[Peritonitis caused by Nocardia farcinica in a patient undergoing continuous ambulatory peritoneal dialysis (note)].

We report here an isolation of bacteria genus Nocardia, Nocardia farcinica, in a dialysis liquid of a renal insufficiency patient treated by continuous ambulatory peritoneal dialysis (C.A.P.D.). Real peritonitis was due to infection with this bacteria, eliminated after an adapted antibiotherapy. This observation is interesting first because of the rare occurrence of Nocardia's peritonitis in the patients on CAPD, and secondly in relation with the species of Nocardia, Nocardia farcinica, less known than Nocardia asteroides but not less pathogenic.


Is multiple sclerosis in Brazil and Asia alike?

The clinical picture of 67 consecutive patients with definite multiple sclerosis in Belo Horizonte, Brazil, was analysed. There were 20 patients with the predominant optic-spinal form of the disease and eight with Devic's disease. Visual loss occurred in 43% of the cases as the presenting symptom and in 84% in the course of the illness. Symptoms related to spinal cord and cerebellar involvement were observed in 64% and 52% respectively. The high prevalence of visual and spinal abnormalities at onset and during the course of the illness, the lesser common involvement of the cerebellum and the relative frequency of Devic's disease make multiple sclerosis in Brazil similar to that observed in Eastern countries in distinction to the clinical pattern seen in the United States and Europe.


Idiopathic dystonia. Clinical profile of 76 Brazilian patients.

Dystonia may be classified by age of onset (childhood, adolescence, adult onset), body distribution of the abnormal movements (focal, segmental, unilateral, multifocal and generalized) and etiology (idiopathic and symptomatic). We studied 76 patients with idiopathic dystonia among 122 cases of dystonic syndrome (62.3% of the total). There were 48 female and 28 male patients. Adult-onset focal dystonia was the most frequent feature (37 patients). The onset of generalized dystonia was more frequently seen under the age of 20, whereas focal and segmental dystonia usually started over this age. Postural tremor of the hands was observed in 19.7% of the patients. Spasmodic torticollis was the most prevalent form of dystonia overall. Except for writer's cramp, which occurred more frequently in males, and generalized dystonia, which was equally divided between sexes, all other forms were more frequent in females. Our data suggest that differences in racial origin, social and economical status and environmental factors do not account for a different manifestation in dystonia pattern.


[Early differentiation between Duchenne and Becker muscular dystrophy: clinical, laboratory, electrophysiology, histochemical, and immunohistochemical study of 138 cases].

194 clinical, laboratory, electrophysiologic, histological, histochemical and immunohistochemical parameters were studied through statistical analysis in 112 cases of Duchenne muscular dystrophy (DMD) and in 26 cases of Becker muscular dystrophy (BMD). It was found a significant statistical difference (p < 0.05) between the two groups concerning the age of evaluation, beginning of symptoms, difficulty in walking, running, climbing and going downstairs, frequent falling down, support to walk, localized muscle pain, stopping climb stairs, and inability to walk. Muscle biopsy showed statistically significant (p < 0.05) differences between the two groups regarding the intensity of connective tissue and focal adipose tissue proliferation, presence of diffuse rounded atrophic and angulated fibers, diffuse hypertrophic and splitting fibers. There were also differences regarding excessive internal fibers nuclei, hypertrophic types 1 and 2 fibers, angulated atrophic fibers and focal increasing in the NADH-TR, angulated atrophic fibers in non-specific esterase, and accumulated NBT in the periphery of fibers in succinic dehidrogenase. Isolatedly muscle biopsy gave the correct diagnosis in 52.7% of DMD cases and in 69.2% of BMD cases. Dystrophin detection by immunofluorescence (60 cases) showed: absence in 87.0% of fibers in DMD cases, and sarcolemmal membrane discontinuites in all BMD cases. The muscle biopsy diagnosis had an agreement with the dystrophin results in 82.6% of DMD cases and 71.4% of BMD cases.


Carrier detection of Duchenne and Becker muscular dystrophy using muscle dystrophin immunohistochemistry.

To ascertain whether dystrophin immunohistochemistry could improve DMD/BMD carrier detection, we analyzed 14 muscle biopsies from 13 DMD and one BMD probable and possible carriers. All women were also evaluated using conventional methods, including genetic analysis, clinical and neurological evaluation, serum CK levels, EMG, and muscle biopsy. In 6 cases, there was a mosaic of dystrophin-positive and dystrophin-deficient fibers that allowed to make the diagnosis of a carrier state. Comparing dystrophin immunohistochemistry to the traditional methods, it was noted that this method is less sensitive than serum CK measurements, but is more sensitive than EMG and muscle biopsy. The use of dystrophin immunohistochemistry in addition to CK, EMG and muscle biopsy. improved the accuracy of carrier detection. This method is also helpful to distinguish manifesting DMD carriers from patients with other neuromuscular diseases like limb-girdle muscular dystrophy and spinal muscular atrophy.


[Psychosocial approach of the epileptic patient: analysis of 30 adults].

Psychosocial aspects of patient's life were analysed in 30 patients with epilepsy aged over 18 years old. At this age people usually are apt by themselves to exert several psychosocial activities. Professional, familial, social, scholar, affective, sexual and religious activities were evaluated in our group of patients. Results evidence that epileptic patients studied show several kinds of difficulties in their psychosocial adjustment.


CSF in 85 patients with AIDS and CNS cryptococcosis.

In an eight years time period (July 1984-June 1992) CSF samples of 40718 patients were studied, and 610 were from patients with AIDS clinically diagnosed and immunologically confirmed through HIV antibodies detection. Among opportunistic infections detected in them 85 were CNS cryptococcosis. For the purpose of this study the CSF of these 85 patients are the AIDS group of CNS cryptococcosis. For comparison, CSF data from 50 patients with CNS cryptococcosis but without AIDS were taken (non-AIDS group); in this group, 22 patients were immunosuppressed after renal transplant. In AIDS group, the more frequent CSF findings were: yeast presence at direct exam (Fuchs-Rosenthal cell counting chamber), growing of the yeast in cultures, and gamma globulins increase. In non-AIDS group were more frequent: hypercytosis, neutrophil cells presence, and total protein increase. Differences between the two groups are discussed taking into account CNS/CSF immune changes induced by HIV infection. It is concluded that in CNS cryptococcosis of patients with AIDS the CSF evidenced more extensive signs of the fungal opportunistic infection than signs of inflammatory response to the infection. The latter were more prominent among patients of the non-AIDS group of CNS cryptococcosis.


Neurotoxoplasmosis and AIDS. Cerebrospinal fluid analysis in 96 patients.

The behavior of CSF inflammatory pattern in patients with AIDS and/or toxoplasmosis of the CNS is studied in 176 patients, divided in three groups. In the first group, 96 patients with toxoplasmosis and AIDS are considered; in the second group, 50 patients with toxoplasmosis without AIDS; in the third group, 30 AIDS patients without toxoplasmosis nor any other opportunistic infection. It is possible to conclude that patients with toxoplasmosis associated to AIDS exhibit CSF inflammatory pattern similar to patients with neurotoxoplasmosis without AIDS, except in respect to gamma globulin rates for which a cumulative effect can be detected.


[Conservative treatment of laminar extradural hematomas in children].

We successfully treated by nonsurgical methods 15 children with laminar epidural hematomas (EH), with minimal neurological symptoms and no signs of brain herniation. These EH were discovered 30 minutes to 5 days after head injury. The majority were localized in the parietal region. All children recovered without surgery from 2 to 12 days after hospitalization and all had evidence on CT scan of spontaneous clot reabsorption. We discuss the criteria for patient selection for this kind of treatment on EH in children.


[Primary necrosis of the corpus callosum (Marchiafava-Bignami's disease): report of 5 cases].

The clinicopathological study of five autopsied cases of Marchiafava-Bignami disease is reported. The demyelination of the central portion of the corpus callosum is the major characteristics of the disease. In two cases the demyelination also involved the anterior and posterior commissures. There is a clear relationship to alcoholism and malnutrition in the cases reported in the present study.


[Basal ganglia calcifications in children].

We report the study of four children with bilateral basal ganglia calcifications (BGC) visualized on CT scan. Epilepsy was the clinical manifestation of three patients whose laboratory investigation revealed abnormal calcium metabolism. The first aim of this paper is to call attention to a treatable entity that can cause epileptic syndromes in infancy and childhood. The second purpose is to review the literature comparing with our fourth child who presented encephalopathy with BGC.


Diffuse encephalic calcification. A case report.

The basal ganglia calcification is known since the last century but with the new neuroimage techniques (CT scan) its diagnosis became more frequent specially in asymptomatic patients. The authors report a case with non-familial primary diffuse encephalic calcification with exuberant calcifications on cerebral hemispheres, cerebellum and brain stem, seen on CT scan.


[Intracranial saccular aneurysm: report of 3 cases in a same family].

The authors report the cases of three patients from the same family, all with intracranial saccular aneurysm (left carotid artery, anterior communicating artery, and middle cerebral artery). All three patients were operated on with good recovery and no complications. The authors call attention for some etiopathogenic aspects of familial saccular aneurysms.


Segmental myoclonus and basilar artery. Giant aneurysm. Case report.

A 70 years-old man was admitted at our hospital because of unstable angina pectoris. He had essential hypertension and right hemiplegia from a ischemic stroke two years before admission. On neurologic examination, it was found mental disorientation, unstable emotionality, right spastic hemiparesis with right Babinski sign, and segmental myoclonus affecting the superior lip and the palate (palatal nystagmus) on the right side. On the CT scan, a giant aneurysm of the basilar artery was detected. We conclude that the segmental myoclonus could be explained by ischemic lesions in the Guillain-Mollaret triangle.


[Parkinsonism secondary to ethylene oxide exposure: case report].

Ethylene oxide is a gas widely used in the production of industrial chemicals. It is also used to sterilize heat-sensitive medical supplies. Previous reports of acute and chronic exposure have described neurotoxic effects like peripheral neuropathy and cognitive impairment. We describe a pure parkinsonian syndrome following acute ethylene oxide intoxication. A 39-years-old male was referred to our Movement Disorders Clinic for evaluation of a parkinsonian syndrome. He was acutely exposed to ethylene oxide four years before and remained comatose for three days, and gradually regained consciousness. At that time he showed a global parkinsonian syndrome including bradykinesia, rigidity and rest tremor, with a severe motor disability; no other neurological disorders were found. The symptomatology was partially controlled with biperidene and levodopa plus carbidopa. Two years later he developed L-dopa induced dyskinesias. Four years after the intoxication he was evaluated at our clinic. General examination showed no abnormalities. Neurologic examination revealed a normal mental status. Motor evaluation disclosed moderate bradykinesia, rigidity and rest tremor, shuffling gait, poor facial mimic, stooped posture, and his speech was low and monotonous; deep tendon reflexes were brisk. The Hoehn-Yahr disability score was degree IV. Routine laboratory and radiological exams showed results within normal limits. The CSF examination was normal. Brain computed tomography and magnetic ressonance were normal. A trial with bromocriptine and levodopa plus carbidopa did not improve dyskinesia, and he was put on a schedule including amantadine and biperidene with improvement to grade III in Hoehn-Yahr scale. In the present case there was a clear relation between the acute exogenous intoxication and irreversible parkinsonism.(ABSTRACT TRUNCATED AT 250 WORDS)


Granuloma formation and arterial thrombosis following cotton wrapping of an intracranial aneurysm. A case report.

