Lack of modulation of Ib inhibition during antagonist contraction in spasticity.

OBJECTIVE: To examine the modulation of non-reciprocal group I (Ib) inhibition during tonic contraction of antagonist muscles in patients with spasticity vs normal subjects. METHODS: The authors studied 10 patients with spastic paraplegia due to cervical compression myelopathy and 16 age-matched normal subjects. Ib inhibition to soleus motoneurons was recorded as the change in size of the H-reflex of the soleus, evoked by conditioning stimulus to the nerve innervating the medial gastrocnemius muscle. The extent of inhibition was studied at rest and during tonic contraction of the pretibial muscles of variable strength. RESULTS: In the resting state, the extent of inhibition in the patients did not differ from normal controls. During antagonist contraction, the extent of inhibition increased both in the normal subjects and patients. The increment was smaller in the patients, especially in those with severe spastic gait. The smaller increment in the inhibition was correlated with the time required to walk 10 m in the patients. CONCLUSION: The authors observed a lack of modulation of Ib inhibition during tonic antagonist contraction in patients with spasticity, especially those with gait disturbance. Disturbed central modulation of non-reciprocal (Ib) interneurons may be responsible for spasticity.

Microsatellite instability and alternative genetic pathway in intrahepatic cholangiocarcinoma.

BACKGROUND/AIMS: Intrahepatic cholangiocarcinoma (ICC) arises from intrahepatic bile duct epithelium and is the second most prevalent among primary liver cancers. The aim of this study was to clarify the mechanism of cholangiocarcinogenesis. METHODS: We studied the incidence of microsatellite instability (MSI) involving eight highly polymorphic microsatellite markers and alternations of the K-ras, p53 and mdm-2 genes in human ICC tissues. Overexpression of mdm-2 oncoprotein was also immunohistochemically studied. RESULTS: Of all 65 cases examined, K-ras gene mutation was found in three cases (4.6%) at codon 12. Analysis of p53 alterations was performed in 28 cases including 22 frozen samples and mutations were found in three cases (10.7%). Overexpression of mdm-2 protein was observed in 25 (41.7%) out of 60 cases analyzed. In 22 frozen samples, seven (31.8%) cases showed mdm-2 amplification and four (18.2%) cases revealed MSI-positive phenotype. Among the cases analyzed, all the tumors with mdm-2 amplification/overexpression harbored the wild-type p53 gene and all the microsatellite instability-positive cases were from mass-forming (MF) + periductal-infiltrating (PI) subtype. CONCLUSIONS: These results suggest that mdm-2 plays a role, which might be partially through inhibiting p53 activity, in cholangiocarcinogenesis and that M

Comprehensive allelotyping of human intrahepatic cholangiocarcinoma.

We performed a genome-wide scan for loss of heterozygosity (LOH) in 22 intrahepatic cholangiocarcinoma (ICC) cases using 168 polymorphic microsatellite markers throughout all of the human chromosomes and 48 markers of which LOH is reportedly characteristic of hepatocellular carcinoma (HCC). Markers with LOH in more than 30% of informative cases were observed at 21 loci. Among these, eight markers on 6q (three loci), 4q (two loci), 9q, 16q, and 17p shared high frequencies of LOH with HCC in our previous study. As for gross appearance, mass-forming type tumors showed higher frequency of LOH (P < 0.001) compared with other types. Compared by tumor size (< or =5 cm versus >5 cm), number (multiple versus solitary), and the International Union Against Cancer TNM classification (stage IVB versus II-IVA), LOH was observed more frequently in advanced stages (P < 0.01, respectively). However, LOH frequency does not differ regardless of lymph node status (pN0 versus pN1). Frequent LOH on 1p36 including the p73 locus was noted in large tumors without lymph node metastasis. These suggest that ICC shares some common carcinogenic steps with HCC such as LOH of 4q and 6q and that inactivation of tumor suppressor genes on chromosome 1p36 contributes to progression of ICC but not to metastatic traits.

Increased expression of cytosolic chaperonin CCT in human hepatocellular and colonic carcinoma.

