Biomedical subjects

N L Banik

Publications and source records attributed to N L Banik.

At least 55 records · Page 3Linked to original sources

PubMed1997

Increased calpain content and progressive degradation of neurofilament protein in spinal cord injury.

Spinal cord injury was induced in rat by weight drop. The extent of degradation of neurofilament proteins in the lesion following trauma was examined and served as a measure of calpain activity. Calpain was identified in the samples by myelin mcalpain antibody and the content was estimated from the immunoblot. There was progressive degradation of both 68 kDa and 200 kDa neurofilament proteins in the cord lesion at intervals after injury. At 30 min after injury there was 20% degradation of both neurofilament proteins while the breakdown of 68 kDa and 200 kDa NFPs amounted to more than 60% at 24 h and beyond. Calpain content progressively increased in the lesion by 22% at 30 min to 91% at 4 h after trauma compared to control and then decreased but remained elevated for up to 72 h following injury. These results suggest that calpain is a primary responder synthesized early in injury and involved initially in the breakdown of cytoskeletal proteins in spinal cord trauma. Later in the injury cascade, increased calpain activity is derived from inflammatory as well as endogenous cells supporting a pivotal role for calpain throughout the process of secondary and evolving tissue damage in spinal cord trauma.

Animals↗

PubMed1997

Immunolocalization of cytoplasmic and myelin mcalpain in transfected Schwann cells: II. Effect of withdrawal of growth factors.

We have examined the reversal of the regulatory effect of growth factors on calpain/calpastatin activity in transfected Schwann cells (tSc) after their subsequent withdrawal. Removal of nerve growth factor (NGF) or cyclic adenosine monophosphate (cAMP) from tSc resulted in a smaller loss of mu calpain (37%) and mcalpain (36.5 %) activity compared to treated cells from which the growth factors were not withdrawn. The mu calpain activity increased approximately 12% following withdrawal of acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) at 24 hr, while the increased mcalpain activity was more than 30-40% compared with that of cells that were continuously treated. The activity of both isoforms returned to their normal levels (untreated) at 48-72 hr following withdrawal of various growth factors, including NGF, cAMP, aFGF, bFGF, platelet-derived growth factor aa (PDGFaa), and PDGFbb. The inhibitory activity of calpastatin was greater than control following withdrawal of NGF, cAMP, PDGFaa, or PDGFbb at 24 hr and this inhibitory activity was less with treatment by aFGF and bFGF. The control activity was restored at 48 hr following withdrawal of these factors. The intensity of the cytoplasmic calpain immunoreactivity was significantly decreased in the nuclear and non-nuclear regions of the cytoplasm, respectively, following withdrawal of cAMP at 144 hr. Removal of bFGF from the medium resulted in an increase of cytoplasmic calpain immunoreactivity in the nuclear regions and cytoplasm, while there was dramatic loss of myelin calpain immunoreactivity from both the nuclear region and cytoplasm. The changes in calpain activity and immunoreactivity in tSc following withdrawal of growth factors suggest that release of calpain from membrane to cytosol may be regulated by these factors.

Animals↗

PubMed1997

Immunolocalization of cytoplasmic and myelin mcalpain in transfected Schwann cells: I. Effect of treatment with growth factors.

We have examined the effect of growth factors on the activity and localization of calpain in transfected Schwann cells (tSc). Axolemma-enriched fraction, cAMP, or NGF showed concentration-dependent inhibition of both mu calpain and mcalpain activity. In contrast, both acidic FGF and basic FGF stimulated mu calpain (37%) and mcalpain (58%) of tSC while PDGF-aa and PDGF-bb inhibited both calpain activities. The inhibitor (calpastatin) activity was approximately 90% following treatment with NGF, cAMP, PDGF-aa, and PDGF-bb compared to control while this activity was 40% with FGF-treated samples. Immunofluorescence studies indicated localization of cytoplasmic calpain in the nuclear region following growth factor treatment in the cytoplasm. Growth factor treatment caused a decrease in the intensity of calpain immunoreactivity. Treatment with cAMP or FGF resulted in strong immunoreactivity of mcalpain in the nuclear region and cytoplasm compared to untreated. The growth factors did not cause translocation of calpain to the outer surface of the cell membrane. The increased immunoreactivity seen with myelin calpain antibody was greater than cytosolic antibody. The changes seen in calpain activity and immunoreactivity following treatment with growth factors suggest that these factors may regulate calpain-calpastatin expression and translocation to the membrane for interaction with lipids for enzyme activation.

