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Biomedical subjects

M Solimena

Publications and source records attributed to M Solimena.

At least 37 records · Page 2Linked to original sources
PubMed1998

Stabilization of the receptor protein tyrosine phosphatase-like protein ICA512 in GH4C1 cells upon treatment with estradiol, insulin, and epidermal growth factor.

Treatment with 1 nM estradiol, 300 nM insulin, and 5 nM epidermal growth factor induces secretory granule accumulation and prolactin storage in GH4C1 rat pituitary tumor cells. The same triple treatment induced more than 6-fold accumulation of both the precursor (100 kDa, pro-ICA512) and the mature forms (60-70 kDa, ICA512 transmembrane fragment) of ICA512, a receptor protein tyrosine phosphatase-like protein that is preferentially localized in secretory granule membranes. Accumulation of ICA512 resembles that of prolactin storage, for the combination of all three, estradiol, insulin, and epidermal growth factor, gave the greatest induction, which was maximal at 4 days. This effect was specific, as the levels of the small GTP-binding protein Rab3, which is also associated with secretory granule membranes, were unaffected by the triple hormone/growth factor treatment. Increased transcription and translation of ICA512 could only partially account for its 6-fold accumulation, as ICA512 messenger RNA and ICA512 synthesis levels were 1.8 +/- 0.35- and 1.6 +/- 0.17-fold in triple treated GH4C1 cells compared with those in untreated cells, respectively. Pulse-chase procedures showed that pro-ICA512 was more stable in treated cells. These results indicate that the enlargement of the secretory granule compartment results in the stabilization of ICA512 and raise the possibility that trafficking of secretory granules may affect ICA512's function and vice versa.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

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.

Anti-Inflammatory Agents↗
PubMed1998

Expression of amphiphysin I, an autoantigen of paraneoplastic neurological syndromes, in breast cancer.

Amphiphysin I is a 128 kD protein highly concentrated in nerve terminals, where it has a putative role in endocytosis. It is a dominant autoantigen in patients with stiff-man syndrome associated with breast cancer, as well as in other paraneoplastic autoimmune neurological disorders. To elucidate the connection between amphiphysin I autoimmunity and cancer, we investigated its expression in breast cancer tissue. We report that amphiphysin I was expressed as two isoforms of 128 and 108 kD in the breast cancer of a patient with anti-amphiphysin I antibodies and paraneoplastic sensory neuronopathy. Amphiphysin I was also detectable at variable levels in several other human breast cancer tissues and cell lines and at low levels in normal mammary tissue and a variety of other non-neuronal tissues. The predominant amphiphysin I isoform expressed outside the brain in humans is the 108 kD isoform which represents an alternatively spliced variant of neuronal amphiphysin I missing a 42 amino acid insert. Our study suggests a link between amphiphysin I expression in cancer and amphiphysin I autoimmunity. The enhanced expression of amphiphysin I in some forms of cancer supports the hypothesis that amphiphysin family members may play a role in the biology of cancer cells.

Alternative Splicing↗
PubMed1997

Targeted expression of the neuropeptide calcitonin gene-related peptide to beta cells prevents diabetes in NOD mice.

To investigate whether the immunosuppressive neuropeptide calcitonin gene-related peptide (CGRP) was a potential candidate for tissue-specific gene therapy, we engineered nonobese diabetic (NOD) mice to produce CGRP in beta cells by placing the modified calcitonin gene under the control of the rat insulin promoter. CGRP inhibits CD4 T cell production of the cytokines that have been implicated in the pathogeny of type I diabetes. Three transgene-positive mouse lines were obtained, two of which expressed immunoreactive CGRP in beta cells (NOD-CGRP mice). Isolated islets from one of these two transgene-positive founders produced active CGRP, whereas islets from transgene-negative littermates did not. The production of CGRP by beta cells prevented insulin-dependent diabetes mellitus in male and reduced its incidence by 63% in female mice. This prevention was due to a local immunosuppressive effect of CGRP as no difference was detected between NOD-CGRP and NOD littermate lymph node, spleen, and thymus cells by either FACS analysis or proliferative response to stimulation by Ag, alloantigen or anti-CD3. These data suggest that CGRP is a potential therapeutic molecule to prevent or treat diabetes and possibly other diseases and conditions in which immune cells are involved. These data also suggest that endogenous CGRP concentrated in sensory nerve endings may regulate locally the immune response, further strengthening the importance of the functional neuroimmune link.

Animals↗
PubMed1997

STEP: a family of brain-enriched PTPs. Alternative splicing produces transmembrane, cytosolic and truncated isoforms.

