Structure of the DNA-Eco RI endonuclease recognition complex at 3 A resolution.

The crystal structure of the complex between Eco RI endonuclease and the cognate oligonucleotide TCGCGAATTCGCG provides a detailed example of the structural basis of sequence-specific DNA-protein interactions. The structure was determined, to 3 A resolution, by the ISIR (iterative single isomorphous replacement) method with a platinum isomorphous derivative. The complex has twofold symmetry. Each subunit of the endonuclease is organized into an alpha/beta domain consisting a five-stranded beta sheet, alpha helices, and an extension, called the "arm," which wraps around the DNA. The large beta sheet consists of antiparallel and parallel motifs that form the foundations for the loops and alpha helices responsible for DNA strand scission and sequence-specific recognition, respectively. The DNA cleavage site is located in a cleft that binds the DNA backbone in the vicinity of the scissile bond. Sequence specificity is mediated by 12 hydrogen bonds originating from alpha helical recognition modules. Arg200 forms two hydrogen bonds with guanine while Glu144 and Arg145 form four hydrogen bonds to adjacent adenine residues. These interactions discriminate the Eco RI hexanucleotide GAATTC from all other hexanucleotides because any base substitution would require rupture of at least one of these hydrogen bonds.

Exposition of a family of RNA m(5)C methyltransferases from searching genomic and proteomic sequences.

The Escherichia coli fmu gene product has recently been determined to be the 16S rRNA m(5)C 967 methyltransferase. As such, Fmu represents the first protein identified as an S -adenosyl-L-methionine (AdoMet)- dependent RNA m(5)C methyltransferase whose amino acid sequence is known. Using the amino acid sequence of Fmu as an initial probe in an iterative search of completed DNA sequence databases, 27 homologous ORF products were identified as probable RNA m(5)C methyltransferases. Further analysis of sequences in undeposited genomic sequencing data and EST databases yielded more than 30 additional homologs. These putative RNA m(5)C methyltransferases are grouped into eight subfamilies, some of which are predicted to consist of direct genetic counterparts, or orthologs. The enzymes proposed to be RNA m(5)C methyltransferases have sequence motifs closely related to signature sequences found in the well-studied DNA m(5)C methyltransferases and other AdoMet-dependent methyltransferases. Structure-function correlates in the known AdoMet methyltransferases support the assignment of this family as RNA m(5)C methyltransferases.

Pseudobulbar laughter as a levodopa off phenomenon exacerbated by subthalamic deep brain stimulation.

Pseudobulbar affect is a common symptom in neurodegenerative diseases and can also result from lesions in cortical, subcortical and brainstem regions. In Parkinson's disease (PD), pseudobulbar affect (PBA) can occur as a wearing off phenomenon, manifested usually as crying without emotionality. In addition, subthalamic (STN) deep brain stimulation (DBS) has been reported to induce PBA in PD patients with no prior history of such episodes. We present a case of inappropriate laughter lacking mirth as a levodopa OFF phenomenon in a patient with PD, whose laughter also worsened with STN-DBS in his non-medicated state. Levodopa ameliorated his PBA both with and without stimulation. The case demonstrates pseudobulbar laughter as a levodopa OFF phenomenon that is also exacerbated by STN-DBS.

Expression, purification, and characterization of thymidylate synthase from Lactococcus lactis.

The thymidylate synthase (TS) gene from Lactococcus lactis has been highly expressed in Escherichia coli. The TS protein was purified by sequential chromatography on Q-Sepharose and phenyl-Sepharose. Six grams of cell pellet yielded 140 mg of homogeneous TS. TS is a highly conserved enzyme, and several of the conserved amino acid residues that have been implicated in catalytic function are altered in L. lactis TS. By use of a 3-dimensional homology model, we have predicted covariant changes that might compensate for these differences. With the large amounts of L. lactis TS now available, studies can be pursued to understand the structure-function relationships of this enzyme compared to other TSs and to confirm the presumed roles of the compensatory changes predicted in the homology model.

