Stiff-man syndrome in a woman with breast cancer: an uncommon central nervous system paraneoplastic syndrome.

We report a patient who developed stiff-man syndrome, including disabling shoulder subluxation and wrist ankylosis, in association with breast cancer. Immunologic investigations disclosed autoimmunity directed against not only glutamic acid decarboxylase but also amphiphysin, a 128-kd protein located in the presynaptic compartment of neurons. The patient improved after surgery and corticosteroid treatment and has been stable for nearly 4 years on only anti-estrogenics. The triad of stiff-man syndrome, breast cancer, and autoantibodies against amphiphysin identifies a new autoimmune paraneoplastic syndrome of the CNS.

Atypical stiff-person syndrome with spinal MRI findings, amphiphysin autoantibodies, and immunosuppression.

In an atypical case of stiff-person syndrome (SPS), spinal T2-weighted MRI revealed a hyperintense lesion extending from C2 to C7 that corresponded with the clinical symptoms and signs. CSF showed lymphocytic pleocytosis and oligoclonal bands. Amphiphysin autoantibodies were detected in serum and CSF; however, unlike other reported cases, no malignancy occurred during a 3-year observation period. Methylprednisolone and cyclophosphamide pulse therapy led to a marked reduction of symptoms.

Distribution of alpha latrotoxin receptor in the rat brain by quantitative autoradiography: comparison with the nerve terminal protein, synapsin I.

Tissue slice autoradiography was employed to reveal the brain distribution of the receptor for alpha latrotoxin, the presynaptic neurotoxin of the black widow spider venom. The receptor distribution pattern was compared with that of a marker protein for nerve endings, synapsin I, a phosphoprotein known to be present within nerve terminals. The alpha latrotoxin receptor and synapsin I were detected in gray matter-containing regions but their relative amounts were not constant. In the cerebral cortex and in the caudatum their distribution was similar, while in the hippocampus they were both abundant, but their distribution varied: synapsin I labeling was heavier in CA4 and CA3, alpha latrotoxin receptor labeling in CA1 and dentate gyrus. A dissociation was also observed in the globus pallidus and in the lateral thalamic nuclear complex, where alpha latrotoxin receptor labeling was very weak. The most striking dissociation occurred in the cerebellum, where the molecular layer was strongly labeled for synapsin I, but almost unlabeled for the alpha latrotoxin receptor, which was more concentrated in the granular layer. Taken as a whole, the data appear compatible with a widespread localization of the alpha latrotoxin receptor at synapses. However, they also suggest that either some nerve terminals are insensitive to alpha latrotoxin, or the receptor for the toxin is not present at a similar concentration in all presynaptic plasma membranes.

Cell-specific localization of the alpha-subunit of calcium/calmodulin-dependent protein kinase II in Purkinje cells in rodent cerebellum.

Brain calcium/calmodulin-dependent protein kinase type II, a multimeric 600-650 kDa enzyme composed of alpha- (50 kDa) and beta/beta' (60 and 58 kDa) subunits, may be formed by alpha- and beta-subunits combining in variable proportions in different types of neurons. This study presents evidence, using cerebella from mutant mice, that the alpha-subunit displays a restricted localization in the rodent cerebellum, being detectable only in Purkinje cells. Immunocytochemical analysis of normal rat cerebellum with an antibody selective for the alpha-subunit confirmed that this subunit was detectable only in Purkinje cells. In contrast, the beta/beta'-subunits appeared to be present in all types of cerebellar mutants examined. These results indicate that different cells of the cerebellum express distinct isozymic forms of the multifunctional calcium/calmodulin-dependent protein kinase type II. It appears that Purkinje cells primarily contain an isoenzyme formed by both alpha- and beta/beta'-subunits, and that non-Purkinje cells contain an isoenzyme formed primarily by beta/beta'-subunits.

Pericytes, like vascular smooth muscle cells, are immunocytochemically positive for cyclic GMP-dependent protein kinase.

