Subcellular localization of the inositol 1,4,5-triphosphate receptor, P400, in the vestibular complex and dorsal cochlear nucleus of the rat

The subcellular localization of the inositol 1,4,5-trisphosphate receptor protein, P₄₀₀, was studied in the vestibular complex, an area to which Purkinje cells project, as well as in neurons of the dorsal cochlear nucleus and in ectopic Purkinje cells of adult rat brain. The receptor was demonstrated by electron microscopical immunocytochemistry using the avidin-biotin peroxidase complex procedure, with the monoclonal antibody 4C11 raised against mouse cerebellar inositol 1,4,5-trisphosphate receptor protein. Immunoreactivity was found in preterminal fibres and terminal boutons in the nuclei of the vestibular complex, generally associated with the subsurface systems and stacks or fragments of smooth endoplasmic reticulum. Ectopic Purkinje cells and cartwheel cells of the dorsal cochlear nucleus also displayed immunoreactivity, but this was much less intense in the latter. The results of the present study suggest that this receptor protein, involved in the release of Ca²⁺, is located in sites that enable it to influence the synthesis, transport and release of neurotransmitters.     

Long-term Outcomes of Patients Undergoing War-related Amputations of the Foot and Ankle

Long-term clinical and functional outcomes for patients undergoing foot and ankle amputations are not well documented. We attempted to document long-term outcomes for patients who required lower extremity amputations as a result of wounds suffered during wartime. For this study, 27 Iranian soldiers who had wounds requiring amputation of the foot and ankle were selected for follow-up. The participants' wartime medical records were reviewed, a clinical examination was performed, and each participant completed a questionnaire. Postamputation follow-up averaged 17.5 years. The most prevalent (66.6%) cause of injury was a land mine. The prevalences of different clinical symptoms reported by the amputees at the time of the last follow-up were as follows: 11 (40.7%) with phantom sensation, 6 (22.2%) with phantom pain, 12 (44.4%) with stump pain, 12 (44.4%) with back pain, 9 (33.3%) with contralateral knee pain, and 4 (14.8%) with ipsilateral knee pain; 20 (74%) reported treatment for psychological conditions. In regard to social conditions, 13 (48.1) were currently employed, or had been employed, for a number of years after the amputation; 26 (96%) had children, and all of the patients were married. The results of this observational study indicate that individuals have significant long-term pain and discomfort after war-related lower extremity amputation. Although all 27 (100%) of the amputees were able to maintain satisfactory family functioning, only 13 (48.1%) of the study participants were able to remain productively employed after undergoing amputation, and 20 (74%) reported long-term psychological problems in addition to their physical pain.     

Synthesis of [3H] vardenafil,Levitra®, using a new labeling technique

Tritium was introduced into the new orally active, selective phosphodiesterase type V (PDE V) inhibitor vardenafil (Levitra^®), by reduction of a suitable amide precursor with freshly prepared lithium aluminum tritide. A specific activity of 52.7 Ci/mmol (1.95 TBq/mmol) was achieved. In order to overcome the usual technical difficulties during the preparation of complex tritides a new and easy labeling technique which has considerable potential for various tritia‐tion procedures, was developed. Copyright © 2003 John Wiley & Sons, Ltd.     

Differential generation of class I H-2D- versus H-2K-restricted cytotoxicity against a demyelinating virus following central nervous system infection

Despite the fact that both H-2K and D molecules are up-regulated in the central nervous system (CNS) following Theiler's murine encephalomyelitis virus (TMEV) infection, resistance in this virus model of multiple sclerosis maps exclusively to D. To address this paradox, we examined the ability of the K and D molecules to present viral antigens to cytotoxic T lymphocytes (CTL). Whereas no virus-specific CTL were detected in the CNS of susceptible B10.Q and B10.S mice 7 days post-infection, D-restricted CTL were identified readily in the CNS of resistant B10 animals. There was no evidence of K-restricted CTL in the CNS of B10 mice at day 7 post-infection. The presence of both K- and D-restricted virus-specific CTL in the spleen of immunized B10 mice demonstrates that the exclusive use of D molecules by CTL in the CNS of mice 7 days post-infection is not due to the inability of the K molecules to present viral peptides to lymphocytes. We conclude that the prominent role of the D locus in determining resistance or susceptibility to TMEV-induced demyelination is determined by factors governing the regulation of the immune response, and not by the presence or absence of CTL precursors capable of recognizing viral peptides presented by the K and D antigen-presenting molecules, or by differences in the ability of the K and D molecules to present viral peptides.     