Pharmacokinetics of recombinant human superoxide dismutase in healthy volunteers.

We studied the pharmacokinetics and effects of recombinant human superoxide dismutase (rhSOD) in 32 normal human volunteers after intravenous bolus doses from 1 mg/kg to 45 mg/kg in a single-blind, placebo-controlled, crossover design. The drug was well tolerated. Neither cardiovascular nor renal function, such as the echocardiographically determined cardiac index, insulin or para-aminohippurate clearance, or the urinary excretion of beta 2-microglobulin or N-acetylglucosaminidase, was affected. Pharmacokinetic analysis by use of noncompartmental methods showed an overall half-life of rhSOD to be about 4 hours for doses from 3 mg/kg to 45 mg/kg. The peak concentrations ranged from 24 to 837 mg/L, and urinary excretion increased from 3% to 57% of total dose after single intravenous bolus administrations of the drug from 1 mg/kg to 45 mg/kg. The mean renal clearance of rhSOD initially increased with dose then plateaued at the highest dose, whereas the nonrenal clearance decreased with dose to a plateau; total clearance remained essentially constant. The progressive increase in renal clearance may be explained by saturation of the tubular reabsorption and degradation of the protein, a mechanism previously described in animal models.

Positive-selection vectors utilizing lethality of the EcoRI endonuclease.

The construction and use of a series of positive-selection vectors are described. These plasmids encode EcoRI endonuclease, the synthesis of which is under the control of the lacUV5 promoter. The pKG2 plasmid encodes a wild-type EcoRI endonuclease. In the absence of EcoRI methylase, the endonuclease is lethal. Cloning into any of the unique restriction sites within the endonuclease-coding gene allows survival of the transformed EcoRI-methylase-less host. The pKGW and pKGS plasmids encode an altered EcoRI endonuclease which, when repressed in a lacIQ host, allows survival in the absence of the methylase. Induction with IPTG, however, results in cell death as a result of high-level EcoRI synthesis. Cloning into any of the unique restriction sites within the EcoRI gene of pKGW or pKGS allows survival of derepressed transformed cells. These vectors strongly select for cloning events which inactivate the endonuclease gene.

Comparison of the nucleotide and amino acid sequences of the RsrI and EcoRI restriction endonucleases.

The RsrI endonuclease, a type-II restriction endonuclease (ENase) found in Rhodobacter sphaeroides, is an isoschizomer of the EcoRI ENase. A clone containing an 11-kb BamHI fragment was isolated from an R. sphaeroides genomic DNA library by hybridization with synthetic oligodeoxyribonucleotide probes based on the N-terminal amino acid (aa) sequence of RsrI. Extracts of E. coli containing a subclone of the 11-kb fragment display RsrI activity. Nucleotide sequence analysis reveals an 831-bp open reading frame encoding a polypeptide of 277 aa. A 50% identity exists within a 266-aa overlap between the deduced aa sequences of RsrI and EcoRI. Regions of 75-100% aa sequence identity correspond to key structural and functional regions of EcoRI. The type-II ENases have many common properties, and a common origin might have been expected. Nevertheless, this is the first demonstration of aa sequence similarity between ENases produced by different organisms.

Cloning, expression and characterization of thymidylate synthase from Cryptococcus neoformans.