Pericytes are elongated cells with multiple processes and are located within the basement membrane of capillaries and postcapillary venules. Because of their shape, distribution, and morphological characteristics, pericytes are considered to be contractile cells. Cyclic GMP-dependent protein kinase (cGK) is an enzyme which mediates the effect of guanosine 3':5'-monophosphate in a second messenger cascade resulting in the phosphorylation of specific substrates. This enzyme is present in high concentration in both vascular and visceral smooth muscle as well as cerebellar Purkinje cells. Using an indirect immunoperoxidase technique, cGK was localized to both capillary and postcapillary venule pericytes in a variety of microvascular beds. Endothelial cells and connective tissue fibroblasts were consistently negative at the level of sensitivity of the procedure used. The specificity of reaction to pericytes and vascular smooth muscle within the vasculature permits cGK localization to be used as an efficient and selective staining procedure and as a criterion for identification. The presence of similar levels of cGK in both pericytes and vascular smooth muscle indicates a close biochemical relationship between these two cell types and provides further suggestive evidence that pericytes may have a contractile function similar to that of vascular smooth muscle cells.

High-affinity binding of the regulatory subunit (RII) of cAMP-dependent protein kinase to microtubule-associated and other cellular proteins.

Interaction of the regulatory subunit of the type II cAMP-dependent protein kinase (RII) with tissue-specific cellular binding proteins has been demonstrated by two independent methods. Complexes of RII and its binding proteins were isolated on a cAMP analog-Sepharose affinity column, eluted from the column, and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Alternatively, nitrocellulose blots made from polyacrylamide gels containing samples of tissue extracts or affinity column eluates were treated with sequential overlays of RII, monospecific antibody, and radioiodinated protein A. In bovine cerebrum, specific high-affinity interactions between RII and several binding proteins, including major proteins of 300, 80, and 68 kDa, were recognized by the two methods. The 300-kDa and 68-kDa proteins were identified as microtubule-associated protein 2 (300 kDa) and a protein of lower molecular weight (68 kDa) that copurifies with it. The additional major binding protein of 80 kDa requires further characterization. In addition, several binding proteins distinct from those observed in bovine cerebrum were found in bovine heart. Many of the RII binding proteins from brain and heart served to differing extents as substrates for the purified catalytic subunit of cAMP-dependent protein kinase. One hypothesis of the significance of the protein kinase regulatory subunit interaction with cellular binding proteins is that this may control the protein kinase holoenzyme localization and, thereby, define the substrate targets most accessible for phosphorylation by the activated protein kinase catalytic subunit. Alternatively, RII binding to a variety of cellular proteins might regulate their function--i.e., RII could be a regulator for multiple proteins in addition to the catalytic subunit of the cAMP-dependent protein kinase.

Synaptojanin 2, a novel synaptojanin isoform with a distinct targeting domain and expression pattern.

Synaptojanin (synaptojanin 1) is a recently identified inositol 5'-phosphatase, which is highly enriched in nerve terminals and is implicated in synaptic vesicle recycling. It is composed of three domains: an amino-terminal SacI homology region, a central inositol 5'-phosphatase homology region, and a carboxyl-terminal proline-rich region. We have now identified and characterized a novel form of synaptojanin, synaptojanin 2, which has a broader tissue distribution. Synaptojanin 2 cDNA from rat brain library encodes a protein of 1,248 amino acids with a predicted Mr of 138,268. The two synaptojanin isoforms share 57.2 and 53.8% amino acid identity in their SacI and phosphatase domains, respectively. In marked contrast, their carboxyl-terminal proline-rich regions bear little homology. Expression of synaptojanin 2 in COS7 cells produced a 140-kDa protein with inositol 5'-phosphatase actvity. Protein binding assays demonstrated that among the major src homology 3-proteins known to bind to the proline-rich region of synaptojanin 1, Grb2, amphiphysin, and members of SH3p4/8/13 protein family, only Grb2 bound to that of synaptojanin 2. Furthermore, subcellular fractionation studies in transfected Chinese hamster ovary cells revealed that synaptojanin 2 was predominantly associated with the particulate fraction while synaptojanin 1 was mainly localized in the soluble fraction. This observation suggests that the proline-rich regions of synaptojanins 1 and 2 are implicated in different protein-protein interactions and direct the two isoforms to different subcellular compartments. Our results demonstrate the presence of a family of synaptojanin-type inositol 5'-phosphatases with different tissue and subcellular distributions, which may be involved in distinct membrane trafficking and signal transduction pathways in mammalian cells.

cGMP-dependent protein kinase is present in high concentrations in contractile cells of the kidney vasculature.

