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.

Large-scale purification of two forms of active lac operator from plasmids.

A practical procedure is described for obtaining milligram quantities of a small (29 nucleotide) Eco RI restriction fragment of DNA containing the Escherichia coli lac operator. A yield of 10--15 mg of operator is obtained from 1 kg of wet cell paste. The resultant operator is shown to be homogeneous and competitively active in filter assays. Two separable but interconvertible forms of lac operator exist in solution, probably linear duplex and hairpin isomers. Only the presumed linear form is active in binding lac repressor by competition assay, but the two isomers are interconvertible by heating to 80 degrees C. The methods described here should be generally applicable for purifying other restriction fragments from plasmids.

Dynamic contributions to the DNA binding entropy of the EcoRI and EcoRV restriction endonucleases.

Molecular Dynamics simulations on DNA-EcoRI and DNA-EcoRV complexes suggest that the DNA within these complexes is significantly more ordered than free DNA. Similarly, both the protein and the DNA are more ordered in the specific (cognate) DNA-EcoRV complex than they are in the non-cognate DNA-protein complex, consistent with recently proposed analogies between protein folding and sequence-specific DNA-protein recognition. Analysis of the trajectories shows that the net entropy gain upon specific binding to be the result of opposing contributions. Solvent release, which increases entropy versus configurational terms (as measured by the magnitude of the atomic fluctuations), and collective terms from tight coupling between the motions of the protein and the DNA.

Molecular dynamics simulation study of DNA dodecamer d(CGCGAATTCGCG) in solution: conformation and hydration.

A molecular dynamics simulation of the dodecamer duplex d(CGCGAATTCGCG) using the particle mesh Ewald sum assumed a B-conformation remarkably close to the observed X-ray structure. The Ewald summation method effectively eliminates the usual "cut-off" of long-range interactions and allowed us to evaluate the full effect of the electrostatic forces. This simulation showed remarkable agreement with the Dickerson X-ray structure in both average structure and B-factors; within the EcoRI site itself, the rms deviation between the average theoretical and observed structures was 1.1 A. The width of the minor groove fluctuated between a wide and narrow configuration with the latter corresponding closely to the X-ray structure. The simulation also suggested a strong sequence-dependent signature on the minor groove width in both wide and narrow conformers. Hydration shells in both the major and minor grooves were observed. The "spine of hydration" in the minor groove was clear. In the major groove the first hydration shell appears to be a ribbon-like structure that reproduces the principal features of observed X-ray structures; subtle variations of this hydration pattern suggest sequence dependencies. Sequence-dependent features were also examined for helical and other geometric parameters. The successful reproduction of many experimentally observed fine structural features shows that the Ewald summation significantly improves the fidelity of the calculations.

An in vivo study of phosphorus and glucose metabolism in Alzheimer's disease using magnetic resonance spectroscopy and PET.

OBJECTIVES: To study phosphorus and glucose metabolism in whole-brain slices of otherwise healthy patients with dementia of the Alzheimer type (DAT) and healthy controls. DESIGN: We used proton nuclear magnetic resonance imaging phosphorus spectroscopy and positron emission tomography to study in vivo brain phosphorus and glucose metabolism. PATIENTS: Whole-brain slice phosphorus metabolism was studied in nine drug free patients with mild to moderately severe dementia of the Alzheimer type (DAT) and in eight age- and sex-matched healthy controls. Mean ages (+/- SD) of the patients and controls were 60 +/- 10 years and 64 +/- 16 years, respectively. Positron emission tomography was used to study cerebral glucose metabolism in seven of the patients with DAT and seven of the healthy controls. RESULTS: Patients with DAT had significant brain glucose hypometabolism compared with controls, but there was no significant group difference in any phosphorus metabolite concentration or ratio in the same volume of brain tissue. Also, within patients with DAT there was no correlation between any phosphorus metabolite concentration or ratio and either severity of dementia or glucose metabolism. CONCLUSIONS: We suggest glucose metabolism is reduced early in DAT (reflecting decreased basal synaptic functioning) and is unrelated to a rate limitation in glucose delivery, abnormal glucose metabolism, or abnormal coupling between oxidation and phosphorylation. Normal or near-normal levels of phosphorus metabolites are maintained in mild, moderate, and severe DAT. Therefore, altered high-energy phosphate levels are not a consequence of reduced glucose metabolism in DAT, and do not play a major role in the pathophysiology of the disorder, at least in whole-brain sections.

