Antiepileptic drug-induced visual hallucinations in a child.

The neurologic signs and symptoms of carbamazepine and phenytoin toxicity, such as ataxia, dysarthria, and nystagmus, are well known. The psychiatric manifestations of toxicity, such as psychosis and hallucinations, however, are less widely recognized. This study reports the case of a 9-year-old male with seizures who developed intermittent complex visual hallucinations after therapy with antiepileptic drugs was begun. This study considered seizures, migraine, underlying psychiatric diathesis, and drug toxicity as possible etiologies but after extensive investigation concluded that his symptoms were most likely a drug side effect.

Utility of early single photon emission computed tomography (SPECT) in neonatal gelastic epilepsy associated with hypothalamic hamartoma.

Gelastic epilepsy, or laughing seizures, is a rare seizure manifestation often associated with hypothalamic hamartoma. This seizure type is well described in older children and adults, but has only rarely been reported in neonates, oftentimes recognized in retrospect when the children are older. We report a child diagnosed at 3 months of age with a large hypothalamic mass after evaluation for spells occurring since birth. The spells were characterized by bursts of hyperpnea, followed by repeated "cooing" respirations, giggling, and smiling. These spells were recognized soon after birth in the delivery room, and occurred at 15-20 minute intervals. They did not interrupt feeding and occurred during sleep. On referral to our center, the patient was noted to be thriving, with normal medical and neurologic examinations except for his spells. The laboratory evaluation was normal, as were endocrine and ophthalmologic evaluations. Neuroimaging was performed, with magnetic resonance imaging demonstrating a large 2.8-cm isodense, nonenhancing hypothalamic mass. Electroencephalogram was abnormal, demonstrating bi-frontal sharp and spike-wave discharges. Video-EEG did not demonstrate ictal discharges associated with the patient's spells. Single photon emission computed tomography (SPECT) demonstrated dramatic ictal uptake in the area of the tumor, with normalization during the interictal phase. Partial excision of hamartomatous tissue has minimally improved the spells. In conclusion, this patient manifested an unusual, early presentation of a rare seizure type. SPECT scanning confirmed the intrinsic epileptogenesis of the hamartoma, further justifying a surgical approach to such patients. Early surgical intervention is probably indicated in an attempt to minimize or prevent the cognitive and behavioral sequelae commonly seen with this seizure type.

Kinetic analysis of the effects of mutagenesis of W501 and V432 of the hepatitis C virus NS3 helicase domain on ATPase and strand-separating activity.

Two hydrophobic residues, W501 and V432, in the nucleic acid (NA) binding pocket of the HCV helicase domain (E) were mutagenized in an effort to investigate contributions of these residues to substrate affinities and to enzymatic activities. The affinities of wild-type [hE(wt)] and mutant enzymes [hE(W501F), hE(W501A), and hE(V432A)] for NA and ATP were determined by monitoring changes in the intrinsic protein fluorescence, in the fluorescence of fluorescently tagged nucleic acid, and in the enzymatic activity. The steady-state kinetic parameters of the mutant enzymes for ATP hydrolysis (at saturating concentrations of NA) were similar to those of hE(wt). hE(W501F), hE(W501A), and hE(V432A) had strand-separating activities that were 136%, 3.8%, and 3.1% of that of hE(wt). The processivities of hE(W501F), hE(W501A), and hE(V432A) were reduced relative to that of hE(wt). The reduced processivities of hE(W501F) and hE(W501A) were primarily due to an increase in the rate of dissociation of E. ATP from E.ATP.NA. The reduced processivity of hE(V432A) was primarily due to a reduction in the intrinsic forward rate constant for strand separation. This result suggested that V432 may constitute part of the forward "stepping" motor of E. hE(W501A) and hE(V432A) did not display a dominant negative phenotype in a steady-state helicase assay with hE(wt). hE(wt) stored in the presence of beta-mercaptoethanol was covalently modified at three cysteinyl residues. The biological significance of the potential reactivity of these cysteinyl residues on hE(wt) is unknown.

