in the absence of chemotherapy, these cells had no antitumor effect in the secondary recipients. The antitumor effect of cells generated by IFN plus CsA therapy following chemotherapy could be blocked by normal spleen cells. These data suggest that treatment with IFN plus CsA following nonmyeloablative chemotherapy generates cells with MHC-unrestricted cytotoxicity; this effect may be related to abolition of suppressor influences by the chemotherapy.


A retrovirus in chinook salmon (Oncorhynchus tshawytscha) with plasmacytoid leukemia and evidence for the etiology of the disease. A plasmacytoid leukemia (PL) has caused mortalities in chinook salmon (Oncorhynchus tshawytscha) reared in seawater netpens in western British Columbia, Canada, since 1988. Kidney or eye tissues from 11 of 13 fish from netpens with clinical PL had reverse transcriptase (RT) activity. This RT activity was associated with virus particles of retrovirus morphology and buoyant density. In a transmission experiment, PL-positive donor fish tissues also had RT activity and virus particles of retrovirus morphology and buoyant density, as did recipient fish tissues following development of the disease 6 weeks postinjection with a tissue homogenate from the donor fish. Kidney and spleen tissues from fish that developed PL following injection with an inoculum that was passed through a 0.22-micron filter, in a separate experiment (M. L. Kent and S. C. Dawe. Further evidence for a viral etiology in the plasmacytoid leukemia of chinook salmon Oncorhynchus tshawytscha. Dis. Aquat. Org., in press, 1992), also exhibited RT activity. The virus particles observed by electron-microscopic examination of tissues or sucrose fractions from PL-positive fish were enveloped and were about 110-nm diameter with a central electron-dense core. Polypeptides of about M(r) 120,000, 80,000, 42,000, 27,000, 25,000, 22,000, and 19,000 were observed when purified virus particles were examined by polyacrylamide gel electrophoresis analysis. Many infectious neoplasms of animals, including fishes, are caused by retroviruses. The evidence in this study shows the presence of a retrovirus in chinook salmon with PL and further suggests a retroviral etiology of the disease. We are tentatively calling this virus salmon leukemia virus.


Angiogenesis, microvascular architecture, microhemodynamics, and interstitial fluid pressure during early growth of human adenocarcinoma LS174T in SCID mice. To date, most quantitative information on tumor angiogenesis, microcirculation, and transport has been derived from rodent tumors grown in transparent chamber preparations. In this paper we present a chamber technique adapted to immunodeficient mice for the study of human tumor xenografts. Microcirculatory parameters in severe combined immunodeficient mice bearing a dorsal skin fold chamber preparation were quantified using intravital microscopy and image analysis. The take rate of the human colon adenocarcinoma LS174T in the chamber preparation was 100%, and the tumor area doubling time was 6.5 days. Three days following implantation of 2 x 10(5) tumor cells onto the striated skin muscle, capillary sprouts were noted in the tumor cell mass. Microvasculature in the tumors was established after 10 days. Capillary density, vessel diameter, red blood cell velocity, and blood flow rates in individual microvessels measured on days 10, 14, 18, and 22 showed no statistical difference in the striated muscle (capillaries) and subcutaneous tissue (arterioles and venules) of the skin of tumor-free animals (N = 6), whereas these parameters increased slightly, but not significantly, in the LS174T tumors (N = 7). Mean interstitial fluid pressure (+/- SD) in these small tumors was 4.6 +/- 1.7 mmHg (N = 4) on day 10 and 5.1 +/- 0.9 mmHg (N = 4) on day 22 and significantly elevated compared to that in the subcutaneous and skin tissue (-0.9 +/- 0.8 mmHg) (N = 4) (P < 0.001). To our knowledge, this is the first model enabling intravital microscopic studies of human tumor xenografts in a transparent chamber preparation in severe combined immunodeficient mice. Studies on angiogenesis, microcirculation, and transport using such a preparation should provide new insights into microcirculation-mediated mechanisms for cancer treatment.