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.

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.

Differences in the inhibition of translation by cisplatin, transplatin, and certain related compounds.

The non-therapeutic cisplatin congeners transplatin and chloroethylenetriamine platinum (dien) inhibited translation to a similar extent as cisplatin did. The IC50 values were: cisplatin 23 microM, transplatin 54 microM, and dien 117 microM. Unlike certain heavy metal inhibitors of translation, the effect of neither cisplatin nor the congeners was reversed by 3':5'-cyclic adenosine monophosphate (cAMP). This suggests that the effect of these platinum compounds does not occur by the heavy metal mechanism. Polyribosomes and ribosomal subunits formed in transplatin-inhibited reactions differed from those in reactions inhibited by cisplatin. Specifically, large polyribosomes and complete 80S ribosomal subunits accumulated in the presence of transplatin. This indicates that while cisplatin slowed initiation of peptide synthesis, the trans-isomer slowed elongation. Substantive differences were not found between cisplatin and the monofunctional compound dien. This congener increased the non-peptidyl disintegrations per minute in the acid precipitates of assays containing [35S]methionine. The high background indicated that an interaction between the label and a precipitable component of the system was induced by dien. However, consumption of methionine by this interaction did not appear to be the cause of the inhibition. Although there may be differences in the mechanisms of the effects, the finding that the non-therapeutic congeners inhibit translation at similar concentrations as cisplatin suggests that this inhibition is not responsible for the anticancer effect. On the other hand, the possibility that decreased translation could play an important role in the toxicity of these compounds in certain quiescent cells cannot be ruled out.

Effect of vancomycin infusion on cardiac function in patients scheduled for cardiac operation.

Patients scheduled for cardiac operation often receive vancomycin before the operation to decrease postoperative staphylococcal wound infections. In animal studies, vancomycin depressed cardiac function approximately 15%. Because of the potentially serious consequences of myocardial depression in patients undergoing cardiac operation, we examined the effect of vancomycin infusion on cardiac hemodynamics in patients scheduled for cardiac operation. Patients who were scheduled for cardiac operation and vancomycin prophylaxis were enrolled in our study. After baseline cardiac output, mean arterial pressure, central venous pressure, and pulmonary capillary wedge pressure were measured, 1 gm of vancomycin HCl was infused over 1 hour. Cardiac output, mean arterial pressure, central venous pressure, and pulmonary capillary wedge pressure were measured at 15, 30, 60, 90, and 120 minutes after the start of the infusion. In the 46 patients that completed the study, no significant change was observed in cardiac output or systemic vascular resistance at any time when compared with baseline. Mean arterial pressure increased significantly (p = 0.03) between baseline (90.8 +/- 2.4 standard error of mean) and 90 minutes (94.1 +/- 2.4 standard error of mean). One patient had a transient 30% fall in mean arterial pressure and systemic vascular resistance with facial flushing during the infusion. In conclusion, we found that vancomycin infusion over 1 hour in patients before cardiac operation is safe and not associated with cardiac depression.

Echocardiographic diagnosis and surgical treatment of postpartum pulmonary embolism.

Pulmonary embolism remains a problem in the United States in terms of both morbidity and mortality. New diagnostic modalities to make rapid diagnosis are now available, and allow for bedside diagnosis of pulmonary embolism without the use of pulmonary angiography. As a reference, a case involving a postpartum patient is reviewed. Use of echocardiography, a device readily available even in small institutions, allowed for early diagnosis and institution of therapy in this particular case and in others. Diagnostic features of pulmonary embolism are discussed and the literature is reviewed.

Blunt injuries to the aortic arch vessels.