The authors report the case of a patient whose left middle cerebral artery aneurysm was wrapped with cotton. Occlusion of the middle cerebral artery, probably secondary to a foreign-body inflammatory reaction, developed late in the postoperative course. A computerized tomography scan revealed cerebral infarct, and an enhancing expansive lesion at the site of the aneurysm suggesting the formation of a granuloma. These findings are discussed and the literature is reviewed.


[Schwannomatosis: report of a new case].

Schwannomatosis is a rare disorder, still not quite well defined, seldom described in the literature. In this paper we report the case of male. Patient, 52 years old, who in the last 30 years developed five subcutaneous tumors within his limbs peripheral nerves, which histologically proved to be schwannomas. A brain computed tomography showed a partially calcified tumor in the left temporal lobe which most likely was a meningioma. A thorough clinical examination was unable to find signs of type I or type II neurofibromatosis. The present condition, probably a form of phacomatosis, has to be distinguished from neurofibromatosis and is considered as an independent clinical entity whose origin still awaits further detailed investigations.


Kleptomania, mood disorder and lithium.

Kleptomania has been found in association with major depression in a fairly large number of reports in recent years. We describe a patient with concurrent DSM-III-R Bipolar Mood Disorder and Kleptomania, whose symptoms remitted completely, apparently in response to lithium therapy, which raised the possibility that pharmacological treatment may benefit kleptomania. Further studies are needed to establish the possible relationship between kleptomania, mood disorders and lithium therapy.


[Definitively: epilepsy is not a disease].

The author analyses past concepts which still hold good today. Such concepts regard epilepsy as a mental disease and the epileptic is usually taken as someone suffering from a severe mental disorder, tending to criminal behaviour. The author proves, taking into account various statements from prominent personalities in the scientific world, that epilepsy is neither a mental disease nor a disease in its own sense, and that the epileptic has not an aggressive behaviour.


Adenine nucleotide concentrations and energy charge in muscle of chronic haemodialysis patients.

Adenine nucleotide concentrations and energy charge ratios were measured in muscle samples collected during transplant surgery of 7 patients suffering from chronic renal failure and undergoing haemodialysis. The energy charge ratio of 0.75 in the muscle of transplant patients was significantly lower (p less than 0.01) than that of 0.89 found in muscle from controls. The lower energy charge ratio and increased concentrations of adenosine diphosphate and adenosine monophosphate may contribute to the patients' reduced exercise ability and their poor metabolic state.


Mycobacterial spindle cell pseudotumor of lymph nodes.

Two cases of spindle cell pseudotumor in the lymph nodes of patients with acquired immunodeficiency syndrome caused by mycobacterial infection are reported and the literature reviewed. The lesions mimicked neoplasms because they were composed predominantly of spindle cells arranged in a storiform pattern. Most of the spindle cells were phagocytic cells that contained large amounts of mycobacteria. It is important for the pathologist to recognize the lesion so that a prompt tissue diagnosis can be provided because specific therapy is available.


Brachial plexus palsy: an old problem revisited.

It is an almost assumption in obstetric literature that brachial palsy is due to extreme lateral traction on the fetal head during the last phase of delivery. In contrast, there have been reports in the neurologic literature of probable intrauterine origin of brachial plexus palsy. Data to dispute or support the latter view were sought. With this overview in mind, our perinatal data base was searched for all instances of shoulder dystocia and independently for all diagnoses of brachial plexus impairment. Seventeen instances of brachial plexus impairment associated with shoulder dystocia were found. Twenty-two instances of brachial plexus impairment without mention of shoulder dystocia were ascertained. The characteristics of the two groups were remarkably different, especially in birth weight and in maternal age and parity. The data are strongly suggestive that intrauterine maladaptation may play a role in brachial plexus impairment. Brachial plexus impairment should not be taken as prima facie evidence of birth process injury.


Inflammatory myoglandular polyps of the colon and rectum. A clinicopathological study of 32 pedunculated polyps, distinct from other types of polyps.

Hitherto unclassified colorectal polyps were identified in 32 patients (23 men and 9 women; mean age, 53 years). The only symptom, which was observed in less than half the patients, was passage of blood or occult blood. Endoscopic examination revealed solitary pedunculated, red polyps with a smooth surface. These polyps were found in the left colon, especially in the sigmoid. Their characteristic features were inflammatory granulation tissue in the lamina propria mucosae, proliferation of smooth muscle, and hyperplastic glands with occasional cystic dilatation. The etiology of this type of polyp is unknown, but it could involve chronic trauma from the fecal stream and from peristalsis of the bowel. These polyps can be differentiated from juvenile polyps and inflammatory polyps by the presence of abundant smooth-muscle cells in the inflamed lamina propria mucosae. They also can be differentiated from Peutz-Jeghers polyps, which appear as hamartomatous structures with tree-like proliferation of muscularis mucosae covered by colonic mucosa without inflammatory granulation tissue. Their locations and macroscopic appearance distinguish these polyps from mucosal prolapse syndrome and polyps developed after colostomy. In addition, these new polyps differ from inflammatory cap polyps in that they lack a fibrin cap. We propose the name inflammatory myoglandular polyps for these polyps, which are distinct clinicopathologically from other types of colorectal polyps.


Reevaluation of secular trends in depression rates.

Results of numerous community surveys of psychiatric illness suggest a striking change in the occurrence of depression, with younger generations experiencing higher lifetime risk and earlier age of onset. Data from the National Institute of Mental Health Epidemiologic Catchment Area Survey (a cross-sectional survey of psychiatric morbidity in five US communities conducted between 1980 and 1984) were reexamined for evidence of methods effects which might contribute to these unexpected findings. A pattern of higher lifetime risk and earlier age of onset among recent birth cohorts was observed for every psychiatric disorder examined, with schizophrenia, major depression, and panic disorder showing equally strong trends. For respondents of all ages, reported first onset of major depression clustered in the 10-year period prior to the study interview, in contrast to the expectation that older respondents would report onset in early adulthood. Examination of individual psychiatric symptoms revealed a nearly universal pattern of decreasing lifetime prevalence among older respondents, a reversal of the expected accumulation of lifetime symptoms with age. These findings suggest that effects of study methods may contribute to the apparent temporal trends in prevalence of depression and that cross-sectional surveys may underestimate lifetime psychiatric morbidity among older respondents. Generational changes in the lifetime risk of depression or other psychiatric disorders may not be reliably assessed by cross-sectional survey data.


The laryngeal mask airway for intraocular surgery: effects on intraocular pressure and stress responses.

Tracheal intubation, performed routinely during general anaesthesia in patients undergoing intraocular surgery, may have adverse effects on cardiovascular function and intraocular pressure. This study assessed the suitability of the laryngeal mask airway (LMA) as a substitute for tracheal intubation. Intraocular and systemic pressor effects, heart rate changes and catecholamine concentrations were measured in two groups of 10 patients receiving standardized anaesthesia with either a tracheal tube (TT) or a LMA. There were significantly smaller changes in the pressor responses to insertion and in concentrations of catecholamines at critical times in the anaesthetic sequence in the LMA group. Mean (SEM) rate-pressure product was significantly smaller in the LMA group compared with the TT group after both insertion (8276 (730) vs 13307 (1348), P < 0.01) and removal (10152 (595) vs 14137 (1044), P < 0.01) of the airway device. The change in intraocular pressure was significantly less in the LMA group at all time points after airway instrumentation than that in the TT group, with the greatest difference after extubation (-2.3 (2.4) mm Hg vs 14.5 (3.4) mm Hg, P < 0.01).


Mechanistic investigation of medium-chain fatty acyl-CoA dehydrogenase utilizing 3-indolepropionyl/acryloyl-CoA as chromophoric substrate analogues.

The CoA derivative 3-indolepropionyl-CoA (IPCoA) serves as a competent pseudosubstrate for the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-catalyzed reaction. The reaction product trans-3-indoleacryloyl-CoA (IACoA) exhibits a characteristic UV-vis absorption spectrum with lambda max = 367 nm and epsilon 367 = 26,500 M-1 cm-1. The chromophoric nature of IACoA allows us to measure the direct conversion of substrate to product (at 367 nm) without recourse to absorption signals for either the enzyme-bound flavin or the coupling electron acceptors, as well as probe the enzyme site environment. The interaction of IACoA with medium chain fatty acyl-CoA dehydrogenase (MCAD)-FAD is characterized by resultant (spectra of the mixture minus the individual components) absorption peaks at 490, 417, and 355 nm. These absorption peaks increase in magnitude as the pH of the buffer media decreases. Transient kinetic analysis for the interaction of MCAD-FAD with IACoA suggests that the formation of the enzyme-IACoA complex proceeds in two steps. The first (fast) step involves the formation of an E-IACoA collision complex, which [formula: see text] is isomerized (concomitant with changes in the protein structure) to an E*-IACoA complex in the second (slow) step. We have studied the effect of pH on Kc, k2, and k-2. While Kc shows practically no dependence on pH (within a 2-fold variation between pH 6.0 and 9.5), k2 and k-2 show a strong dependence on pH. Both k2 and k-2 exhibit a sigmoidal dependence on the pH of the buffer media, with pKa's of 7.53 and 8.30, respectively. In accordance with the model presented herein, the pKa of 7.53 represents an enzyme site group which is involved in the interaction with IACoA within the E-IACoA collision complex. This pKa is perturbed to 8.30 upon isomerization of the collision complex. The pH-dependent changes in k2 and k-2 are such that the equilibrium distribution between E-IACoA and E*-IACoA is favored to the latter complex (by about 20-fold) at lower pH than at higher pH. A cumulative account of the spectral, kinetic, and thermodynamic properties of the enzyme-IACoA complexes has allowed us delineate the microscopic pathway by which the E-IACoA isomerization (presumably via protein conformational changes) is coupled to the proton equilibration steps.


Right bundle branch block, persistent ST segment elevation and sudden cardiac death: a distinct clinical and electrocardiographic syndrome. A multicenter report.