Ex vivo differentiation therapy as a method of leukemic cell purging in murine bone marrow expansion cultures. We investigated the use of differentiation therapy as a method of purging bone marrow (BM) of leukemic cells in ex vivo murine BM expansion cultures (delta-cultures). In clonal cultures and in suspension cultures a combination of the differentiation-inducing agents granulocyte colony-stimulating factor (G-CSF), interleukin (IL)-6, and all-trans-retinoic acid (ATRA) was found to be most effective in inducing the differentiation of the murine myelomonocytic leukemic cell line WEHI 3B D+ LacZ clone 2.8 (clone 2.8). Furthermore, we investigated the activity of a mutant form of IL-6, mutein, and found it to have a greater specific activity in cell proliferation assays and in a clone 2.8 differentiation assay than the native form of IL-6. Coculture of clone 2.8 and BM in IL-1 and kit-ligand-stimulated delta-cultures showed that the added stimuli, G-CSF, mutein, and ATRA, decreased the expansion of leukemic cells. Mice transplanted with G-CSF, mutein, and ATRA-purged BM had an increased survival time relative to nonpurged controls. The addition of G-CSF, mutein, and ATRA to delta-cultures did not result in any impairment of hematopoietic stem cells when measured 5 wk after transplantation.


Loss of keratin expression in anaplastic carcinoma cells due to posttranscriptional down-regulation acting in trans. Rat keratin K5 and vimentin complementary DNAs have been isolated, identified, and used to study keratin and vimentin expression as markers for cell differentiation. Isologous rat neoplastic epithelial cell lines used were based on a clonal benign epithelial line (A5P/B10) and a clonal anaplastic malignant derivative line (T952/F7). Stable cytoplasmic mRNA was detected for keratin but not vimentin in the benign cells. The anaplastic derivative cells expressed vimentin but showed a 1000-fold reduction in the keratin message, which nuclear run-on assays identified as being due to posttranscriptional down-regulation. An identical pattern of posttranscriptional down-regulation was found in independent malignant somatic cell hybrids of the benign and anaplastic cells. trans-acting regulatory mechanisms implicated in posttranscriptional (pretranslational) keratin down-regulation in these anaplastic malignant cells may play a role in the apparent loss of differentiation evident in tumor progression.


Human glucocorticoid receptor gene deletion following exposure to cancer chemotherapeutic drugs and chemical mutagens. The sensitivity of the human glucocorticoid receptor (hGR) gene to mutagenesis by the cancer chemotherapeutic drugs adriamycin, bleomycin, and chlorambucil was evaluated using glucocorticoid-sensitive (dexs) subclones of the human leukemic cell line CEM-C7. Treatment of cells with either bleomycin or chlorambucil increased the frequency of glucocorticoid-resistant (dexr) clones 3.3- and 10-fold, respectively. Measurement of steroid-binding activity in intact dexr cells demonstrated that the predominant phenotype of drug-induced dexr clones was receptorless (r-). dexs CEM cells express only one functional hGR allele and, in addition, are heterozygous for a BclI restriction fragment length polymorphism in the hGR gene (L. A. Palmer and J. M. Harmon, Cancer Res., 51:5224-5231, 1991). To determine the basis of the r- phenotype, BclI digests of genomic DNA isolated from r+ and r- cell lines were examined for the presence of the polymorphic 2.4- and 4.4-kilobase digestion products. A deletion of all or part of the hGR gene was demonstrated by the absence of the 4.4-kilobase fragment in one of two bleomycin-induced dexr clones, as well as the ICR191-induced dexr cell line ICR27TK.3. Cytogenetic analysis of ICR27TK.3 showed that the distal portion of the long arm of one chromosome 5 had been replaced with a portion of chromosome 15. Thus, in at least two dexr cell lines, deletions and/or chromosome breaks in the hGR locus appear to account for the r- phenotype.


Antiangiogenic agents potentiate cytotoxic cancer therapies against primary and metastatic disease. The formation of a blood supply (angiogenesis) is critical to the growth of solid tumors. The naturally occurring steroid tetrahydrocortisol, the synthetic cyclodextrin derivative beta-cyclodextrin tetradecasulfate, and the tetracycline derivative minocycline have antiangiogenic activity. Tetrahydrocortisol and beta-cyclodextrin tetradecasulfate in a 1:1 molar ratio by continuous infusion over 14 days and minocycline administered i.p. over 14 days from day 4 to day 18 postimplantation of the Lewis lung carcinoma significantly increased the growth delay of the primary tumor after treatment with cis-diamminedichloroplatinum(II), melphalan, cyclophosphamide, Adriamycin, bleomycin, and radiation therapy administered in standard regimens. Addition of the antiangiogenic agents to treatment with the cytotoxic therapies not only reduced the number of lung metastases formed from the primary tumor but also reduced the number of large metastases. Five of 12 animals treated with the antiangiogenic modulators and cyclophosphamide were long-term survivors (> 120 days). Thus, antiangiogenic therapies can potentiate the efficacy of standard anticancer therapies.