Annexin I degradation in bronchoalveolar lavage fluids from healthy smokers: a possible mechanism of inflammation.

Annexin I is a glucocorticoid-inducible, phospholipase A2-inhibitory protein and is proposed to have an anti-inflammatory role. Although annexin I is a cytosolic protein, it is found extracellularly in secreted fluids such as semen. We have examined the expression of annexin I in bronchoalveolar lavage fluids (BALF) from smokers and nonsmokers to investigate the role of annexin I in the airway. We find that annexin I is secreted in BALF. This secretion is not due to cell death or damage, because a cytosolic protein, 3-phosphoglycerate kinase, is not seen in BALF. We observed that BALF from smokers (n = 10) had high protein concentrations as compared with BALF from nonsmokers (n = 11). Annexin I levels were higher in BALF from smokers compared with nonsmokers. However, in smokers, annexin I was exclusively found in the Mr 34,000 form that lacks the Mr 3,000 N-terminal anti-inflammatory peptide. In nonsmokers, both the Mr 37,000 native annexin I and the Mr 34,000 proteolytically cleaved form are present, with the Mr 37,000 form being most abundant. The NH2-terminal Mr 3,000 peptide of annexin I exhibits anti-inflammatory actions (G. Cirino et al, Br. J. Pharmacol., 108: 573-574, 1993). Previous studies have implicated neutrophil elastase as the protease cleaving annexin I to the Mr 34,000 protein. We observed increased elastase levels in BALF from smokers. However, we find no correlation between bronchial sample percent of neutrophils in BALF and the relative amount of the Mr 34,000 band generated. Our data clearly demonstrate that annexin I is degraded in BALF from smokers, and we propose that proteolytic cleavage of annexin I in BALF from smokers may be a mechanism by which polymorphonuclear neutrophils infiltrate sites of inflammation; thus, inactivation of annexin I in smokers' lungs may lead to chronic and uncontrolled inflammation.

Increased tissue neutral endopeptidase 24.11 activity in spontaneously hypertensive hamsters.

The purpose of this study was to determine whether tissue neutral endopeptidase (NEP) 24.11 activity, a membrane-bound metalloenzyme widely distributed in the peripheral circulation that cleaves and inactivates vasodilator peptides, is increased in spontaneously hypertensive hamsters relative to genetically/age-matched normotensive hamsters. Mean arterial pressure and heart rate were 163 +/- 11 mm Hg and 312 +/- 7 beats/min in spontaneously hypertensive hamsters and 99 +/- 3 mm Hg and 302 +/- 10 beats/min in normotensive hamsters, respectively (mean +/- SEM). NEP 24.11 activity is significantly increased in the kidney, cheek pouch, and spinotrapezius muscle, and significantly decreased in the heart and aorta of spontaneously hypertensive hamsters relative to controls (P < .05). Lung and brain NEP 24.11 activity is similar in both groups. Renal NEP 24.11 activity increases and to a similar extent in spontaneously hypertensive and normotensive hamsters as chloride anion concentration in the assay buffer is increased. Substituting citrate for chloride anion significantly attenuates renal NEP 24.11 activity. Taken together, these data indicate that NEP 24.11 activity in spontaneously hypertensive hamsters is increased in two organs that contribute appreciably to peripheral vascular resistance, skeletal muscle, and kidney. We suggest that the spontaneously hypertensive hamster is a suitable model to study the role of skeletal muscle and renal NEP 24.11 in regulating vasomotor tone in essential hypertension.

Children with headache: clinical predictors of surgical space-occupying lesions and the role of neuroimaging.

