Development of a model to investigate the effects of prolonged ischaemia on the muscles of mastication of male Sprague Dawley rats.

OBJECTIVE: The aims of this study were to develop a novel rodent model of masticatory muscle ischaemia via unilateral ligation of the external carotid artery (ECA), and to undertake a preliminary investigation to characterize its downstream effects on mechanosensitivity and cellular features of the masseter and temporalis muscles. DESIGN: The right ECA of 18 male Sprague-Dawley rats was ligated under general anaesthesia. Mechanical detection thresholds (MDTs) at the masseter and temporalis bilaterally were measured immediately before ECA ligation and after euthanasia at 10-, 20-, and 35-days (n = 6 rats/timepoint). Tissue samples from both muscles and sides were harvested for histological analyses and for assessing changes in the expression of markers of hypoxia and muscle degeneration (Hif-1α, VegfA, and Fbxo32) via real time PCR. Data were analyzed using mixed effect models and non-parametric tests. Statistical significance was set at p < 0.05. RESULTS: MDTs were higher in the right than left hemiface (p = 0.009) after 20 days. Histological changes indicative of muscle degeneration and fibrosis were observed in the right muscles. Hif-1α, VegfA, and Fbxo32 were more highly expressed in the masseter than temporalis muscles (all p < 0.05). Hif-1α and, VegfA did not change significantly with time in all muscles (all p > 0.05). Fbxo32 expression gradually increased in the right masseter (p = 0.024) and left temporalis (p = 0.05). CONCLUSIONS: ECA ligation in rats induced hyposensitivity in the homolateral hemiface after 20 days accompanied by tissue degenerative changes. Our findings support the use of this model to study pathophysiologic mechanisms of masticatory muscle ischaemia in larger investigations.

Rapid disease course in African Americans with multiple sclerosis.

OBJECTIVE: To investigate utility of a Multiple Sclerosis Severity Scale (MSSS)-based classification system for comparing African American (AA) and white American (WA) multiple sclerosis (MS) subpopulations in the New York State Multiple Sclerosis Consortium (NYSMSC) database. MSSS is a frequency-rank algorithm relating MS disability to disease duration in a large, untreated reference population. Design/ METHODS: Distributions of patients in 6 MSSS-based severity grades were calculated for AA and WA registrants. RESULTS: There were 419 AA and 5,809 WA patients in the NYSMSC, who had EDSS recorded during years 1-30 since symptom onset. Median EDSS was not different in AA and WA (3.5 vs 3.0, p = 0.60), whereas median MSSS in AA was higher than in WA (6.0 vs 4.8, p = 0.001). AA patients were overrepresented in the 2 most severe grades (41.5% vs 29.3% for WA) and underrepresented in the 2 lowest grades (23.4% vs 35.4%; p < 0.001). In multivariable analysis (ordered logistic and median regression), MSSS for AA remained significantly higher than in WA after adjusting for age, gender, disease duration, disease type distribution, and treatment with disease-modifying therapies. CONCLUSIONS: The 6-tiered MSSS grading system is a powerful tool for comparing rate of disease progression in subpopulations of interest. MSSS-based analysis demonstrates that African ancestry is a risk factor for a more rapidly disabling disease course.

[Oculographic findings in 145 patients with benign paroxysmal positional vertigo]. / Hallazgos oculográificos en 145 pacientes con vértigo posicional paroxístico benigno.

OBJECTIVE: Benign paroxysmal positional vertigo (BPPV) can be produced by specific manoeuvres and be studied by electrooculography (EOG). It allows an exhaustive study of features in the positional nystagmus. Although most of the patients with BPPV express typical nystagmus, there exists a group of them that exhibit a non typical form. In this work, a comparative study was carried out between two groups of patients. MATERIAL AND METHODS: We have studied retrospectively 145 patients with BPPV and Dix-Hallpicke (D-H) positive manoeuvre registered by EOG techniques. All patients were explored in a complete EOG tests. Two groups were identified: typical response/atypical response according to the features of nystagmus response. Possible alterations in other EOG tests in each group were investigated and differences between both types of response were analyzed. RESULTS: 83.5% of cases showed typical nystagmus and atypical in the rest. Other EOG alterations observed in these patients, were (group typical response/atypical response): caloric hypofunction (17.3%/23%), positional nystagmus (12.4%/62.5%), alterations in visual-oculomotor function tests (10%/50%). CONCLUSIONS: Frequent alterations were observed in the classic manoeuvres and also in the rest of EOG tests. Probably atypical response in the first is due to or can explain the existence of the second.