Therapeutic effects of arotinolol, a beta-adrenergic blocker, on tremor in MPTP-induced parkinsonian monkeys. The effect of arotinolol, a peripherally acting beta-adrenergic-blocking agent, on postural or kinetic tremor was studied in monkeys with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism. Male cynomolgus monkeys (Macaca fascicularis) were treated with three injections of MPTP hydrochloride (0.3 mg/kg, i.v.) at an interval of 3-4 days, followed by several injections of the same dose every 7 days. Four monkeys with persistent parkinsonian symptoms manifested for greater than 1 year were used. The animals developed mild to moderate degrees of postural or kinetic tremor, and their motor activity was reduced. Arotinolol (20-30 mg/kg, s.c.) significantly suppressed postural tremor in a dose-dependent manner. Propranolol (20-30 mg/kg) was also effective in suppressing the tremor. However, the application of propranolol induced emesis, whereas arotinolol had no adverse effects. These results suggest that arotinolol is a useful adjunct to dopaminergic therapy for tremor in Parkinson's disease.


Structure of the polysaccharide O-antigen of Salmonella riogrande O:40 (group R) related to blood group A activity. The structure of the Salmonella O:40 (Group R) antigen was determined from an analysis of the antigenic O-polysaccharide component of the lipopolysaccharide produced by Salmonella riogrande O:40. Using 1H- and 13C-NMR spectroscopy, methylation analysis, and periodate degradation methods, the O-polysaccharide was found to be a high molecular weight branched polymer of repeating pentasaccharide units having the structure: [formula: see text] The reported human blood group A activity was concluded to reside in an epitope of a terminal trisaccharide portion of the O-chain involving alpha-D-GalpNAc and beta-D-GlcpNAc residues linked (1----3) and (1----2), respectively, to beta-D-Manp branched residues in which the alpha-D-GalpNAc residue would appear to be the critical antigenic factor recognized by polyclonal blood group A antisera.


Comparison of cyclic GMP in human internal mammary artery and saphenous vein: implications for coronary artery bypass graft patency. The aim was to examine the capacity of the human saphenous vein (native and surgically prepared) and the internal mammary artery to generate cyclic GMP, the second messenger that mediates smooth muscle relaxation following production of nitric oxide. 209 vessel segments were used from 22 patients undergoing coronary revascularisation. Isolated vessel segments were stimulated with a range of endothelium dependent and endothelium independent agonists and flash frozen for radioimmunoassay for cyclic GMP. Control/basal levels of cyclic GMP were significantly higher in the internal mammary artery than either native or distended saphenous vein. Endothelium dependent agonist stimulation with acetylcholine, bradykinin, or substance P induced significant increases in cyclic GMP in internal mammary artery and native saphenous vein, whereas distended veins showed non-significant changes in response to agonist stimulation. Endothelium removal abolished agonist stimulated increases in cyclic GMP. Glyceryl trinitrate and sodium nitroprusside elicited significant further increases in cyclic GMP in native vein and internal mammary artery. All values obtained were significantly greater in arterial than in venous tissue. Differences in basal and stimulated cyclic GMP activity in arteries and veins have been shown. This could represent an additional protective mechanism against constrictor influences in arterial bypass grafts, which may explain their documented better long term performance.


