Insulin binding and internalization in cultured fibroblasts from myotonic muscular dystrophy

The binding and internalization of insulin receptors were investigated in 8 adult form and 6 early-onset myotonic muscular dystrophy (MMD) patients and in age- and sex-matched controls, using cultured skin fibroblasts to avoid the in vivo milieu of donors. The specific insulin binding in the presence of [¹²⁵I]insulin alone at pH 7.4 and pH 8.0 in MMD patients was not significantly different from that of the controls. The competition curves of insulin binding and the Scatchard plots in MMD and control fibroblasts were similar. Insulin receptor affinity in MMD patients was not different from that in the controls. In the presence of chloroquine, a lysosomotropic agent, the rate of increase in cell-associated radioactivity was similar in both MMD groups and controls. Thus, the normal binding and internalization of the insulin receptors in cultured skin fibroblasts from MMD patients suggest that the insulin receptors are not determined by the pathological genetic factors in MMD. Furthermore, the abnormal insulin binding to freshly isolated cells, reported previously, may be a reflection of environmental factors rather than a genetically determined cellular abnormality in MMD.     

Platelet membrane phospholipids in euthymic bipolar disorder patients: are they affected by lithium treatment?

BACKGROUND: Abnormalities in cell membrane processes and intracellular signal transduction pathways may be implicated in the pathophysiology of bipolar disorder. In this study, we attempted to investigate, in euthymic bipolar patients: 1) in vivo signal transduction abnormalities of the phosphatidylinositol pathway in platelets; and 2) possible in vivo effects of lithium treatment on platelet membrane phospholipids. METHODS: We determined the relative absorbances of eight individual classes of platelet membrane phospholipids, using two-dimensional thin-layer chromatography in high-performance plates, followed by scanning laser densitometry, in a group of 10 lithium-treated euthymic bipolar patients and 11 normal controls. RESULTS: The mean relative absorbance of phosphatidyl-inositol-4,5-bisphosphate (PIP2) was lower in the patient group (0.29 +/- 0.08% vs. 0.39 +/- 0.12%; t = 2.35, df = 19, p = .03); no significant differences between patients and controls were found for the other phospholipids. CONCLUSIONS: This study provides in vivo evidence that bipolar patients on lithium treatment exhibit a decreased relative amount of PIP2 in the platelet cell membranes compared to normal controls.     

Diminished epidermal growth factor binding by neurofibromatosis fibroblasts

Neurofibromatosis (NF) is an autosomal dominant disease characterized by growth abnormalities of epithelial, mesothelial, and endothelial elements. We recently reported abnormal growth and morphology of NF fibroblasts in tissue culture. Because epidermal growth factor (EGF) is known to stimulate the growth of fibroblasts in tissue culture, we studied the binding of commercial iodine 125-labeled EGF to age- and passage-matched confluent NF (N = 6) and normal (N = 4) fibroblasts. Fibroblasts were maintained at 37 degrees C for 2, 30, 60, 120, and 240 minutes in a medium in which the cells grow slowly (Dulbecco's Eagle medium) and one in which they grow normally (Ham's F-12 medium). Binding assays were done in both serum-free media according to accepted procedures. The EGF binding did not differ in the two media, and pooled data are presented. These data demonstrate no significant differences in the early binding of EGF to normal and NF fibroblasts (4,682 +/- 1,092 versus 3,441 +/- 826 cpm/10(6) cells; 20,000 cpm/ng; p > 0.15 at 30 minutes). At one hour, however, differences suggestive of abnormal EGF binding become apparent (12,495 +/- 1,989 versus 3,172 +/- 853 cpm/10(6) cells; 20,000 cpm/ng; p < 0.0025). We conclude that there may be a membrane defect in NF which is reflected by diminished EGF binding.     

Atypical adult GM1 gangliosidosis: biochemical comparison with other forms of primary beta-galactosidase deficiency

We studied beta-galactosidase in skin fibroblasts from patients with different forms of beta-galactosidase deficiency: adult GM1 gangliosidosis, type 1 GM1 gangliosidosis, and Morquio B syndrome. Enzyme properties in the adult cases differed from the other disorders and also from normal controls. Genetic hybridization studies indicated that all three forms belong to the same complementation group. Therefore, the adult disorder must be due to a mutation of the structural gene for beta-galactosidase, which is allelic to the mutations in type 1 GM1 gangliosidosis and Morquio B syndrome.     