[Benign paroxysmal positional vertigo of the horizontal semicircular canal]. / Vértigo posicional paroxístico benigno del canal horizontal.

OBJECTIVE: Assesment of 8 new cases of benign paroxysmal positional vertigo of the horizontal semicircular canal (BPPV-HC), since this variant of benign positional vertigo occurs rarely. An oculographic study of features in the positional response was carried out. Electrooculograpy (EOG) allows an exhaustive study of positional nystagmus. MATERIAL AND METHODS: We have studied retrospectively 8 patients with BPPV-HC and positive head rotation manoeuvre registered by EOG techniques. Possible alterations in other EOG tests in the group of patients were analyzed. RESULTS: Most of the patients (7/8) expressed bilateral horizontal geotropic changing-direction nystagmus. One patient exhibited apogeotropic horizontal nystagmus. 5/8 cases showed caloric hypofunction. CONCLUSIONS: We have proven that BPPV-HC is an uncommon disorder. In most of the cases, provocative manoeuver generates bilateral horizontal geotropic changing-direction nystagmus that probably is due to canalitiasis of the horizontal semicircular canal. The rest, a few cases, exhibit apogeotropic horizontal response that can be secondary to cupulolitiasis or location particles in the anterior portion of the horizontal canal. A caloric test showed abnormal in many and can help to locatize the affected ear.

[Electrooculography. Its value in the diagnosis of the patient with a balance disorder]. / Electrooculografía: aportación al diagnóstico del paciente con alteraciones del equilibrio.

INTRODUCTION: Controversy persists on the value of electrooculography (EOG) in the diagnosis of the unbalanced patient. The aim of this study has been to know the utility of EOG in the diagnosis of patients with equilibrium disorders. MATERIAL AND METHODS: We have examined 1000 patients in whom EOG test has been performed for unbalance symptoms. Results have been classified in peripheric or central pattern. Those patients included in the central pattern group have been compared with the results of imaging techniques. RESULTS: 45.7% of EOG performed showed pathological signs, 29.2% were of peripheral characteristics and 16.5% of central ones, of whom 6% showed different pathologies in the imaging test. DISCUSSIONS AND CONCLUSIONS: EOG in now a days of high value in the diagnosis of unbalanced patient, specially in those cases in which other clinical explorations were normal.

[Determination of postural behaviour patterns in the Spanish healthy population]. / Determinación de los patrones de comportamiento postural en población sana española.

INTRODUCTION: Postural control in a standing position is externally reflected by the displacement of the centre of pressure (COP). It is necessary to determine COP movement normal patterns in order to obtain an accurate balance assessment. MATERIALS AND METHODOLOGY: A non pathological subject group was studied using NED/IBV s. v. e. evaluation equipment. Static trials based on Romberg's test were undertaken in addition to the analysis trials of voluntary postural control. RESULTS: Normal patterns have been obtained for the COP displacement and for the maximum forces produced in the anteroposterior and midlateral axes. CONCLUSIONS: COP movement comparative analysis, related to postural behaviour patterns of non-pathological persons, represents an accurate method to assess postural balance in a standing position. This information enables the design of individual and customised rehabilitation strategies in order to reduce the risk of falling.

[Analysis of the interaction between visual and vestibular influence in postural control]. / Análisis de la interacción visuo-vestibular y la influencia visual en el control postural.

INTRODUCTION: Postural control depends on a coordinate function of the visual, somatosensory and vestibular systems. A correct analysis of each of them is necessary, in order to obtain an adequate postural evaluation. METHODS: A normal and a pathological vestibular group of subjects are study. A dynamometric platform studied was performed for every subject in both groups. A scalogram, the centre of pressure variation velocity, and the area, were the parameters analyzed. RESULTS: In both groups, parameters showed higher values when propioceptive and visual system were altered. A pattern of visual dependence was identified in both groups. The scalogram in the normal group showed a minimal centre of pressure variation. In contrast, the pathological group showed a major variation, with differences depending on the compensation status. CONCLUSIONS: Our study protocol with posturography is an adequate instrument of postural evaluation, which discriminates the visual dependence and aids to evaluate the compensation.

[Study of 1,000 patients with disequilibrium]. / Estudio de 1.000 pacientes con desequilibrio.

We present a series of one thousand patients tested with electronystagmography (ENG) for evaluation of complaints of unstability. Personal data and electronystagmographic results are analysed.

Solution structure of the TAZ2 (CH3) domain of the transcriptional adaptor protein CBP.