Ontogeny of substance P-, CGRP-, and VIP-containing nerve fibers in the amphibian carotid labyrinth of the bullfrog, Rana catesbeiana. An immunohistochemical study. The ontogeny of substance P, CGRP (calcitonin gene-related peptide), and VIP (vasoactive intestinal polypeptide) containing nerve fibers in the carotid labyrinth of the bullfrog, Rana catesbeiana, was examined by the peroxidase-antiperoxidase method. The time of appearance of these three peptides was different for each. First, CGRP fibers appeared in the wall of the carotid arch and external carotid arteries, and in a thin septum between these two arteries at an early stage of larval development (stage III). At stage V, substance P immunoreactive fibers appeared, and VIP fibers were detected at the early metamorphic stage (stage XXII). Up to the completion of metamorphosis, the number of these fibers remained low. From 1 to 5 weeks after metamorphosis, substance P, CGRP, and VIP fibers increased in number to varying degrees. By 8 weeks after metamorphosis, the distribution and abundance of these fibers closely resembled those of the adults. Some CGRP and VIP immunoreactive glomus cells were found at the stages immediately before and after the completion of metamorphosis. These findings suggest that substance P, CGRP, and VIP fibers during larval development and metamorphosis may be nonfunctional, and start to participate in vascular regulation only after metamorphosis. The transient CGRP and VIP in some glomus cells may be important for the development of the labyrinth, or may take part in vascular regulation through the close apposition of the glomus and smooth muscle cells (g-s connection).


Projections and pathways of submucous neurons to the mucosa of the guinea-pig small intestine. Double-labelling immunohistochemistry and retrograde transport of the carbocyanine dye, DiI, were used to establish the pathways of submucous neurons to the mucosa of the guinea-pig small intestine. Following the application of DiI to a villus, DiI-labelled nerve cell bodies were found in the submucous plexus up to 8.3 mm circumferentially and 3.8 mm longitudinally. The size of each of the four characterised classes of submucous neurons was determined and their distributions and projections mapped. Cells characterised by vasoactive intestinal polypeptide immunoreactivity accounted for 52% of DiI-labelled cells and had the longest projections. Cells characterised by neuropeptide Y (19%) or by calretinin immunoreactivity (13% of all DiI-labelled neurons) had relatively short projections and cells with substance P immunoreactivity (20%) had intermediate lengths of projection. When DiI was applied directly to the submucous plexus, filled neurons of all classes had significantly shorter projections, indicating that they must run for considerable distances in other pathways to the mucosa, probably via the non-ganglionated plexus. On average, each villus is innervated by at least 70 submucous neurons. From quantitative estimates there are 9 submucous neurons per villus. Thus, each submucous neuron is likely to supply about 8 villi. This demonstrates a high degree of convergence and divergence in the innervation of the mucosa.


Splenic primary sensory afferents in the guinea pig demonstrated with anterogradely transported wheat-germ agglutinin conjugated to horseradish peroxidase. The distribution of primary visceral afferents to the spleen of the guinea pig was studied after injections of wheat-germ agglutinin conjugated to horseradish peroxidase (HRP) into the left dorsal root ganglia at levels T7-T12. After anterograde transport of the tracer, labeled fibers were found in the nerves around the splenic artery in the hilus region and in the splenic parenchyma. The majority of labeled fibers in the spleen were detected in the white pulp. HRP-positive fibers were also observed in the red pulp and in the trabeculae. The distribution of the HRP-labeled fibers was in part similar to those of substance P-immunoreactive and calcitonin gene-related peptide-immunoreactive nerve structures. The results show that the anterograde tracing technique can be used successfully to investigate splenic primary afferent innervation.


Comparative study of the olfactory epithelium of the three-spined stickleback (Gasterosteus aculeatus) and the nine-spined stickleback (Pungitius pungitius). The olfactory epithelium of the three-spined stickleback (Gasterosteus aculeatus) and the nine-spined stickleback (Pungitius pungitius) has been studied with a conventional histochemical and a novel immunological staining technique. In both species, the sensory epithelium is arranged in folds separated by non-sensory epithelial tissue. In the nine-spined stickleback, intrinsic folds consisting of non-sensory cells are found in the apical part of the sensory epithelium where they divide the surface of the sensory epithelium into small islets. These non-sensory cells are non-ciliated, flattened and piled on top of each other; they contain numerous electron-translucent vesicles. The intrinsic folds are absent from the sensory epithelium of the three-spined stickleback. In both species, axons of receptor cells form a layer of fibers in the sensory epithelium immediately above the basal cells. In the three-spined stickleback, thick branches of the olfactory nerve are frequently found in this layer. These branches are only occasionally observed in the sensory epithelium of the nine-spined stickleback. Thus, the three-spined stickleback and the nine-spined stickleback show considerable differences in the organization of the sensory regions of the olfactory epithelium.


