Long-term follow-up of IgM monoclonal gammopathy of undetermined significance.

The current study was conducted to determine the risk of adverse outcomes among patients with monoclonal gammopathy of undetermined significance (MGUS) of the IgM class. Two hundred thirteen patients with IgM MGUS were identified in southeastern Minnesota from 1960 to 1994. The primary end point was progression to lymphoma or a related disorder assessed by the Kaplan-Meier method. Patients were followed for a total of 1,567 person-years (median, 6.3 years per subject). Seventeen patients developed lymphoma (relative risk [RR], 14.8) and six progressed to Waldenstrom's macroglobulinemia (RR, 262), while three developed primary amyloidosis (RR, 16.3) and three others had chronic lymphocytic leukemia (RR, 5.7). The relative risk of progression was 16-fold higher in the IgM MGUS patients compared to the white population of the Iowa Surveillance, Epidemiology, and End Results (SEER) program. The risk of progression of MGUS of IgM type to lymphoma or related disorders averaged 1.5% per year throughout the period of observation.

Rehearsal by eye movement improves visuomotor performance in cerebellar patients.

In order to assess the effect of rehearsal by eye movement alone on visuomotor performance, the eye movements and visually guided stepping of two cerebellar patients were monitored before and after a first and second batch of eye-movement rehearsals, in which patients made saccadic eye movements to the first 6 footfall targets (in a sequence of 18) whilst standing stationary at the start of the walkway. There was a marked improvement in oculomotor and locomotor performance following the second batch of eye-movement rehearsal. Both patients showed reduced occurrence of saccadic dysmetria, evident as a significant increase in the proportion of single to multi-saccadic eye movements (from 46 to 77% for DB and from 75 to 94% for TP). This was accompanied by increased regularity and accuracy of stepping in both patients, and decreased stance and double support phase durations (one patient only). Separate testing confirmed that these improvements in eye movements and stepping did not result from simple repetition of the task. This is the first demonstration of a technique--rehearsal by eye movement--that improves the visuomotor performance of cerebellar patients. It is compelling evidence for our proposal that during visually guided stepping the locomotor control system is dependent on assistance from the oculomotor control system.

A high bone marrow plasma cell labeling index in stable plateau-phase multiple myeloma is a marker for early disease progression and death.

The plasma cell labeling index (PCLI) is a measure of plasma cell proliferative activity and is an important prognostic factor in newly diagnosed multiple myeloma (MM). Occasionally patients have been observed with stable, plateau phase MM with minimal numbers of residual light-chain-restricted monoclonal plasma cells, but a high PCLI. No data are available on the outcomes for such patients. Data from 57 patients with plateau phase MM and a marrow PCLI of more than 1.0% were compared with 105 matched control patients with MM with a marrow PCLI of less than 1.0%. All patients had less than 10% total plasma cells on marrow aspirate and biopsy. The median time to progression and overall survival were 8 months and 20 months, respectively, in the high PCLI group versus 39 months and 56 months, respectively, in the low PCLI group (P < .0001). These findings suggest that a high PCLI in patients with apparently stable, plateau phase MM is an adverse parameter that may predict a short time to disease progression and death.

Molecular cloning and expression of an inwardly rectifying K(+) channel from bovine corneal endothelial cells.

PURPOSE: To determine the presence of a putative inwardly rectifying K(+) channel in bovine corneal endothelial (BCE) cells and to characterize its molecular and electrophysiological properties. METHODS: An RT-PCR strategy was used to clone an IRK1 channel sequence from BCE mRNA. Northern blot analysis was used to confirm expression of this sequence in cultured BCE cells. Two-electrode voltage-clamp and whole-cell patch-clamp recordings were used to characterize the cloned channel expressed in Xenopus oocytes and the native channels in cultured BCE cells, respectively. RESULTS: A full-length (1284 bp) coding sequence that shares 99.7% nucleotide sequence and 100% amino acid sequence identity to bovine lens IRK1 (Kir2.1) was cloned. The authors designate this sequence BCE IRK1 or BCIRK1. Northern blot analysis indicated that BCIRK1 mRNA is expressed in cultured BCE cells with two major transcripts of 7.5 and 5.5 kb. BCIRK1 cDNA was subcloned into the vector, pcDNA3.1(-), and cRNA transcribed from the BCIRK1 cDNA clone was injected into Xenopus oocytes. Two-electrode voltage-clamp recordings from injected oocytes revealed inwardly rectifying K(+) currents that were blocked by external Ba(2+) and Cs(+) in a concentration- and voltage-dependent manner. Whole-cell patch-clamp recordings from dissociated cultured BCE cells revealed strongly inwardly rectifying K(+) currents with similar properties. CONCLUSIONS: Corneal endothelial cells express IRK1 (Kir2.1) inwardly rectifying K(+) channels. Consistent with the properties of IRK1 channels, BCIRK1 is likely involved in regulating membrane potential and possibly other cellular functions in corneal endothelial cells.

Structural requirement of the calcium-channel subunit alpha2delta for gabapentin binding.