The objectives of this study were to present data on eight patients with recurrent episodes of aborted sudden death unexplainable by currently known diseases whose common clinical and electrocardiographic (ECG) features define them as having a distinct syndrome different from idiopathic ventricular fibrillation. Among patients with ventricular arrhythmias who have no structural heart disease, several subgroups have been defined. The present patients constitute an additional subgroup with these findings. The study group consisted of eight patients, six male and two female, with recurrent episodes of aborted sudden death. Clinical and laboratory data and results of electrocardiography, electrophysiology, echocardiography, angiography, histologic study and exercise testing were available in most cases. The ECG during sinus rhythm showed right bundle branch block, normal QT interval and persistent ST segment elevation in precordial leads V1 to V2-V3 not explainable by electrolyte disturbances, ischemia or structural heart disease. No histologic abnormalities were found in the four patients in whom ventricular biopsies were performed. The arrhythmia leading to (aborted) sudden death was a rapid polymorphic ventricular tachycardia initiating after a short coupled ventricular extrasystole. A similar arrhythmia was initiated by two to three ventricular extrastimuli in four of the seven patients studied by programmed electrical stimulation. Four patients had a prolonged HV interval during sinus rhythm. One patient receiving amiodarone died suddenly during implantation of a demand ventricular pacemaker. The arrhythmia of two patients was controlled with a beta-adrenergic blocking agent. Four patients received an implantable defibrillator that was subsequently used by one of them, and all four are alive. The remaining patient received a demand ventricular pacemaker and his arrhythmia is controlled with amiodarone and diphenylhydantoin. Common clinical and ECG features define a distinct syndrome in this group of patients. Its causes remain unknown.


Medications in older patients.

Adverse drug reactions are common in persons aged 65 and older and are associated with increased morbidity and mortality. A heightened susceptibility to adverse reactions is due to a number of factors, including an increased incidence of disease, multiple drug use, and altered pharmacokinetic and pharmacodynamic properties of many drugs. The risk of drug interactions increases with the number of medications taken. Adverse drug reactions can be prevented through prudent prescribing practices, patient education, and adequate monitoring of drug efficacy and side effects. Several types of medications are of particular concern, including many antihypertensive agents, drugs with anticholinergic effects, psychoactive medications, and nonsteroidal anti-inflammatory drugs. Some drugs, such as histamine H2-receptor antagonists, are relatively safe but are overprescribed. Data regarding the risks associated with these problem drugs are presented, with recommendations for safe and effective treatment alternatives.


Hypotonous maculopathy after trabeculectomy with subconjunctival 5-fluorouracil.

Long-term success in trabeculectomy has been enhanced in recent years by postoperative subconjunctival 5-fluorouracil injections and intraoperative mitomycin C applications. During 1990 and 1991, after trabeculectomy with a small scleral flap (2 x 3 mm) augmented by antimetabolite therapy, hypotonous maculopathy developed in eight eyes of six patients. The maculopathy was characterized by loss in visual acuity, retinal striae, and choroidal folds without evidence of vascular leakage. The average loss in visual acuity was four Snellen lines. Visual acuity did not return to preoperative levels even when the hypotony could be reversed. Reversing the hypotony with various strategies aimed at stimulating subconjunctival scarring has been relatively ineffective. During that same period, an additional seven eyes in six patients had prolonged hypotony but without development of maculopathy. Features common to patients who developed maculopathy included age (mean age, 46 years; range, 32 to 60 years) and myopia (mean, -7.5 diopters; range, -0.75 to -11.75 diopters). The patients with hypotony but no maculopathy were older (mean age, 73 years; range, 63 to 82 years) and were closer to emmetropia (mean, -1.11 diopters; range, +1.50 to -9.00 diopters). The means of the ages and refractive errors were statistically significantly different in the two groups (P = .007 and .04, respectively). Trabeculectomy with adjunctive antifibrosis therapy should be used with caution in young myopic patients.


Cerebral palsy in twins.

Eighty-six children with cerebral palsy (CP) born as a product of twin pregnancies were studied. Data regarding their co-twins were also gathered. The authors' findings suggest that (1) monozygotic twins have a higher risk of CP than dizygotic twins, (2) twin order at birth does not seem to be a relevant factor in the etiology of CP, (3) mothers less than 24 or more than 34 years of age had the highest incidence of children with CP as a product of a twin pregnancy, and (4) the data do not support a genetic basis for the disease.


Pyogenic granuloma of the cornea after penetrating keratoplasty.

An 81-year-old man with metastatic prostate carcinoma underwent a penetrating keratoplasty for phlyctenular keratitis. Two years later he developed a fleshy, vascular mass in the superotemporal corneal graft wound, at the site of prior graft sutures. An excisional biopsy of the mass was performed to rule out metastatic carcinoma. Histopathological findings were consistent with pyogenic granuloma. To our knowledge there have been no prior case reports illustrating pyogenic granuloma as a late complication of penetrating keratoplasty.


Restoring sensation after trigeminal nerve injury: a review of current management.

After a year, even the most expert peripheral nerve repair carries a poor prognosis. Early referral and intervention offer the best management of trigeminal nerve injuries.


Cross-talk regulation between cyclic AMP production and phosphoinositide hydrolysis induced by prostaglandin E2 in osteoblast-like cells.

In cloned osteoblast-like MC3T3-E1 cells, PGE2 stimulated both cAMP accumulation and the formation of inositol trisphosphate (IP3) dose dependently. The cAMP accumulation showed the peak value at 5 min and decreased thereafter, whereas the IP3 formation reached a plateau almost within 10 min and sustained it up to 30 min. The effect of PGE2 on cAMP accumulation (EC50 was 80 nM) was more potent than that on IP3 formation (EC50 was 0.8 microM). 12-O-Tetradecanoyl-phorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, reduced the PGE2-induced cAMP accumulation, whereas 4 alpha-phorbol 12,13-didecanoate, a PKC-nonactivating phorbol ester, had little effect on the cAMP accumulation. 1-Oleoyl-2-acetyl-glycerol, a specific activator for PKC, inhibited PGE2-induced cAMP accumulation. TPA had little effect on cAMP accumulation induced by forskolin or NaF, a GTP-binding protein activator. So, the effect of TPA is presumed to be exerted at the point between the PGE2 receptor and Gs. On the other hand, forskolin and dibutyryl cAMP had little effect on the IP3 formation stimulated by PGE2. H-7, a PKC inhibitor, enhanced the PGE2-induced cAMP accumulation in comparison with HA1004, a control for H-7. Our data suggest that PGE2 regulates cAMP production through self-induced activation of PKC. These results strongly suggest that there is an autoregulatory mechanism in PGE2 signaling, and PGE2 modulates osteoblast functions through a cross-talk interaction between cAMP production and phosphoinositide hydrolysis in osteoblast-like cells.


Receptors for N-acetylated sugars may stimulate adenylate cyclase to sensitize and tune mechanoreceptors involved in triggering nematocyst discharge.

In fishing tentacles of sea anemones, cnidocyte/supporting cell complexes (CSCCs) trigger the discharge of nematocysts following stimulation by swimming prey of specific mechanoreceptors and chemoreceptors located on the supporting cells. Two types of mechanoreceptors have been identified: a contact-sensitive mechanoreceptor (CSM), and a vibration-sensitive mechanoreceptor (VSM). The CSMs become predisposed to initiate nematocyst discharge into static (i.e., nonvibrating) test probes in the presence of submicromolar free and conjugated N-acetylated sugars, a process referred to as sensitization. In seawater, the VSMs cause maximal discharge in response to test probes vibrating at 30, 50-55, and 75 Hz, whereas in the presence of submicromolar N-acetylated sugars the VSMs cause maximal discharge into test probes vibrating at 5, 15, 30, and 40 Hz, a process referred to as tuning. Tuning of the VSMs is accompanied by elongation of the stereocilium bundles comprising the VSMs. We report that dibutyryl cyclic-AMP sensitizes CSMs and tunes VSMs to the lower frequencies of 5, 15, 30, and 40 Hz, while cyclic-AMP has no such effects. Endogenous adenylate cyclase activity at the apical plasma membrane of the supporting cells is detectable by cytochemical methods in the presence of N-acetylated sugars but not in seawater alone. By activating adenylate cyclase with L858051, an analogue of forskolin, or by activating the stimulatory form of G proteins (Gs) with cholera toxin, CSCCs are induced to sensitize CSMs and to tune VSMs to the lower frequencies of 5, 15, 30, and 40 Hz. Caged GTP-gamma S also sensitizes CSMs but tunes VSMs to 5, 15, 30, 40, 55, 65, and 75 Hz, suggesting that VSM tuning may be regulated both by Gs and inhibitory G-proteins. Together, these results implicate cAMP as the second messenger for activated supporting cell chemoreceptors involved in sensitizing the CSMs and tuning the VSMs to lower frequencies.


Diagnosis of nasopharyngeal carcinoma by DNA amplification of tissue obtained by fine-needle aspiration.

In nasopharyngeal carcinoma the primary lesion is often difficult to find. Metastatic lesions occur frequently but are difficult to distinguish from other head and neck tumors. The viral genome of the Epstein-Barr virus (EBV) can be identified in the cells of this carcinoma. We used the polymerase chain reaction (PCR) to test for the presence of EBV genomes in 15 samples of metastatic squamous-cell carcinoma of the neck obtained by fine-needle aspiration and in 26 samples obtained by biopsy of lymph nodes. For controls we used disease-free lymph nodes from 10 patients with various head and neck tumors, tonsillar tissue from 46 subjects, blood from 59 EBV-seropositive blood donors, and mononuclear cells from 8 patients with fatal lymphoproliferative lesions. Of the 41 malignant lesions examined, only the nine nasopharyngeal carcinomas (one primary lesion and eight metastases) contained EBV genomes. None of the 20 nodes with other types of cancer, the 10 disease-free nodes, or any of the 105 normal control samples contained detectable EBV. In two patients with suspected metastases from occult primary tumors, the presence of EBV was predictive of nasopharyngeal carcinoma; in both cases overt nasopharyngeal carcinoma developed within one year. In patients with suspected nasopharyngeal carcinoma, fine-needle aspiration can provide tissue for diagnosis by DNA amplification of EBV genomes. The presence of EBV in metastases from an occult primary tumor is predictive of the development of overt nasopharyngeal carcinoma.


Detection of hepatitis C virus antibody in the absence of viral RNA in patients with autoimmune hepatitis.

