Structure of E. coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase reveals similarity to the purine nucleoside phosphorylases.

BACKGROUND: 5'-methylthioadenosine/S-adenosyl-homocysteine (MTA/AdoHcy) nucleosidase catalyzes the irreversible cleavage of 5'-methylthioadenosine and S-adenosylhomocysteine to adenine and the corresponding thioribose, 5'-methylthioribose and S-ribosylhomocysteine, respectively. While this enzyme is crucial for the metabolism of AdoHcy and MTA nucleosides in many prokaryotic and lower eukaryotic organisms, it is absent in mammalian cells. This metabolic difference represents an exploitable target for rational drug design. RESULTS: The crystal structure of E. coli MTA/AdoHcy nucleosidase was determined at 1.90 A resolution with the multiwavelength anomalous diffraction (MAD) technique. Each monomer of the MTA/AdoHcy nucleosidase dimer consists of a mixed alpha/beta domain with a nine-stranded mixed beta sheet, flanked by six alpha helices and a small 3(10) helix. Intersubunit contacts between the two monomers present in the asymmetric unit are mediated primarily by helix-helix and helix-loop hydrophobic interactions. The unexpected presence of an adenine molecule in the active site of the enzyme has allowed the identification of both substrate binding and potential catalytic amino acid residues. CONCLUSIONS: Although the sequence of E. coli MTA/AdoHcy nucleosidase has almost no identity with any known enzyme, its tertiary structure is similar to both the mammalian (trimeric) and prokaryotic (hexameric) purine nucleoside phosphorylases. The structure provides evidence that this protein is functional as a dimer and that the dual specificity for MTA and AdoHcy results from the truncation of a helix. The structure of MTA/AdoHcy nucleosidase is the first structure of a prokaryotic nucleoside N-ribohydrolase specific for 6-aminopurines.

Cloning and expression of Escherichia coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase: identification of the pfs gene product.

The enzyme 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (EC 3.2.2.9) is responsible for cleavage of the glycosidic bond in both 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH). Based on amino acid sequence analysis of this enzyme from Klebsiella, we recently speculated that an open reading frame found in E. coli (designated pfs) encoded MTA/SAH nucleosidase. To explore this possibility, we amplified, cloned, and expressed the complete pfs gene from E. coli genomic DNA. The recombinant protein exhibited a molecular weight and Michaelis constants for MTA that are in agreement with those reported for native enzyme. From this biochemical evidence we confirm our original assignment of the pfs gene as encoding MTA/SAH nucleosidase.

Adenosine- and adenine-nucleotide-mediated inhibition of normal and transformed keratinocyte proliferation is dependent upon dipyridamole-sensitive adenosine transport.

Extracellular adenosine and its related nucleotides have been referred to as retaliatory metabolites that can be released into the extracellular environment during inflammation, wounding, and other pathologic states. We have previously reported that these compounds reversibly inhibit the proliferation of normal keratinocyte cultures and we now demonstrate that these compounds also arrest the proliferation of transformed keratinocytes. Although our study shows that keratinocytes express mRNA corresponding to the A2B purinoreceptors and that adenosine or AMP treatment elevates intracellular cAMP in these cells, our study also demonstrates that dipyridamole-inhibitable transport of adenosine into the keratinocyte is central to the mechanism by which adenosine and adenine nucleotides arrest proliferation in these cells. In support of this mechanism, our results demonstrate that human keratinocytes express mRNA corresponding to the recently cloned dipyridamole-sensitive human equilibrative nucleoside transporter. Interestingly, coincubation with adenosine deaminase reverses the antiproliferative action of adenosine and exerts no effect on the antiproliferative activity of the adenine nucleotides, thus supporting a model in which adenine nucleotides are enzymatically converted to adenosine and transported into the keratinocyte in a tightly coupled and adenosine-deaminase-resistant manner. Analysis of adenosine- and adenosine-monophosphate-treated keratinocytes demonstrated that quiescence is induced within 12-24 h, and fluorescence-activated cell sorter analysis suggests that treatment with these compounds may result in the inhibition of keratinocyte proliferation at both G1 and S phases of the cell cycle. In addition to their documented antiproliferative action on other cell types, adenosine, adenine nucleotides, and related analogs may also represent a potential new class of pharmacologic regulators of keratinocyte proliferation in vivo.

