Laryngeal chondronecrosis after radiation therapy in a dog.

A 5-year-old pug presented with a soft tissue swelling on the ventral neck and moderate stridor with associated respiratory effort. This patient received hypofractionated radiotherapy for metastatic upper lip mast cell tumour and to the submandibular lymph nodes 6 months before presentation. Oral examination showed moderate elongation of the soft palate, stage III laryngeal collapse with only the right laryngeal saccule mildly everted and exuberant pale epiglottal and left pharyngeal mucosa. Staphylectomy, resection of the epiglottal mucosa and left arytenoid lateralisation were performed. One day after surgery, temporary tracheostomy was performed after respiratory distress due to the severe laryngeal and pharyngeal oedema. A third oral exam showed pale and redundant caudal pharyngeal mucosa obstructing the rima glottis, soft and collapsible arytenoid cartilage with pale mucosa and bilateral everted laryngeal saccules. Permanent tracheostomy was elected and laryngeal cartilage biopsies were taken. Histologic diagnosis showed cartilage necrosis and abundant tissue oedema. The patient was euthanased 1 week later.

An EMG-driven biomechanical model of the canine cervical spine.

Due to the frequency of cervical spine injuries in canines, the purpose of this effort was to develop an EMG-driven dynamic model of the canine cervical spine to assess a biomechanical understanding that enables one to investigate the risk of neck disorders. A canine subject was recruited in this investigation in order to collect subject specific data. Reflective markers and a motion capture system were used for kinematic measurement; surface electrodes were used to record electromyography signals, and with the aid of force plate kinetics were recorded. A 3D model of the canine subject was reconstructed from an MRI dataset. Muscles lines of action were defined through a new technique with the aid of 3D white light scanner. The model performed well with a 0.73 weighted R2 value in all three planes. The weighted average absolute error of the predicted moment was less than 10% of the external moment. The proposed model is a canine specific forward-dynamics model that precisely tracks the canine subject head and neck motion, calculates the muscle force generated from the twelve major moment producing muscles, and estimates resulting loads on specific spinal tissues.

Computational strategies for the design of new enzymatic functions.

In this contribution, recent developments in the design of biocatalysts are reviewed with particular emphasis in the de novo strategy. Studies based on three different reactions, Kemp elimination, Diels-Alder and Retro-Aldolase, are used to illustrate different success achieved during the last years. Finally, a section is devoted to the particular case of designed metalloenzymes. As a general conclusion, the interplay between new and more sophisticated engineering protocols and computational methods, based on molecular dynamics simulations with Quantum Mechanics/Molecular Mechanics potentials and fully flexible models, seems to constitute the bed rock for present and future successful design strategies.

Theoretical studies of the hydrolysis of antibiotics catalyzed by a metallo-ß-lactamase.

In this paper, hybrid QM/MM molecular dynamics (MD) simulations have been performed to explore the mechanisms of hydrolysis of two antibiotics, Imipenen (IMI), an antibiotic belonging to the subgroup of carbapenems, and the Cefotaxime (CEF), a third-generation cephalosporin antibiotic, in the active site of a mono-nuclear ß-lactamase, CphA from Aeromonas hydrophila. Significant different transition state structures are obtained for the hydrolysis of both antibiotics: while the TS of the CEF is an ionic species with negative charge on nitrogen, the IMI TS presents a tetrahedral-like character with negative charge on oxygen atom of the carbonyl group of the lactam ring. Thus, dramatic conformational changes can take place in the cavity of CphA to accommodate different substrates, which would be the origin of its substrate promiscuity. Since CphA shows only activity against carbapenem antibiotic, this study sheds some light into the origin of the selectivity of the different MbL and, as a consequence, into the discovery of specific and potent MßL inhibitors against a broad spectrum of bacterial pathogens. We have finally probed that a re-parametrization of semiempirical methods should be done to properly describe the behavior the metal cation in active site, Zn(2+), when used in QM/MM calculations.

Successful wound healing over exposed metal implants using vacuum-assisted wound closure in a dog.

An eight-month-old Labrador retriever was presented with a grade IIIb open shearing injury of the left tarsus. Acute severe surgical site infection developed 2 days after pan-tarsal arthrodesis, resulting in wound dehiscence. Vacuum-assisted wound therapy was carried out for 12 days to treat an extensive full-thickness soft tissue defect with exposure of metal implants. New granulation tissue formation covering most of the defect was achieved by day 10 of this therapy. Epithelialization was achieved by second intention healing with open wound management. To the authors' knowledge, this is the first veterinary clinical case report documenting complete healing over exposed metal implants without any requirement for surgical revision.