The TAZ2 (CH3) domain of the transcriptional adapter protein CBP has been implicated in direct functional interactions with numerous cellular transcription factors and viral oncoproteins. The solution structure of the TAZ2 domain of murine CBP has been determined by nuclear magnetic resonance (NMR). The protein adopts a novel helical fold stabilized by three zinc ions, each of which is bound to one histidine and three cysteine ligands in HCCC-type motifs. Each zinc-binding site is formed from the carboxy terminus of an alpha-helix, a short loop, and the amino terminus of the next alpha-helix. A peptide derived from the N-terminal transactivation domain of p53 binds specifically to one face of the TAZ2 domain. The close similarities between the TAZ2 and TAZ1 (CH1 domain of CBP/p300) sequences suggest that both domains will adopt similar three-dimensional structures.

NMR structure of the HIV-1 nucleocapsid protein bound to stem-loop SL2 of the psi-RNA packaging signal. Implications for genome recognition.

The RNA genome of the human immunodeficiency virus type-1 (HIV-1) contains a approximately 120 nucleotide Psi-packaging signal that is recognized by the nucleocapsid (NC) domain of the Gag polyprotein during virus assembly. The Psi-site contains four stem-loops (SL1-SL4) that possess overlapping and possibly redundant functions. The present studies demonstrate that the 19 residue SL2 stem-loop binds NC with affinity (K(d)=110(+/-50) nM) similar to that observed for NC binding to SL3 (K(d)=170(+/-65) nM) and tighter than expected on the basis of earlier work, suggesting that NC-SL2 interactions probably play a direct role in the specific recognition and packaging of the full-length, unspliced genome. The structure of the NC-SL2 complex was determined by heteronuclear NMR methods using (15)N,(13)C-isotopically labeled NC protein and SL2 RNA. The N and C-terminal "zinc knuckles" (Cys-X(2)-Cys-X(4)-His-X(4)-Cys; X=variable amino acid) of HIV-1 NC bind to exposed guanosine bases G9 and G11, respectively, of the G8-G9-U10-G11 tetraloop, and residues Lys3-Lys11 of the N-terminal tail forms a 3(10) helix that packs against the proximal zinc knuckle and interacts with the RNA stem. These structural features are similar to those observed previously in the NMR structure of NC bound to SL3. Other features of the complex are substantially different. In particular, the N-terminal zinc knuckle interacts with an A-U-A base triple platform in the minor groove of the SL2 RNA stem, but binds to the major groove of SL3. In addition, the relative orientations of the N and C-terminal zinc knuckles differ in the NC-SL2 and NC-SL3 complexes, and the side-chain of Phe6 makes minor groove hydrophobic contacts with G11 in the NC-SL2 complex but does not interact with RNA in the NC-SL3 complex. Finally, the N-terminal helix of NC interacts with the phosphodiester backbone of the SL2 RNA stem mainly via electrostatic interactions, but does not bind in the major groove or make specific H-bonding contacts as observed in the NC-SL3 structure. These findings demonstrate that NC binds in an adaptive manner to SL2 and SL3 via different subsets of inter and intra-molecular interactions, and support a genome recognition/packaging mechanism that involves interactions of two or more NC domains of assembling HIV-1 Gag molecules with multiple Psi-site stem-loop packaging elements during the early stages of retrovirus assembly.

NMR structure of stem-loop SL2 of the HIV-1 psi RNA packaging signal reveals a novel A-U-A base-triple platform.

The genome of the human immunodeficiency virus type-1 (HIV-1) contains a stretch of approximately 120 nucleotides known as the psi-site that is essential for RNA packaging during virus assembly. These nucleotides have been proposed to form four stem-loops (SL1-SL4) that have both independent and overlapping functions. Stem-loop SL2 is important for efficient recognition and packaging of the full-length, unspliced viral genome, and also contains the major splice-donor site (SD) for mRNA splicing. We have determined the structure of the 19-residue SL2 oligoribonucleotide by heteronuclear NMR methods. The structure is generally consistent with the most recent of two earlier secondary structure predictions, with residues G1-G2-C3-G4 and C6-U7 forming standard Watson Crick base-pairs with self-complementary residues C16-G17-C18-C19 and A12-G13, respectively. However, residue A15, which is located near the center of the stem, does not form a predicted bulge, and residues A5 and U14 do not form an expected Watson-Crick base-pair. Instead, these residues form a novel A5-U14-A15 base-triple that appears to be stabilized by hydrogen bonds from A15-H61 and -H62 to A5-N1 and U14-O2, respectively; from A5-H61 to U14-O2, and from C16-H42 to U14-O2'. A kink in the backbone allows the aromatic rings of the sequential U14-A15 residues to be approximately co-planar, adopting a stable "platform motif" that is structurally similar to the A-A (adenosine) platforms observed in the P4-P6 ribozyme domain of the Tetrahymena group I intron. Platform motifs generally function in RNA by mediating long-range interactions, and it is therefore possible that the A-U-A base-triple platform mediates long-range interactions that either stabilize the psi-RNA or facilitate splicing and/or packaging. Residue G8 of the G8-G9-U10-G11 tetraloop is stacked above the U7-A12 base-pair, and the remaining tetraloop residues are disordered and available for potential interactions with either other RNA or protein components.

