Biomedical subjects
Publications and source records attributed to D Nandi.
Post-herpetic neuralgic affects up to 20% of patients after an attack of trigeminal Herpes Zoster infection. Past medical and surgical treatments have been unrewarding. We report the successful treatment of such a case with deep brain stimulation into the region of the contralateral periventricular grey area (PVG) and ventral posterior lateral thalamic nucleus (VPL).
In the current era of functional surgery for movement disorders, deep brain stimulation (DBS) of the globus pallidus internus (GPi) is emerging as the favoured intervention for patients with dystonia. Here we report our results in 20 patients with medically intractable dystonia treated with GPi stimulation. The series comprised 14 patients with generalized dystonia and six with spasmodic torticollis. Although comparisons were limited by differences in their respective neurological rating scales, chronic DBS clearly benefited both patient groups. Data conveying the rate of change in neurological function following intervention are also presented, demonstrating the gradual but progressive and sustained nature of improvement following stimulation of the GPi in dystonic patients.
OBJECTIVES: To investigate the oscillatory activity in the Parkinsonian subthalamic nucleus using the macro-electrodes for deep brain stimulation. METHODS: During bilateral deep brain stimulating electrode implantation, spontaneous and evoked field potentials were recorded from the subthalamic nucleus (STN) in two patients with Parkinson's disease (PD) during spontaneous resting tremor, passive manipulation of the wrist, and following electrical stimulation of the contralateral STN. RESULTS: Frequency analysis of the STN field potentials recorded during spontaneous resting tremor showed significant coherence with electromyographic activity in the contralateral arm, suggesting a close involvement of the STN in the generation of resting tremor in PD. The STN responded to passive movement of the contralateral wrist, but not to ipsilateral movement. High frequency (100 Hz) electrical stimulation of the STN induced tremor (4 Hz) in both forearms, and also oscillation of the contralateral STN (4 Hz). In contrast, low frequency (5 Hz) stimulation induced contralateral arrhythmic involuntary movement (3 Hz), but without altering the contralateral STN activity. CONCLUSIONS: We propose that the functional connection between the STN and arm muscles is mainly contralateral, but cross talk may occur between bilateral STN via a frequency-dependent pathway.
Lesioning or chronic deep brain stimulation (DBS) of the nucleus ventralis intermedius results in abolition of tremor in the contralateral limbs in Parkinson's disease (PD) and also in essential tremor. Recently, chronic DBS of the subthalamic nucleus has also proved to be very effective in reducing contralateral limb tremor in PD. These targets have been less effective in controlling the complex limb tremor often seen in multiple sclerosis (MS). Consequently, other targets have been sought in cases of MS with tremor. We describe a patient with MS with disabling proximal and distal involuntary arm movements in whom we were able to obtain sustained control of contralateral arm tremor and achieve functional improvement of the affected arm by chronic DBS of the region of the zona incerta. We also highlight the important role played by local field potentials recorded from the brain, with simultaneous recording of corresponding EMGs, in target localisation.
The rostral areas of the brainstem have been extensively studied in higher mammals and to a lesser extent in humans in the last two decades, looking for anatomical, electrophysiological and neurochemical evidence of involvement in the initiation and control of voluntary movement. This has come with the realisation that the axial symptoms of advanced Parkinson's disease (PD), like akinesia, postural impairment and gait freezing, are relatively less responsive to current medical and surgical treatments directed primarily at the basal ganglia and thalamus. The pedunculopontine nucleus (PPN) is one such area of interest. We have found that lesioning and electrical stimulation at high frequencies of the PPN region in the normal behaving primate induces akinesia, and low frequency stimulation can induce tremor. Micro-injections of gamma-aminobutyric acid (GABA) receptor A agonist, muscimol, into the PPN decreases activity. In the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treated Parkinsonian primate model, bicuculline, a GABA(A) antagonist, can alleviate akinesia when infused into the PPN region. This may suggest new targets for treating the intractable akinetic symptoms of advanced PD.