The thymidylate synthase (TS)-encoding gene from Cryptococcus neoformans (Cn) has been isolated from cDNA and genomic libraries. The 1127-bp gene contains three introns and a 951-bp open reading frame encoding a 35,844-Da protein. The cDNA clones lack 324 bp of the 5' coding region of the gene. The complete coding sequence was assembled as an expression cassette in pUC19 using parts of the coding sequence from the cDNA and genomic DNA and completing the sequence using synthetic DNA. Production of active TS from Cn (CnTS) was first demonstrated by complementation of a thymine(Thy)-requiring Escherichia coli strain. The expression cassette was subsequently subcloned into the T7 polymerase vector pET15-b. In this construct, CnTS is produced as approximately 10% of the total soluble protein in E. coli. Homogeneous enzyme was obtained at a 36% yield after consecutive chromatography on DEAE-cellulose, Q-Sepharose, phenyl-Sepharose and Affi-Gel Blue. Steady-state kinetic analysis showed that the Km values for dUMP and CH2H4.folate were 2.7 +/- 0.5 microM and 38.2 +/- 2.5 microM, respectively, and the kcat was 5.1 s-1. The enzyme was stable upon storage at -80 degrees C in Tris.HCl pH 7.4 and thiol.

Purification and characterization of the restriction endonuclease RsrI, an isoschizomer of EcoRI.

Rhodobacter sphaeroides strain 630 produces restriction enzyme RsrI which is an isoschizomer of EcoRI. We have purified this enzyme and initiated a comparison with the EcoRI endonuclease. The properties of RsrI are consistent with a reaction mechanism similar to that of EcoRI: the position of cleavage within the -GAATTC-site is identical, the MgCl2 optimum for the cleavage is identical, and the pH profile is similar. Methylation of the substrate sequence by the EcoRI methylase protects the site from cleavage by the RsrI endonuclease. RsrI cross-reacts strongly with anti-EcoRI serum indicating three-dimensional structural similarities. We have determined the sequence of 34 N terminal amino acids for RsrI and this sequence possesses significant similarity to the EcoRI N terminus.

Construction and characterization of new cloning vehicles. II. A multipurpose cloning system.

In vitro recombination techniques were used to construct a new cloning vehicle, pBR322. This plasmid, derived from pBR313, is a relaxed replicating plasmid, does not produce and is sensitive to colicin E1, and carries resistance genes to the antibiotics ampicillin (Ap) and tetracycline (Tc). The antibiotic-resistant genes on pBR322 are not transposable. The vector pBR322 was constructed in order to have a plasmid with a single PstI site, located in the ampicillin-resistant gene (Apr), in addition to four unique restriction sites, EcoRI, HindIII, BamHI and SalI. Survival of Escherichia coli strain X1776 containing pBR313 and pBR322 as a function of thymine and diaminopimelic acid (DAP) starvation and sensitivity to bile salts was found to be equivalent to the non-plasmid containing strain. Conjugal transfer of these plasmids in bi- and triparental matings were significantly reduced or undetectable relative to the plasmid ColE1.

Approach to the treatment of limb disorders with botulinum toxin A. Experience with 187 patients.

OBJECTIVE: To determine the dosing, response expectation, efficacy, and most rational strategy for using intramuscular injections of botulinum toxin A (BTX) for limb disorders. DESIGN: Open-label prospective analysis of outcome after BTX treatment in patients with limb disorders. PROCEDURE: Botulinum toxin A prepared from lyophilized botulinum toxin was injected into selected upper and lower limb muscles under electromyographic guidance. Booster injections were given every 10 to 14 days during the first month (if needed) until optimal effects were achieved. Clinical data and muscle strength testing were obtained before the first injections and repeated at each visit. Level of disability, global functional improvement, and relief of pain were evaluated 6 to 8 weeks after the first set of injections. Practical and meaningful BTX doses by muscle, limb, or condition according to specified levels of efficacy were developed. MAIN OUTCOME MEASURES: Botulinum toxin A efficacy was calculated as an arithmetic combination of changes in the 3 clinical ratings before and after administration of BTX. RESULTS: Botulinum toxin A injections were given to 187 patients with limb disorders during an 8-year period (136 with dystonia, 37 with parkinsonian, essential, and cerebellar tremors, and 14 with spasticity). Four overall outcomes from no effect to almost complete improvement in the use of the limb or relief of pain were found, and determined the strategy for follow-up injections. Average BTX efficacy for all patients was 65% and ranged from 83.5% for focal hand dystonia to 35.7% for parkinsonian tremor. Botulinum toxin A injections relieved pain, independent of motor function, in 82.7% of patients with painful muscle spasms. CONCLUSIONS: Botulinum toxin A was found to be a safe and useful treatment of various limb conditions. Botulinum toxin A was significantly more effective when only a few muscles needing low doses were injected, and tended to be more useful in dystonia and spasticity than tremor. Candidates for BTX injection could be categorized functionally into 3 groups independent of the underlying disorders. The only significant adverse effect of BTX injection in limbs was transient weakness in injected or neighboring muscles.