To determine whether laboratory findings showing antibodies to hepatitis C virus (HCV) in patients with autoimmune hepatitis represent false-positive results and to identify possible explanations for true-positive results in these patients. Cross-sectional. University-based hospital. Fifty-two patients with non-A, non-B chronic hepatitis as a control group and 26 patients with classic chronic active autoimmune hepatitis. Comparison of the results of five kinds of assays of HCV antibodies and HCV RNA. Of 52 patients with non-A, non-B chronic hepatitis, HCV antibodies (anti-HCV) were detected in 42 patients (81%; 95% CI, 67% to 90%) by a first-generation enzyme-linked immunosorbent assay (ELISA-I), in 39 patients (75%) by Sp42 ELISA, in 37 patients (71%) by RIA-I, in 49 patients (94%) by ELISA-II, and in 48 patients (92%) by RIBA-II. We found HCV RNA in 47 patients (90%; CI, 79% to 97%). Of the 26 patients with autoimmune hepatitis, anti-HCV were detected in 23 patients (88%; CI, 70% to 98%) by ELISA-I, in 12 (46%) by both RIA-I and Sp42 ELISA, in 20 (77%) by ELISA-II, and in 9 (35%) by RIBA-II. However, HCV RNA was found in only five of these patients (19%; CI, 7% to 39%). None of our patients, including controls, had antibodies to superoxide dismutase. Of the 21 patients who had autoimmune hepatitis that was completely responsive to steroid therapy, 18 had anti-HCV by ELISA-I, but 13 of these patients had negative results by RIBA-II, and only two patients had HCV RNA. Of the five patients who did not respond to steroid treatment, all had anti-HCV by ELISA-I, four had negative results by RIBA-II, and three had HCV RNA. Testing for HCV antibodies in patients with autoimmune hepatitis frequently elicits positive results when the ELISA-I or ELISA-II tests are used. Most of these appear to represent false-positive results because HCV RNA is usually absent from the serum. Such false positivity may result from previous infection with HCV or from cross-reaction of an epitope of HCV. Other patients with apparent autoimmune hepatitis who fail to respond to corticosteroid therapy may actually have chronic hepatitis C (or other non-A, non-B hepatitis) infection.


Reducing the risk for transfusion-transmitted cytomegalovirus infection.

To define the groups of patients at risk for transfusion-transmitted cytomegalovirus infection and to define the methods to reduce this risk. English-language publications on transfusion medicine. Studies were selected that described cytomegalovirus infection in transfusion-dependent patients. Special attention was paid to reports that included observations about the prevalence and clinical manifestations of cytomegalovirus infection and recommendations for the prevention of infection. Some patients with impaired immune responses who have never been exposed to cytomegalovirus are at risk for transfusion-transmitted cytomegalovirus infection. This infection, which is associated with substantial morbidity and mortality, can be avoided by additional screening of blood donors or by special processing of components for transfusion. Transfusion products that are unlikely to transmit cytomegalovirus infection can be prepared by filtration to remove leukocytes or can be obtained by selecting donors who are seronegative for antibodies to cytomegalovirus. These products are indicated for certain groups of immunosuppressed patients, including pregnant women who are cytomegalovirus seronegative, premature infants of low birth weight who are born to cytomegalovirus-seronegative mothers, cytomegalovirus-seronegative recipients of allogeneic bone marrow transplants from cytomegalovirus-seronegative donors, and cytomegalovirus-seronegative patients with the acquired immunodeficiency syndrome (AIDS).


Heterogeneity of radiosensitivity in a human glioma cell line.

Sixteen clones were isolated from an early-passage human glioma cell line (IN859) and have been found to show variation in several biological characteristics including DNA content, modal chromosome number, and morphology. In addition, heterogeneity of radiosensitivity was detected: the doses that gave a surviving fraction of 0.01 varied by a factor of approximately 1.5. The most sensitive (clone 6) and the most resistant (clone 9) clones were selected for further study; their surviving fractions at 2Gy (SF2) were 0.37 and 0.64, respectively. When compared at a fixed radiation dose the sensitive clone surprisingly demonstrated greater split-dose recovery than the resistant clone; it also showed greater low dose-rate sparing.


Malignant astrocytomas treated with iodine-125 labeled monoclonal antibody 425 against epidermal growth factor receptor: a phase II trial.

Twenty-five patients with primary presentation of malignant astrocytoma, astrocytoma with anaplastic foci, and glioblastoma multiforme were treated with surgical resection and definitive radiation therapy followed by intravenous or intra-arterial administration of Iodine-125 labeled monoclonal antibody-425, which binds specifically to human epidermal growth factor receptor. The patients presented with primary untreated disease, positive contrast enhanced computed tomography scans of the brain, and compatible clinical symptoms. In this Phase II clinical trial, the patients had surgical debulking or biopsy followed by definitively administered external beam radiation therapy and one or multiple doses (35 to 90 mCi per infusion) of radiolabeled antibody. The total cumulative doses ranged from 40 to 224 mCi. The administrations of the radiolabeled antibody were performed in most cases 4-6 weeks following completion of the primary surgery and radiation therapy. Ten patients had astrocytoma with anaplastic foci and 15 had glioblastoma multiforme. No significant life-threatening toxicities were observed during this trial. At 1 year 60% of the patients with astrocytoma with anaplastic foci or glioblastoma multiforme are alive. The median survival for both groups was 15.6 months.


Long term tolerance of thoracic organs to intraoperative radiotherapy.

The tolerance of mediastinal structures to intraoperative radiotherapy (IORT) was investigated in 3 separate animals trials using 49 adult foxhounds and one limited Phase I trial in 4 patients with Stage II or III non-small cell lung cancer (NSCLC). The 1- to 2-year results of these trials have been previously reported with significant toxicity found at dose levels over 20 Gy. We now report the results of five dogs reserved for long term studies and one Stage II NSCLC patient alive at 5 years. Two dogs received 20 Gy IORT and one received 30 Gy IORT to the esophagus, all three to a single 6 cm field with 9 MeV electrons. One control dog underwent surgery without irradiation. One dog received 20 Gy IORT to a single 5 cm mediastinal field with 13 MeV electrons following left pneumonectomy. At 5 years, all five dogs reserved for a long term evaluation were alive and evaluable with minimal endoscopic and radiographic abnormalities. The one patient alive at 5 years for evaluation received 25 Gy IORT to two matched 6 cm fields with 13 MeV electrons. She has stable dyspnea on exertion and there is no evidence of cancer by endoscopy. We conclude, based on these limited data, that IORT in the mediastinum may be safe at dose levels that do not exceed 20 Gy, and further careful evaluation at these lower treatment doses is warranted to determine efficacy.


Calcium channels and nifedipine inhibition of serotonin-induced [3H]thymidine incorporation in cultured cerebral smooth muscle cells.

Cultures of smooth muscle cells were prepared from the basilar artery of adult guinea pigs. Passaged cultures (10-30 passages) that expressed serotonin receptors were studied using [3H]thymidine incorporation. When tested in quiescent medium, serotonin potently stimulated [3H]thymidine incorporation (EC50 of 31 nM) by as much as 400% at 24 h. The number of cells was not significantly increased at 24 or 48 h. At concentrations of 10(-8)-10(-5) M 5-HT, [3H]thymidine uptake was reduced 40-50% by the dihydropyridine Ca2+ channel blocker, nifedipine (1 microM). To demonstrate a possible mechanism for the sensitivity to nifedipine, Ca2+ currents were measured using the whole cell patch clamp technique. The cells expressed dihydropyridine-sensitive L-type Ca2+ channels, but not other subtypes of Ca2+ channels, as indicated by the kinetic and voltage-dependent characteristics of the current and by the stimulatory effect of Bay K 8644. The magnitude of the Ca2+ currents was related exponentially to the membrane surface area, measured as cell capacitance. These data support the association of dihydropyridine-sensitive Ca2+ channels with mitogenesis in vascular smooth muscle, and suggest an alternate mechanism of action for the beneficial effect of dihydropyridines in prophylaxis of cerebral vasospasm.


Successful management of inferior vena cava and right atrial nephroblastoma tumor thrombus with preoperative chemotherapy.

Involvement of inferior vena cava (IVC) by tumor thrombus occurs in up to 10% of patients with nephroblastoma. Right atrial involvement by tumor thrombus is much less frequent. Four patients presenting with advanced nephroblastoma were diagnosed as having IVC involvement with tumor thrombus. Two of these patients had in addition thrombus extending up to the right atrium. All 4 patients were treated with preoperative chemotherapy (vincristine, actinomycin D, 4-epi-Adriamycin). Nephrectomy was subsequently performed without undue difficulty on all 4 patients. The intravascular tumor thrombus had completely cleared in all 4 patients and most of the primary renal tumor was necrotic. The results obtained with preoperative chemotherapy as given to these patients mediates strongly against difficult surgery being undertaken as primary treatment for such patients.


Hepatocellular carcinoma associated with arteriohepatic dysplasia in a 4-year-old girl.

Hepatocellular carcinoma and obliterated hepatic bile duct were found at postmortem examination in a 4-year-old girl with arteriohepatic dysplasia (Alagille's syndrome). AFP level was extremely high. Liver cirrhosis was present on percutaneous needle biopsy 9 months before she succumbed in progressive liver failure. Episodes of repeated gastrointestinal, life-threatening hemorrhages occurred during the last 6 months of her life. Histopathologic findings of the eyes were documented at autopsy.


Nitric oxide as a mediator of relaxation of the corpus cavernosum in response to nonadrenergic, noncholinergic neurotransmission.

Nitric oxide has been identified as an endothelium-derived relaxing factor in blood vessels. We tried to determine whether it is involved in the relaxation of the corpus cavernosum that allows penile erection. The relaxation of this smooth muscle is known to occur in response to stimulation by nonadrenergic, noncholinergic neurons. We studied strips of corpus cavernosum tissue obtained from 21 men in whom penile prostheses were inserted because of impotence. The mounted smooth-muscle specimens were pretreated with guanethidine and atropine and submaximally contracted with phenylephrine. We then studied the smooth-muscle relaxant responses to stimulation by an electrical field and to nitric oxide. Electrical-field stimulation caused a marked, transient, frequency-dependent relaxation of the corpus cavernosum that was inhibited in the presence of N-nitro-L-arginine and N-amino-L-arginine, which selectively inhibit the biosynthesis of nitric oxide from L-arginine. The addition of excess L-arginine, but not D-arginine, largely reversed these inhibitory effects. The specific liberation of nitric oxide (by S-nitroso-N-acetylpenicillamine) caused rapid, complete, and concentration-dependent relaxation of the corpus cavernosum. The relaxation caused by either electrical stimulation or nitric oxide was enhanced by a selective inhibitor of cyclic guanosine monophosphate (GMP) phosphodiesterase (M&B 22,948). Relaxation was inhibited by methylene blue, which inhibits cyclic GMP synthesis. Our findings support the hypothesis that nitric oxide is involved in the nonadrenergic, noncholinergic neurotransmission that leads to the smooth-muscle relaxation in the corpus cavernosum that permits penile erection. Defects in this pathway may cause some forms of impotence.


The treatment of lower extremity sarcomas with wide excision, radiotherapy, and free-flap reconstruction.