Identification of a cell-surface DNA receptor and its association with systemic lupus erythematosus.

We have previously reported the existence of a cell-membrane-associated molecule on human PBMC, which binds DNA and has the characteristics of a receptor. Monoclonal antibodies have been made to this receptor and have been used successfully for the purification of this cell-surface molecule. Preliminary studies have indicated a receptor for DNA on murine kidney and spleen cells which is similar in molecular weight to the human DNA receptor (30 kD). The occurrence of autoantibodies to cell-surface receptors has been described in several autoimmune diseases and we have noted that the serum of patients with lupus and similar disorders inhibit the binding of labeled DNA to human leukocytes. Using a "dot-blot" assay with affinity-purified human DNA receptor, sera from patients with various CTD and from healthy volunteers were screened for anti-receptor antibodies; anti-receptor antibodies were found in many patients with CTD and some of their first-degree relatives. The prevalence of anti-receptor antibodies in normal blood donors was less than 2%. It is hypothesized that anti-receptor antibodies represent an early immune response in lupus and kindred disorders and that anti-DNA antibodies may arise from the corresponding anti-idiotypic response.

Immunization of BALB/c mice with a monoclonal anti-DNA antibody induces an anti-idiotypic antibody reactive with a cell-surface DNA binding protein.

DNA binds to cell-surface proteins on human and murine leukocytes and induces secretion of the cytokine interleukin 6 (IL-6). Cell-surface DNA binding molecules have been shown to serve as target antigens for the production of autoantibodies in patients with systemic lupus erythematosus (SLE), and in lupus-prone mice. Recent studies have demonstrated that a subset of anti-anti-DNA antibodies, isolated from patients with SLE, are idiotypically related to antibodies reactive with a cell-surface DNA binding molecule. We now report that immunization of normal mice with a murine monoclonal anti-DNA antibody induces an anti-idiotypic response which has reactivity with a cell-surface DNA binding molecule. An anti-idiotypic anti-DNA monoclonal antibody (LB17) was isolated from the spleen of an immunized mouse. This monoclonal antibody blocked the binding of DNA to murine splenocytes and mimicked the functional effect of DNA by stimulating the secretion of IL-6. These experiments provide further evidence for an idiotypic connectivity between antibodies to cell-surface DNA binding proteins and anti-DNA antibodies. It is hypothesized that this idiotypic system is part of the network of natural autoantibodies and that its perturbation may give rise to pathogenic antibodies.

Imaging of spontaneous canine mammary tumors using fluorescent contrast agents.

We present near-infrared frequency-domain photon migration imaging for the lifetime sensitive detection and localization of exogenous fluorescent contrast agents within tissue-simulating phantoms and actual tissues. We employ intensity-modulated excitation light that is expanded and delivered to the surface of a tissue or tissue-simulating phantom. The intensity-modulated fluorescence generated from within the volume propagates to the surface and is collected using a gain-modulated image-intensified charge-coupled device camera. From the spatial values of modulation amplitude and phase of the detected fluorescent light, micromolar volumes of diethylthiatricarbocyanine iodide (tau = 1.17 ns) and indocyanine green (ICG) (tau = 0.58 ns) embedded 1.0 cm deep in a tissue phantom are localized and discriminated on the basis of their lifetime differences. To demonstrate the utility of frequency-domain fluorescent measurements for imaging disease, we image the fluorescence emitted from the surface of in vivo and ex vivo canine mammary gland tissues containing lesions with preferential uptake of ICG. Pathology confirms the ability to detect spontaneous mammary tumors and regional lymph nodes amidst normal mammary tissue and fat as deep as 1.5 cm from the tissue surface.

Vena caval occlusion after bird's nest filter placement.