Subthalamic nucleus stimulation is an effective therapy for alleviating parkinsonian tremor, rigidity and bradykinesia. Although microelectrode recording is said to be essential for accurate targeting, this often prolongs the operation and the multiple recording tracts required may increase the incidence of complications, particularly haemorrhage. We describe a technique for implantation of deep brain electrodes in the subthalamic nucleus using MRI/CT fusion for anatomical localisation followed by bipolar recording of focal field potentials via the implanted stimulating electrode for neurophysiological confirmation of the stimulation site. The technique is effective, safe and requires much less time, and can be used as an alternative method to microelectrode recording.
Ever since it was demonstrated about twenty years ago by two independent groups (Aziz et al. and Bergman et al.) that the cardinal clinical features of MPTP-induced Parkinson's disease (PD) in non-human primate models can be alleviated by lesions of the subthalamic nucleus (STN), this structure has been the focus of interest for functional neurosurgeons involved in the treatment of PD. Initially lesioning and later chronic high frequency stimulation of the STN has become the standard surgical target of akinetic PD. In this brief report we present our experience with 14 STN lesions (8 unilateral and 3 bilateral) confirmed by post-operative imaging. We found significant improvement in OFF rigidity and in ON tremor following unilateral lesions. The major complications were speech disturbance and L-Dopa resistant limb dystonia. Functional disability scores showed inconsistent reduction. There was insufficient data to comment on the significance of bilateral lesions; however, there was a similar pattern of improvement in tremor and speech disturbance. In addition, there was worsening of gait. We comment on the lower degree of improvement in motor scores in our series compared to the few others in recent literature and stress that even in these studies the UPDRS benefits did not translate directly into functional benefit for the patients.
The small size and surrounding neuronal structures and fibre tracts make the STN a difficult stereotactic target. In this article we present the technique used by us to target the STN. Our combined experience from two centres comprises 18 lesions and 27 stimulator implants in the STN. Our criteria for patient selection and the use of MRI, frame-on CT and volumetric image fusion are presented. The role of a movement disorder specialist neurologist in the operating theatre, local field potential recording, impedance monitoring, macrostimulation, post-operative CT/MRI and test stimulation are detailed.
Purified gamma-aminobutyric acid aminotransferase (GABA-AT) from pig brain under certain conditions gave a single band on 12% NaDodSO(4)-PAGE, whereas two or three distinct bands were observed on 7.5% native PAGE. These multiple active species were isolated by 5% preparative gel electrophoresis and characterized by N-terminal sequencing and MALDI-TOF mass spectrometry. The results indicate that these active enzyme species are not GABA-AT isozymes in pig brain, but are the products of proteolysis of the N-terminus of GABA-AT, differing by 3, 7, and 12 residues from the full sequence (as deduced from the cDNA), respectively. Conditions for obtaining the nontruncated GABA-AT were found, and the potential cause for the proteolysis was determined. It was found that Na(2)EDTA inhibits the N-terminal cleavage during GABA-AT preparation from pig brain. The presence of Triton X-100 in the homogenization step is partially responsible for this proteolysis, and Mn(2+) strongly enhances the protease activity, suggesting the presence of a membrane-bound matrix metalloprotease that causes the N-terminal cleavage.
The proteasome is a large multicatalytic proteinase that plays a role in the generation of peptides for presentation by major histocompatibility complex class I molecules. The 20S proteolytic core of mammalian proteasomes is assembled from a group of 17 protein subunits that generate a distinctive pattern of spots upon two-dimensional gel electrophoresis. The genes for most of these subunits have been cloned from humans and rats. We isolated cDNA clones for the mouse orthologues of ten of the subunits [PSMA1 (C2), PSMA2 (C3), PSMA3 (C8), PSMA4 (C9), PSMA5 (ZETA), PSMA6 (IOTA), PSMA7 (C6-I), PSMB2 (C7-I), PSMB3 (C10-II), and PSMB5 (X)] to complete the cloning of all of the mouse subunits. Using antisera raised against these subunits or their orthologues, we verified the identity of these proteins by two-dimensional NEPHGE-PAGE.