Cultured skin fibroblasts as a cell model for investigating schizophrenia

Cultured skin fibroblasts, among other non-neuronal cells (e.g. platelets, lymphocytes, red blood cells), provide an advantageous system for investigating dynamic molecular regulatory processes underlying abnormal cell growth, metabolism, and receptor-mediated signal transduction, without the confounding effects of disease state and its treatment in a variety of brain disorders, including schizophrenia, and are useful for studies of systemic biochemical defects with predominant consequences for brain function. These cells are also useful for studying aspects of neurotransmitter functions because the cells express enzymes involved in their metabolism, as well as their receptors with complete machinery for signal transduction. These processes also function predictably with receptors that are transfected in fibroblasts. This review will focus on the use of cultured skin fibroblasts for studies of molecular mechanisms underlying the pathogenesis of schizophrenia, some of which have also been studied in post-mortem brains. These mechanisms might involve DNA processing and mitogenesis, cell-cell adhesion molecules, actions of growth factors, oxidative damage, and membrane phospholipid derived second messengers. This review will further discuss the implications of these processes to clinical and structural brain abnormalities. An understanding of these biochemical processes might help establish therapeutic implications and identify the risk for illness through experimental strategies such as epidemiology, family pedigree and high risk populations. Finally, despite some methodological limitations, skin fibroblasts are relatively easy to grow and maintain as primary cultures or as immortalized cell lines for long periods of time for use in investigating newly identified biochemical abnormalities.     

Collagenase activity in skin fibroblasts of patients with amyotrophic lateral sclerosis

In the current study we have measured collagenase activity released from skin explants and fibroblasts of patients with both Guam-type and sporadic amyotrophic lateral sclerosis and controls. The rationale for such a study derives from work reported more than 20 years ago demonstrating abnormalities in skin collagen metabolism in patients with the disease. We were not able to find significant differences in collagenase activity when fibroblasts were compared relative to the total protein secreted. This is explained, in part, by our finding of an increase in total protein released from fibroblasts of the amyotrophic lateral sclerosis patient group. Increased collagenase release did occur when activity was expressed per number of cells plated but was not statistically significant. In addition, increased release followed a 3-day lag period in skin organ culture. These results suggest that collagenase and other enzymes known to activate collagenase, such as plasminogen activator, capable of degrading extracellular matrix components might be responsible for the increased collagenolytic activity previously observed in amyotrophic lateral sclerosis patients' skin. Further evaluation of extracellular-acting degradative enzymes from skin, muscle, nerve and central nervous system may be important to follow-up such leads in understanding the pathogenesis of this enigmatic and fatal disorder.     

心境障碍的神经生物学研究进展

近年的研究发现，心境障碍患者在脑、神经细胞和信号分子水平都存在异常。边缘—丘脑—皮质环路和边缘—皮质—纹状体—苍白球—丘脑环路参与了心境障碍行为的发生，这些部位的糖代谢和脑血流量、皮质容量、神经元和胶质细胞的数量和形态均发生改变，同时心境障碍患者脑内磷酸肌醇环路、Wnt信号通路和神经营养因子下游信号转导通路也有相应变化。     

Signal transduction abnormalities in Alzheimer's disease: evidence of a pathogenic stimuli

Hippocampal and select cortical neuronal populations in Alzheimer's disease exhibit phenotypic changes characteristic of cells re-entering the cell division cycle. Therefore, in this study, we investigated whether components, known to trigger cellular proliferation and differentiation, upstream of the ras/mitogen-activated kinase pathway, could contribute to the activation of a signal transduction cascade in Alzheimer's disease. We found that proteins implicated in signal transduction from cell surface receptors via the ras pathway, namely Grb2 and SOS-1, were altered in cases of Alzheimer's disease in comparison to age-matched controls. SOS is increased in susceptible pyramidal neurons, while Grb2 shows more subtle alterations in subcellular distribution. Importantly, both SOS-1 and Grb2 show considerable overlap with early cytoskeletal abnormalities suggesting that the alteration in signal transduction molecules is a concurrent, if not preceding, event in the pathogenesis of Alzheimer's disease. Taken together with the cell cycle abnormalities previously reported, these findings suggest that a signal derived from the cell surface contributes to a stimulus for neurons in Alzheimer's disease to re-enter the cell cycle.     