Delayed-onset cerebellar syndrome.

BACKGROUND: Delayed-onset involuntary movements, including dystonia and myoclonus, have been reported after stroke or head trauma. Moreover, there have been reports of delayed-onset isolated intention tremor and, in several of these cases, gait ataxia. OBJECTIVE: To further define the clinical features of a delayed-onset cerebellar syndrome. DESIGN: Subjects with cerebellar tremor and either head trauma or stroke were identified using a computerized database, providing detailed demographic and clinical information of 4002 patients with involuntary movements other than Parkinson's disease seen at our center between 1983 and 1995. Medical records and videotaped neurological examinations were retrospectively reviewed. SETTING: The Center for Parkinson's Disease and Other Movement Disorders at Columbia-Presbyterian Medical Center, New York, NY. PATIENTS: Five patients with delayed-onset cerebellar syndromes. RESULTS: Five patients with stroke or head trauma developed a cerebellar syndrome 3 weeks to 2 years after the initial insult. The syndrome, characterized by intention tremor, ataxic dysarthria, nystagmus, dysmetria, dysdiadochokinesis, and gait ataxia, was progressive in at least one patient. In four patients, lesions were present on neuroimaging in the thalamus or brain stem (especially in the midbrain). CONCLUSIONS: A delayed-onset cerebellar syndrome may follow head trauma or stroke. The syndrome is sometimes progressive and often disabling. The delayed onset implies that the syndrome is not caused by the initial lesion itself but may be caused by development of post-synaptic supersensitivity or secondary reorganization of involved pathways.

Use of intrathecal baclofen in the treatment of patients with dystonia.

BACKGROUND: Continuous infusion of intrathecal (IT) baclofen is a highly effective standard therapy for severe spasticity of spinal origin. By contrast, there is limited clinical experience regarding the use of IT baclofen in treating patients with dystonia, and little is known regarding the indications for treatment, efficacy, and safety of IT baclofen in this disorder. OBJECTIVE: To study retrospectively the effects of IT baclofen in treating 25 patients with severe segmental or generalized dystonia. SETTING: Neurological Institute, Columbia-Presbyterian Medical Center, New York, NY. PATIENTS: Twenty-five patients with severe segmental or generalized dystonia that was refractory to oral medications underwent IT baclofen test dosing. In addition to dystonia, 17 patients had spasticity or painful spasms. Thirteen of 25 patients responded to the test doses of IT baclofen, according to unblinded neurological assessments that included the patient's subjective report; all 13 underwent implantation of a pump for continuous IT baclofen infusion. RESULTS: In contrast to reports of patients with spasticity of spinal origin, those with dystonia in the present series had a lower response rate to bolus IT baclofen doses and a smaller degree of clinical improvement. For 10 of the 13 responders to the test doses of IT baclofen, dystonia rating scale scores of videotaped examinations by blinded observers detected no significant change (P < .07) in severity of dystonia. Retrospective data from 11 of 13 patients with implantable pumps, followed up for a mean interval of 21 months after pump insertion, showed continuing efficacy in 6 individuals (55%), based on a determination of patient satisfaction; however, only 3 patients (27%) reported a sustained improvement in functional capacity. Five (38%) of the 13 patients with implantable pumps experienced severe complications that required hospitalization. CONCLUSIONS: Despite recent reports that have described the benefit in small numbers of patients with dystonia, we concluded that the role of IT baclofen in treating severe dystonia remains uncertain. Intrathecal baclofen may be more effective when dystonia is associated with spasticity or pain. In the present series, we detected no significant difference in the response to IT baclofen in patients with or without spasticity or pain, perhaps owing to the small sample size.