The NMR structure of the nucleocapsid protein from the mouse mammary tumor virus reveals unusual folding of the C-terminal zinc knuckle.

The nucleocapsid protein (NC) from the mouse mammary tumor virus (MMTV) has been overexpressed in Escherichia coli and purified to homogeneity for structural studies by nuclear magnetic resonance (NMR) spectroscopy. The protein contains two copies of a conserved zinc-coordinating "CCHC array" or "zinc knuckle" motif common to the nucleocapsid proteins of nearly all known retroviruses. The residues comprising and adjacent to the zinc knuckles were assigned by standard two-dimensional (1)H and three-dimensional (1)H-(15)N NMR methods; the rotational dynamic properties of the protein were determined from (15)N relaxation experiments, and distance restraints derived from the nuclear Overhauser effect (NOE) data were used to calculate the three-dimensional structure. The (1)H-(1)H NOE and (15)N relaxation data indicate that the two zinc knuckles do not interact with each other, but instead behave as independently folded domains connected by a flexible 13-residue linker segment. The proximal zinc knuckle folds in a manner that is essentially identical to that observed previously for the two zinc knuckles of the human immunodeficiency virus type 1 nucleocapsid protein and for the moloney murine leukemia virus nucleocapsid zinc knuckle domain. However, the distal zinc knuckle of MMTV NC exhibits a rare three-dimensional fold that includes an additional C-terminal beta-hairpin. A similar C-terminal reverse turn-like structure was observed recently in the distal zinc knuckle of the Mason-Pfizer monkey virus nucleocapsid protein [Gao, Y., et al. (1998) Protein Sci. 7, 2265-2280]. However, despite a high degree of sequence homology, the conformation and orientation of the beta-hairpin in MMTV NC is significantly different from that of the reverse turn in MPMV NC. The results support the conclusion that structural features of NC zinc knuckle domains can vary significantly among the different genera of retroviridae, and are discussed in terms of the recent and surprising discovery that MMTV NC can facilitate packaging of the HIV-1 genome in chimeric MMTV mutants.

Dynamical behavior of the HIV-1 nucleocapsid protein.

The HIV-1 nucleocapsid protein (NC) contains two CCHC-type zinc knuckle domains that are essential for genome recognition, packaging and infectivity. The solution structure of the protein has been determined independently by three groups. Although the structures of the individual zinc knuckle domains are similar, two of the studies indicated that the knuckles behave as independently folded, non-interacting domains connected by a flexible tether, whereas one study revealed the presence of interknuckle NOE cross-peaks, which were interpreted in terms of a more compact structure in which the knuckles are in close proximity. We have collected multidimensional NMR data for the recombinant, isotopically labeled HIV-1 NC protein, and confirmed the presence of weak interknuckle NOEs. However, the NOE data are not consistent with a single protein conformation. 15N NMR relaxation studies reveal that the two zinc knuckle domains possess different effective rotational correlation times, indicating that the knuckles are not tumbling as a single globular domain. In addition, the 1H NMR chemical shifts of isolated zinc knuckle peptides are very similar to those of the intact protein. The combined results indicate that the interknuckle interactions, which involve the close approach of the side-chains of Phe16 and Trp37, are transitory. The solution behavior of NC may be best considered as a rapid equilibrium between conformations with weakly interacting and non-interacting knuckle domains. This inherent conformational flexibility may be functionally important, enabling adaptive binding of NC to different recognition elements within the HIV-1 psi-RNA packaging signal.

Structure of the HIV-1 nucleocapsid protein bound to the SL3 psi-RNA recognition element.

The three-dimensional structure of the human immunodeficiency virus-type 1 (HIV-1) nucleocapsid protein (NC) bound to the SL3 stem-loop recognition element of the genomic Psi RNA packaging signal has been determined by heteronuclear magnetic resonance spectroscopy. Tight binding (dissociation constant, approximately 100 nM) is mediated by specific interactions between the amino- and carboxyl-terminal CCHC-type zinc knuckles of the NC protein and the G7 and G9 nucleotide bases, respectively, of the G6-G7-A8-G9 RNA tetraloop. A8 packs against the amino-terminal knuckle and forms a hydrogen bond with conserved Arg32, and residues Lys3 to Arg10 of NC form a 310 helix that binds to the major groove of the RNA stem and also packs against the amino-terminal zinc knuckle. The structure provides insights into the mechanism of viral genome recognition, explains extensive amino acid conservation within NC, and serves as a basis for the development of inhibitors designed to interfere with genome encapsidation.