Gabapentin [Neurontin, 1-(aminomethyl)cyclohexaneacetic acid] is a novel anticonvulsant drug with a high binding affinity for the Ca(2+)-channel subunit alpha(2)delta. In this study, the gabapentin-binding properties of wild-type and mutated porcine brain alpha(2)delta proteins were investigated. Removal of the disulphide bonds between the alpha(2) and the delta subunits did not result in a significant loss of gabapentin binding, suggesting that the disulphide linkage between the two subunits is not required for binding. Singly expressed alpha(2) protein remained membrane associated. However, alpha(2) alone was unable to bind gabapentin, unless the cells were concurrently transfected with the expression vector for delta, suggesting that both alpha(2) and delta are required for gabapentin binding. Using internal deletion mutagenesis, we mapped two regions [amino acid residues 339-365 (DeltaF) and 875-905 (DeltaJ)] within the alpha(2) subunit that are not required for gabapentin binding. Further, deletion of three other individual regions [amino acid residues 206-222 (DeltaD), 516-537 (DeltaH) and 583-603 (DeltaI)] within the alpha(2) subunit disrupted gabapentin binding, suggesting the structural importance of these regions. Using alanine to replace four to six amino acid residues in each of these regions abolished gabapentin binding. These results demonstrate that region D, between the N-terminal end and the first putative transmembrane domain of alpha(2), and regions H and I, between the putative splicing acceptor sites (Gln(511) and Ser(601)), may play important roles in maintaining the structural integrity for gabapentin binding. Further single amino acid replacement mutagenesis within these regions identified Arg(217) as critical for gabapentin binding.

The novel anticonvulsant drug, gabapentin (Neurontin), binds to the alpha2delta subunit of a calcium channel.

Gabapentin (1-(aminomethyl)cyclohexane acetic acid; Neurontin) is a novel anticonvulsant drug, with a mechanism of action apparently dissimilar to that of other antiepileptic agents. We report here the isolation and characterization of a [3H]gabapentin-binding protein from pig cerebral cortex membranes. The detergent-solubilized binding protein was purified 1022-fold, in a six-step column-chromatographic procedure, with a yield of 3.9%. The purified protein had an apparent subunit Mr of 130,000, and was heavily glycosylated. The partial N-terminal amino acid sequence of the Mr 130,000 polypeptide, EPFPSAVTIK, was identical to that reported for the alpha2delta subunit of the L-type Ca2+ channel from rabbit skeletal muscle (Hamilton, S. L., Hawkes, M. J., Brush, K., Cook, R., Chang, R. J., and Smilowitz, H. M. (1989) Biochemistry 28, 7820-7828). High levels of [3H]gabapentin binding sites were found in membranes prepared from rat brain, heart and skeletal muscle. Binding of [3H]gabapentin to COS-7 cells transfected with alpha2delta cDNA was elevated >10-fold over controls, consistent with the expression of alpha2 delta protein, as measured by Western blotting. Finally, purified L-type Ca2+ channel complexes were fractionated, under dissociating conditions, on an ion-exchange column; [3H]gabapentin binding activity closely followed the elution of the alpha2 delta subunit. [3H]Gabapentin is the first pharmacological agent described that interacts with an alpha2delta subunit of a voltage-dependent Ca2+ channel.

Class I and IV antiarrhythmic drugs and cytosolic calcium regulate mRNA encoding the sodium channel alpha subunit in rat cardiac muscle.

Previous studies have shown that chronic in vivo treatment with the antiarrhythmic drug mexiletine produces an increase in sodium channel number. We examined whether chronic mexiletine treatment would similarly regulate the level of mRNA encoding the cardiac sodium channel. RNA isolated from cardiac tissue was probed with a 2.5-kilobase cRNA transcribed with T7 RNA polymerase from the clone Na 8.4, which encodes nucleotides 3361-5868 of the alpha subunit of the RIIA sodium channel subtype. Chronic mexiletine treatment produced a 3-fold increase in the level of mRNA encoding sodium channel alpha subunits. Previous studies of cultured skeletal muscle cells had suggested that chronic sodium channel blockade may mediate an increase in sodium channel mRNA by changes in cytosolic Ca2+ concentration. To address this issue, we assessed whether verapamil would also produce up-regulation of the level of mRNA encoding the sodium channel and whether the calcium ionophore A23187 would produce the opposite effect on mRNA level. Verapamil treatment increased sodium channel mRNA level up to 3-fold, whereas in vitro A23187 treatment decreased the mRNA level 5-fold. The combination of verapamil and mexiletine produced no further increase in the mRNA level, compared with that seen with the single agents, suggesting a convergent second messenger pathway for the actions of these two drugs. These data show that the level of mRNA encoding sodium channels is substantially increased during antiarrhythmic drug treatment and suggest that change in cytosolic Ca2+ concentration is the second messenger involved in the regulation of levels of mRNA encoding the alpha subunit of the cardiac sodium channel.

Primary structure and functional expression of the beta 1 subunit of the rat brain sodium channel.