PURPOSE: To determine clinical predictors useful in differentiation of surgical lesions from medically treated disorders and the role of neuroimaging in children with headache. MATERIALS' AND METHODS: In a 4-year retrospective study, 315 patients with headache and no known neurologic disorder underwent brain magnetic resonance (MR) imaging. Sixty-nine patients also underwent brain computed tomography (CT). Clinical data were correlated with findings from MR imaging and CT and the final diagnosis by means of logistic regression. RESULTS: Thirteen (4%) patients had surgical space-occupying lesions. Seven independent multivariate predictors of a surgical lesion were identified. Sleep-related headache and no family history of migraine were the strongest predictors. Other predictors included vomiting, absence of visual symptoms, headache of less than 6 months duration, confusion, and abnormal neurologic examination findings. A positive correlation between number of predictors and risk of surgical lesion was noted (P < .0001). No difference between MR imaging and CT was noted in detection of surgical space-occupying lesions, and there were no false-positive or false-negative surgical lesions detected with either modality on the basis of clinical follow-up. CONCLUSION: Children at high risk on the basis of these criteria usually require neuroimaging, while children at low risk may be safely followed up clinically without neuroimaging.

Altered expression of annexin II in human B-cell lymphoma cell lines.

Annexin II is a growth-regulated gene, whose expression is significantly increased in various human cancers. We examined annexin II expression in II human B-cell lymphoma cell lines and in normal B-cells. Wide variation was observed in the levels of annexin II in these cell lines. Annexin II overexpression was observed in 5 cell lines, while significantly reduced expression was observed in Raji, OMA-BL-1 and REH cell lines. Analysis of the annexin II gene, mRNA and protein in Raji and OMA-BL-1 cell lines indicated that annexin II gene was unaltered and that a low level of annexin II transcripts are produced in these cells. Down-regulation of annexin II expression was at the transcriptional level, and no reexpression of annexin II was observed after treatment of cells with demethylating agents. Thus methylation of the annexin II gene does not appear to be responsible for annexin II down-regulation. A slow migrating altered form of annexin II was detected in Raji and OMA-BL-1 cells, which was detected with the anti-chicken annexin II antiserum, but not with the anti-human annexin II antiserum. The slow migrating annexin II species was found to be sensitive to dephosphorylation by calf intestinal alkaline phosphatase, resulting in reduction of the size of the protein on SDS-polyacrylamide gels. The phosphorylated annexin II was also observed in nuclear extracts of human K562 and HeLa cells. Thus, Raji and OMA-BL-1 cells exclusively produce a phosphorylated form of annexin II, and phosphorylated annexin II may be important for cell survival and proliferation.

Detection of secreted and intracellular annexin II by a radioimmunoassay.

A simple radioimmunoassay for detection of secreted and intracellular annexin II in human cells is presented. Annexin II is a multifunctional protein in human cells and may have a role in several types of cancers. No enzymatic activity has been associated with the protein, thus making its detection difficult. Using purified annexin II from human placenta, we have developed a sensitive radioimmunoassay protocol. A linear response was observed up to a concentration of 0.5 microgram purified protein in the assay. Using this radioimmunoassay protocol, annexin II can be detected in undiluted clinical human samples such as bronchoalveolar lavages and various tissue extracts. We demonstrate the applicability of this technique to measure intracellular annexin II in extracts of a human adenocarcinoma cell line (HeLa) and secreted annexin II from bronchoalveolar lavage fluid from a human patient. Using HeLa cell extracts and BAL, we observed a linear response with up to 10 micrograms total protein in the assay. We further demonstrate the applicability of this technique to measure differences in intracellular and secreted annexin II in the human pancreatic adenocarcinoma cell lines CD-11, CD-18 and Capan-2. While CD-11 and CD-18 do not secrete annexin II, the cell line Capan-2 secretes high levels of the protein.

Synthesis and anticonvulsant activity of N-benzylpyrrolo[2,3-d]-, -pyrazolo[3,4-d]-, and -triazolo[4,5-d]pyrimidines: imidazole ring-modified analogues of 9-(2-fluorobenzyl)-6-(methylamino)-9H-purine.