Treatment of extremity sarcomas has evolved into a multidisciplinary approach utilizing surgery, radiotherapy, and, in some cases, chemotherapy. Limb-sparing surgery has maintained low rates of local recurrence when supplemented with early postoperative radiotherapy (brachytherapy). Leg defects that result from resection resemble those caused by trauma and appear ideally suited to free-flap reconstruction. However, the resection site is subjected to 4500 cGy of radiation given within 2 weeks of surgery. It has not been demonstrated that free flaps can endure early postoperative radiation without adverse effects. Three patients are presented with locally recurrent leg sarcomas treated by wide excision, brachytherapy, and free-flap reconstruction. All flaps survived, and the wounds healed uneventfully. This study reviews the current multidisciplinary approach to the treatment of lower extremity sarcomas and demonstrates the durability of free-flap reconstruction in the presence of early postoperative radiation therapy.


Imaging of nephroblastomatosis: an overview.

Nephroblastomatosis is an abnormality of nephrogenesis characterized by incomplete maturation of primitive nephrogenic cells. The resultant individual lesions have variable histologic findings and distribution in the kidney. There is wide acceptance of nephroblastomatosis as a precursor lesion to Wilms tumor. Consequently, the role of imaging in assessing patients for neoplastic transformation of nephroblastomatosis should be emphasized. Computed tomography (CT) has been shown to be superior to ultrasound (US) in this regard. Nonetheless, the low cost and lack of radiation of US make it attractive for serial screening studies. Although magnetic resonance imaging shows promise in assessment of the kidney for nephrogenic rests, experience is limited in its use in evaluating patients for nephroblastomatosis. The authors present protocols for assessing patients identified as being at either high or low risk for the development of Wilms tumor due to nephroblastomatosis. These protocols take into account the advantages of CT and US. Scanning intervals are determined from epidemiologic data on the occurrence of Wilms tumor; the most intense screening occurs during the younger ages of life, when the likelihood of Wilms tumor development is highest.


Hepatocellular carcinoma: reduced frequency of typical findings with dynamic contrast-enhanced CT in a non-Asian population.

Four computed tomographic (CT) findings have been described as typical or diagnostic of hepatocellular carcinoma: Primary morphologic findings include tumor encapsulation and fatty metamorphosis and secondary findings include venous invasion and arterioportal shunting. However, virtually all of the articles reporting these findings with significant frequency have been from Japan and thus represent an almost purely Asian population. The authors analyzed a series of 93 patients with hepatocellular carcinoma, only eight of whom were Asian. With the exception of venous invasion (48%), the analysis showed that while the four CT features may be typical of hepatocellular carcinoma, three features occurred with low frequency in a non-Asian population: tumor encapsulation, 12%; fatty metamorphosis, 0%; and arterioportal shunting, 4%. In addition, a review of the literature showed that all of the findings have occurred in other hepatic neoplasms, including benign and metastatic tumors.


Hepatocellular carcinoma: evaluation with color Doppler US and MR imaging.

Fifteen patients with hepatocellular carcinoma underwent examination with color Doppler ultrasound (US), magnetic resonance (MR) imaging, dynamic computed tomography (CT), and angiography. Doppler signals ranging from 0.22 to 3.48 kHz could be obtained from within the tumor in 14 of 15 patients. The resistive index was 0.38-0.77. Color Doppler signals were visualized in nine of 15 lesions with a Doppler shift greater than 0.7 kHz. The Doppler signals and the resistive index of the tumor vessels became lower as the vessels progressed into the center of the lesion. The appearance of tumor vessels recognized on MR images obtained with gradient-recalled acquisition in the steady state (GRASS) in 11 of 15 lesions was compared with that on CT scans and angiograms. Tumors that were hyperintense on GRASS images obtained with a flip angle of 15 degrees transmitted Doppler signals of considerably higher amplitude compared with the isointense lesions. Both color Doppler US and MR imaging provided useful information for characterizing intratumoral blood flow.


Small hepatocellular carcinoma: diagnosis with US angiography with intraarterial CO2 microbubbles.

Ultrasonographic (US) angiography enhanced with intraarterial CO2 microbubbles, a contrast material used in US imaging, was performed of 103 histologically proved hepatocellular carcinomas (HCCs) smaller than 3 cm in diameter in 95 patients. The detection rate for hypervascular HCC with US angiography was compared with the rate of detection with conventional angiography, digital subtraction angiography (DSA), and computed tomography (CT) after intraarterial injection of iodized oil. Sensitivity in detection of hypervascular HCCs with US angiography was 86% (89 of 103 HCCs), compared with 63% (44 of 70 HCCs) detected with conventional angiography, 70% (23 of 33 HCCs) with DSA, and 82% (75 of 91 HCCs) with CT with iodized oil. US angiography depicted small hypervascular HCCs, especially those less than 1 cm in diameter, and helped clarify vascularity as isovascular or hypovascular in angiographically undetectable HCCs. Findings at US angiography assisted the choice of a therapeutic strategy for treatment of HCC, such as transarterial therapy, percutaneous ethanol injection therapy, or resection.


Small hepatocellular carcinoma: differentiation from adenomatous hyperplastic nodule with color Doppler flow imaging.

Liver nodules smaller than 25 mm in diameter (19 hepatocellular carcinomas [HCCs] and nine adenomatous hyperplastic nodules [AHNs]) were examined with color Doppler flow imaging and hepatic angiography. Angiography revealed a tumor vessel in eight (42%) of the 19 HCCs, while color Doppler flow imaging revealed an arterial pulsating afferent tumor vessel in 10 (53%) of the 19 HCCs but in none of the AHNs. In addition, color Doppler flow imaging revealed a constant-flow efferent tumor vessel continuing to a portal branch in 10 (53%) of the 19 HCCs but in none of the AHNs. In 15 (79%) of the 19 HCCs, a pulsating afferent tumor vessel or a constant-flow efferent tumor vessel or both were observed. Therefore, in this series, color Doppler flow imaging was of value in distinguishing between these two lesions.


Focal liver lesions: MR imaging with Mn-DPDP--initial clinical results in 40 patients.

Manganese (II) N,N'-dipyridoxylethylenediamine-N,N'-diacetate-5,5'-bis(phosphate) (DPDP) was evaluated as a contrast agent for magnetic resonance (MR) imaging (1.5 T) of focal liver lesions in 40 patients. Doses of 5 and 10 mumol/kg were administered intravenously. Mn-DPDP-enhanced T1-weighted images were compared quantitatively and subjectively with standard T1- and T2-weighted nonenhanced images. Use of Mn-DPDP resulted in a statistically significant increase in signal intensity of liver parenchyma in T1-weighted images at both doses. No enhancement was seen in metastases, cholangiocarcinomas, or lymphomas, while all hepatocellular carcinomas were enhanced. Enhancement was seen in focal nodular hyperplasia and in regenerative nodules. The lesion-to-liver contrast in Mn-DPDP-enhanced gradient-recalled-echo images was superior to that of all precontrast images (P less than .01). The number of nonenhancing malignant liver lesions detected in spin-echo (SE) images was increased (272 in T2-weighted SE images vs 390 in T1-weighted Mn-DPDP-enhanced SE images). Image interpretation (eg, visualization and demarcation of the lesions) was markedly better in Mn-DPDP-enhanced images than in all precontrast images (P less than .001).


Sonography with intraarterial infusion of carbon dioxide microbubbles (sonographic angiography): value in differential diagnosis of hepatic tumors.

Differential diagnosis of small liver tumors is important, but is not always possible, even with angiography. To solve this problem, we introduced sonographic angiography, which combines sonography and angiography. The vascular pattern of a variety of hepatic nodules was evaluated with sonographic angiography, and the results were compared with those of conventional angiography. Sonographic angiography (sonography performed during intraarterial infusion of carbon dioxide microbubbles) was performed in 184 patients with a total of 222 hepatic nodules: 139 hepatocellular carcinomas, nine adenomatous hyperplasias, seven regenerative nodules, 21 hemangiomas, 33 metastases, seven lymphomas, one granuloma, and five focal nodular hyperplasias. Sonographic angiography detected a hypervascular pattern with peripheral blood supply in cases of hepatocellular carcinoma (sensitivity, 90%; specificity, 89%). Typical vascular patterns of adenomatous hyperplasia, hemangioma, metastasis, and focal nodular hyperplasia on sonographic angiography were hypovascularity (sensitivity, 100%; specificity, 91%), spotty pooling (sensitivity, 100%; specificity, 100%), peripheral hypervascularity (sensitivity, 64%; specificity, 100%), respectively. The detectability of hypervascularity was greater with sonographic angiography than with conventional angiography in hepatocellular carcinoma, metastasis, and hemangioma. Our experience indicates that sonographic angiography depicts characteristic vascular features that reflect the vascular anatomy of specific types of hepatic tumors, and thus is useful in the differential diagnosis of these lesions.


Energy-dependent processes involved in reduced drug accumulation in multidrug-resistant human lung cancer cell lines without P-glycoprotein expression.

Mechanisms contributing to reduced cytotoxic drug accumulation were studied in two multidrug-resistant (MDR) human lung cancer cell lines without P-glycoprotein expression. In these (non-small cell) SW-1573/2R120 and (small cell) GLC4/ADR MDR cells, the steady-state accumulation of [14C]daunorubicin was 30 and 12%, respectively, of that in the parent cells. When cells, at steady state, were permeabilized with digitonin, the amount of daunorubicin binding increased only in the resistant cells. The reduced accumulation of daunorubicin in the SW-1573/2R120 and GLC4/ADR cells was accompanied by a lower initial (2 min) uptake rate of this drug. No difference in initial efflux rate of daunorubicin from preloaded cells could be detected between sensitive and resistant SW-1573 cells. However, daunorubicin was extruded 5-fold faster from GLC4/ADR cells than from the parental cells. In the presence of the energy metabolism inhibitors sodium azide and deoxyglucose, the reduced daunorubicin accumulations in the SW-1573/2R120 and GLC4/ADR MDR cells were (almost) completely reversed. The effects of these inhibitors on drug uptake were already apparent during the earliest measured time points (less than 15 s). Also, the enhanced efflux of daunorubicin from GLC4/ADR cells was inhibited. In ATP-depleted cells, the intracellular pH was lowered by approximately 0.3 units in resistant as well as in sensitive cells. The lower intracellular pH, however, could not account for the increase in daunorubicin accumulation in the resistant cells. Also, for vincristine and etoposide, the increases in drug accumulation under energy-deprived conditions were more pronounced in the resistant SW-1573/2R120 cells than in the parent SW-1573 cells. These results suggest that accumulation of drugs in the non-P-glycoprotein MDR human lung carcinoma cell lines SW-1573/2R120 and GLC4/ADR is reduced by an energy-dependent drug export mechanism which prevents efficient transport of drug to the target. Since P-glycoprotein expression in lung tumors is generally low, these MDR lung cancer cell lines can be used as a model to study alternative mechanisms leading to multidrug resistance in this tumor type.


ras transformation of simian virus 40-immortalized rat hepatocytes: an in vitro model of hepatocarcinogenesis.