Thirty patients with 33 vascular injuries from blunt trauma to the brachiocephalic branches of the aortic arch are reported. To our knowledge, this is the largest series reported to date of blunt injuries to these vessels. Mechanisms of injury included deceleration, traction, and crush. Half of the injured vessels were the innominate artery, and a quarter each were the common carotid and subclavian arteries. Common associated injuries were head injuries, hemopneumothorax, lung contusion, long bone fractures, and brachioplexus injuries. Widened mediastinum and extrapleural hematoma were common radiographic findings, and aortic rupture was frequently suspected. Angiography was performed in all patients to identify precisely the nature and site of the injury. Surgical approaches varied with the anatomical site of the injury and required consideration of vascular control in chest, neck, and upper extremity. Twenty-seven patients are alive 6 months to 10 years after injury. Eighteen of 20 vascular reconstructions were patent at follow-up. No patient with brachioplexus injury had return of neurological function.

Clinical experience with sotalol in the treatment of cardiac arrhythmias.

The treatment of cardiac arrhythmias has changed over the past several years, as concerns have grown about proarrhythmias and increased mortality associated with certain antiarrhythmic agents. Sotalol, a unique racemic compound with both Class II (beta-blocking) and Class III (repolarization-prolonging) properties, effectively controls life-threatening arrhythmias and has a favorable safety profile, especially when compared with traditional antiarrhythmic drugs. A review of clinical trials demonstrates that sotalol is an effective treatment for premature ventricular complexes, ventricular tachycardia, and ventricular fibrillation. Moreover, it significantly reduces cardiac and all-cause mortality in these patient populations. Although sotalol currently is approved in the United States only for the treatment of life-threatening ventricular arrhythmias, it also is active against several supraventricular arrhythmias. Sotalol is safe and generally well tolerated; most of the adverse effects experienced with its use are related to its beta-blocking activity. However, sotalol has been associated with life-threatening proarrhythmias, including torsades de pointes, but the incidence of this adverse effect is low. Unlike pure Class II agents, sotalol usually is well tolerated in patients with mild-to-moderate left ventricular dysfunction and rarely causes new or worsening congestive heart failure. Sotalol represents an advance in the treatment and prevention of symptomatic ventricular arrhythmias and appears to be a reasonable first-line choice when the arrhythmia is life threatening.

Calcium deficiency enhances cadmium accumulation in the central nervous system.

Weanling male rats were administered 1 of 4 diets for 40 days: control (CONT), low Ca (LOCA), control plus Cd (CONT + Cd) or low Ca plus Cd (LOCA + Cd). After 40 days, Cd was analyzed in 7 brain regions, spinal cord, serum, liver, kidney, muscle and femur by atomic absorption spectrophotometry with Zeeman background correction. No significant difference in Cd between CONT and LOCA was found except in femur, where it was increased. In CONT + Cd rats, peripheral tissues showed an increase in Cd of 30-71 fold above CONT rats. Brain regions exhibited a more modest 7-10 fold change, and serum Cd was 8.5 times above control. LOCA + Cd rats showed a 25-fold increase of Cd above LOCA in serum, 25-100 fold in peripheral tissues, and a 14-20 times in brain. These findings show that brain Cd is increased during Ca deficiency, but that central nervous system Cd changes less than peripheral tissue Cd. This increase in brain Cd could alter brain function.

Hepatocyte growth factor (HGF/SF) in Alzheimer's disease.