The chaperonin-containing t-complex polypeptide 1 (CCT) is a hetero-oligomeric molecular chaperone that assists in the folding of actin, tubulin, and other cytosolic proteins. We recently reported that the expression level of CCT is closely correlated with growth rates of mammalian cultured cells. Here we examine the levels of CCT subunits and other molecular chaperones in tumor tissues of patients with hepatocelluar and colonic carcinoma, and compare them with nontumor tissues in the same patients. Expression levels of CCTbeta in tumor tissues was significantly higher than in nontumor tissues in all patients with hepatocellular carcinoma (n = 15) and 83% of patients with colonic carcinoma (n = 17). The increased level of CCT expression in colonic cancer cells was confirmed by immunohistochemistry with anti-CCTbeta antibody. The levels of CCTbeta were highly correlated (r = 0.606) with those of the proliferating cell nuclear antigen (PCNA), which was used as an indicator of cell growth. CCTalpha gave similar results, although the correlation with PCNA levels was weaker. Other cytosolic and endoplasmic reticulum chaperones also showed higher expression in significant numbers of tumor tissues but less frequently than that observed with CCT. These results suggest that CCT is up-regulated in rapidly proliferating tumor cells in vivo to effectively produce proteins required for growth, and may serve as a useful tumor marker because it is widely distributed in the cytosol.

Comprehensive allelotype study of hepatocellular carcinoma: potential differences in pathways to hepatocellular carcinoma between hepatitis B virus-positive and -negative tumors.

To examine the role of the loss of heterozygosity (LOH) in hepatitis-related carcinogenesis, we performed a genome-wide scan of LOH in 44 tumors of hepatocellular carcinoma (HCC) using 216 microsatellite markers throughout all human chromosomes. A high frequency of LOH (>30% of informative cases) was observed at 33 loci on chromosome arms 4q, 6q, 8p, 8q, 9p, 9q, 13q, 16p, 16q, 17p, and 19p. LOH on 19p has not yet been reported, and that appears to be a new candidate in the search for tumor suppressor genes. High rates of LOH are correlated with hepatitis B virus (HBV) positivity, poorly differentiated tumors, vascular invasion, and intrahepatic metastasis (P <.0001). LOH on 13q and 16q occurred more frequently in HBV(+) patients (P <.0001), and LOH on 6q occurred more frequently in virus-negative patients (P <.001). The frequency of LOH on 4q and 13q was significantly lower in well-differentiated tumors than in moderately and poorly differentiated tumors (P <.01). In contrast, LOH on 6q was frequently detected in well-differentiated tumors compared with other histological subclasses (P <.001). Our results suggest that LOH on 6q may play an important role in the early stage of hepatocarcinogenesis in virus-negative patients, but different mechanisms might underlie the initial step to carcinogenesis in HBV(+) patients. LOH on 13q and 16q may play an essential role in the progression of HBV(+) tumors. Further studies of fine deletion mapping on chromosomes 13q and 16q are required to define the genomic segments on which putative tumor suppressor genes responsible for HBV(+) tumors exist.

Analysis of genetic changes in intrahepatic cholangiocarcinoma induced by thorotrast.

Thorotrast, a colloidal suspension of radioactive (232)ThO(2) that emits alpha particles, was used as a radiographic contrast agent in the 1930s-1950s. Several decades after injection, Thorotrast causes liver cancers, among which intrahepatic cholangiocarcinoma (ICC) is prominent. We investigated mutations of the RAS and the TP53 genes in archival sections of ICC induced by Thorotrast. Compared to ICC that was not associated with Thorotrast, the frequency of mutation of the KRAS gene was lower, while that of the TP53 gene was more than two times higher. The most common mutation of the TP53 gene was A-G transitions. Interestingly, TP53 mutations were also found in noncancerous areas of livers in which Thorotrast had been deposited. Furthermore, mutations tended to accumulate in tissues from more advanced tumors. These results suggest that deposited Thorotrast continuously damages DNA in liver cells in some way, resulting in A-G transitions of the TP53 gene. However, we have not been able to rule out the possibility that genetic insults occur indirectly in the proliferating cells adjacent to the necrosis rather than being a direct effect of alpha particles.

Licorice-induced hypokalemic myopathy and hypokalemic renal tubular damage in anorexia nervosa.

A patient with a history of anorexia nervosa developed licorice-induced hypokalemic myopathy. With potassium replacement, high CPK blood level and myopathic signs returned to normal. However, the patient manifested persistent hypokalemia and impaired renal function to concentrate and acidify the urine. Renal biopsy demonstrated intense degeneration and vacuolation of tubules with a normal glomerus which was consistent with hypokalemic nephropathy. Prolonged hypokalemia in anorexia nervosa is sometimes attributed to surreptitious purging or taking diuretics, but it is necessary to check the urine pH, the urine-specific gravity, and the urine potassium level in order to find underlying renal damage even after hypokalemic myopathy is treated successfully.