Voltage-sensitive sodium channels are responsible for the initiation and propagation of the action potential and therefore are important for neuronal excitability. Complementary DNA clones encoding the beta 1 subunit of the rat brain sodium channel were isolated by a combination of polymerase chain reaction and library screening techniques. The deduced primary structure indicates that the beta 1 subunit is a 22,851-dalton protein that contains a single putative transmembrane domain and four potential extracellular N-linked glycosylation sites, consistent with biochemical data. Northern blot analysis reveals a 1,400-nucleotide messenger RNA in rat brain, heart, skeletal muscle, and spinal cord. Coexpression of beta 1 subunits with alpha subunits increases the size of the peak sodium current, accelerates its inactivation, and shifts the voltage dependence of inactivation to more negative membrane potentials. These results indicate that the beta 1 subunit is crucial in the assembly, expression, and functional modulation of the heterotrimeric complex of the rat brain sodium channel.

Functional properties of rat brain sodium channels expressed in a somatic cell line.

Transfection of Chinese hamster ovary cells with complementary DNA encoding the RIIA sodium channel alpha subunit from rat brain led to expression of functional sodium channels with the rapid, voltage-dependent activation and inactivation characteristic of sodium channels in brain neurons. The sodium currents mediated by these transfected channels were inhibited by tetrodotoxin, persistently activated by veratridine, and prolonged by Leiurus alpha-scorpion toxin, indicating that neurotoxin receptor sites 1 through 3 were present in functional form. The RIIA sodium channel alpha subunit cDNA alone is sufficient for stable expression of functional sodium channels with the expected kinetic and pharmacological properties in mammalian somatic cells.

Electrical activity, cAMP, and cytosolic calcium regulate mRNA encoding sodium channel alpha subunits in rat muscle cells.

The number of sodium channels increases sharply during development of rat skeletal muscle cells in vitro. An 8.5 kb mRNA encoding sodium channel alpha subunit rises to a peak on day 13 in vitro and falls to a value of 50% of the peak by day 18, consistent with the conclusion that mRNA abundance is a major determinant of the rapid rise in sodium channel number. Electrical activity and increased cytosolic calcium decrease the level of alpha subunit mRNA, and cAMP increases its level in parallel with changes in the number of sodium channels. The similarity between the changes in mRNA levels and sodium channel density indicates that the regulation of alpha subunit mRNA level is an important mechanism of feedback regulation of sodium channel density by electrical activity in developing rat muscle cells.

Coital risk factors for cervical cancer. Sexual activity among white middle class women.

This study reports the sexual history of a group of young women participating in an epidemiologic project. The methods used to recruit and interview the participants minimized self-selection bias. Results of over 11,000 interviews with 1,892 participants showed that 72.3% should be considered at high risk for developing cervical cancer and its precursors and should be screened regularly for this disease.

Characterization of actin mRNA levels during BC3H1 cell differentiation.

The expression of a vascular smooth muscle specific alpha-actin isoform can be induced in mouse BC3H1 smooth muscle cells by treating confluent monolayers with serum-free medium (Strauch, A. R., and Rubenstein, P. A. (1984) J. Biol. Chem. 259, 3152-3159; 7224-7229). Using blot hybridization techniques, two size classes of actin RNA were identified in BC3H1 cells with the relative amount of RNA in each size class varying according to the developmental state of the cells; a 2100-nucleotide actin RNA was most abundant in myoblasts, whereas a smaller 1500-nucleotide actin RNA was found predominantly in fully differentiated myocytes. Results of in vitro translation experiments suggested that the 2100-nucleotide actin RNA on blots of myoblast total RNA corresponded to a mixture of similar size transcripts encoding both beta- and gamma-actin, while the 1500-nucleotide actin RNA in myocytes was an alpha-actin mRNA. Cell-cell contact and serum withdrawal initiated a 6-fold increase in the level of alpha-actin mRNA in BC3H1 cells that was followed by a 3-fold decrease in the amount of beta- and gamma-actin mRNA when confluent cells were exposed to serum-free medium for prolonged periods. Vascular smooth muscle alpha-actin was the major alpha-actin isoform synthesized in L-[35S]cysteine-labeled BC3H1 myocytes, indicating that the 1500-nucleotide actin mRNA size class in these cells may be enriched for vascular smooth muscle alpha-actin transcripts.

High-dose methotrexate with a safe rescue program.

23 patients with osteogenic sarcoma were observed during 142 6-hour high-dose infusions of methotrexate (MTX, 3,000--8,200 mg/m2). Calcium leukovorin was given by intravenous injection at 3-hour intervals beginning 2 h after the completion of each MTX infusion with extension of the intervals to 6 h following the first day of rescue. All patients also received continuous intravenous infusions of alkalinized fluids for the entire duration of leukovorin rescue. No larger doses of leukovorin were given to any patient. Three of the 142 MTX infusions resulted in mild cytotoxic side effects. Plasma MTX clearance ranged from 90 to 600 ml/min among the 62 infusions where plasma clearance could be accurately calculated. The 3 patients with mild toxicity had low drug clearance, but others with similar low MTX clearance experienced no apparent toxic effects beyond the expected transient nausea.