Analogues of 9-(2-fluorobenzyl)-6-(methylamino)-9H-purine (1) containing isosteric replacements of the imidazole ring atoms were synthesized and tested for anticonvulsant activity. The pyrrolo[2,3-d]-, pyrazolo[3,4-d]-, and triazolo[4,5-d]pyrimidines were less active than 1 against maximal electroshock-induced seizures (MES) in rats when given po. The differences in anti-MES activity for these analogues was not explained by differences in pKa or lipophilicity. However, the four classes of heterocycles have distinctly different calculated electrostatic isopotential maps, which may be related to optimum anticonvulsant activity.

1-(Fluorobenzyl)-4-amino-1H-1,2,3-triazolo[4,5-c]pyridines: synthesis and anticonvulsant activity.

A series of (fluorobenzyl)triazolo[4,5-c]pyridines was synthesized and tested for activity against maximal electroshock-induced seizures in rodents. The most promising compound, 14 (BW 534U87), which is a carbon-nitrogen isoster of a purine anticonvulsant, has a profile in rodents that suggests 14 will be free of emesis and useful in the treatment of seizure disorders for which phenytoin is presently indicated.

Differential expression of annexins I and II in bovine bronchial epithelial cells.

Annexins I and II have been proposed to participate in regulation of inflammation and cell proliferation. In the present study, we examined the expression of annexins I and II in various levels of the bovine respiratory tract and in cultured bovine bronchial epithelial cells. Immunoblot analysis of whole-tissue extracts revealed low-level expression of annexins I and II in bronchial mucosa mainstem to fourth generation. In contrast, high levels of annexins I and II (15- to 20-fold higher than in the bronchial mucosa) were seen in the distal lung parenchyma. Immunohistochemical staining of tracheobronchial tissue sections with anti-annexin I antibody revealed an uneven positive reaction of about 30 to 40% of the columnar differentiated ciliated and secretory cells, with no obvious positivity within the nondifferentiated basal epithelial cell layer. In contrast, anti-annexin II antibody reacted nearly uniformly within the basal cell layer, with no obvious decoration of the differentiated cell types. These results were confirmed by immunoblot analysis of annexin I and II in density fractionated populations of basal and secretory/ciliated epithelial cells. Both annexins I and II were expressed at constitutive levels in bovine bronchial epithelial cells grown in glucocorticoid- and serum-free medium, and dexamethasone increased expression of both proteins in a concentration-dependent manner. We conclude that annexins I and II are differentially expressed in the differentiated ciliated cells and undifferentiated basal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of bone cement powder on human adherent monocytes/macrophages in vitro.

This study reports the effects of Simplex bone cement powder (BC) on the proliferation and production of bone resorbing factors in vitro by human adherent monocytes/macrophages. Adherent peripheral blood cells were isolated from seven healthy individuals and exposed to a dispersion of BC powder (1 mg/mL), phytohemagglutinin (PHA, 40 micrograms/mL), or medium alone at different periods of cell incubation (days 0-2, 0-7, 5-7, or 10-12). Cell proliferation was quantified by incorporation of 3H-thymidine uptake. Culture supernatants were evaluated for levels of interleukin 1-like activity (IL-1) by murine thymocyte proliferation assay, prostaglandin E2 (PGE2) by radioimmunoassay, lysosomal enzyme activity (N-acetyl-beta-D-glucosaminidase and beta-glucuronidase using fluorometry, and collagen and casein degrading activity using radioactive substrates. Human adherent peripheral blood cells showed a proliferative response to PHA that coincided with cell maturation; BC did not inhibit PHA-induced cell proliferation of either adherent or nonadherent blood cells, indicating the non-toxic nature of these particles at the concentrations tested. BC stimulated increased release of the lysosomal enzyme N-acetyl-beta-D-glucosaminidase; the levels of PGE2, IL-1, collagenase, and caseinase were unchanged.