A gustatory cyclic nucleotide-gated channels CNGgust, is expressed in the retina

Cyclic nucleotide-gated (CNG) channels are essential proteins that contribute to the intracellular signal transduction of the senses of sight and smell. Recently, we found a novel CNG channel (CNGgust) in rat taste buds, and demonstrated its possible involvement in taste signal transduction. In the present study, we used RT-PCR and immunostaining to prove that this gustatory CNG channel is expressed in the outer segments of rat cone photoreceptor cells. The study strongly suggests that the senses of taste and sight share, at least in part, a common signal transduction pathway.     

Abnormalities in protein kinase C signaling and the pathophysiology of bipolar disorder

Protein kinase C (PKC) is a group of calcium and phospholipid-dependent enzymes, which plays a pivotal role in cell signaling systems. Recently accumulated evidence indicates that alterations in PKC activity play a significant role in the pathophysiology of bipolar disorder. A number of laboratories investigated the effect of mood stabilizers on the regulation of PKC activity in bipolar patients, in animals, and in cultured cells. Following chronic lithium treatment, PKC activation was significantly reduced in rat brains, as measured by the translocation of cytoplasmic PKC to the membrane compartment, or by quantitative binding of the PKC ligand, PDBu. The effect of the therapeutic concentration of lithium in attenuating PKC-dependent intracellular parameters was also demonstrated in cultured cells. More importantly, alterations in platelet PKC was shown in bipolar patients during the manic state of the illness. In comparison to patients with major depressive disorder, schizophrenia, or healthy controls, PKC activity was significantly increased in manic patients, suggesting that changes in PKC may be an illness-specific marker. Interestingly, enhanced PKC activity during mania was suppressed following mood-stabilizer treatment as manic symptoms improved. In parallel to the findings in platelets, postmortem studies demonstrate that membrane-associated PKC and stimulation-induced translocation of cytosolic enzyme to the membrane were also increased in frontal cortex of bipolar patients. Other studies suggest alterations in other signal transduction mechanisms in bipolar disorder. These include alterations in G protein activation, phosphatidylinositol (PI) signaling, cyclic AMP formation, and intracellular calcium homeostasis. The alterations of PKC activity in bipolar disorder may be related to changes in these other intracellular signaling mechanisms. Alternatively, the changes of PKC activity may be the core pathology of the illness. More studies are required to further characterize the association of PKC changes with bipolar disorder, using a proper neuronal model.     

Concanavalin A binding to fibroblasts from Duchenne muscular dystrophy patients and age-matched controls

An investigation of [125I]Con A binding to skin fibroblasts from Duchenne muscular dystrophy patients and age-matched controls was carried out. The age groups examined were 5-6 years, 11-12 years, and 15-17 years. Only small differences in binding abilities were observed between dystrophic cells and matched controls. When data was examined as micrograms Con A bound/micrograms protein, dystrophic fibroblasts bound slightly more lectin compared to controls with the 5-6 and 11-12 year age groups, whereas the 15-17 years age group bound slightly less Con A compared to normal controls. However, analysis of binding data as lectin bound/cell showed slightly reduced binding of Con A to dystrophic cells from all age groups when compared to matched controls. It was also found that the amount of Con A bound by both normal and dystrophic fibroblasts markedly increased with the age of the donor. Obviously several factors must be taken into account when analyzing lectin binding data obtained with human fibroblasts. Taken as a whole, our studies do not provide evidence for significant modification of cell surface Con A receptors on fibroblasts from Duchenne muscular dystrophy patients.     

Diagnosis of adult type of Niemann-Pick disease (type C) in two brothers by filipin staining of bone marrow smears]

Niemann-Pick disease, type C (NPC) is a neurometabolic genetic disorder that is distinguished from other types of Niemann-Pick disease by its later onset, more insidious progression, variable visceromegaly, and abnormalities of intracellular cholesterol metabolism. We report cases in 18-year-old and 20-year-old brothers who presented with disinhibition and involuntary movement of their hands. Both brothers presented various signs such as dementia, vertical supranuclear ophthalmoplegia (VSO), dysarthria, axial and limb dystonia, hyperreflexia, pathologic reflex, cerebellar ataxia, as reported. They also presented startle response. Brain MRI showed diffuse cerebral atrophy and abdominal CT reveals hepato-splenomegaly in both patients. These cases were suspected to be NPC based on dementia, VSO, cerebellar ataxia, hepato-splenomegaly and foam cells in the bone marrow. Generally, the diagnosis of NPC is based on deficient cholesterol esterification and excessive lysosomal filipin staining in cultured skin fibroblasts. However, culture of fibroblasts obtained from a biopsied skin samples is slow. We have rapidly made the diagnosis of NPC in our patients by filipin staining of foam cells from bone marrow. This diagnostic process using a bone marrow smear is more convenient and rapid than previous methods using cultured skin fibroblasts.     