Hepatocyte growth factor (HGF/SF), is a heparin-binding polypeptide which stimulates DNA synthesis in a variety of cell types and also promotes cell migration and morphogenesis. HGF/SF mRNA has been found in a variety of tissues, including brain. In a previous study, we showed that basic fibroblast growth factor (bFGF), another heparin-binding protein is increased in Alzheimer's disease (AD), and appears to be associated with the heparan-sulfate proteoglycans bound to B/A4 amyloid (Biochem. Biophys. Res. Commun. 171 (1990) 690-696). In the present study, we examined the distribution of HGF/SF in 4% paraformaldehyde fixed samples of prefrontal cortex from control and Alzheimer patients, in order to assess the possibility that HGF/SF may be found in association with the pathologic changes which occur in Alzheimer's disease. A specific polyclonal antibody directed against HGF/SF revealed widespread HGF/SF-like immunoreactivity in both the cerebral cortex and white matter. Confocal microscopy confirmed that HGF/SF could be found in both GFAP positive astrocytes and LN3 positive microglia cells, as well as rare scattered cortical neurons. In the AD cases studied, the immunoreactivity was increased within both the astrocytes and microglial cells surrounding individual senile plaques. No staining was seen within the neurofibrillary tangles. Western blot analysis confirmed the normal molecular form of HGF/SF in Alzheimer's disease. Quantitative ELISA assay demonstrated a significant increase in HGF/SF in AD relative to age matched controls. These studies confirm the presence of HGF/SF immunoreactivity within neurons, astrocytes and microglial cells. They also indicate that HGF/SF may be increased within senile plaques as a function of the gliosis and microglial proliferation which occurs in association with these structures in Alzheimer's disease.

Computerized tomographic localization of clinically-guided sacroiliac joint injections.

OBJECTIVE: The goal of this study was to use computed tomographic (CT) scanning to localize clinically guided sacroiliac (SI) joint injections and identify other structures affected by this procedure. DESIGN: A prospective, double-blind, correlational outcome study design was used. Injection of 39 SI joints with a mixture of bupivacaine (0.25%), methylprednisolone (40 mg), and iohexol (Omnipaque; 180 mg/dl) using a clinically guided technique, (i.e., no image guidance) was performed. Patients had CT scans obtained both immediately after needle placement and after contrast injection. Neither the patients nor their clinicians were aware of the CT findings at the time of injection. SETTING: Academic multidisciplinary pain center. PATIENTS: Patients with SI disease by clinical criteria. RESULTS: Intra-articular injection was accomplished in 8 of 37 (22%) patients. Injected material was identified within 1 cm of the joint 68% of the time. Epidural (spinal canal) injected material was seen 24% of the time. CONCLUSIONS: The low rate of intra-articular injection seen with this clinically-guided technique suggests restraint in its use for injection therapy. Some image guidance (e.g., fluoroscopy, CT) is probably necessary to reliably inject the SI joint. Perhaps in clinical settings, where image guidance is not readily available, a clinically-guided technique could initially be tried in patients at low risk for complications from such injections. This study also provides an anatomic explanation for the occasional weakness observed after SI joint injection.

The effect of gabapentin on neuropathic pain.

OBJECTIVE: To evaluate the effects of gabapentin on pain scores and opiate use. DESIGN: Retrospective review of patients charts who received gabapentin for at least 30 days. Data were collected concerning patients' diagnosis, current drug use, concurrent drug use, gabapentin dose, pain scores, and patient-reported side effects. Patients were divided into three groups based on their pain diagnosis; low back, neuropathic, and myofascial pain. The neuropathic group was subdivided into postherpetic neuralgia, diabetic neuropathy, sympathetically maintained pain, and phantom pain. SETTING: Two tertiary referral teaching hospitals in southeastern Michigan. RESULTS: A total of 122 charts were reviewed and included in this study. A significant decrease in pain scores with gabapentin was seen in the neuropathic pain group (paired t-test, p < .0001) but not in the low back pain group. Of the neuropathic pain group, patients with postherpetic neuralgia had the greatest decrease in pain scores. Ten patients showed a > 75% decrease in pain scores, of these: nine had a direct nerve injury, and one had postherpetic neuralgia. Opiate use was unchanged in all groups. Patients who were taking opiates had significantly less benefit with gabapentin use in terms of pain score. Patient-reported side effects were similar to those reported in a nonchronic pain population. CONCLUSION: Gabapentin may be a useful adjunct for treating neuropathic pain with a minimum of side effects. Particular advantage may be gained with the use of this drug for postherpetic neuralgia and direct peripheral nerve injuries.