Clinical presentation and pharmacological therapy in corticobasal degeneration.

BACKGROUND: To date, to our knowledge, there is no systematic presentation of treatment outcome in large series of patients clinically diagnosed as having corticobasal degeneration. OBJECTIVE: To evaluate the clinical presentation and treatment outcome of patients clinically diagnosed as having corticobasal degeneration. SUBJECTS: We gathered case patients seen in 8 major movement disorder clinics during the last 5 years who were diagnosed as having corticobasal ganglionic degeneration. METHODS: Using a chart review method, we recorded the clinical presentation, medications used, response to medications, and adverse effects. RESULTS: A total of 147 case patients were reviewed, 7 were autopsy proven. Parkinsonian features were present in all, other movement disorders in 89%, and higher cortical dysfunction in 93%. The most common parkinsonian sign was rigidity (92%), followed by bradykinesia (80%), gait disorder (80%), and tremor (55%). Other movement disorders were dystonia in 71% and myoclonus in 55%. Higher cortical dysfunction included dyspraxia (82%), alien limb (42%), cortical sensory loss (33%), and dementia (25%). Ninety-two percent of the case patients received dopaminergic drugs, which resulted in a beneficial effect for 24%. Parkinsonian signs were the elements improving the most and levodopa was the most effective drug. Benzodiazepines, primarily clonazepam, were administered to 47 case patients, which resulted in improvement of myoclonus in 23% and dystonia in 9%. The most frequent disabling adverse effects of drug trials in these case patients were somnolence (n = 24), gastrointestinal complaints (n = 23), confusion (n = 16), dizziness (n =12), hallucinations (n = 5), and dry mouth (n = 5). CONCLUSIONS: Pharmacological intervention was largely ineffective in the management of corticobasal degeneration, and new treatments are needed for ameliorating the symptoms of this syndrome.

Oral and genital tardive pain syndromes.

We report the development of chronic painful oral or genital sensations in 11 patients with tardive akathisia, tardive dyskinesias, or tardive dystonia. In each case, the pain syndrome became a source of profound distress for the patient, overshadowing all other concurrent neuropsychiatric symptoms, and requiring treatment. Agents effective in treating tardive dyskinesia and tardive akathisia, such as catecholamine depletors, proved effective in reducing the painful sensations. Our observations support the concept of tardive pain as a complication of chronic neuroleptic exposure.

Double-blind, placebo-controlled trial of botulinum toxin injections for the treatment of spasmodic torticollis.

We enrolled 55 patients in a double-blind, placebo-controlled, parallel design study of the effectiveness of botulinum toxin (Botox) injections for the treatment of spasmodic torticollis. Patients received a standard series of injections, either placebo or Botox. We determined the sites of injection and dose per muscle by the nature of head deviation. Compared with placebo, Botox produced statistically significant improvement in the severity of torticollis, disability, pain, and degree of head turning. There were no serious side effects. During the double-blind phase, 61% of patients injected with Botox improved; 74% of patients subsequently improved during a later open phase at a higher dose of Botox. Direction of head turning, severity of torticollis, and presence or absence of jerky movements did not significantly influence the response rate. We conclude that Botox is a valuable treatment for spasmodic torticollis.

Falling asleep at the wheel: motor vehicle mishaps in persons taking pramipexole and ropinirole.

The authors report a new side effect of the dopamine agonists pramipexole and ropinirole: sudden irresistible attacks of sleep. Eight PD patients taking pramipexole and one taking ropinirole fell asleep while driving, causing accidents. Five experienced no warning before falling asleep. The attacks ceased when the drugs were stopped. Neurologists who prescribe these drugs and patients who take them should be aware of this possible side effect.