Primary rat hepatocytes were transfected with simian virus 40 DNA and cultured in a chemically defined medium. Proliferating colonies developed after 2-3 weeks. Three cell lines were established by cloning albumin-secreting colonies, as identified by an immunooverlay assay. Two of the cell lines, ALB-6 and ALB-8, expressed all five liver-specific mRNAs studied, albumin, alpha-1-antitrypsin, fibrinogen, alpha-1-acid glycoprotein, and histidase. ALB-6 cells were nontumorigenic in nude mice while ALB-8 cells were weakly tumorigenic with only one of four injected nude mice developing a slowly growing tumor. Further transfection of ALB-6 and ALB-8 cells with an activated c-Ha-ras or N-ras oncogene resulted in strongly tumorigenic cells. The tumors induced by ras-transformed ALB-6 cells were moderately differentiated hepatocellular carcinomas. The tumors derived from ras-transformed ALB-8 cells were poorly differentiated, while the slowly growing tumors induced by untransfected or control DNA-transfected ALB-8 cells were well-differentiated trabecular hepatocellular carcinomas, suggesting histological dedifferentiation of cells following ras transformation. However, the synthetic capabilities of the cells were not lost in that the ras-transfected cultures and the tumors induced by ras-transformed cells retained the ability to synthesize the five liver-specific mRNAs. Thus we have developed an in vitro model of carcinogenesis in which, by sequential exposure to SV40 DNA and a ras oncogene, primary rat hepatocytes are transformed.


Relative activity of structural analogues of amsacrine against human leukemia cell lines containing amsacrine-sensitive or -resistant forms of topoisomerase II: use of computer simulations in new drug development.

Anilino analogues of amsacrine showed increased activity against amsacrine (AMSA)-resistant cell lines when compared with the parent compound, but the mechanisms of amsacrine resistance in these lines were unknown (Finlay, G. J., Baguley, B. C., Snow, K., and Judd, W., J. Natl. Cancer Inst., 82: 662-667, 1990). We tested the cytotoxic and DNA-cleaving activities of two amsacrine analogues which were derivatives of 9-anilinoacridine (1'-methylcarbamate and 1'-benzenesulfonamide) against an amsacrine-resistant human leukemia cell line (HL-60/AMSA) whose resistance is due to an amsacrine-resistant topoisomerase II. Neither agent could overcome the amsacrine resistance of HL-60/AMSA. Neither agent could induce HL-60/AMSA topoisomerase II-mediated cleavage of DNA in an isolated biochemical system, although at high concentrations the two analogues could inhibit HL-60/AMSA topoisomerase II-mediated DNA strand passage. Both analogues were at least as active, if not more active, than amsacrine against amsacrine-sensitive HL-60 and its topoisomerase II. Comparison of the cellular and biochemical results with those from computer simulation of the energy-minimized structures of amsacrine, its inactive isomer o-AMSA, and the two new active analogues suggests the following possibilities: (a) the positioning of the potential topoisomerase II-binding site (1'-anilino group) of the two new drugs resembles the positioning of this site in amsacrine; (b) the HL-60 topoisomerase II has a binding site which interacts with amsacrine and the two anilino analogues but not with o-AMSA, an analogue with altered positioning of the methoxy group; (c) the HL-60/AMSA topoisomerase II interacts with reduced affinity with amsacrine and the two anilino analogues, although HL-60/AMSA topoisomerase II still interacts with the structurally distinct topoisomerase II-reactive nonintercalator, etoposide; (d) because of their higher DNA binding affinity or the greater possible positions of their side groups in comparison to amsacrine, the two analogues can, at high concentrations, inhibit the strand-passing activity of HL-60/AMSA topoisomerase II.


Antitumor activity of basic fibroblast growth factor-saporin mitotoxin in vitro and in vivo.

Many cancer cell lines express basic fibroblast growth factor (FGF) receptors, making them potential targets for the delivery of FGF-based cytotoxic compounds. To this end, we have investigated the antitumor activity of a novel mitotoxin, Fibroblast Growth Factor-saporin (FGF-SAP), a conjugate of FGF and the ribosome-inactivating protein, saporin. In vitro, FGF-SAP is cytotoxic for human melanoma, teratocarcinoma, and neuroblastoma cells expressing FGF-receptors. Mice treated with FGF-SAP i.v., on a variety of schedules, showed dramatic tumor growth inhibition with minimal toxicity. Thus, FGF-SAP appears to be a well-tolerated and potent antitumor agent. The potential of FGF-targeted cytotoxicity is discussed.


Topoisomerase II alpha and topoisomerase II beta genes: characterization and mapping to human chromosomes 17 and 3, respectively.

Human cells contain two topoisomerase II isozymes named topo II alpha and topo II beta. The complementary DNAs for both enzymes have been cloned. The topo II alpha and topo II beta complementary DNAs hybridized to unique sequences of human, rodent, and chicken DNAs in Southern blots. The human topo II alpha gene has previously been mapped to chromosome 17. We confirmed the chromosomal location of topo II alpha and mapped the topo II beta gene to chromosome 3. In addition, topo II beta exhibits genetic polymorphism as has been reported for topoisomerases I and II alpha.


Neuropeptide signal transduction in lung cancer: clinical implications of bradykinin sensitivity and overall heterogeneity.

To define the role of neuropeptides in lung cancer biology, we evaluated the effect of seven peptide classes on signal transduction and growth in human lung and breast cancer cell lines. Flow cytometric methods were used to quantitate the calcium response in individual cells produced by these peptides alone or in combination. The effects on growth were assessed by [3H]thymidine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and soft agarose colony assays. All lung cancer cells demonstrated calcium responses to one or more peptides with classic small cell lines displaying the greatest responsiveness, followed by variant small cell lines and non-small cell lines. Breast cancer cell lines demonstrated little or no response. There was great variability in the magnitude of calcium response and pattern of response between lung cancer cell lines to individual neuropeptides. Bradykinin was the most potent peptide and produced responses in the highest fraction of lung cancer cell lines. Combinations of peptides produced greater intracellular calcium release than the single peptides, although in less than a quantitatively additive manner. Each peptide produced a refractory period which was peptide class specific. The growth stimulating effects of these neuropeptides were absent or small in magnitude and did not correlate with calcium signal transduction. These results imply that lung cancer cells display a wide sensitivity to neuropeptides but in a very heterogeneous manner. Knowledge of this heterogeneity should be incorporated into the design of antitumor strategies based on this autocrine pathway.


High levels of enhanced reactivation of herpes simplex virus in skin fibroblasts from various hereditary cancer-prone syndromes.

The dose response of the enhanced reactivation (ER) of herpes simplex virus type 1 has been studied in UV-irradiated normal human skin fibroblasts and fibroblasts from the following hereditary cancer-prone syndromes: retinoblastoma, aniridia, polyposis coli, neurofibromatosis type 1 and 2, dysplastic nevus syndrome, Von Hippel-Lindau syndrome, multiple endocrine neoplasia type 2, and Bloom's syndrome. Surprisingly, much higher levels of ER were observed in all these genetically heterogeneous hereditary disorders than in normal human skin fibroblasts. These results suggest that loss of one allele of putative tumor suppressor genes may activate cellular processes that result in the induction of the ER response, and they support our previous observation suggesting that ER may somehow be related to the process of carcinogenesis (P. J. Abrahams et al., Cancer Res., 48: 6054-6057, 1988).


Measurement of SO2 effects on the 218Po ion mobility spectrum by alpha-track detection.

A number of investigators have reported the formation of radiolytic ultrafine particles produced by the interaction of ionizing radiation with atmospheric trace gases. Previous studies have suggested that a very high localized concentration of the OH radical produced by the radiolysis of water can react with atmospheric trace gas, like SO2, to produce lower vapor pressure compounds that can then nucleate. To determine trace-gas dependence of the active, positively charged, first decay product of radon, the mobility spectrum of the decay products in the range of 0.06-3.0 cm2 V-1s-1 was measured in a flow-through radon chamber using a specially designed mobility spectrometer. Measurements were taken for different relative humidities and concentrations of SO2 in purified laboratory compressed air. The resultant mobility spectra were compared with radiolytic ultrafine particle activity distribution data. In the case of low humidities, the reduction of available OH concentration and hence neutralization rate led to the increasing intensity of the shoulder around 1.35 cm2 V-1s-1 with increasing SO2, suggesting SO2 clustering around the PoOx+ ion. For high humidity conditions (5 ppm SO2, 30% RH), there was clear droplet formation, H2SO4, clustering around the ion, and also an increase in the background level between the high mobility peak and droplet peak.


Increased cerebrospinal fluid dopamine and 3,4-dihydroxyphenylacetic acid levels in Huntington's disease: evidence for an overactive dopaminergic brain transmission.

Levels of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), noradrenaline (NA), 3-methoxy-4-hydroxyphenylglycol (MHPG), and 5-hydroxyindoleacetic acid (5-HIAA) in the CSF of patients with Huntington's disease (HD) were measured by HPLC. CSF DA, DOPAC, and MHPG levels were found to be increased in HD patients. Levels of HVA, 5-HIAA, and NA in the CSF of HD patients did not differ from those of controls. Changes in CSF DA and DOPAC levels were consistent with previous findings of increased DA tissue content in some brain areas of patients with HD. These results suggest that CSF DOPAC levels could be a more reliable index of overactive dopaminergic brain systems in HD than CSF HVA levels.


Terminal autoreceptor control of 5-hydroxytryptamine release as measured by in vivo microdialysis in the conscious guinea-pig.

In vivo microdialysis in the frontal cortex of the freely moving guinea-pig was used to measure extracellular 5-hydroxytryptamine (5-HT) and study terminal autoreceptor control of its release. The indoleamine levels were determined by HPLC with electrochemical detection. Release of extracellular 5-HT and, to a lesser extent, 5-hydroxyindoleacetic acid was sensitive to tetrodotoxin, confirming the neuronal origin of measured neurotransmitter levels. Both systemic and local administration of the 5-HT1 agonist 5-carboxamidotryptamine caused inhibition of extracellular 5-HT levels, confirming the regulatory role of the terminal, and possibly also the somatodendritic, 5-HT autoreceptor on neuronal 5-HT release. Levels of extracellular 5-hydroxyindoleacetic acid were not affected by 5-carboxamidotryptamine following either central or peripheral administration.


Differential responses of the phosphorylation of ribosomal protein S6 to nerve growth factor and epidermal growth factor in PC12 cells.