Mexiletine in the treatment of spasmodic torticollis.

We studied the clinical efficacy of mexiletine, a derivative oral form of lidocaine, for treatment of spasmodic torticollis. One of the nine subjects of this study was previously reported. Before starting oral mexiletine, normal saline was first injected intravenously as placebo control; lidocaine infusion then followed and clinical evaluation was provided by dystonia rating scale scores, videotape recordings, and surface electromyographic recording. In all patients, lidocaine injection resulted in a decrease of dystonic muscle contractions within 5 minutes and the effect lasted approximately 1 hour. With gradual increase of mexiletine dose, similar clinical improvement was obtained with oral doses ranging from 450-1200 mg/day for more than 6 months. Side effects in six of nine patients included upper gastrointestinal symptoms, dizziness, ataxia, and dysarthria. These were tolerable or medically manageable; only one patient required a small reduction in mexiletine dose. Strong positive correlation was found between serum and cerebrospinal fluid (CSF) mexiletine concentrations with a CSF/serum ratio of 0.6 (r = 0.96, p = 0.0005) suggesting its effective penetrance into the central nervous system. We suggest that oral mexiletine therapy may be a safe and effective treatment for spasmodic torticollis.

Inhibitory projections from pronator teres to biceps brachii motoneurones in human.

Neural projections from the pronator teres (PT) muscle to biceps brachii (BB) motoneurones were studied in three healthy human subjects using a post-stimulus time histogram method. In 25 BB motor units, electrical stimulation to the PT nerve with intramuscular needle electrodes induced inhibition in nine units (36%), whereas facilitation was produced in 18 units (72%) by stimulation to the median nerve trunk with surface electrodes at the distal end of the intermuscular septum of the arm or in the cubital fossa. Six motor units (24%) received both inhibition (PT nerve stimulation) and facilitation (median nerve trunk stimulation). In the six, the latency of the inhibition was, on average, 1.2 ms longer than that of the facilitation. The stimulation site for the inhibition was, on average, 4.8 cm distal to that for the facilitation. The inhibition was evoked with an intensity well below the motor threshold. These findings suggest that BB motoneurones receive oligosynaptic inhibition of group I afferents from PT in human.

Progressive decrease in heteronymous monosynaptic Ia facilitation with human ageing.

To evaluate functional change in the spinal reflex pathway with ageing, we studied heteronymous Ia facilitation from the quadriceps to soleus muscle in 30 normal volunteers (aged 24-68 years). The size of the test H-reflex of the soleus muscle was adjusted to 25% that of the maximal M-response. The conditioning stimulus was adjusted to 1.5-fold the motor threshold to stimulate all the Ia fibres in the femoral nerve. Facilitation was quantified as the slope of the very early part of facilitation, within 0.8 ms of onset. This procedure enabled us to evaluate the extent of monosynaptic Ia facilitation without contamination by other effects. The extent of facilitation decreased linearly with age. This decrease in facilitation could reflect a decrease in the number of Ia fibres and in their conduction velocities, and an increase in presynaptic inhibition on Ia terminals. The increase in presynaptic inhibition may be an adaptive phenomenon in the ageing of the neuromuscular system or, alternatively, a deteriorating process with decreasing flexible supraspinal modulation.

A fission yeast gene encoding a protein that preferentially associates with curved DNA.

We searched for fission yeast (Schizosaccharomyces pombe) proteins that preferentially bind to a synthetic curved DNA sequence, by means of a DNA-binding gel shift assay in the presence of an excess amount of a non-curved DNA sequence as a competitor. We identified such a protein in S. pombe. The protein, thus purified, has an apparent molecular weight of 42,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was suggested that this protein (42 K-protein) recognizes and binds to a curved DNA structure in a given nucleotide sequence, although it also binds to a non-curved DNA sequence with lower affinity. As its putative coding sequence, a 1.9-kilobase genomic DNA from S. pombe was cloned and sequenced. Sequencing of a cDNA clone also revealed the existence of an open reading frame, with no intron, encoding a 381-amino-acid protein with a calculated molecular mass, 41,597. This protein appears to be located in the nucleus. The predicted protein sequence revealed that the 42 K-protein exhibits no significant similarity to any other known proteins, except to a hypothetical protein of Caenorhabditis elegans.

Sequence of a fission yeast gene encoding a protein with extensive homology to eukaryotic elongation factor-1 gamma.