There is increasing evidence that the recently discovered Atrial Natriuretic Factor, a potent vasorelaxing, natriuretic and diuretic hormone, may achieve some of its diverse physiological effects by regulating the cellular level of cGMP. Since activation of a cGMP-dependent protein kinase is an established effect of cGMP, we have studied the cellular localization of cGMP-dependent protein kinase within the rat kidney cortex using both immunocytochemical and immunochemical procedures. cGMP-dependent protein kinase is selectively concentrated in contractile cells of the kidney vasculature, including both intra- and extraglomerular mesangial cells, vascular smooth muscle cells and microvascular pericytes, as well as in interstitial myofibroblasts. cGMP-dependent protein kinase is not detectable in significant amounts in tubular epithelial cells, podocytes, or endothelial cells. These results support the hypothesis that Atrial Natriuretic Factor may achieve some, and maybe all, of its renal effects by regulating hemodynamic parameters via activation of cGMP-dependent protein kinase within vascular contractile cells.

Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the beta1-adrenergic receptor.

Several G-protein coupled receptors, such as the beta1-adrenergic receptor (beta1-AR), contain polyproline motifs within their intracellular domains. Such motifs in other proteins are known to mediate protein-protein interactions such as with Src homology (SH)3 domains. Accordingly, we used the proline-rich third intracellular loop of the beta1-AR either as a glutathione S-transferase fusion protein in biochemical "pull-down" assays or as bait in the yeast two-hybrid system to search for interacting proteins. Both approaches identified SH3p4/p8/p13 (also referred to as endophilin 1/2/3), a SH3 domain-containing protein family, as binding partners for the beta1-AR. In vitro and in human embryonic kidney (HEK) 293 cells, SH3p4 specifically binds to the third intracellular loop of the beta1-AR but not to that of the beta2-AR. Moreover, this interaction is mediated by the C-terminal SH3 domain of SH3p4. Functionally, overexpression of SH3p4 promotes agonist-induced internalization and modestly decreases the Gs coupling efficacy of beta1-ARs in HEK293 cells while having no effect on beta2-ARs. Thus, our studies demonstrate a role of the SH3p4/p8/p13 protein family in beta1-AR signaling and suggest that interaction between proline-rich motifs and SH3-containing proteins may represent a previously underappreciated aspect of G-protein coupled receptor signaling.

Antibodies against glutamic acid decarboxylase: prevalence in neurological diseases.

UNLABELLED: High prevalence of autoantibodies against glutamic acid decarboxylase (GAD-Ab) in stiff man syndrome (SMS) not only helps diagnosis, but also suggests immune mediated impairment of GABAergic functions. However, the presence of GAD-Ab has also been reported in other neurological syndromes. Therefore the prevalence of GAD-Ab was investigated in SMS, progressive encephalomyelitis with rigidity and myoclonus (PERM), and in other neurological diseases (OND). Serum antibodies against the GAD isoforms, GAD65 and GAD67, were investigated with radioimmunoassays in 13 patients with SMS, nine with PERM, 279 consecutive patients with OND, and in 100 normal controls. RESULTS: Prevalence of GAD65Ab was around 80% in patients with SMS/PERM compared with 5% in patients with OND and 1% in normal controls. Prevalence of GAD67Ab was 60% in SMS/PERM, 2% in patients with OND, and 1% in normal controls. Raised GAD-Ab clustered in an OND subgroup with sporadic progressive ataxia, but not in OND subgroups with recognised neuroimmunological diseases. In conclusion, increased GAD-Ab is neither a non-specific epiphenomenon of neuronal damage nor a common feature of recognised neuroimmunological disorders. In neurological diseases, GAD-Ab may be a pathogenetic agent or a marker for an ongoing autoimmune process, or both.