Co-localization of vimentin and cytokeratins in M-cells of rabbit gut-associated lymphoid tissue (GALT). The occurrence of cytokeratins, vimentin, and desmin in the dome epithelia and adjacent non-dome epithelia in four locations of gut-associated lymphoid tissues (GALT) of adult and newborn rabbits (Peyer's patches, sacculus rotundus, caecal lymphoid patches and appendix) was studied with monoclonal antibodies, using the indirect immunoperoxidase technique. In all locations investigated in adult animals, antibodies specific for vimentin labelled (1) M-cells, which engulf intra-epithelial lymphocytes, (2) columnar epithelial cells at the base of the domes lacking an apparent contact with lymphocytes ("immature" M-cells), and (3) flat cells, which lie in the lamina propria under the dome epithelium, and which line the basal lamina with thin cytoplasmic processes. In newborn rabbits, columnar epithelial cells resembling the immature M-cells of adults were selectively stained with vimentin antibodies. In M-cells, the strongest immunoreactivity was present in the perinuclear region and close to the pocket membrane, whereas the most apical and most basal parts of the cytoplasm showed no vimentin-immunoreactivity. Enterocytes in the dome epithelium and in the non-dome epithelium were vimentin-negative. M-cells and enterocytes bound antibodies against cytokeratin peptides 18 and 19 in adult and newborn animals. Compared with enterocytes, M-cells showed less intense staining for cytokeratins. Dome epithelia and no-dome epithelia did not contain desmin-immunoreactive cells. The results suggest that vimentin is a sensitive marker for M-cells in rabbit GALT.


Nucleolar morphology and rDNA in situ hybridisation in monocytes. The aim of this study was to correlate morphological changes of nucleoli of non-proliferating monocytes to their functional activity, since nucleolar morphology is currently considered as a diagnostic marker for cell proliferation. Monocytes from healthy donors were fractionated by current counterflow centrifugation and kept in culture for 6 days. Cells were stimulated by the addition of 200 units/ml interferon gamma (IFN gamma). Under this stimulus the monocytes show no proliferation but a strongly augmented expression of type I Fc IgG receptor, human leucocyte antigen DR, human leucocyte antigen DP and human leucocyte antigen DQ. Morphological changes after stimulation included the appearance of multinucleated cells, typical signs of the activation of rRNA synthesis indicated by an increase in nucleolar size, and changes in nucleolar structure such as the appearance of reticulate and compact nucleoli. The number of nucleolus organiser regions (NORs) visualised by in situ hybridisation was compared with the position and number of nucleoli visualised by silver staining in interphase cells. In comparison with control cultures, activated monocytes show a distinct increase in the number of those NORs that take part in the formation of nucleoli. Our results show that, in non-proliferating activated monocytes, the morphology of nucleoli and the increase of NOR activity are similar to those in proliferating cells. NOR activation is therefore an indicator for cellular activity, but is not necessarily correlated with proliferation.