Secondary dystonia and the DYTI gene.

Early-onset (< 28 years) primary dystonia in most Ashkenazi Jews is due to a single founder mutation in the DYT1 gene on chromosome 9q34, as determined by very strong linkage disequilibrium with a haplotype of 9q34 alleles at surrounding marker loci. The role of this mutation in individuals with secondary causes for dystonia has never been tested, although environmental insults, such as neuroleptic exposure or perinatal asphyxia, are proposed to precipitate dystonia in genetically predisposed individuals. We assessed 9q34 haplotypes in 40 Ashkenazi patients with secondary dystonia; 25 had early onset of symptoms, including 15 with exposure to neuroleptic medication or perinatal asphyxia. Of the 25 patients with early onset, 9 were considered phenocopies of DYT1 having normal examinations except for dystonia, normal radiographic and other laboratory studies, and onset in a limb or the neck. Only one individual whose dystonia developed in the setting of a measles infection carried the associated haplotype. Our findings indicate that clinical diagnostic criteria that include historical information to detect tardive dystonia and perinatal asphyxia discriminate primary dystonia due to the DYT1 founder mutation. We found no evidence that the DYT1 founder mutation contributes to secondary dystonia.

Trypanosoma brucei dihydrofolate reductase-thymidylate synthase: gene isolation and expression and characterization of the enzyme.

The gene encoding the bifunctional dihydrofolate reductase (DHFR) and thymidylate synthase (TS) of Trypanosoma brucei brucei has been isolated and expressed in Escherichia coli, and the enzyme has been purified and characterized. The coding sequence of the DHFR-TS is 1581 nt, encoding a 527-amino-acid protein of 58,505 Da. The gene was expressed under control of the trc promoter in pKK233-2. The resulting expression plasmid conferred trimethoprim resistance to E. coli DH5 alpha and complemented the TS deficiency in chi 2913recA cells indicating the presence of active DHFR and TS. DHFR-TS was purified by methotrexate-Sepharose chromatography. In addition to the full-length enzyme, the purified enzyme contained 31 and 31.5-kDa forms of the enzyme that cross-reacted with anti-L. major DHFR-TS antibodies; one was truncated at the N- and C termini, and the other at only the C terminus. Despite the presence of sufficient TS for complementation, TS activity was not detectable in the crude extract or in the final purified enzyme preparation. Although the majority of the enzyme appears to be full length, it is possible that the TS domain has been degraded by one of more residues, which would inactivate the ability to synthesize thymidylate. Kinetic analysis of DHFR yielded kcat and Km values similar to those of related enzymes. The T. brucei DHFR has Ki values for antimicrobial antifolates pyrimethamine and trimethoprim which are significantly lower than the closely related T. cruzi or L. major DHFRs or than human DHFR.

Inhibition of complement, evoked antibody, and cellular response prevents rejection of pig-to-primate cardiac xenografts.

Complement (C) inhibition alone using a recombinant soluble form of complement receptor type 1 (sCR1) prevents hyperacute rejection but not subsequent irreversible accelerated acute rejection of discordant pig-to-cynomolgus monkey cardiac xenografts, which occurs within 1 week. To inhibit accelerated acute rejection, which is associated with a rise in serum xenoreactive antibody (Ab) and a cellular infiltrate, triple therapy with standard immunosuppressive agents (cyclosporine, cyclophosphamide, and steroids [CCS]) was combined with continuous C inhibition using sCR1. Each of two monkeys that received sCR1 + CCS showed minimal evidence of rejection when killed on days 21 and 32 in comparison to a monkey that received sCR1 + subtherapeutic CCS (rejected at 11 days) and a control that received CCS alone (rejected at 38 min). Prolonged xenograft survival was associated with low Ab levels and a minimal cellular infiltrate, suggesting that combined inhibition of C, xenoreactive Ab responses, and cellular immunity may be a useful approach in overcoming the immune barriers to discordant xenotransplantation.