BACKGROUND: Inferior vena caval thrombosis as a result of intracaval barrier devices occurs in 6.5% of patients with Greenfield filters. The incidence is less well defined in patients in whom bird's nest filters have been placed. We reviewed our experience with bird's nest filters to determine the incidence of filter-induced caval thrombosis. METHODS: The records of 140 patients with bird's nest filters were reviewed, living patients were interviewed, and the inferior vena cava examined in 37 patients by duplex scanning. RESULTS: Ninety-three patients were available for evaluation. Five of these patients were found to have caval thrombosis by duplex scanning and 2 had clinical symptoms and signs compatible with caval thrombosis. The majority of these patients were on anticoagulants at the time of filter thrombosis. CONCLUSION: The incidence of filter-induced vena caval thrombosis in patients with bird's nest filters (7%) is comparable with that of Greenfield filters. Because of the catastrophic manifestations of this complication and the increasing application of vena caval filters, the role of these filters in the treatment of thrombotic disease needs further critical evaluation.

Imbrication versus excision for fascial healing.

BACKGROUND: The purpose of this study was to determine whether imbrication of native fascia versus excision and closure of new raw fascia forms a stronger union. METHODS: We utilized the anterior rectus sheath fascia of Sprague-Dawley rats as the model. Sixty rats underwent fascial tightening procedures. Each rat had the anterior rectus sheath shortened by 1 cm, 30 by imbrication and 30 by excision and closure. Ten of each group were harvested at 7 days' healing, 10 at 14 days, and 10 of each group at 28 days' healing. The anterior rectus sheath was removed, a "dumbbell" shape constructed, and the cross sectional area at the point of interest determined. The fascia was placed on an Instron tensiometer to determine the breaking strength. Tensile strength was calculated and the data analyzed by ANOVA and the Kruskall-Wallis test. Tissue samples of the closures were histologically analyzed for fibroblast counts, degree of inflammation, and presence of dense fibrous connective tissue. Tissue samples were also analyzed for enzymatic collagen crosslinking. RESULTS: There was a statistically significant difference in tensile strength between the two groups at 7, 14, and 28 days. Results show that at 7 days the mean tensile strength of excision was 0.133 kg/mm2 +/- 0.056 and the mean tensile strength of imbrication was 0.083 kg/mm2 +/- .048 (P < 0.05); at 14 days the mean tensile strength of excision was 0.105 kg/mm2 +/- 0.033 and the mean tensile strength of imbrication was 0.057 kg/mm2 +/- 0.014 (P < 0.002), and at 28 days the mean tensile strength of excision was 0.279 kg/mm2 +/- 0.143 and the mean tensile strength of imbrication was 0.145 kg/mm2 +/- 0.061 (P < 0.03). Histologic findings showed no statistical significance between the two closure methods when comparing degree of inflammation or the number of fibroblasts present. However, at 7 and 14 days there is a significantly greater presence of dense fibrous connective tissue in the excision group (P < 0.03 at 7 days and P < 0.044 at 14 days by ANOVA). Collagen crosslink analysis showed that by day 28 there is a significantly greater amount (P < 0.05 by ANOVA) of the difunctional crosslink dihydroxylysinonorleucine (DHLNL) and a greater ratio between DHLNL and the difunctional crosslink hydroxylysinonorleucine (HLNL) in the excision and closure group. CONCLUSION: We conclude that at 7, 14, and 28 days healing, excision provides a significantly stronger closure than imbrication. According to the crosslinking analysis, it is likely that this strength advantage may continue to increase over time. These findings suggest that excision and closure may be the preferred method for fascial tightening procedures.

Nicotine alters catecholamines and electrocortical activity in perfused mouse brain.

Nicotine differentially altered electrocortical (ECoG) activity and brain catecholamine metabolism in mice (C3H and C57BL) known to differ in behavioral response to nicotine. Nicotine appeared to produce a concentration dependent desynchronization to ECoG activity in isolated perfused mouse brain (IPMB) from C3H mice. Homovanillic acid (HVA) production was unchanged in C3H perfused brains while an apparent reduction in 3-methoxy-4-hydroxyphenethyleneglycol (MPHG) was observed. Brain content of norepinephrine and dopamine remained relatively constant in the various regions tested. In IPMB from C57BL mice, nicotine elicited an enhancement of ECoG amplitude which was accompanied by decreased HVA production rates. A downward trend in MHPG production was also observed. These effects were associated with increased levels of norepinephrine and dopamine in various brain regions.