Neuronal changes in the basolateral complex during development of the amygdala of the rat

Neuronal changes in the amygdala basolateral complex were studied during development and maturation in fetal and postnatal rat brains using morphometrical methods. Forty brains of animals of various ages were fixed in formalin, frozen and cut into 25 μm thick sections and stained with cresyl violet or haematoxylin and eosin (H&E). In cresyl violet preparations, the complex appeared for the first time on embryonic day (E)17 and was composed of two homogeneous nuclei—lateral and basolateral. On about the seventh postnatal day, each of these nuclei was divided into two parts—the first one into the dorsolateral and ventromedial and the second one into the anterior and posterior. Morphometric investigations showed a different increase of the neuronal and nuclear size in various parts of the basolateral complex up to postnatal day (P)14; after that time these parameters did not change significantly. The neuronal density and the total number of neurons stabilized at P7 in all parts of this complex, except for the dorsolateral part of the lateral nucleus in which a 30% decrease of the total number of cells was observed. From P14, in all nuclei under study, the total number of neurons did not change significantly.     

Splenic nonenhancement at computed tomography: A sign of the hypoperfusion complex

When splenic nonenhancement is seen at computed tomography, one should look for signs of vascular pedicle injury; if injury to the vascular pedicle is not present, nonenhancement of the spleen could be secondary to severe vasoconstriction and may be considered an additional sign of the hypoperfusion complex. The presence of splenic nonenhancement may also help differentiate the hypoperfusion complex from other types of bowel injury.     

Biochemical analysis of plasma-soluble invariant chains and their complex formation with soluble HLA-DR

The invariant chain (CD74) is preferentially localized in the cytoplasm and regulates the loading of exogenous derived peptides into HLA class II heterodimers. In addition, a small proportion of CD74:class II complexes is also expressed on the cell surface. We identified and quantified soluble CD74 (sCD74) molecules in the plasma and sCD74:sHLA-DR complexes by ELISA. EDTA plasma samples from 86 healthy probands were analyzed. sCD74 could be detected in all samples with a mean concentration of 1.14 relative units±1.04 SD (range 0.17-4.31). Approximately 10% of the samples had increased amounts of sCD74 (3.0 relative units). Complexes of sCD74 and sHLA-DR were detected in all samples and their quantities were positively correlated (r=0.83, p0.001) with the sCD74 concentrations. SDS-PAGE analysis of plasma samples with high sCD74 concentrations (3.0 relative units) revealed four isoforms of sCD74 with molecular weights of 45, 43, 35, 31 kDa corresponding to known sizes of intracellular CD74. However, only molecular weights of the 45 and 43 kDa isoforms of sCD74 are found complexed with sHLA-DR. Our data demonstrate, that CD74 molecules are present in their soluble form in the plasma of healthy probands and form complexes with soluble HLA-DR molecules.     

Interactions between membrane IgM and the cytoskeleton involve the cytoplasmic domain of the immunoglobulin receptor

Cross-linking induced interactions between the membrane form of immunoglobulin (mIg) and the cytoskeletal matrix have been described by several groups. To date, the function of mIgM association with the cytoskeleton is not yet understood. Delineation of the molecular basis of these interactions will be instrumental in elucidating their function. We have previously shown that the Igα/β heterodimer is not required for ligand-induced mIgM binding to the cytoskeleton. In this study, we have investigated the role of other B cell-specific proteins in mediating these interactions. For this, we expressed mIgM in the non-hematopoietic human cervical carcinoma cell line HeLa S3 and verified the capacity of the surface-expressed IgM to interact with the cytoskeletal matrix upon cross-linking with anti-μ chain antibodies. We show here that only the mIgM molecule itself and no other B cell-specific protein(s) is required in mediating mIgM interactions with actin filaments. In an attempt to determine the cytoskeleton-binding site of mIgM we investigated the role of the cytoplasmic tail of mIgM (KVK) in binding the receptor to actin-based microfilaments. Using mutated forms of mIgM expressed in J558L cells, we show here that KVK plays a role in mediating these interactions. The absence of KVK did not, however, completely abrogate mIgM-cytoskeletal interactions, suggesting that there are additional molecular requirements for the ligand-induced mIgM binding to the cytoskeletal matrix.     