The family of striatal enriched phosphatases (STEPs) consists of protein tyrosine phosphatases (PTPs) that are enriched within the central nervous system. Previous biochemical studies have shown that the STEP family includes transmembrane, as well as soluble, cytosolic proteins. We now extend these findings with the isolation and characterization of a new, truncated member of this family, termed STEP38. The cDNA of STEP38 encodes a protein of 346 amino acids with a predicted mobility of 38 kDa. In contrast to the cytosolic variants, it contains two hydrophobic amino acid sequences at its N-terminus, two sequences enriched in Pro, Glu, Asp, Ser and Thr residues (PEST sequences), and two polyproline domains. We have used differential centrifugation, continuous sucrose gradients, and transfection experiments to clarify the subcellular localization of STEP38 within membrane compartments. These experiments suggest that a pool of STEP38 is targeted to membrane compartments of the endoplasmic reticulum. The STEP38 cDNA contains a stop codon upstream of the catalytic phosphatase domain that is normally present in other STEP variants, and enzymatic assays conform that STEP38 is inactive. Thus, the STEP family consists of cytosolic, transmembrane, and truncated isoforms. These findings are similar to what has been found for some members of the protein tyrosine kinase (PTK) family that uses alternative splicing mechanisms to produce active and inactive variants. By analogy with suggested mechanisms of action for the truncated PTKs, inactive STEP isoforms may participate in signaling events by protecting potential substrates from dephosphorylation by other members of this family.

Alternative Splicing↗
PubMed1996

STEP61: a member of a family of brain-enriched PTPs is localized to the endoplasmic reticulum.

The STEP family of protein tyrosine phosphatases is highly enriched within the CNS. Members of this family are alternatively spliced to produce both transmembrane and cytosolic variants. This manuscript describes the distinctive intracellular distribution and enzymatic activity of the membrane-associated isoform STEP61. Transfection experiments in fibroblasts, as well as subcellular fractionations, sucrose density gradients, immunocytochemical labeling, and electron microscopy in brain tissue, show that STEP61 is an intrinsic membrane protein of striatal neurons and is associated with the endoplasmic reticulum. In addition, structural analysis of the novel N-terminal region of STEP61 reveals several motifs not present in the cytosolic variant STEP46. These include two putative transmembrane domains, two sequences rich in Pro, Glu, Asp, Ser, and Thr (PEST sequences), and two polyproline-rich domains. Like STEP46, STEP61 is enriched in the brain, but the recombinant protein has less enzymatic activity than STEP46. Because STEP46 is contained in its entirety within STEP61 and differs only in the extended N terminus of STEP61, this amino acid sequence is responsible for the association of STEP61 with membrane compartments and may also regulate its enzymatic activity.

Amino Acid Sequence↗
PubMed1996

ICA 512, an autoantigen of type I diabetes, is an intrinsic membrane protein of neurosecretory granules.

Islet cell autoantigen (ICA) 512 is a novel autoantigen of insulin-dependent diabetes mellitus (IDDM) which is homologous to receptor-type protein tyrosine phosphatases (++PTPases). We show that ICA 512 is an intrinsic membrane protein of secretory granules expressed in insulin-producing pancreatic beta-cells as well as in virtually all other peptide-secreting endocrine cells and neurons containing neurosecretory granules. ICA 512 is cleaved at its luminal domain and, following exposure at the cell surface, recycles to the Golgi complex region and is sorted into newly formed secretory granules. By immunoprecipitation, anti-ICA 512 autoantibodies were detected in 15/17 (88%) newly diagnosed IDDM patients, but not in 10/10 healthy subjects. These results suggest that tyrosine phosphorylation participates in some aspect of secretory granule function common to all neuroendocrine cells and that a subset of autoantibodies in IDDM is directed against an integral membrane protein of insulin-containing granules.

Amino Acid Sequence↗
PubMed1995

Targeting of the 67-kDa isoform of glutamic acid decarboxylase to intracellular organelles is mediated by its interaction with the NH2-terminal region of the 65-kDa isoform of glutamic acid decarboxylase.

The two isoforms of glutamic acid decarboxylase (GAD), GAD67 and GAD65, synthesize the neurotransmitter gamma-aminobutyric acid in neurons and pancreatic beta-cells. Previous studies suggest that GAD67 is a soluble cytosolic protein, whereas GAD65 is membrane-associated. Here, we study the intracellular distribution of GAD67 in neurons, pancreatic beta-cells, and fibroblasts transfected either with GAD65 and GAD67 together or with GAD67 alone. Neuronal GAD67 is partially recovered with GAD65 in membrane-containing pellet fractions and Triton X-114 detergent phases. The two proteins co-immunoprecipitate from extracts of brain and GAD65-GAD67 co-transfected fibroblasts, but not when extracts of GAD65 and GAD67 transfected fibroblasts were mixed and used as a starting material for immunoprecipitation. GAD67 is concentrated in the Golgi complex region in GAD65-GAD67 co-transfected fibroblasts, but not in fibroblasts transfected with GAD67 alone. A pool of neuronal GAD67 co-localizes with GAD65 in the Golgi complex region and in many synapses. The two proteins also co-localize in the perinuclear region of some pancreatic beta-cells. GAD67 interacts with the NH2-terminal region of GAD65, even in the absence of palmitoylation of this region of GAD65. Taken together, our results indicate that GAD65-GAD67 association occurs in vivo and is required for the targeting of GAD67 to membranes.