Previous studies from this laboratory have shown that the phosphorylation of the S6 protein of the ribosomes is catalyzed by at least two different and separable kinase activities in PC12 cells. One of these activities is increased by treatment of the cells with nerve growth factor, the other by treatment of the cells with epidermal growth factor. The present work shows that these two factors stimulate the phosphorylation of S6 with quite different kinetics, and that both the number of phosphates incorporated into S6 and the phosphopeptide pattern of S6 are different in cells treated with nerve growth factor than in cells treated with epidermal growth factor. The characteristics of the nerve growth factor-sensitive S6 kinase and of the epidermal growth factor-sensitive kinase were also clearly different. Substrate specificity and inhibitor studies indicated that neither was identical to cyclic AMP-dependent kinase, kinase C, or the calcium/calmodulin-dependent kinases. However, two major phosphopeptides produced by S6 phosphorylation in nerve growth factor-treated cells were also seen on phosphorylation of S6 by cyclic AMP-dependent kinase in vitro. In addition, when rat liver 40S ribosomal subunits were pretreated with cyclic AMP-dependent kinase in vitro, the action of the nerve growth factor-sensitive S6 kinase was increased about twofold.


Human brain lectin: a soluble lectin that binds actin.

A biotinylated probe was used for detection of endogenous ligands of a human brain lectin on blotted human brain soluble proteins. Of the various proteins from brain extract resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, five reacted with the biotinylated probe. After elimination of saccharidic moieties by periodic treatment of the same extract, a single band with Mr approximately 43,000 was recognized by the lectin. This band was identified as actin using an anti-actin antibody. These results were confirmed by binding of biotinylated lectin to purified actin.


Forskolin stimulation of cyclic AMP accumulation in rat brain cortex slices is markedly enhanced by endogenous adenosine.

Stimulation of cyclic AMP (cAMP) accumulation in rat cortex slices by 1 microM forskolin (F) was markedly reduced (96%) by treatment with adenosine deaminase (ADA). The effect of ADA was progressively less at higher concentrations of F, but still inhibited the response by 50% at 100 microM F. ADA-mediated inhibition of the cAMP response to 1 microM F was completely reversed by 5 microM 2-chloroadenosine (CA), an ADA-resistant analogue. Stimulation by F (controls) and F plus CA (ADA treated) in cortex slices was significantly inhibited by 200 microM caffeine (CAF) and by 10 microM 8-phenyltheophylline. cAMP accumulation in ADA-treated cortex slices stimulated with CA at concentrations from 5 to 100 microM was markedly enhanced by 1 microM F. Neither ADA treatment nor 200 microM CAF significantly affected cAMP accumulation in slices stimulated by 1 microM vasoactive intestinal polypeptide or adenylate cyclase in membranes stimulated by 1 microM F. CAF (1 mM) did not significantly increase basal cAMP levels in cortex slices, whereas 1 mM 3-isobutyl-1-methylxanthine caused a significant 80% increase and 100 microM rolipram enhanced cAMP levels by 4.5-fold. F-stimulated cAMP accumulation (1 microM) in cortex slices was inhibited 98% by 1 mM CAF and 49% by 1 mM 3-isobutyl-1-methylxanthine, and was enhanced 2.5-fold by 100 microM rolipram. These data have been interpreted to indicate that the stimulation of cAMP accumulation in rat cortex slices by 1 microM F is predominantly due to synergistic interaction with endogenous adenosine and that the inhibition of this response by CAF is largely due to blockade of adenosine receptors.


Expression of functional bradykinin receptors in Xenopus oocytes.

mRNA prepared from various tissues and cultured cells was injected into Xenopus laevis oocytes. Three to five days after injection, the response of the oocytes to the peptide bradykinin was monitored. The oocytes were voltage clamped and the membrane currents generated on application of agonist were recorded. mRNA from NG108-15, rat uterus, and human fibroblast cell line WI38 gave similar responses to bradykinin (1 microM), with an initial inward current (10-20 nA) followed by a prolonged period of membrane current oscillations. The same pattern of response was given by total RNA from rat dorsal root ganglia. No response to bradykinin (10 microM) was recorded from oocytes injected with rat brain mRNA, although these oocytes gave peak inward currents of about 75 nA in response to serotonin (10 microM). mRNA from both NG108-15 cells and rat uterus was fractionated on sucrose gradients. This resulted in an approximately five-fold increase in the size of the response compared to that given by unfractionated mRNA. The largest responses were given by mRNA fractions with a size of approximately 4.5 kb. Data were obtained consistent with the expression of both B1 and B2 receptors by WI38 human fibroblasts and with the expression of only the B2 type of receptor by NG108-15 cells.


Lithium effects on inositol phospholipids and inositol phosphates: evaluation of an in vivo model for assessing polyphosphoinositide turnover in brain.

Administration of lithium chloride to rats injected intracerebrally with [3H]inositol led to time- and dose-dependent increases in levels of labeled inositol monophosphates in brain. Quantitative analysis of the inositol phosphates by ion chromatography revealed 37- and 20-fold increases in the mass of myo-inositol 1-phosphate and 4-phosphate, respectively, at 4 h intraperitoneal after injections of 6 mEq/kg of lithium chloride. Albeit to a much lesser extent, lithium administration also resulted in an increase in the level of myo-inositol, 1,4-bisphosphate in brain. The lithium-induced increase in content of labeled inositol monophosphates was marked by a concomitant decrease in content of labeled inositol, and after injections of high doses of lithium, e.g., 10 mEq/kg, this was followed by a general decrease in labeling of the inositol phospholipids. In general, animals injected with [3H]inositol but not lithium did not reveal obvious differences in labeling of inositol monophosphates on stimulation by mecamylamine or pilocarpine. However, when animals were injected with [3H]inositol and then lithium, there were large increases in the levels of labeled inositol monophosphates on administration of these compounds. Administration of atropine to the lithium-treated mice led to a partial reduction in the amount of labeled inositol monophosphates accumulated due to the administration of lithium alone. Furthermore, atropine was able to block the pilocarpine-induced increase in level of labeled inositol monophosphates. These results demonstrate the suitable use of the radiotracer technique together with lithium administration for assessing the effects of drugs and receptor agonists on the signaling system involving polyphosphoinositide turnover in brain.


Characterization of [3H]Ro 5-4864 binding sites in rat vas deferens.

The presence of benzodiazepine binding sites in rat vas deferens was detected using [3H]Ro 5-4864 as a radioligand. The binding of [3H]Ro 5-4864 to the mitochondrial sites is saturable, reversible, and temperature and time dependent. The association rate constant (k1) was 8.7 +/- 0.7 x 10(7) M-1 min-1, and the dissociation rate constant (k-1) was 0.031 +/- 0.003 min-1. The dissociation constant (KD) determined by saturation binding was 5.22 +/- 0.56 nM. The density of binding was 4,926 +/- 565 fmol/mg of protein. The Hill coefficient of binding was 0.99 +/- 0.01, an indication that [3H]Ro 5-4864 binds to a single site. The [3H]Ro 5-4864 binding was inhibited competitively by Ro 5-4864 and 2-hydroxy-5-nitrobenzyl-6-thioguanosine and noncompetitively by PK 11195, nitrendipine, alpha,beta-methylene-ATP, and carboxyatractyloside and was not affected by clonazepam, dicyclohexylcarbodiimide, or protoporphyrin IX. Our data indicate that [3H]Ro 5-4864 binding sites are not identical to those labeled by PK 11195. These binding sites are modulated by the ADP/ATP mitochondrial carrier, and an interaction of dihydropyridines and [3H]Ro 5-4864 binding sites in rat vas deferens is suggested.


The epithelial phenotype of human neuroblastoma cells express bradykinin, endothelin, and angiotensin II receptors that stimulate phosphoinositide hydrolysis.

The neuroblastoma line SK-N-SH consists of distinct and interconverting cell types, which include a neuroblast phenotype (SH-SY5Y), an epithelial phenotype (SH-EP), and an intermediate cell type (SH-IN). In SH-SY5Y cells, only muscarinic receptor activation produced stimulation of phosphoinositide turnover, whereas in SH-EP cells, where muscarinic receptors are not present, the peptides bradykinin, endothelin, and angiotensin II stimulated phosphoinositide hydrolysis with EC50 values of 16, 6, and 0.7 nM, respectively, and a rank order of maximal effects of bradykinin greater than endothelin greater than angiotensin II. Fetal calf serum at concentrations between 1 and 10% was also a potent stimulator of phosphoinositide hydrolysis in SH-EP cells but not in SH-SY5Y cells. In the intermediate cell clone, SH-IN, phosphoinositide hydrolysis was stimulated not only by muscarinic receptors, but also by endothelin, bradykinin, and serum, an indication that this cell type harbors all the kinds of receptors that are differentially expressed in the other two cell types. The effects of the three peptides--bradykinin, endothelin, and angiotensin II--on phosphoinositide hydrolysis in SH-EP cells were additive, a result suggesting that the three kinds of receptors may activate distinct transducer proteins and/or phospholipase C subtypes. Pretreatment of intact SH-EP cells with pertussis toxin under conditions sufficient to ADP-ribosylate 90-95% of the endogenous guanine nucleotide regulatory protein substrates did not impair the ability of any of the receptors to stimulate phosphoinositide hydrolysis in any of the cell types. In contrast, short-term exposure to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (1 microM) abolished the stimulation of phosphoinositide hydrolysis mediated by peptide receptors in SH-EP cells and partially inhibited that by muscarinic receptors in SH-SY5Y cells. Prolonged incubation of SH-EP cells with phorbol ester resulted in a recovery of receptor responsiveness, the extent and rate of which were different for each receptor type. In contrast, there was no recovery of responsiveness for muscarinic receptors in SH-SY5Y cells. The pattern of phorbol ester-mediated effects depended on the cell rather than on the receptor type. In fact, muscarinic receptor responsiveness in SH-IN, the intermediate cell type, was desensitized by and recovered from treatment with phorbol esters in a manner more similar to peptide receptors in SH-EP than to muscarinic receptors in SH-SY5Y. These data suggest that the transduction mechanisms by which distinct receptor types are coupled to phosphoinositide hydrolysis in the three cell phenotypes differ in sensitivity to feedback regulation by protein kinase C.


Quantitative immunoprecipitation studies with anti-gamma-aminobutyric acidA receptor gamma 2 1-15 Cys antibodies.

Antibodies raised against the synthetic peptide NH2-QKSDDDYEDYASNKTC-COOH (gamma 2 1-15 Cys), which corresponds to the N-terminal amino acid sequence with a C-terminal cysteine of the human gamma 2 subunit of the gamma-aminobutyric acidA (GABAA) receptor, were used to study the quantitative immunoprecipitation of agonist benzodiazepine binding sites from bovine brain. Anti-gamma 2 1-15 Cys antibodies were found to immunoprecipitate specifically in parallel [3H]flunitrazepam- and [3H]muscimol-reversible binding sites in a dose-dependent manner. The maximum percentages of [3H]flunitrazepam binding sites immunoprecipitated from detergent extracts of bovine cerebral cortex, cerebellum, and hippocampus were 68, 77, and 83%, respectively. Immunoprecipitation studies with anti-alpha 1 324-341 antibodies carried out in parallel with anti-gamma 2 1-15 Cys antibodies provided evidence for the promiscuity of the gamma 2 subunit within native GABAA receptors. These results substantiate the association of the gamma 2 polypeptide with native GABAA receptors.