The distribution and colocalization of neuropeptides in perivascular nerves innervating the large arteries and veins of the snake, Elaphe obsoleta. Single- and dual-labelling immunohistochemistry were used to determine the distribution and coexistence of neuropeptides in perivascular nerves of the large arteries and veins of the snake, Elaphe obsoleta, using antibodies for vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide, neuropeptide Y, galanin, somatostatin, and leu-enkephalin. Blood vessels were sampled from four regions along the body of the snake: region 1, arteries and veins anterior to the heart; region 2, central vasculature 5 cm anterior and 10 cm posterior to the heart; region 3, arteries and veins in a 30-cm region posterior to the liver; and region 4, dorsal aorta and renal arteries, renal and intestinal veins, 5-30 cm cephalad of the vent. A moderate to dense distribution of vasoactive intestinal polypeptide-like immunoreactive fibres was found in most arteries and veins of regions 1-3, but fibres were absent from the vessels of region 4. The majority of vasoactive intestinal polypeptide-like immunoreactive fibres contained colocalized substance P-like immunoreactivity, and these fibres were unaffected by either capsaicin or 6-hydroxydopamine (6-OHDA) pretreatment. In the anterior section of the snake, the vagal trunks contained many cell bodies with colocalized vasoactive intestinal polypeptide and substance P-like immunoreactivity. It is suggested that the vasoactive intestinal polypeptide/substance P-like immunoreactive cell bodies and fibres are parasympathetic postganglionic nerves. Neuropeptide Y-like immunoreactive fibres were observed in all arteries and veins, being most dense in regions 3 and 4. The majority of these fibres also contained colocalized galanin-like immunoreactivity, and were absent in tissues from 6-OHDA pretreated snakes, suggesting that neuropeptide Y and galanin are colocalized in adrenergic nerves. A small number of neuropeptide Y-like immunoreactive fibres contained vasoactive intestinal polypeptide but not galanin, and were unaffected by 6-OHDA treatment. All calcitonin gene-related peptide-like immunoreactive fibres contained colocalized substance P-like immunoreactivity, and these fibres were observed in all vessels, being particularly dense in the carotid artery and jugular veins. All calcitonin gene-related peptide/substance P-like immunoreactive fibres appeared damaged after capsaicin treatment suggesting they represent fibres from afferent sensory neurons. A sparse plexus of somatostatin-like immunoreactive fibres was observed in the vessels only from region 4. No enkephalin-like immunoreactive fibres were found in any blood vessels from any region. This study provides morphological evidence to suggest that there is considerable functional specialization within the components of the rat snake peripheral autonomic system controlling the circulation, in particular the regulation of venous capacitance.


Light- and electron-microscopic immunochemical analysis of nerve fibre types innervating the taenia of the guinea-pig caecum. The present work was undertaken to determine by immunocytochemical methods which of the putative enteric neurotransmitters are contained in axons supplying the guinea-pig taenia coli and what proportion of axons is accounted for by the presence of these substances. Numerous fibres displayed immunoreactivity for dynorphin (DYN), enkephalin (ENK), gamma-aminobutyric acid (GABA), nitric oxide synthase (NOS), substance P (SP) and vasoactive intestinal peptide (VIP), but, in contrast to other gut regions, fibres showing immunoreactivity for gastrin-releasing peptide, galanin and neuropeptide Y were rare in the taenia. Fibres reactive for calbindin, calcitonin gene-related peptide, cholecystokinin, 5-hydroxytryptamine and somatostatin were also rare. Tyrosine hydroxylase-like immunoreactivity (TH-LI) was present in numerous fibres that disappeared after extrinsic denervation, a procedure that did not detectably affect any of the other major groups of fibres. Simultaneous staining of extrinsically denervated preparations revealed that SP-LI and VIP-LI were located in separate fibres, and ultrastructural studies showed these to be 58% and 33% of intrinsic fibres supplying the muscle. Immunoreactivity for the general marker, neuron-specific enolase, was located in 95-98% of axons. ENK-LI and DYN-LI were in the same axons, and similar proportions of the fibres with either SP-LI or VIP-LI, about 85%, contained immunoreactivity for ENK and DYN. All VIP-LI fibres, but no SP-LI fibres, were reactive for NOS. The results imply that the taenia of the guinea-pig caecum is innervated by two major groups of enteric neurons: (i) excitatory neurons that contain ACh, SP, other tachykinins, and, in most cases, DYN-LI and ENK-LI; and (ii) inhibitory neurons that contain NOS-LI, VIP-LI, in most cases, the two opioids and, quite probably, ATP as a transmitter. GABA-LI is contained in a smaller population of intrinsic axons. Even though the taenia represents one of the simplest tissues for examining transmission from enteric neurons to intestinal muscle, it shares some of the complexity of other regions, in that four major axon types supply the muscle and both the enteric excitatory and enteric inhibitory neurons contain multiple transmitters.