Molecular dynamics simulations suggest that the Eco RI kink is an example of molecular strain.

The energy surface in the vicinity of the "Eco RI kink" was investigated by conducting both in vacuo molecular dynamics simulations as well as a simulation with explicit solvent. The in vacuo simulations used the "all atom" AMBER 3.0 force field with a distant dependent dielectric function and "hydrated" counter ions while the simulation with explicit solvent used the AMBER 4.0 force field, fully charged phosphates and counter ions and a dielectric constant of 1.0. The thrust of the simulations was to discriminate between two models of the energy surface of the deformed DNA as found in the recognition complex with Eco RI endonuclease. In the intrinsic model, the kinked DNA is a local minimum of the energy surface intrinsic to the DNA itself while in the strained model there is no significant energy barrier separating kinked and regular B-DNA. The two models have significant implications for theories of indirect recognition of DNA based on sequence-dependent deformability. The simulations suggest that the Eco RI-kinked structure is an example of molecular strain because it is not near a minimum of any of the potential energy functions examined. The simulations leave the question of an energy barrier somewhat open and raise the possibility that the Eco RI kink is at (or near) a point of dynamic instability of the energy surface (either a true maximum or a saddle point).

Spin-labeled nucleotide mobility in the boundary of the EcoRI endonuclease binding site.

A complex consisting of the EcoRI endonuclease site-specifically bound to spin-labeled DNA 26mers was prepared to provide a model system for studying possible conformational changes resulting from protein binding. EPR was used to monitor the mobility of the spin labels that were strategically placed in position 6, 9, or 11 with respect to the dyad axis of the 26mer. These positions are located within the flanking region on either side of the EcoRI hexamer binding site. This allows the monitoring of potential distal structural changes in the DNA helix caused by protein binding. The spectral line shapes indicate that the spin label closest to the EcoRI endonuclease binding site, i.e., in position 6, is most influenced by the binding event. The EPR data are analyzed according to a model that distinguishes between spectral effects due to a change in the hydrodynamic shape of the complex and those resulting from local variations in the spin-label mobility as characterized by a local order parameter S. S reflecting the motional restriction of the spin-labeled base is 0.20 +/- 0.01 for all three oligomers as well as for the two complexes with the label in position 9 or 11, while the position 6 labeled complex yields S = 0.25. To further evaluate the origin of the slightly larger EPR effect observed with position 6 labeled material, molecular dynamics (MD) simulations were used to explore the space accessible to the probes in positions 6, 9, and 11. MD results gave similar nitroxide trajectories for all three labeled 26mers in the absence or presence of EcoRI. Thus, the small position 6 effect is attributed to a structural distortion in the major groove of the DNA at this location possibly corresponding to a bend induced by protein binding. The observation that the spectral changes are small indicates the absence of any significant structural disruption being propagated along the helix as a result of protein binding. Also, the fact that the line shape of the 26mers did not change as expected from hydrodynamic theory in view of the significant increase in molecular volume upon protein binding suggests that there are additional relaxation processes involving the protein and nucleic acid.

Elevation of brain manganese in calcium-deficient rats.

Rats, 3 weeks of age, consumed diets low or normal in calcium (Ca) with or without supplemental manganese (Mn) as Mn (II) acetate in drinking water. After 6 weeks, the animals were killed and [Mn] was determined in 8 brain regions, spinal cord, liver, serum, kidney, femur, and skeletal muscle. Serum [Mn] increased 1.5-, 4-, and 40-fold respectively, in normal Ca-supplemented Mn rats, low Ca rats, and low Ca-supplemented Mn rats. Elevation of tissue [Mn] occurred in all experimental groups with the greatest changes in the low Ca-extra Mn group: 6 - 12 fold in brain and spinal cord, and 2.5 - 140 fold in muscle, liver, kidney, and femur. Ratios of serum [Mn]/tissue [Mn] decreased as serum [Mn] increased suggesting saturable distribution. The findings suggest that Ca deficiency may cause Mn neurotoxicity by increasing dietary Mn absorption and brain [Mn].