Three pathways of Epstein-Barr virus gene activation from EBNA1-positive latency in B lymphocytes.

Previous studies on Epstein-Barr virus (EBV)-positive B-cell lines have identified two distinct forms of virus latency. Lymphoblastoid cell lines generated by virus-induced transformation of normal B cells in vitro, express the full spectrum of six EBNAs and three latent membrane proteins (LMP1, LMP2A, and LMP2B); furthermore, these lines often contain a small fraction of cells spontaneously entering the lytic cycle. In contrast, Burkitt's lymphoma-derived cell lines retaining the tumor biopsy cell phenotype express only one of the latent proteins, the nuclear antigen EBNA1; such cells do not enter the lytic cycle spontaneously but may be induced to do so by treatment with such agents as tetradecanoyl phorbol acetate and anti-immunoglobulin. The present study set out to determine whether activation of full virus latent-gene expression was a necessary accompaniment to induction of the lytic cycle in Burkitt's lymphoma lines. Detailed analysis of Burkitt's lymphoma lines responding to anti-immunoglobulin treatment revealed three response pathways of EBV gene activation from EBNA1-positive latency. A first, rapid response pathway involves direct entry of cells into the lytic cycle without broadening of the pattern of latent gene expression; thereafter, the three "latent" LMPs are expressed as early lytic cycle antigens. A second, delayed response pathway in another cell subpopulation involves the activation of full latent gene expression and conversion to a lymphoblastoidlike cell phenotype. A third response pathway in yet another subpopulation involves the selective activation of LMPs, with no induction of the lytic cycle and with EBNA expression still restricted to EBNA1; this type of latent infection in B lymphocytes has hitherto not been described. Interestingly, the EBNA1+ LMP+ cells displayed some but not all of the phenotypic changes normally induced by LMP1 expression in a B-cell environment. These studies highlight the existence of four different types of EBV infection in B cells, including three distinct forms of latency, which we now term latency I, latency II, and latency III.


Induction of erythropoietin responsiveness in murine hematopoietic cells by the gag-myb-ets-containing ME26 virus.

ME26 virus, which was generated by inserting the coding region of the acute avian leukemia-inducing virus E26 into a murine retrovirus vector, encodes a 135-kDa gag-myb-ets fusion protein. Amphotropic murine leukemia virus pseudotypes of ME26 virus induce a high incidence of erythroleukemia 2 to 4 months after injection into newborn NFS/N mice. Spleen cells from the majority of these mice proliferate to high levels in the presence of the erythroid hormone erythropoietin (Epo) and can easily be established as permanent Epo-dependent cell lines. The cell lines contain multiple copies of ME26 viral DNA and express viral message and protein. An Epo receptor mRNA of normal size can be detected in these cells, and binding studies reveal a single class of lower-affinity Epo receptor with an affinity for Epo that is in the range of that previously reported for erythroid cells. The ME26 virus-induced Epo-dependent cell lines, however, appear more immature than previously described erythroid cell lines and more closely resemble early hematopoietic precursor cells, suggesting that the virus may be activating the Epo receptor in hematopoietic cells that do not normally express it. Consistent with this idea, we are able to infect an interleukin-3-dependent myeloid cell line, FDC-P2, with ME26 virus and convert it to Epo dependence. The ME26 virus-infected FDC-P2 cells, even before growth on Epo, showed a large increase in the amount of Epo receptor mRNA. However, no ME26 viral integrations can be detected adjacent to the Epo receptor gene, indicating that the virus is not activating the Epo receptor gene by promoter/enhancer insertion. Our results are more consistent with the hypothesis that the gag-myb-ets-encoded viral fusion protein, which is known to bind DNA, is directly or indirectly activating the expression of the Epo receptor gene in these cells.


cis-acting regulatory regions in the long terminal repeat of simian foamy virus type 1.

Simian foamy virus type 1 (SFV-1), a member of the Spumavirinae subfamily of retroviruses, encodes a transcriptional transactivator (taf) that strongly augments gene expression directed by the viral long terminal repeat (LTR) (A. Mergia, K. E. S. Shaw, E. Pratt-Lowe, P. A. Barry, and P. A. Luciw, J. Virol. 65:2903-2909, 1991). This report describes cis-acting regulatory elements in the LTR that control viral gene expression. A series of LTR mutants and hybrid promoter constructs have been analyzed in transient expression assays for responsiveness to Taf. The targets for transactivation have been mapped to two regions of the U3 domain of the LTR, between positions -1196 and -880 and between positions -403 and -125 (+1 represents the transcription initiation site). No significant nucleotide sequence homology between these two regions is noted; thus, the SFV-1 taf gene acts through at least two distinct sequence elements in the LTR. The target contained between positions -403 and -125 acts independently of orientation, in different cell types and species, and in the context of a heterologous promoter. Thus, the target element between positions -403 and -125 has properties of a transcriptional enhancer. The observation that two distinct elements in the SFV-1 LTR are targets for transcriptional transactivation is novel with respect to observations for other retroviral systems. The R-U5 region of the SFV-1 LTR down-regulates transactivation by severalfold. Computer analysis of the R-U5 region revealed a secondary structure with a free-energy level of -74 kcal (ca. -310,000 J); this structural feature may account for the inhibitory effect on gene expression directed by the LTR. Taf of SFV-1 had no effect on gene expression directed by the LTR of the related human foamy virus, whereas Taf transactivates gene expression directed by the LTRs of the human and simian immunodeficiency viruses. Comparative functional analysis of Taf on homologous and heterologous LTRs may facilitate elucidation of the mechanism of transactivation of foamy viruses.


Deletion of the VP16 open reading frame of herpes simplex virus type 1.

VP16 (also called Vmw65 and alpha TIF) is a structural protein of herpes simplex virus type 1 (HSV-1) that trans-induces HSV-1 immediate-early gene transcription. This report describes an HSV-1 VP16 deletion mutant that was constructed and propagated in a cell line transformed with a VP16 expression vector. The VP16 deletion mutant replicated like wild-type HSV-1 during infection of the VP16-expressing cell line. Deletion mutant virions propagated in this cell line contained wild-type, cell-derived VP16 protein that was recruited during virion assembly and was functional for immediate-early gene trans-induction. The mutant failed to replicate during subsequent infection of cells that do not express VP16, as determined in plaque assays and single-step replication assays. The deletion mutant induced nearly normal levels of viral DNA synthesis and capsid production during these infections, but it induced slightly lower levels of viral DNA encapsidation and appeared by transmission electron microscopy to be defective in further steps of virion maturation. A genetic revertant of the deletion mutant that was restored for VP16-coding sequences exhibited fully wild-type replication properties in both VP16-expressing and nonexpressing cells. The absence of VP16 protein synthesis at late times of HSV-1 infection prevents the production of infectious progeny virus and correlates with a profound defect in HSV-1 particle assembly.


Structural organization, expression, and functional characterization of the murine cytomegalovirus immediate-early gene 3.

We have previously defined ie3 as a coding region located downstream of the ie1 gene which gives rise to a 2.75-kb immediate-early (IE) transcript. Here we describe the structural organization of the ie3 gene, the amino acid sequence of the gene product, and some of the functional properties of the protein. The 2.75-kb ie3 mRNA is generated by splicing and is composed of four exons. The first three exons, of 300, 111, and 191 nucleotides (nt), are shared with the ie1 mRNA and are spliced to exon 5, which is located downstream of the fourth exon used by the ie1 mRNA. Exon 5 starts 28 nt downstream of the 3' end of the ie1 mRNA and has a length of 1,701 nt. The IE3 protein contains 611 amino acids, the first 99 of which are shared with the ie1 product pp89. The IE3 protein expressed at IE times has a relative mobility of 88 kDa in gels, and a mobility shift to 90 kDa during the early phase is indicative of posttranslational modification. Sequence comparison reveals significant homology of the exon 5-encoded amino acid sequence with the respective sequence of UL 122, a component of the IE1-IE2 complex of human cytomegalovirus (HCMV). This homology is also apparent at the functional level. The IE3 protein is a strong transcriptional activator of the murine cytomegalovirus (MCMV) e1 promoter and shows an autoregulatory function by repression of the MCMV ie1/ie3 promoter. The high degree of conservation between the MCMV ie3 and HCMV IE2 genes and their products with regard to gene structure, amino acid sequence, and protein functions suggests that these genes play a comparable role in the transcriptional control of the two cytomegaloviruses.


Herpes simplex virus type 1 recombination: role of DNA replication and viral a sequences.

During the course of infection, elements of the herpes simplex virus type 1 (HSV-1) genome undergo inversion, a process that is believed to occur through the viral a sequences. To investigate the mechanism of this recombinational event, we have developed an assay that detects the deletion of DNA segments flanked by directly repeated a sequences in plasmids transiently maintained in Vero cells. With this assay, we have observed a high frequency of recombination (approximately 8%) in plasmids that undergo replication in HSV-1-infected cells. We also found a low level of recombination between a sequences in plasmids introduced into uninfected cells and in unreplicated plasmids in HSV-1-infected cells. In replicating plasmids, recombination between a sequences occurs at twice the frequency seen with directly repeated copies of a different sequence of similar size. Recombination between a sequences appears to occur at approximately the same time as replication, suggesting that the processes of replication and recombination are closely linked.


Yeast cells are incapable of translating RNAs containing the poliovirus 5' untranslated region: evidence for a translational inhibitor.

We have expressed in the yeast Saccharomyces cerevisiae a full-length poliovirus cDNA clone under the control of the GAL10 promoter to better characterize the effect of poliovirus on host cell metabolism. We find that yeast cells are unable to translate poliovirus RNA in vivo and that this inhibition is mediated through the 5' untranslated region of the viral RNA. The in vivo inhibition of translation of poliovirus RNA and P2CAT RNA (which contains the 5' untranslated region fused upstream of the bacterial chloramphenicol transferase gene) can be mimicked in vitro in yeast translation lysates. In fact, a trans-acting inhibitor present in yeast lysates can inhibit translation of either poliovirus or P2CAT RNA in HeLa cell translation lysates. In contrast, when the inhibitor is added to translations programmed with chloramphenicol acetyltransferase RNA, yeast prepro-alpha-factor RNA, or an RNA containing the internal ribosome entry site of encephalomyocarditis virus, no inhibition is seen. The inhibitory activity has been partially purified by DEAE-Sephacel chromatography. The partially purified inhibitor is heat stable, escapes phenol extraction, is resistant to proteinase K and DNase I treatment, and is sensitive to RNase A digestion, suggesting that the inhibitor is an RNA. In an in vitro translation assay, the inhibitory activity can be overcome by increasing the concentration of HeLa cell lysate but not P2CAT RNA, suggesting that the inhibitor interacts (directly or indirectly) with one or more components of the HeLa cell translational machinery rather than with the viral RNA.