Management of degenerative lumbosacral disease in cats by dorsal laminectomy and lumbosacral stabilization.

In this case series we describe the diagnosis and surgical treatment of five cats affected by clinical cauda equina syndrome as a result of degenerative lumbosacral stenosis. Radiographic and magnetic resonance imaging findings confirmed the suspected diagnosis of disc-associated lumbosacral disease. Cauda equina decompression was achieved by dorsal laminectomy followed by dorsal annulectomy and nuclear extirpation. Dorsal stabilization was achieved using miniature positive-profile pins inserted into the vertebral body of L7 and the wings of S1 with the free ends of the pins being embedded in a bolus of gentamicin-impregnated polymethylmethacrylate. Reassessment two years postoperatively using a previously validated feline specific owner questionnaire indicated satisfactory outcome with complete return to normal activity and resolution of signs of pain in all cases.

Theoretical QM/MM studies of enzymatic pericyclic reactions.

The chorismate to prephenate enzyme catalyzed reaction has been used in this review as the conduit to show different theoretical approaches that have been used over the years in our laboratory to explain its molecular mechanism. This pericyclic reaction has the advantage that other protein scaffolds such as catalytic antibodies or some promiscuous enzymes present certain chorismate mutase activity. The obtained results on all these protein environments, by comparison with the uncatalyzed reaction in solution, have been used to propose, as a general conclusion, that the origin of enzyme catalysis is in the relative electrostatic stabilization of the transition state with respect to the Michaelis complex. This feature implies that reactants of catalyzed reaction were closer to the transition state than those of the non-catalyzed reaction. From this hypothesis, and considering the features of the wild type chorismate mutases as the optimal catalyst for the reaction, some mutations on both kinds of alternative proteins have been proposed which would presumably enhance the rate constant of the chemical step.The studies presented in this paper demonstrate that the improvements and developments of the methods and techniques of theoretical and computational chemistry are now mature enough to model physic-chemical properties of biological systems with good accuracy. The combination of a potent computational protocol with molecular engineering techniques can be a promising methodology to develop novel enzymes with new or more efficient catalytic functions.

Simultaneous bilateral contracture of the infraspinatus muscle.

A case of bilateral fibrotic contracture of the infraspinatus muscles in a five-year-old Belgian Shepherd dog is described. The dog was presented with progressive forelimb lameness with postural and gait abnormalities three months after an episode of overexertion. When walking, the lower part of both forelimbs swung in a lateral arc causing a circumduction movement and in the standing position, the dog showed elbow adduction with external rotation of the distal part of both front limbs. Orthopaedic examination revealed bilateral atrophy of both infraspinatus and supraspinatus muscles and restriction in the range of motion of both shoulders, especially when attempting abduction and flexion. No specific findings were observed in the shoulder or elbow radiographs but hyperechogenic areas were evident in the ultrasonographic examination of both infraspinatus muscles. A diagnosis of fibrotic contracture of both infraspinatus muscles was established and bilateral tenectomy of the insertion tendons of the infraspinatus muscles was performed. Complete recovery of the animal was achieved after the surgery, which was confirmed in a long-term follow-up (10 months). In conclusion, physical examination and ultrasonography allowed a proper diagnosis of the condition, and tenectomy of the infraspinatus muscles resulted in a complete recovery of the patient even with bilateral involvement.

y+ LAT-1 mediates transport of the potent and selective iNOS inhibitor, GW274150, in control J774 macrophages.

This study has characterised the transport mechanism(s) for the novel and selective inhibitor of inducible nitric oxide synthase (iNOS), GW274150, in murine macrophage J774 cells. Transport of GW274150 was saturable (K(m) = 0.24 +/- 0.01 mM and V(max) of 8.5 +/- 0.12 pmol.microg protein(-1) min(-1)), pH-insensitive and largely Na(+)-independent. Transport was also susceptible to trans-stimulation and was significantly inhibited by a 10-fold excess of L-arginine, L-lysine, L-leucine, L-methionine, L-glutamine and 6-diazo-5-oxo-L-norleucine but not by other amino acids or by N-ethylmaleimide. More importantly, the inhibitions caused by the neutral amino acids were critically dependent on Na(+). These results strongly implicate system y(+)L in the transport of GW274150. Northern blot analysis confirmed this by revealing the presence of transcripts for y(+)LAT-1 but not y(+)LAT-2. Thus, taken together, our data show for the first time that J774 macrophages express y(+)LAT-1 transporters and that these carriers mediate transport of GW2741500 at least in these cells.