The organization of trigeminotectal and trigeminothalamic neurons in rodents: a double-labeling study with fluorescent dyes

Retrogradely transported fluorescent dyes (fast blue and diamidino-dihydrochloride yellow) were used to compare the distributions of trigeminofugal neurons that project to the superior colliculus and/or the thalamus in three rodent species. The objective was to determine what the projection and collateralization patterns of these trigeminofugal pathways are and whether they are similar among different species. In each anesthetized animal, one dye was injected into the superior colliculus and the other into the topographically congruent area of the thalamus. Counts of the numbers of yellow, blue, and double-labeled neurons were made throughout the trigeminal complex: principalis, pars oralis, pars interpolaris, and pars caudalis. Trigeminothalamic projections were similar in each of the rodent species studied. The densest concentration of retrogradely labeled neurons was in principalis, with substantially fewer neurons in pars interpolaris, and fewer still in pars oralis and pars caudalis. These neurons were generally small and tended to have round or fusiform somata. A common pattern was also noted among the three species for trigeminotectal neurons. Most trigeminotectal projections originated from neurons in pars interpolaris, somewhat fewer from pars oralis, and the fewest from principalis and pars caudalis. These neurons tended to be the largest in each subdivision and were often multipolar. Following paired injections of the tracers, double-labeled neurons were scattered throughout the sensory trigeminal complex and had morphologies characteristic of single-labeled trigeminotectal neurons. Although comparatively few double-labeled neurons were observed in any species, most of those seen were restricted to the ventrolateral portion of pars interpolaris, a position that corresponds to the representation of the vibrissae. These data indicate that, regardless of the rodent species, the vast majority of labeled trigeminal neurons project either to the superior colliculus or the thalamus, but not to both targets. This might be expected on the basis of the very different behavioral roles these structures play. On the other hand, a subpopulation of trigeminal neurons exists (mainly in pars interpolaris) that does project to both the superior colliculus and the thalamus, perhaps because both structures require some of the same somatosensory information to perform their behavioral functions.     

Ia-expressing microglial cells in experimental allergic encephalomyelitis in rats

Summary Monoclonal antibodies (MRC OX-6 and OX-17) recognized three types of cells expressing Ia antigen during the course of acute experimental allergic encephalomyelitis (EAE) in rats. In earlier stages of the disease, in animals with or without paralysis, Ia antigens were mostly localized to subarachnoidal and perivascular lymphocytic and histiocytic cell infiltrates, possibly serving as antigen-presenting cells. On the other hand, in convalescent rats, Ia antigens were expressed in a large number of cells with dendritic processes heavily populating the spinal gray matter. The appearance of these Ia-expressing cells in the convalescent stage coincided with the development of degenerating axon terminals in the spinal gray matter. These Ia-expressing cells possessed morphological features characteristic of microglia and were positive for ML-1 lectin but did not express glial fibrillary acidic protein. Immune electron microscopy disclosed the presence of Ia reaction products in the Golgi apparatus, endoplasmic reticulum and plasma membrane of these cells with dendritic processes, indicating active synthesis of Ia molecules in microglia. In addition, Ia antigens were localized to the cells with ultrastructural features of macrophages. Thus, Ia-expressing cells in EAE seems to play dual roles: the induction of immunological reactions during earlier stages and the participation in reparative processes during convalescence.Supported by Grants-in-aid from the Ministry of Health and Welfare for Intractable Neuroimmunological Diseases and from the Ministry of Education, Science and Culture (Project 61570380 to HK)