Animals↗
PubMed1995

Stiff man syndrome: neurophysiological findings in eight patients.

The neurophysiological findings in eight patients with the stiff man syndrome (SMS), including four of six tested with autoantibodies against glutamic acid decarboxylase, are presented. Neurophysiological findings did not make it possible to discriminate between patients with and those without autoimmunity against GABAergic neurons. Investigation of mono- and polysynaptic reflexes revealed abnormal results in a variable number of SMS patients, the abnormalities largely corresponding to those seen in spastic paresis. A stereotyped motor response to electrical stimulation of peripheral nerves was recorded from the trunk muscles of all patients investigated. This response was termed spasmodic reflex myoclonus and consisted of a sequence of 1-3 synchronous myoclonic bursts, 60-70 ms after median nerve stimulation, followed by a tonic decrescendo activity over a number of seconds. The recruitment order of muscles along the neuraxis in spasmodic reflex myoclonus suggested that the latter was generated in the spinal cord and conveyed via propriospinal tracts. It is thought that spasmodic reflex myoclonus may serve not only as a diagnostic tool, but also as a key to understanding some aspects of the pathophysiology of both spasms and stiffness in SMS. It is speculated that stiffness is a fragment of spasms, both being generated by common neuronal mechanisms tentatively ascribed to interneurons in the spinal grey matter.

Electromyography↗
PubMed1994

Autoimmunity in stiff-Man syndrome with breast cancer is targeted to the C-terminal region of human amphiphysin, a protein similar to the yeast proteins, Rvs167 and Rvs161.

Amphiphysin, a neuronal protein first identified in chicken synaptic membranes, is the autoantigen of Stiff-Man Syndrome (SMS) associated with breast cancer. We have now cloned human amphiphysin and found the N- and C-terminal domains of the protein to be highly conserved between chicken and human. Patient autoantibodies have a distinct pattern of reactivity with amphiphysin, and the dominant autoepitope is located in its C-terminal region, which contains an SH3 domain. Portions of chicken and human amphiphysin are also homologous to portions of Rvs167 and Rvs161, two yeast proteins which are involved in cell entry into stationary phase upon exposure to unfavourable growth conditions.

Amino Acid Sequence↗
PubMed1994

Stiff man syndrome: clinical and laboratory findings in eight patients.

The clinical, biochemical, neuroimaging and neurophysiological findings of eight patients with stiff man syndromes (SMS) [four of six being tested with autoantibodies against glutamic acid decarboxylase (GAD)] are presented. In two patients (one GAD-positive, one GAD-negative), transient oculomotor disturbances suggested progressive encephalomyelitis with rigidity and myoclonus (PERM) as differential diagnosis. The catalogue of characteristic clinical symptoms of SMS is extended by three new symptoms: (1) an aura-like feeling reported by five patients to precede spontaneous spasmodic attacks; (2) a stereotyped motor pattern seen in seven patients during spasmodic jerks, consisting of brief opisthotonos, stiffening of the slightly abducted legs and inversion of the plantar-flexed feet; (3) a paroxysmal fear when crossing a free space unaided, or even thinking of it. Clinical findings did not enable us to discriminate between patients tested GAD-positive or GAD-negative. Cerebrospinal fluid contained elevated immunoglobulin levels or cell counts, or both, in the majority of patients. Autopsy of one patient revealed scattered lymphocyte cuffs around leptomeningeal, intracerebral and particularly intraspinal vessels, suggesting a mild inflammatory process. Whether SMS and PERM are closely related is discussed; they are possibly both manifestations of a spectrum of encephalomyelopathies having autoimmunity against GABAergic neurons in common.

Adult↗
PubMed1994

A signal located within amino acids 1-27 of GAD65 is required for its targeting to the Golgi complex region.