Effects of ionization, metal cationization and protonation on 2'-deoxyguanosine: changes on sugar puckering and stability of the N-glycosidic bond.

The influence of oxidation, protonation, and metal cationization with Cu(+) and Cu(2+) on the strength of the N-glycosidic bond in 2'-deoxyguanosine has been studied by means of quantum chemical calculations. In all cases, the N9-C1' bond distance increases (0.03-0.06 A) upon introducing positive charge in the guanine moiety, the observed variations being more important for the dicationic systems. Binding energies show that the effect of X(n)(+) in guanine hinders the homolytic dissociation, whereas it largely favors the heterolytic process. With respect to the deoxyribose ring, it has been found that metal binding, oxidation, and protonation do not significantly change the values of the phase angle of pseudorotation P. However, the glycosyl torsion angle chi varies considerably (from 242.0 degrees to 189.8 degrees) as a consequence of a stabilizing guanine-sugar (H8-O4') interaction due to the increase of acidity of guanine C8-H8 upon cationization.

[Common variable immunodeficiency in children]. / Inmunodeficiencia común variable en la edad pediátrica.

UNLABELLED: Common variable immunodeficiency (CVID) is one of the more frequent primary immunodeficiencies (PID), after IgA deficiency, and affects a heterogeneous group of patients of various ages and with autosomal recessive inheritance. Our objective is to present the group of children diagnosed with CVID treated in our Hospital Infantil Vall d'Hebron and comment on the diagnostic problems that can arise. Sixteen boys and girls were diagnosed between the ages of 7 months and 15 years. The diagnosis is based on low immunoglobulins and a clinical picture of infection. Differential diagnosis in the paediatric age must consider mainly other PIDs: transient hypogammaglobulinaemia of infancy, X chromosome-linked agammaglobulinaemia (XLA), X chromosome-linked hyper IgM syndrome (X-HIM), IgG subclass deficiency and IgA deficiency (IgAD). Other processes that evolve with recurrent respiratory infections, such as cystic fibrosis, must also be discarded. CONCLUSIONS: These patients present a high incidence of respiratory infections and bronchiectasias. We also observe associated allergic and autoimmune processes. Early diagnosis is indispensable to initiate suitable treatment and avoid the consequences of both respiratory and digestive infections.

A hybrid potential reaction path and free energy study of the chorismate mutase reaction.

We present a combination of two techniques--QM/MM statistical simulation methods and QM/MM internal energy minimizations--to get a deeper insight into the reaction catalyzed by the enzyme chorismate mutase. Structures, internal energies and free energies, taken from the paths of the reaction in solution and in the enzyme have been analyzed in order to estimate the relative importance of the reorganization and preorganization effects. The results we obtain for this reaction are in good agreement with experiment and show that chorismate mutase achieves its catalytic efficiency in two ways; first, it preferentially binds the active conformer of the substrate and, second, it reduces the free energy of activation for the reaction relative to that in solution by providing an environment which stabilizes the transition state.

Functional analysis of mutations in SLC7A9, and genotype-phenotype correlation in non-Type I cystinuria.

Cystinuria (OMIM 220100) is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids that results in nephrolithiasis of cystine. Mutations in SLC3A1, which encodes rBAT, cause Type I cystinuria, and mutations in SLC7A9, which encodes a putative subunit of rBAT (b(o,+)AT), cause non-Type I cystinuria. Here we describe the genomic structure of SLC7A9 (13 exons) and 28 new mutations in this gene that, together with the seven previously reported, explain 79% of the alleles in 61 non-Type I cystinuria patients. These data demonstrate that SLC7A9 is the main non-Type I cystinuria gene. Mutations G105R, V170M, A182T and R333W are the most frequent SLC7A9 missense mutations found. Among heterozygotes carrying these mutations, A182T heterozygotes showed the lowest urinary excretion values of cystine and dibasic amino acids. Functional analysis of mutation A182T after co-expression with rBAT in HeLa cells revealed significant residual transport activity. In contrast, mutations G105R, V170M and R333W are associated to a complete or almost complete loss of transport activity, leading to a more severe urinary phenotype in heterozygotes. SLC7A9 mutations located in the putative transmembrane domains of b(o,+)AT and affecting conserved amino acid residues with a small side chain generate a severe phenotype, while mutations in non-conserved residues give rise to a mild phenotype. These data provide the first genotype-phenotype correlation in non-Type I cystinuria, and show that a mild urinary phenotype in heterozygotes may associate with mutations with significant residual transport activity.