CD8+ T lymphocytes recognize antigens as short, MHC class I-associated peptides derived by processing of cytoplasmic proteins. The transporter associated with antigen processing translocates peptides from the cytosol into the ER lumen, where they bind to the nascent class I molecules. To date, the precise location of the class I-TAP interaction site remains unclear. We provide evidence that this site is contained within the heavy chain alpha3 domain. Substitution of a 15 amino acid portion of the H-2Db alpha3 domain (aa 219-233) with the analogous MHC class II (H-2IAd) beta2 domain region (aa 133-147) results in loss of surface expression which can be partially restored upon incubation at 26 degrees C in the presence of excess peptide and beta2-microglobulin. Mutant H-2Db (Db219-233) associates poorly with the TAP complex, and cannot present endogenously-derived antigenic peptides requiring TAP-dependent translocation to the ER. However, this presentation defect can be overcome through use of an ER targeting sequence which bypasses TAP-dependent peptide translocation. Thus, the alpha3 domain serves as an important site of interaction (directly or indirectly) with the TAP complex and is necessary for TAP-dependent peptide loading and class I surface expression.
LMP2, LMP7, and MECL are interferon gamma-inducible catalytic subunits of vertebrate 20S proteasomes, which can replace constitutive catalytic subunits (delta, X, and Z, respectively) during proteasome biogenesis. We demonstrate that MECL requires LMP2 for efficient incorporation into preproteasomes, and preproteasomes containing LMP2 and MECL require LMP7 for efficient maturation. The latter effect depends on the presequence of LMP7, but not on LMP7 catalytic activity. This cooperative mechanism favors the assembly of homogeneous "immunoproteasomes" containing all three inducible subunits, suggesting that these subunits act in concert to enhance proteasomal generation of major histocompatibility complex class I-binding peptides.
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The assembly of individual proteasome subunits into catalytically active mammalian 20S proteasomes is not well understood. Using subunit-specific antibodies, we characterized both precursor and mature proteasome complexes. Antibodies to PSMA4 (C9) immunoprecipitated complexes composed of alpha, precursor beta and processed beta subunits. However, antibodies to PSMA3 (C8) and PSMB9 (LMP2) immunoprecipitated complexes made up of alpha and precursor beta but no processed beta subunits. These complexes possess short half-lives, are enzymatically inactive and their molecular weight is approximately 300 kDa. Radioactivity chases from these complexes into mature, long-lived approximately 700 kDa proteasomes. Therefore, these structures represent precursor proteasomes and are probably made up of two rings: one containing alpha subunits and the other, precursor beta subunits. The assembly of precursor proteasomes occurs in at least two stages, with precursor beta subunits PSMB2 (C7-I), PSMB3 (C10-II), PSMB7 (Z), PSMB9 (LMP2) and PSMB10 (LMP10) being incorporated before others [PSMB1 (C5), PSMB6 (delta), and PSMB8 (LMP7)]. Proteasome maturation (processing of the beta subunits and juxtaposition of the two beta rings) is accompanied by conformational changes in the (outer) alpha rings, and may be inefficient. Finally, interferon-gamma had no significant effect on the half-lives or total amounts of precursor or mature proteasomes.