Protein-RNA recognition.

The x-ray structure of the glutamine aminoacyl tRNA synthetase bound to its cognate tRNA(Gln) and ATP was reported by Steitz and co-workers in 1989, providing the first high resolution structure of a protein-RNA complex. Since then, high resolution structures have been reported for RNA complexes with five other tRNA synthetases, the elongation factor Tu, the bacteriophage MS2 coat protein, the human spliceosomal U1A and U2B"-U1A' proteins, and the HIV-1 nucleocapsid protein. Although the number of high resolution structures of protein-RNA complexes are rather small, some general themes have begun to emerge regarding the nature and mechanisms of protein-RNA recognition.

Zinc ejection as a new rationale for the use of cystamine and related disulfide-containing antiviral agents in the treatment of AIDS.

The highly conserved and mutationally intolerant retroviral zinc finger motif of the HIV-1 nucleocapsid protein (NC) is an attractive target for drug therapy due to its participation in multiple stages of the viral replication cycle. A literature search identified cystamine, thiamine disulfide, and disulfiram as compounds that have been shown to inhibit HIV-1 replication by poorly defined mechanisms and that have electrophilic functional groups that might react with the metal-coordinating sulfur atoms of the retroviral zinc fingers and cause zinc ejection. 1H NMR studies reveal that these compounds readily eject zinc from synthetic peptides with sequences corresponding to the HIV-1 NC zinc fingers, as well as from the intact HIV-1 NC protein. In contrast, the reduced forms of disulfiram and cystamine, diethyl dithiocarbamate and cysteamine, respectively, were found to be ineffective at zinc ejection, although cysteamine formed a transient complex with the zinc fingers. Studies with HIV-1-infected human T-cells and monocyte/macrophage cultures revealed that cystamine and cysteamine possess significant antiviral properties at nontoxic concentrations, which warrant their consideration as therapeutically useful anti-HIV agents.

[Parotid tuberculosis]. / Tuberculosis parotídea.

Two rare cases of parotid gland tuberculosis are presented, which confirms the rising incidence of tuberculosis in Spain. Preoperative diagnosis is difficult, as the disease may be mistaken for a neoplastic process, and complementary tests, fine needle aspiration biopsy, CAT, analyses and chest X-rays are frequently nonspecific, particularly in the absence of a history of lung tuberculosis. As the diagnosis is histopathological, surgery in the form of a parotidectomy with preservation of the facial nerve is the treatment of choice, followed by 9 months of tuberculostatic medication. Only in cases in which the preoperative evidence (antecedents, chest X-rays) suggests the specific origin of the disease may antituberculosis treatment be given. Positive patient response is diagnostic.

[Vestibular compensation. Rehabilitation of a patient with vertigo]. / Compensación vestibular. Rehabilitación del paciente vertiginoso.

Vestibular compensation is the association of neurological reorganization events that allow to recover the balance after peripheral vestibular damage. The aim of rehabilitation of the patient with vertigo is to maximize these events to achieve an earlier return of the patients to active life. An individualized vestibular rehabilitation program is used on the basis of a general protocol that includes the following four elements: a) stabilization of vision and coordination of head and eye movements; b) vestibular habituation exercises adapted on an individual basis; c) postural control exercises via biofeedback techniques; and d) a program to improve general patient conditions. It is concluded that rehabilitation therapy of vertigo should always be taken into account, both in balance alterations caused by peripheral vestibular lesions and in central nervous damage and mixed pathology.

Analysis of DNA content in supraglottic epidermoid carcinoma.

DNA analysis by flow cytometry is considered to be of prognostic value in epidermoid carcinoma of the head and neck. However, few and contradictory studies have been made on laryngeal carcinomas. We studied 48 epidermoid carcinomas in patients subjected to horizontal supraglottic laryngectomy with a 5-year- followup. The technique described by Hedley for fixated and paraffin-embedded tumors was used. Thirteen tumors were excluded on the grounds of presenting variation coefficients in excess of 10. Of the 35 cases analyzed, 28 (80%) were diploid and seven (20%) aneuploid. No correlation was observed between tumor ploidy and patient survival, recurrence, or any of the histopathological variables studied.