The mechanisms involved in the targeting of proteins to different cytosolic compartments are still largely unknown. In this study we have investigated the targeting signal of the 65-kD isoform of glutamic acid decarboxylase (GAD65), a major autoantigen in two autoimmune diseases: Stiff-Man syndrome and insulin-dependent diabetes mellitus. GAD65 is expressed in neurons and in pancreatic beta-cells, where it is concentrated in the Golgi complex region and in proximity to GABA-containing vesicles. GAD65, but not the similar isoform GAD67 which has a more diffuse cytosolic distribution, is palmitoylated within its first 100 amino acids (a.a.). We have previously demonstrated that the domain corresponding to a.a. 1-83 of GAD65 is required for the targeting of GAD65 to the Golgi complex region. Here we show that this domain is sufficient to target an unrelated protein, beta-galactosidase, to the same region. Site-directed mutagenesis of all the putative acceptor sites for thiopalmitoylation within this domain did not abolish targeting of GAD65 to the Golgi complex region. The replacement of a.a. 1-29 of GAD67 with the corresponding a.a. 1-27 of GAD65 was sufficient to target the otherwise soluble GAD67 to the Golgi complex region. Conversely, the replacement of a.a. 1-27 of GAD65 with a.a. 1-29 of GAD67 resulted in a GAD65 protein that had a diffuse cytosolic distribution and was primarily hydrophilic, suggesting that targeting to the Golgi complex region is required for palmitoylation of GAD65. We propose that the domain corresponding to a.a. 1-27 of GAD65, contains a signal required for the targeting of GAD65 to the Golgi complex region.

Amino Acid Sequence↗
PubMed1993

Identification of a dominant epitope of glutamic acid decarboxylase (GAD-65) recognized by autoantibodies in stiff-man syndrome.

Glutamic acid decarboxylase (GAD) is the enzyme that synthesizes the neurotransmitter gamma-aminobutyric acid (GABA) in neurons and in pancreatic beta cells. It is a major target of autoimmunity in Stiff-Man syndrome (SMS), a rare neurological disease, and in insulin-dependent diabetes mellitus. The two GAD isoforms, GAD-65 and GAD-67, are the products of two different genes. GAD-67 and GAD-65 are very similar to each other in amino acid sequence and differ substantially only at their NH2-terminal region. We have investigated the reactivity of autoantibodies of 30 Stiff-Man syndrome patients to GAD. All patient sera contained antibodies that recognize strongly GAD-65, but also GAD-67, when tested by immunoprecipitation on brain extracts and by immunoprecipitation or immunocytochemistry on cells transfected with either the GAD-65 or the GAD-67 gene. When tested by Western blotting, all patient sera selectively recognized GAD-65. Western blot analysis of deletion mutants of GAD-65 demonstrated that autoantibodies are directed predominantly against two regions of the GAD-65 molecule. All SMS sera strongly recognized a fragment contained between amino acid 475 and the COOH terminus (amino acid 585). Within this region, amino acids 475-484 and 571-585 were required for reactivity. The requirement of these two discontinuous segments implies that the epitope is influenced by conformation. This reactivity is similar to that displayed by the monoclonal antibody GAD 6, suggesting the presence of a single immunodominant epitope (SMS-E1) in this region of GAD-65. In addition, most SMS sera recognized at least one epitope (SMS-E2) in the NH2-terminal domain of GAD-65 (amino acids 1-95). The demonstration in SMS patients of a strikingly homogeneous humoral autoimmune response against GAD and the identification of dominant autoreactive target regions may help to elucidate the molecular mechanisms of GAD processing and presentation involved in GAD autoimmunity. Moreover, the reactivity reported here of GAD autoantibodies in SMS partially differs from the reactivity of GAD autoantibodies in insulin-dependent diabetes mellitus, suggesting a link between the pattern of humoral autoimmunity and the clinical condition.

Autoantibodies↗
PubMed1993

The synaptic vesicle-associated protein amphiphysin is the 128-kD autoantigen of Stiff-Man syndrome with breast cancer.

Stiff-Man syndrome (SMS) is a rare disease of the central nervous system (CNS) characterized by progressive rigidity of the body musculature with superimposed painful spasms. An autoimmune origin of the disease has been proposed. In a caseload of more than 100 SMS patients, 60% were found positive for autoantibodies directed against the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD). Few patients, all women affected by breast cancer, were negative for GAD autoantibodies but positive for autoantibodies directed against a 128-kD synaptic protein. We report here that this antigen is amphiphysin. GAD and amphiphysin are nonintrinsic membrane proteins that are concentrated in nerve terminals, where a pool of both proteins is associated with the cytoplasmic surface of synaptic vesicles. GAD and amphiphysin are the only two known targets of CNS autoimmunity with this distribution. This finding suggests a possible link between autoimmunity directed against cytoplasmic proteins associated with synaptic vesicles and SMS.

Autoantigens↗