A polypeptide with an apparent molecular mass of 23 kDa was identified, that exhibited an affinity to a 491-bp DNA derived from one of the Schizosaccharomyces pombe centromeric DNAs (cen1). After determining its N-terminal amino acid (aa) sequence, a Sz. pombe genomic DNA encompassing the coding sequence of the isolated protein was cloned, and a 2.3-kb genomic DNA region sequenced. Further sequence analysis of cDNA clones, originating from this particular genomic region, confirmed the existence of an open reading frame with a short intron, which encodes a 409-aa protein with striking homology to eukaryotic elongation factor-1 gamma.

Distribution of bent DNA structures in the fission yeast centromere.

To gain a clue as to the functional significance of DNA curvature, we experimentally characterized the distribution of bent DNA structures throughout the 35-kb cen1 sequence, one of the isolated functional centromeric DNA of the fission yeast, Schizosaccharomyces pombe. It was revealed that a relatively large central portion of cen1, covering a 2.2-kb DNA sequence, displays a remarkable DNA curvature.

Multiple hemangiopericytomas of the spleen.

Splenic tumors are uncommon. Described is a 58-yr-old man with multiple hemangiopericytomas of the spleen. Hemangiopericytoma is categorized as a benign vascular tumor, but has a relatively high malignant potential. A review of the literature shows that the case we present is only the second ever reported of a tumor originating from the spleen. Radiological findings and the treatment of the tumor are discussed.

Phosphotransfer signal transduction between two regulatory factors involved in the osmoregulated kdp operon in Escherichia coli.

The proteins KdpD and KdpE are regulatory factors critically involved in the osmotic regulation of the kdpABC operon that is responsible for a high-affinity transport system in Escherichia coli. In this study, we obtained biochemical evidence supporting the view that the KdpD protein is a sensory protein kinase that exhibits autophosphorylation and KdpE-phosphotransfer characteristics. During the course of such studies we established a procedure for purifying the KdpE protein in large quantities. We also developed a procedure for preparing cytoplasmic membrane enriched with the KdpD protein that exhibits in vitro ability with regard to phosphorylation of KdpE protein.

Binding of benzo(a)pyrene with peroxidase and its oxidation by peroxidase-H2O2 intermediate.

The oxidation of benzo(a)pyrene (BP) by horseradish peroxidase (HRP) (EC 1.11.1.7) was examined spectrophotometrically by the decomposition of peroxidase-H2O2 intermediate "compound II." The rate constant of the oxidation of BP was 9.5 X 10(4) M-1 sec-1. The oxidation of BP by HRP was inhibited at high BP concentrations, and the hydrogen donor (BP) inhibition constant, KA', was 1.48 microM. The association constant, Kassoc, of the formation of a complex of BP and HRP at 403 nm was 4.37 X 10(4) M-1. The oxidation products of BP have been identified as 1,6-, 3,6- and 6,12-quinone BP. These products showed no mutagenicity in the mutagenicity assay.

Monoclonal antibodies to poly(adenosine diphosphate ribose) recognize different structures.

Two hybridomas producing monoclonal antibodies to poly(adenosine diphosphate ribose) [poly(ADP-Rib)] were established. One antibody, 10H (IgG3, kappa), bound to most of the poly(ADP-Rib) preparation, which consisted of molecules of various sizes of more than 20 ADP-Rib residues. The binding of this antibody was inhibited by not only poly-(ADP-Rib) but also a monomer unit of poly(ADP-Rib), Ado(P)-Rib-P. The sites protected by antibody 10H were isolated and analyzed by hydrolysis with alkaline phosphomonoesterase and then snake venom phosphodiesterase. The sites contained the same amounts of monomer units and branched portions [Ado(P)-Rib(P)-Rib-P] as the original poly(ADP-Rib) molecules but a lower average number of branched portions per molecule than in the original molecules. The other antibody, 16B (IgM, lambda), reacted with only 50% of the radioactive poly(ADP-Rib), and its binding was not inhibited by a monomer unit. This antibody protected 25% of all the poly(ADP-Rib) molecules from hydrolysis by snake venom phosphodiesterase. The protected sites contained twice as many branched portions per molecule as the original poly(ADP-Rib) molecules. These results show that the two monoclonal antibodies recognize different structures of poly-(ADP-Rib); 10H antibody recognizes the linear structure with ribose-ribose linkages, and 16B antibody may recognize specific structures, including the branched portions of poly-(ADP-Rib).