Effects of nicotine on beta-endorphin, alpha MSH, and ACTH secretion by isolated perfused mouse brains and pituitary glands, in vitro.

The effects of nicotine on secretion of the pituitary peptides beta-endorphin, alpha MSH, and ACTH were studied using the isolated perfused mouse brain (IPMB) and isolated superfused pituitaries of C3H mice. Nicotine (6.1 microM) stimulated secretion of beta-endorphin immunoreactivity from C3H IPMB approximately twofold. Secretion of alpha MSH immunoreactivity was stimulated approximately two- and sixfold by 6.1 microM and 12.2 microM nicotine, respectively. However, nicotine (6.1 microM) had no direct effect on the secretion of beta-endorphin, alpha MSH, or ACTH immunoreactivities from the isolated superfused pituitaries. The data suggest nicotine acts in the brain to stimulate pituitary secretion of alpha MSH and beta-endorphin. Electrocorticographic (ECoG) activity of the IPMB was monitored. Nicotine induced characteristic ECoG changes including a reduction of input voltage, a biphasic response of rapid desynchronization followed by prolonged synchronization, and seizure at high doses (12.2 microM).

Complications of enteroplication for the prevention of intussusception recurrence in dogs: 35 cases (1989-1999).

OBJECTIVE: To compare complication and recurrence rates in dogs treated for intussusception that underwent enteroplication to rates in dogs treated for intussusception that did not undergo enteroplication. DESIGN: Retrospective study. ANIMALS: 35 dogs with intestinal intussusception. PROCEDURE: Information on signalment, clinical signs, potential predisposing causes, surgical technique, opioid administration, use of enteroplication, postoperative complications, and whether the intussusception recurred was obtained from the medical records. RESULTS: Dogs ranged from 8 weeks to 10 years old. Opioids were administered in the perioperative period in 34 dogs. Enteroplication was performed in 16 dogs. Complications of enteroplication that required a second surgery were identified in 3 dogs. None of the 16 dogs that underwent enteroplication had a recurrence of intussusception, whereas 1 of the 19 dogs that did not undergo enteroplication had a recurrence. Rate of intussusception recurrence and likelihood that a second surgical procedure would be required were not significantly different between dogs that underwent enteroplication and dogs that did not. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that enteroplication may be associated with life-threatening complications in dogs, but the likelihood of a dog requiring a second surgical procedure following surgical correction of intussusception was not different between dogs that underwent enteroplication at the time of the initial surgery and dogs that did not.

Use of two-dimensional real-time ultrasonography for diagnosing contracture and strain of the infraspinatus muscle in a dog.

A strain injury to the infraspinatus muscle was a potential cause of forelimb lameness in a dog. Ultrasonography was used for evaluation of infraspinatus muscle injury. The ultrasonographic appearance of a typical infraspinatus muscle, contracture of an infraspinatus muscle, acute strain of an infraspinatus muscle, and progression of healing of the infraspinatus muscle are described.

Impaired wound healing in the cancer patient: effects of cytotoxic therapy and pharmacologic modulation by growth factors.

This article reviews the experimental and clinical data regarding the effects of chemotherapy on wound healing. In addition, the role of growth factors in the normal wound healing process and their therapeutic potential to optimize wound healing in the cancer patient are discussed.

Cystotomy, partial cystectomy, and tube cystostomy.