Axoplasmic transport of horseradish peroxidase in single neurons of the dorsal root ganglion studied in vitro by microinjection. The dependence of anterograde axoplasmic transport on cytoskeletal components was investigated using microinjection of horseradish peroxidase (HRP) into the somata of chick dorsal root ganglion cells in vitro. Microinjected HRP was transported anterogradely in the neurites and their branches; this transport was disturbed by colchicine in a drug-dependent and time-dependent manner. Cytochalasin B, a drug that depolymerizes actin, did not inhibit the transport of HRP, despite the formation of local swellings in neurites. The microinjection of polyclonal antibodies directed against tubulin and monoclonal antibodies (mAbs) against 200-kDa neurofilaments disturbed the axoplasmic transport of co-injected HRP, which then exhibited an irregular and discontinuous distribution in the axonal branches. The transport of HRP became discontinuous after the injection of anti-tubulin antibodies and led to the formation of globular deposits of HRP. Polyclonal antibodies against actin and mAbs to 160-kDa and 68-kDa neurofilaments seemed to have no effect on the axoplasmic transport of co-injected HRP. Microinjection of antibodies against tubulin induced formation of perinuclear bundles consisting of cytoskeletal components. The transport of HRP thus appears to be regulated by an intact microtubular system and cross-linker components (200-kDa neurofilaments) of the cytoskeleton. Actin and most intermediate filament proteins do not seem to play an essential role in the transport of HRP.


Enterochromaffin-like cells in the rat stomach: effect of alpha-fluoromethylhistidine-evoked histamine depletion. A chemical, histochemical and electron-microscopic study. In the rat, gastric histamine is stored predominantly in the enterochromaffin-like (ECL) cells, which are located basally in the oxyntic mucosa. The functional significance of histamine in the ECL cells is a matter of speculation. In this study the effect of depletion of histamine on the properties and ultrastructure of the ECL cells was examined. Histamine synthesis was inhibited with alpha-fluoromethylhistidine (3 mg.kg-1.h-1) given via osmotic minipumps over a period of 24 h. The treatment reduced the histidine decarboxylase activity (approximately 20% remaining) and histamine concentration (less than 20% remaining) in the oxyntic mucosa, as well as the intensity of histamine- and chromogranin A-immunostaining in the ECL cells, compared to control rats. The cytoplasmic (secretory) granules/vesicles were greatly reduced in number and size following alpha-fluoromethylhistidine administration. The histamine immunostaining of the mast cells, which occurs at the mucosal surface and in the submucosa, appeared unaffected. We conclude that ECL cell histamine accounts for at least 80% of the total oxyntic mucosal histamine in the rat and that it represents a more mobile pool than mast cell histamine. The reduction in the number and size of the ECL cell granules/vesicles following histamine depletion is in accord with the idea that they represent the storage site for histamine.


Actions of two native GnRHs and protein kinase C modulators on goldfish pituitary cells. Studies on intracellular calcium levels and gonadotropin release. Previous results indicate that the two native gonadotropin (GtH)-releasing hormones of the goldfish, sGnRH and cGnRHII, stimulate GtH secretion in an extracellular Ca2+ ([Ca2+]o) dependent manner. In the present study, sGnRH, cGnRHII, KCI and the protein kinase C (PKC) activators TPA and DiC8, stimulated increases in intracellular Ca2+ ([Ca2+]i) levels in goldfish pituitary cells. Testing in Ca(2+)-deficient medium abolished the [Ca2+]i responses to cGnRHII, TPA and KCI and attenuated responses to sGnRH and DiC8. These results are the first to demonstrate that in teleost pituitary cells both native GnRHs stimulate increases in [Ca2+]i levels via [Ca2+]o entry. sGnRH- and DiC8-stimulated increases in [Ca2+]i also appear to be partially due to mobilization of Ca2+ from intracellular stores. Other results are consistent with a role for PKC in mediating GnRH action especially extracellular Ca2+ entry. Firstly, the PKC inhibitor staurosporine decreased GnRH- and TPA-induced [Ca2+]i responses. Secondly, incubation with Ca(2+)-deficient medium attenuated TPA- and DiC8-stimulated GtH release. Thirdly, GtH release responses to PKC activators were enhanced and reduced by an agonist and an antagonist of Ca2+ channel function, respectively. However, differences in the sensitivity of DiC8- and TPA-elicited responses to manipulations of [Ca2+]o entry indicate that these two PKC activators may have different actions in the goldfish pituitary. A difference in action of the two GnRHs on mobilization of Ca2+ from intracellular stores is also indicated.