Spectroscopic study of the interactions of alkali fluorides with D-xylose.

The interactions of alkali fluorides with D-xylose have been studied by X-ray diffraction (XRD), infrared spectroscopy (IR), nuclear magnetic resonance (NMR, 1H and 13C) and atomic absorption spectrophotometry. KF and CsF form complexes with D-xylose in a 1:1 molar ratio. These complexes can be obtained by solid state milling the reactants in an agate mortar or from methanolic solutions of the sugar and the salt. LiF and NaF do not form complex with D-xylose. IR and XRD prove the identical nature of the complexes obtained by milling and from solution. IR spectra indicate strong perturbation of the OH stretching vibrations with considerable shifts to lower frequencies, which must be caused by strong hydrogen bond formation to the fluorine anion. The perturbations of C-O bond are weak, indicating that cation binding to the oxygen atoms is not the main interaction responsible for the complex formation. 1H NMR spectra of the D-xylose-KF complex dissolved in deuterium oxide is equal to that of pure D-xylose, indicating the destruction of the complex in solution. The complex is stable in DMSO, and 13C spectra of the complex in DMSO-d6 and in solid state (CPMAS) spectra are in accordance with the observed interactions in the IR spectra. As far as we know, this is the first report of a sugar-halide salt complex in which the anion instead of the cation provides the binding forces.

Parkinson's disease, smoking, and family history. EUROPARKINSON Study Group.

There is growing evidence that both genetic and environmental factors play a role in the etiology of Parkinson's disease (PD). The hypothesis of an interaction between genetic and environmental risk factors has been little explored, and never using a population-based case-control study design. Our objective was to investigate the possible interaction between smoking and family history in the etiology of PD, as part of a collaborative population-based case-control study. We included 149 nondemented PD patients ascertained in three European prevalence surveys using a two-phase design. Each patient was matched by age (+/-2 years), gender, and center to three controls drawn from the same populations (n=375). Presence of PD among first-degree relatives and smoking history were assessed through an interview for 127 cases and 306 controls. In the overall sample we found suggestive evidence that family history and ever-smoking interact in determining the risk of PD (P=0.09), with individuals exposed to both risk factors having the highest risk (OR=10.0; 95% CI=2.0-49.6). Analyses were repeated after stratification into two age-groups (cutoff: 75 years). In older patients, the joint exposure to both risk factors was associated with a significant increase in the risk of PD (OR=17.6; 95% CI=1.9-160.5). Among younger subjects, the OR for joint exposure was not significant. In conclusion, our findings suggest that smoking and family history interact synergistically on a multiplicative scale in determining the risk of PD in individuals older than 75 years.

Heteromeric amino acid transporters explain inherited aminoacidurias.

In the past 5 years, the first genes responsible for aminoacidurias caused by defects in renal reabsorption transport mechanisms have been identified. These diseases are type I and non-type I cystinuria and lysinuric protein intolerance. This knowledge came from the molecular characterization of the first heteromeric amino acid transporters in mammals. In 1992, rBAT and 4F2hc (genes SLC3A1 and SLC3A2, respectively, in the nomenclature of the Human Genome Organization) were identified as putative heavy subunits of mammalian amino acid transporters. In 1994, it was demonstrated that mutations in SLC3A1 cause type I cystinuria. Very recently, several light subunits of the heteromeric amino acid transporters have been identified. In 1999, a putative light subunit of rBAT (the SLC7A9 gene; complementary DNA and protein termed amino acid transporter) and a light subunit of 4F2hc (the SLC7A7 gene; cDNA and protein termed y+LAT-1) were shown to be the non-type I cystinuria and lysinuric protein intolerance genes, respectively. In this review, the characteristics of these heteromeric amino acid transporters and their role in these inherited aminoacidurias is described.

Prognostic value of a CCR5 defective allele in pediatric HIV-1 infection.