Knowledge of the regional anatomy and healing characteristics of the urinary bladder are crucial to successful surgical intervention in common disorders of the bladder. Innervation and blood supply enter the neck region of the bladder on the dorsal surface. Surgical approach to the bladder is via a ventral midline incision. Cystotomy is most commonly performed on the ventral surface of the bladder and the incision is closed using absorbable suture material in a single-layer, appositional closure. Removal of urinary calculi is the most common indication for cystotomy and should be accompanied by mucosal biopsy and culture. After cystotomy for removal of calculi, a lateral radiograph should be made to confirm removal of all calculi. Partial cystectomy is indicated for bladder trauma, neoplasia, patent urachus, and urachal diverticula. A large percentage of the bladder wall can be excised with gradual return to near normal function when the trigone region is preserved. Complete cystectomy is not recommended because of the patient morbidity and client dissatisfaction with these procedures. Tube cystostomy is performed routinely for temporary or permanent urinary diversion. Temporary diversion may be performed concurrently with surgical repair of urethral trauma or to relieve acute urethral obstructions. Permanent cystostomy may be performed in cases of neurogenic bladder atony or bladder cancer.

Evaluation of test protocol variables for dental implant fatigue research.

OBJECTIVES: This work begins to explore the influence of cycling rate and environment on fatigue testing of dental implants according to the ISO protocol 14801. METHODS: Twenty-four Straumann implants (4.1 mm x 12 mm) were tested up to five million cycles per ISO 14801: loaded at either 2 or 30 Hz in room air at 25 degrees C or normal saline at 37 degrees C (n=6 per group). Implant displacements/cycle were captured during all testing. Fracture-surface features were examined using scanning electron microscopy (n=12). Two complimentary methods were developed to estimate fatigue crack growth rates. RESULTS: Failures (bulk fracture) were found to be bi-modally distributed, either <350,000 cycles or >1.5 million cycles at both cycling rates. Following initial crack formation, fatigue crack growth required merely 1100-4200 cycles to failure. Initial crack pop-in was statistically more likely under 2 Hz than 30 Hz (chi(2), p<0.05) but testing in air and normal saline were equivalent in terms of likelihood of fracture versus runout (chi(2), p>0.6). On a microscopic level, fatigue crack growth rates appears to be similar at 2 and 30 Hz, but may be slower in the presence of saline versus dry at 2 Hz. SIGNIFICANCE: Implant failure under fatigue conditions involved "classic" damage mechanisms. Failure appears more likely at 2 Hz than 30 Hz for reasons that remain to be elucidated. Saline may enable chemically assisted crack growth involving grain boundaries during the stage of fatigue crack growth, but did not influence likelihood of failure.

Surgical and prosthetic planning for a two-implant-retained mandibular overdenture: a clinical report.

A 2-implant-retained mandibular overdenture is considered by some to be the standard of care for mandibular edentulism. Compared to a conventional complete denture, an implant-retained overdenture requires more thorough planning. Careful consideration is necessary regarding the 3-dimensional orientation of the implants to ensure adequate horizontal and vertical space for prosthetic components. This clinical report describes a patient with a compromised mandibular overdenture in whom the position of the existing implants yielded insufficient space for prosthetic components. This report describes the concepts for treatment planning prior to fabricating a new mandibular overdenture, including considerations for the surgical removal of the existing implants, alveoloplasty to create the necessary space for prosthetic components, and placement of the new implants to ensure an esthetic and functional prosthesis.

Genetic divergence and migration patterns in a North American passerine bird: implications for evolution and conservation.

Like many other migratory birds, the black-throated blue warbler (Dendroica caerulescens) shows pronounced differences in migratory behaviour and other traits between populations: birds in the southern part of the breeding range have darker plumage and migrate to the eastern Caribbean during the winter, whereas those in the north have lighter plumage and migrate to the western Caribbean. We examined the phylogeography of this species, using samples collected from northern and southern populations, to determine whether differentiation between these populations dates to the Pleistocene or earlier, or whether differences in plumage and migratory behaviour have arisen more recently. We analysed variation at 369 bp of the mitochondrial control region domain I and also at seven nuclear microsatellites. Analyses revealed considerable genetic variation, but the vast majority of this variation was found within rather than between populations, and there was little differentiation between northern and southern populations. Phylogeographic analyses revealed a very shallow phylogenetic tree, a star-like haplotype network, and a unimodal mismatch distribution, all indicative of a recent range expansion from a single refugium. Coalescent modelling approaches also indicated a recent common ancestor for the entire group of birds analysed, no split between northern and southern populations, and high levels of gene flow. These results show that Pleistocene or earlier events have played little role in creating differences between northern and southern populations, suggesting that migratory and other differences between populations have arisen very recently. The implications of these results for the evolution of migration and defining taxonomic groups for conservation efforts are discussed.