Prevalence of adult migraine in general practice. In 1988 the International Headache Society presented new criteria for the diagnosis of migraine. We used these criteria in a questionnaire to assist in the diagnosis of migraine. The questionnaire was answered by 230 patients in four general practices. We found a migraine prevalence of 11.7%. More than 70% of migraine patients had 12 or more attacks per year, and the length of attack varied between 4 and 24 h in 80% of them. The majority of migraineurs went to bed during the attack and 55% had stayed away from work due to migraine during the last year. The results of this study are in agreement with others. Migraine is an appreciable economic concern due to frequent short absenteeism from work.


Interpretation of cardiac pathophysiology from pressure waveform analysis: cardiac arrhythmias. Various arrhythmias can produce distorted pressure waveforms, which may be confused with benign physiologic events. Delay in the management of serious arrhythmias can be avoided by vigilant monitoring of systemic pressures.


UNC-5, a transmembrane protein with immunoglobulin and thrombospondin type 1 domains, guides cell and pioneer axon migrations in C. elegans. The unc-5 gene is required for guiding pioneering axons and migrating cells along the body wall in C. elegans. In mutants, dorsal migrations are disrupted, but ventral and longitudinal movements are largely unaffected. The gene was tagged for molecular cloning by transposon insertions. Based on genomic and cDNA sequencing, the gene encodes UNC-5, a transmembrane protein of 919 aa. The predicted extracellular N-terminus comprises two immunoglobulin and two thrombospondin type 1 domains. Except for an SH3-like motif, the large intracellular C-terminus is novel. Mosaic analysis shows that unc-5 acts in migrating cells and pioneering neurons. We propose that UNC-5 is a transmembrane receptor expressed on the surface of motile cells and growth cones to guide dorsal movements.


Mouse P0 gene disruption leads to hypomyelination, abnormal expression of recognition molecules, and degeneration of myelin and axons. We have used homologous recombination in embryonic stem cells to generate mice carrying a mutation in the gene encoding P0, an immunoglobulin-related recognition molecule and the major protein of peripheral nervous system myelin. These mice are deficient in normal motor coordination and exhibit tremors and occasional convulsions. Axons in their peripheral nerves are severely hypomyelinated and a subset of myelin-like figures and axons degenerate. The mutation leads to an abnormal regulation of some, but not all, molecules involved in myelination. These results demonstrate that P0 is essential for the normal spiraling, compaction, and maintenance of the peripheral myelin sheath and the continued integrity of associated axons. They further suggest that this protein conveys a signal that regulates Schwann cell gene expression.


The reverse transcriptase of hepatitis B virus acts as a protein primer for viral DNA synthesis. Hepatitis B viruses (hepadnaviruses) replicate their DNA genomes by reverse transcription of an RNA intermediate. Efforts to examine the biochemical mechanism for viral DNA synthesis have been hampered by the failure to solubilize the reverse transcriptase from virions and to express the polymerase in heterologous systems in an enzymatically active form. Here, we demonstrate that the polymerase of a hepadnavirus synthesized in an in vitro translation reaction exhibits reverse transcriptase activity. Furthermore, our results show that the polymerase acts as a primer for DNA synthesis and remains covalently linked to nascent DNA, a feature that is not known to exist in any other RNA-directed DNA polymerases. Priming of DNA synthesis requires viral RNA but occurs independently of other viral components. The ability to express the hepadnavirus reverse transcriptase in an enzymatically active form will allow detailed biochemical and functional analyses of this complex enzyme, and may facilitate the identification of inhibitors required for antiviral therapy.


The pro region of BPTI facilitates folding. The in vitro folding pathway of bovine pancreatic trypsin inhibitor (BPTI) has been described previously in terms of the disulfide-bonded intermediates that accumulate during folding of the protein. Folding is slow, occurring in hours at pH 7.3, 25 degrees C. In addition, approximately half of the BPTI molecules become trapped as a dead-end, native-like intermediate. In vivo, BPTI is synthesized as a precursor protein that includes a 13 residue amino-terminal pro region. This pro region contains a cysteine residue. We find that, in vitro, both the rate of formation and the yield of properly folded BPTI are increased substantially in a recombinant model of pro-BPTI. The cysteine residue is necessary for this effect. Moreover, a single cysteine residue, tethered to the carboxy-terminal end of BPTI with a flexible linker of repeating Ser-Gly-Gly residues, is sufficient to assist in disulfide formation. Thus, the pro region appears to facilitate folding by providing a tethered, solvent-accessible, intramolecular thiol-disulfide reagent.