Normal phospholipid-related signal transduction in autism

The aim of this study was to test the hypothesis that autism (a neurodevelopmental disorder of unknown etiology) is associated with altered phospholipid-related signal transduction using the niacin skin flush test. This is the first reported use of this test in this disorder. The response to topical aqueous methyl nicotinate solution was recorded at 5-min intervals over 20 min in eight patients with autism and in 16 age- and sex-matched normal individuals with no history of this or any other major neuropsychiatric disorder. There was no significant difference between the mean volumetric niacin response (VNR) (±S.E.M.) in the patients with autism, 27.0 (±2.2) mol s l⁻¹, and the mean VNR of 27.6 (±2.4) mol s l⁻¹ in the control group (P>.8). Therefore, there appears to be no evidence of altered phosopholipid-related signal transduction in autism as assessed by this test. Furthermore, since the VNR is reduced in a related disorder, schizophrenia, these results suggest fatty acid abnormalities in autism are likely to differ from those in schizophrenia.     

Skin biopsy findings in glycogenosis III: clinical, biochemical, and electrophysiological correlations

Electron microscopy of skin specimens was performed in 4 patients (age range, 7 months-40 years) with glycogenosis III and revealed consistent abnormalities. Massive glycogen storage was observed in epithelial secretory cells of eccrine sweat glands and, less markedly, in smooth muscle fibers from the erector pili. Other cells, including Schwann cells of myelinated and unmyelinated fibers, were not affected. The extent of glycogen storage was similar in all patients and unrelated to age or duration of disease. The extralysosomal nature and selectivity of glycogen deposits, sparing fibroblasts and other cells, differ clearly from the findings in skin from patients with glycogenosis II. The purpose of this study was to show that glycogen deposits in glycogenosis III are not restricted to skeletal muscle and liver, and to assess the usefulness of skin biopsy in this disorder.     

Glucocorticoid receptor binding in three different cell types in major depressive disorder: lack of evidence of receptor binding defect.

1. In order to further understand the apparent glucocorticoid resistance in major depressive disorder, circadian variation in cortisol concentration, dexamethasone suppression and glucocorticoid receptor binding in mononuclear leukocytes, polymorphonuclear leukocytes and cultured skin fibroblasts were measured in rigidly defined major depressive disorder patients and non-depressed psychiatric controls. 2. Mononuclear leukocytes binding to glucocorticoid correlated significantly with polymorphonuclear leukocytes binding to glucocorticoid, but both determinations failed to differentiate major depressive disorder and control subjects. 3. Initial and post-dexamethasone in vitro fibroblast binding to glucocorticoid was not different between major depressive disorder and non-depressed control subjects. 4. The phenomenon of glucocorticoid resistance in major depressive disorder remains unexplained.     

Neuraminidase deficiency and accumulation of sialic acid in lymphocytes in adult type sialidosis with partial β-galactosidase deficiency

Neuraminidase deficiency has been demonstrated in cultured skin fibroblasts of patients who have adult type sialidosis with partial β-galactosidase deficiency. A substantial amount of residual enzyme activity has been observed in leukocytes, however. To explain this discrepancy, the nature and distribution of the enzyme were studied. Neuraminidase activity was higher in lymphocytes than in granulocytes of normal controls. In patients' lymphocytes, neuraminidase activity was profoundly decreased and total sialic acid contents were increased 2.3-fold. Two neuraminidases, one sonication-labile and the other sonication-stable, were found in lymphocytes; the former was predominant in cultured skin fibroblasts. The defective enzyme in this disorder was found to be the sonication-labile neuraminidase in both cultured skin fibroblasts and lymphocytes.     

Free cytoplasmic Ca++ at rest and after cholinergic stimulus is increased in cultured muscle cells from Duchenne muscular dystrophy patients

We used a fluorescent dye, quin 2, to measure intracellular free calcium ([Ca++]i) in cultured skeletal muscle cells and skin fibroblasts from five Duchenne muscular dystrophy (DMD) patients and from five controls. We observed an enhanced [Ca++]i level, at rest and after acetylcholine (ACh) stimulation, in DMD muscle cells, but we did not detect any difference between DMD and normal skin fibroblasts. The abnormally higher [Ca++]i transient induced by ACh suggests that it plays a critical role in muscle degeneration. The skin fibroblast results suggests that there is no generalized membrane defect.