Pursuing quality improvement: a social work perspective.

The multidisciplinary approach to the treatment of patients with chronic renal failure has existed since the inception of the Medicare end-stage renal disease (ESRD) Program. Social workers as team members in the various renal outpatient treatment settings have contributed to the planning and assessment of patient needs with the goal of achieving the optimal quality of life available to each individual. Nephrology social workers have also participated in the evolution of quality management through their professional organization. The eight clinical indicators jointly developed by the Council of Nephrology Social Workers and the National Association of Social Workers provide a basis for the implementation of continuous quality improvement concepts. The Health Care Financing Administration, the administrative agency of the ESRD Program, has shifted its quality management approach from quality assurance to the broader concepts of continuous quality improvement. The Health Care Quality Improvement Program provides the structure for all professionals to develop a facility-based system for quality management. By using strategies of quality control and quality improvement, social workers will be able to demonstrate the positive benefit of social service intervention. Identification of useful psychological and functional assessment tools must be incorporated into social work practice to measure outcome. Social workers have the necessary expertise in conflict resolution and group dynamics, which are skills needed for team facilitators used within the conceptual framework of continuous quality improvement. The challenge is to learn these concepts and incorporate them into the daily operation of social work practice.

Effects of 6-hydroxydopamine on brain catecholamines and on acute actions of ethanol in LS/Ibg and SS/Ibg mice.

LS/Ibg (LS) and SS/Ibg (SS) mice differ in ethanol-induced duration of loss of righting response or sleep time, hypothermia, hyperglycemia, and blood ethanol concentrations at regaining righting response. These differences in response to ethanol are a result of differences in central nervous system sensitivity and are mediated by polygenic systems. Studies have indicated that catecholaminergic systems may be involved in the differential effects of ethanol in LS and SS lines of mice (Masserano JM, Weiner N: Investigations into the neurochemical mechanisms mediating differences in ethanol sensitivity in two lines of mice. J Pharmacol Exp Ther 221:404-408, 1982). In this study the neurotoxin, 6-hydroxydopamine (6-OHDA), intracerebroventricular, was used to test this hypothesis. Administration of 6-OHDA markedly altered thermoregulation in LS mice but produced little effect in SS mice, and ethanol-induced hyperglycemia was attenuated in both LS and SS mice by 6-OHDA. Ethanol-induced sleep time was increased in SS mice pretreated with 100 micrograms of 6-OHDA, intracerebroventricular, whereas this response in LS mice was unaffected by 6-OHDA administration. Changes in sleep time were not related to changes in blood ethanol concentrations, indicating that 6-OHDA alters ethanol-induced sleep time by mechanisms other than brain sensitivity. Levels of norepinephrine and dopamine were determined in three brain regions, and the altered capacities for thermoregulation and glucoregulation were associated with changes in hypothalamic catecholamine levels.

Pericardial effusion associated with metastatic disease from an unknown primary tumor in a dog.

A 6.5-year-old, spayed female Siberian husky presented with signs of cardiac tamponade and weakness. Pleural, pericardial, and abdominal effusion were identified with radiographs and ultrasound. Pericardiocentesis relieved signs of tamponade, and the dog was clinically improved. Pericardial effusion recurred, and pericardiectomy was performed. Histopathological examination of excised tissues failed to reveal evidence of infectious or neoplastic disease. After pericardiectomy, clinically apparent thoracic effusion persisted. The dog was euthanized, and postmortem histopathological examination revealed emboli of metastatic carcinoma cells in the epicardium. The location of intrathoracic disease in this dog made antemortem diagnosis difficult, if not impossible.