Rates of peptide proteolysis measured using liquid chromatography and continuous-flow fast atom bombardment mass spectrometry.

An immobilized digestive enzyme assay, which has been used to determine whether orally administered peptide drugs are hydrolyzed by the digestive system, was applied to the measurement of rates of proteolysis of biologically active peptides. In this study, the rates of hydrolysis by trypsin and chymotrypsin of the pressor agent angiotensin II, the peptide hormone [Arg8]vasopressin, and the peptide drug [deamino-Cys1,D-Arg8]vasopressin were measured. Enzyme immobilization prevented autolytic proteolysis and provided a stable enzyme preparation during the assays. For rate determinations, the disappearance of substrate was measured over time by using either flow injection continuous-flow fast atom bombardment (FAB) mass spectrometry with selected ion monitoring or reversed-phase high-performance liquid chromatography (HPLC) with UV absorbance detection. Compared to the HPLC method, continuous-flow FAB was faster, provided more confident identification of the analyte because molecular weight data was obtained, and could be used for all enzymatic reactions instead of only those in which complete chromatographic resolution of substrate from proteolytic fragments was obtained. The in vitro proteolytic rates measured for the vasopressins were compared to data from rat bioassays and confirmed that the limiting factor in the oral bioavailability of [Arg8]vasopressin was rapid hydrolysis by trypsin in the intestinal lumen. The more bioactive compound, [deamino-Cys1,D-Arg8]vasopressin, was more stable to chymotryptic digestion and completely resistant to trypsinization.

Tissue angiotensin I-converting enzyme activity in spontaneously hypertensive hamsters.

The purpose of this study was to measure angiotensin I-converting activity in heart, kidney, lung and cheek pouch tissue homogenates of spontaneously hypertensive and normotensive hamsters. We also determined inhibitor sensitivity and the effects of chloride anion concentration on kidney angiotensin I-converting activity in these animals. We found no significant differences in angiotensin I-converting activity between hypertensive and normotensive hamsters in all tissues tested. Inhibitor sensitivity of kidney angiotensin I-converting activity with captopril and lisonopril was similar in both groups. Finally, kidney angiotensin I-converting activity increased significantly in both groups as chloride anion concentration in the assay buffer increased. Substituting chloride anion for citrate abrogated the increase in angiotensin I-converting enzyme activity.

The role of primer recognition proteins in DNA replication: association with nuclear matrix in HeLa cells.

Primer recognition proteins (PRP) enable DNA polymerase alpha to utilize efficiently DNA substrates with low primer to template ratios. We have previously identified the protein-tyrosine kinase substrate annexin II, and the glycolytic enzyme 3-phosphoglycerate kinase as components of PRP. As a step towards elucidation of the role of PRP in the process of DNA replication, we have investigated the subcellular distribution and specific association of these proteins with the nuclear matrix in HeLa cells. Nuclear extracts prepared from HeLa cells in S phase contain the enzymatic activity of 3-phosphoglycerate kinase (PGK) and phospholipase A2 inhibitory activity of annexin II. Monomer annexin II is approximately equally distributed between the nuclear and cytoplasmic fractions, while a majority of PGK is in the cytoplasm. Immunoblot analyses reveal the presence of these two proteins in nuclei, specifically associated with the nuclear matrix. This is further confirmed by observation of the presence of annexin II and PGK in isolated nuclear matrices by immunoelectron microscopy. The phospholipase A2 inhibitory activity of annexin II colocalizes with the nuclear matrix-bound annexin II. A related protein, annexin I, is not detectable in the nuclear extracts and nuclear matrix. A slower-migrating (perhaps modified) form of annexin II is found to be associated with the nuclear matrix. Attempts to dissociate PGK and annexin II from the nuclear matrix with octyl-beta-glucoside, high salt or metal ion chelators were unsuccessful, suggesting that the interaction is very strong.