BACKGROUND: A deletion of 32 base pairs in the CCR5 gene (delta32 CCR5) has been linked to resistance to HIV-1 infection in exposed adults and to the delay of disease progression in infected adults. MATERIALS AND METHODS: To determine the role of delta32 CCR5 in disease progression of HIV-1 infected children born to seropositive mothers, we studied a polymerase chain reaction in 301 HIV-1 infected, 262 HIV-1 exposed-uninfected and 47 HIV-1 unexposed-uninfected children of Spanish and Italian origin. Infected children were further divided into two groups according to their rate of HIV-1 disease progression: rapid progressors who developed severe clinical and/or immunological conditions within the second year of life, and delayed progressors with any other evolution of disease. Among the latter were the long-term, non-progressors (LTNP) who presented with mild or no symptoms of HIV-1 infection above 8 years of age. Viral phenotype was studied for 45 delayed progressors. RESULTS: No correlation was found between delta32 CCR5 and mother-to-child transmission of HIV-1. However, the frequency of the deletion was substantially higher in LTNP, compared with delayed (p = 0.019) and rapid progressors (p = 0.0003). In children carrying the delta32 CCRS mutation, the presence of MT-2 tropic virus isolate was associated with a severe immune suppression (p = 0.028); whereas, the presence of MT-2 negative viruses correlated with LTNP (p = 0.010). CONCLUSIONS: Given the rapidity and simplicity of the assay, the delta32 CCR5 mutation may be a useful predictive marker to identify children with delayed disease progression who, consequently, may not require immediate antiretroviral treatment.

Kinase mutant Btk results in atypical X-linked agammaglobulinaemia phenotype.

X-linked agammaglobulinaemia (XLA) is a B cell humoral abnormality arising from mutations in the gene encoding Bruton's tyrosine kinase (Btk). The phenotype of XLA can be variable, with some individuals having a less severe immunophenotype, although in most cases this cannot be correlated with the Btk mutation or expression of Btk protein. In this study we describe clinical and immunological heterogeneity within the same pedigree. Analysis of the genetic defect identified a missense mutation in the kinase domain of Btk which, unusually, preserved Btk protein expression but at reduced levels, and also considerably diminished autophosphorylation activity. Structural analysis of the effect of this mutation on the kinase domain suggests that this mutation is not an integral part of the ATP or substrate binding domains but may affect the interaction of the kinase domain with its own kinase domain and other substrates. Together, these data may provide an explanation for the variable XLA phenotype.

Therapeutic effects of docosahexaenoic acid ethyl ester in patients with generalized peroxisomal disorders.

Generalized peroxisomal disorders are severe congenital diseases that involve the central nervous system, leading to severe psychomotor retardation, retinopathy, liver disease, and early death. In these disorders, peroxisomes are not normally formed and their enzymes are deficient. Characteristically, plasmalogen synthesis and beta-oxidation of very-long-chain fatty acids (VLCFAs) are affected. We found that patients with generalized peroxisomal disorders have a profound brain deficiency of docosahexaenoic acid (DHA; 22:6n-3) and low DHA concentrations in all tissues and the blood. Given the fundamental role of DHA in neuronal and retinal membranes, a DHA deficiency of this magnitude might be pathogenic. Thus, we studied the possible therapeutic effect of normalizing DHA concentrations in patients with peroxisomal disorders. We chose the DHA ethyl ester (DHA-EE) because of its high degree of purity at daily oral doses of 100-500 mg. This article summarizes the results of treatment of 13 patients with DHA-EE, with some follow-up evidence of clinical improvement. Supplementation with DHA-EE normalized blood DHA values within a few weeks. Plasmalogen concentrations increased in erythrocytes in most patients and after DHA concentrations were normalized, amounts of VLCFAs decreased in plasma. Liver enzymes returned almost to normal in most cases. From a clinical viewpoint, most patients showed improvement in vision, liver function, muscle tone, and social contact. In 3 patients, normalization of brain myelin was detected by magnetic resonance imaging. In 3 others, myelination improved. In a seventh patient, myelination is progressing at a normal rate. These results suggest a fundamental role of DHA in the pathogenesis of Zellweger syndrome. DHA therapy is thus strongly recommended, not only to alleviate symptoms in patients with life-threatening diseases, but also to clarify remaining questions regarding the role of DHA in health and disease.