We isolated and sequenced a cDNA encoding mouse proteasome subunit LMP3 from a macrophage cDNA library. The gene encodes a 264-amino-acid protein with a calculated molecular mass of 29.11 kDa and an isoelectric point (pI) of 5.44. Comparison of the predicted protein sequence with that of the human and rat homologues, N3, revealed 11 and eight changes, respectively, in the cleaved NH2-terminal presequence of the precursor protein (pre-LMP3), and six and 10 changes, respectively, in the processed product. To corroborate the predicted molecular mass and pI, we analyzed LMP3 by immunoprecipitation with a mAb to human N3 that crossreacts with mouse LMP3. Precursor and processed forms of LMP3 were identified by 2D NEPHGE-PAGE, and their mobilities suggest the Lmp3 clone encodes the entire protein sequence.
The mouse pancreatic beta TC3 and beta TC6-F7 cell lines were used to characterize the effects of interferon-gamma (IFN-y) on beta-cell phenotype and function. Initially, intracellular and secreted insulin were compared in glucose-stimulated cells over time. A significant reduction in insulin content and secretion was observed on a per-cell basis in glucose-stimulated beta TC3 and beta TC6-F7 cells after 12 h of exposure to IFN-gamma. The steadystate level of pre-proinsulin mRNA expression was not affected by IFN-gamma. Thus, we postulate that IFN-gamma's inhibitory actions occur after transcription of pre-proinsulin genes. Time-course analysis of IFN-gamma-regulated mRNA expression of the two intra-MHC-encoded subunits of the proteasome (low-molecular-mass polypeptide [Lmp]-2 and Lmp-7) revealed a correlation between their induction and the inhibitory effects of IFN-gamma on glucose-stimulated insulin production. Increased expression of Lmp-2 and Lmp-7 mRNA was accompanied by a corresponding induction of LMP2 and LMP7 protein expression. Subsequently, major histocompatibility complex (MHC) class I cell-surface expression was significantly increased in IFN-gamma-treated beta TC3 and beta TC6-F7 cells. Exposure of IFN-gamma-treated beta-cells to a peptide aldehyde inhibitor of the proteasome (MG132) significantly attenuated MHC class I cell-surface expression but did not prevent the negative effects of IFN-gamma on glucose responsiveness. Enhanced expression of the MHC class I antigen processing and presentation pathway and diminished insulin production appear to be distinct pathological alterations in beta-cells exposed to the insulitic cytokine IFN-gamma.
We show that six proteasome-associated proteins are induced by IFN-gamma, corresponding to three proteasome beta-type subunits and their precursors: the MHC-linked subunits (LMP-2 and LMP-7) and LMP-10. Concurrently, incorporation of LMP-9, LMP-17, and LMP-19 into proteasomes is reduced. LMP-10 appears to be the product of a previously cloned proteasome subunit gene, MECL-1. MECL-1 transcription is increased in the presence of IFN-gamma, whereas the transcription of two other proteasome genes, Lmp-15 and Lmp-3, is not affected. The three IFN-gamma-inducible subunits and their constitutively expressed counterparts contain most or all of the catalytic sites of the proteasome. Independent assortment of LMP-2, LMP-7, and LMP-10 into different proteasome complexes may thus generate up to 36 unique proteasome subsets. This may increase the repertoire of potentially antigenic peptides for presentation by MHC class I.
LMP-2 and LMP-7, gamma-interferon-inducible subunits of the 20S proteasome, play an important role in antigen processing. To define the molecular basis of their polymorphism, we sequenced Lmp-2 and Lmp-7 cDNA from nine different strains of mice. Three allelic variants of both LMP-2 and LMP-7 were found, but all of the polymorphism in LMP-7 is clustered near the carboxyl terminus of the molecule. We confirmed the nucleotide sequence changes at the protein level in both the unprocessed and processed forms of the molecules by analysis of specific anti-LMP-2, anti-LMP-7 and anti-proteasome immunoprecipitates on two-dimensional PAGE gels. Interestingly, a single amino acid change at position 272 between LMP-7b,d,q and LMP-7k,s,f,x,g7, cas4 from glycine to arginine dramatically affects its migration on SDS-PAGE gels, suggesting the possibility of allele-specific posttranslational modification.