The protein-tyrosine kinase substrate, calpactin I heavy chain (p36), is part of the primer recognition protein complex that interacts with DNA polymerase alpha.

Primer recognition proteins (PRP) stimulate the activity of DNA polymerase alpha on DNA substrates with long single-stranded template containing few primers. Purified PRP from HeLa cells and human placenta are composed of two subunits of 36,000 (PRP 1) and 41,000 (PRP 2) daltons. By amino acid sequence homology, we have identified PRP 2 as the glycolytic enzyme 3-phosphoglycerate kinase. Here we present data that establishes PRP 1 to be the protein-tyrosine kinase substrate, calpactin I heavy chain. Amino acid sequence analysis of six tryptic peptides of PRP 1 followed by homology search in a protein sequence data base revealed 100% identity of all six peptides with the deduced amino acid sequence of human calpactin I heavy chain. The activities of PRP and calpactin I coelute on gel filtration columns, and a high correlation of PRP and calpactin I activities was seen at different stages of purification. A rabbit polyclonal anti-chicken calpactin I antibody was shown to cross-react with PRP 1 polypeptide at various stages of PRP purification, and the homogeneous preparation of PRP exhibits 3-phosphoglycerate kinase (PRP 2) and calpactin I (PRP 1) activities. PRP activity is neutralized by a mouse monoclonal anti-calpactin II antibody although having no effect on the polymerase alpha activity itself. Calpactin II has a 50% amino acid sequence homology with calpactin I. However, PRP 1 is not calpactin II as shown by lack of cross-reaction to a monoclonal anti-calpactin II antibody on Western blots. Calpactin I and 3-phosphoglycerate kinase, purified independently, cannot be efficiently reconstituted into the PRP complex, indicating that their association in the PRP complex involves specific protein-protein interactions that remain to be elucidated. The biochemical and immunological data presented here revealing the identity of PRP 1 as calpactin I provide evidence for one physiological role of calpactin I in the cell.

Suramin affects DNA synthesis in HeLa cells by inhibition of DNA polymerases.

Suramin, a polysulfonated naphthylurea widely used in the treatment of trypanosomiasis and onchocerciasis, is currently being investigated as an antitumor agent for the treatment of advanced cancer. Suramin exerts a wide variety of biological effects. We have shown that suramin inhibits cell proliferation and DNA synthesis in cultured HeLa cells. The replication in vitro of SV40 DNA is completely abolished by 40 microM suramin. The inhibition of DNA replication is due to inhibition of DNA polymerases alpha and delta, the replicative enzymes in eukaryotic cells. DNA polymerase alpha is sensitive to lower concentrations of suramin [concentration to achieve 50% inhibition (IC50) of 8 microM] than is DNA polymerase delta (IC50 36 microM), whereas DNA polymerase beta is relatively insensitive to the drug (IC50 of 90 microM). Suramin inhibits other replicative DNA polymerases such as Escherichia coli polymerase I (Klenow fragment) and Thermus aquaticus polymerase. Suramin is noncompetitive with both substrate deoxyribonucleotides and template-primers with respect to DNA polymerase inhibition. Much lower concentrations (8-30 microM) of the drug are required for 50% inhibition of DNA polymerases than for 50% inhibition of other enzymes such as protein kinase C and reverse transcriptase. These results show an important biological effect of this drug and indicate the need for more studies before its clinical use as an antitumor agent.

Rural hospitals under PPS: a five-year study.

This research examines the impact of prospective payment (PPS) on the financial performance of Kansas hospitals, which are predominantly rural. Financial ratios are presented and regressed on bed size and year. The data suggest that bed size has the strongest effect on financial viability. There are indications of a delayed effect of PPS on the rural, smallest hospitals (fewer than 25 beds), suggesting that non-operating sources of revenue (local property tax mill levies) are being used to subsidize them in the short term. Small hospitals appear to be delaying all capital and long-term costs to survive. The research suggests that the effect of PPS may be long term.