Animals↗

PubMed1997

A new mechanism of methylprednisolone and other corticosteroids action demonstrated in vitro: inhibition of a proteinase (calpain) prevents myelin and cytoskeletal protein degradation.

The affect of methylprednisolone (MP), an anti-inflammatory drug upon purified calpain and the Ca2+-mediated degradation of endogenous proteins of spinal cord homogenate in vitro has been examined. Activity of calpain purified from rabbit muscle was greatly inhibited in a dose-dependent fashion by MP. A 50% inhibition was obtained with 3.2 mM MP concentration and the activity was inhibited further (80%) at 8.1 mM. More potent inhibition of the purified enzyme (70-80%) was produced by dexamethasone (3.9 mM) and prednisolone (4.1 mM). Calpain-mediated degradation of myelin basic protein (MBP) was also inhibited by MP as was cathepsin B-mediated MBP breakdown. The effect of MP and other steroids upon calcium-mediated degradation of spinal cord homogenate was also evaluated. SDS-PAGE analysis revealed significant inhibition of neurofilament protein breakdown by MP and other corticosteroids. This inhibitory effect was much less than that exerted by the calpain inhibitors calpeptin and/or E64-d. These results indicate that MP acts as a proteinase (calpain) inhibitor and define a new mechanism for its actions.

Adrenal Cortex Hormones↗

PubMed1997

Calcium-mediated neurofilament protein degradation in rat optic nerve in vitro: activity and autolysis of calpain proenzyme.

In this study, we examined calcium-mediated degradation of a neurofilament protein (NFP), and autolytic activation of calpain in Lewis rat optic nerve in vitro. After incubation with calcium, homogenized optic nerve samples were analysed by SDS-PAGE in association with ECL immunoblot techniques. 68 kD NFP, calpain, and calpastatin antibodies were used for identification of the respective proteins. The extent of calcium-mediated 68 kD NFP degradation compared to EGTA controls, served to quantify calpain activity, while the extent of calpain autolysis measured the activation of the enzyme. A progressive loss of 68 kD NFP was observed at 15 min (42.1%), 1 hr (52.7%) and 6 hr (73.4%) incubation periods compared to EGTA controls. The immunoreactive calpain bands showed progressive autolysis after 15 min (26.6%), 1 hr (31.4%) and 6 hr (43.4%) incubations. We also found degradation of low molecular weight isoforms of calpastatin (43 kD and 27 kD) in the presence of calcium compared to controls. These results indicate that calpain is present in optic nerve in its inactive form but when calcium is added, it undergoes autolysis and becomes active. Thus, active calpain is capable of degrading endogenous substrates (e.g. cytoskeletal and myelin proteins) and may promote the degeneration of optic nerve in optic neuritis.

Animals↗

PubMed1996

Regulation of brain m calpain Ca2+ sensitivity by mixtures of membrane lipids: activation at intracellular Ca2+ level.

Combinations of certain phospholipids and gangliosides increase the specific activity of m calpain and can activate m calpain at 1 to 10 microM Ca2+ concentration. However, this level of calcium is still greater than the normal intracellular calcium level. We have used combinations of lipids to demonstrate the m calpain activity at the physiological Ca2+ level. GD1a (100 microM) and cerebroside (Cerb; 750 microM; 1:7.5) mixture was the most effective. At 0.5 microM to 1.0 microM Ca2+ concentrations, 15-20% of the maximal activity was detected for the purified myelin and cytosolic m calpains. Other combinations were GD1a (100 microM), GM1 (100 microM), Cerb (750 microM), sulfatide (Sulf; 750 microM), and phosphatidylinositol (PI; 300 microM) at a ratio of 1:1: 7.5:7.5:3, respectively. These lipid mixtures stimulated calpain activity at three- to tenfold less calcium concentration than control. The other mixtures, including GD1a:Sulf (1:9) > GD1a:PI (1:4) > PI:Sulf (1:5) > Cerb:Sulf (1:5) and PI:Cerb (1:2.5), also stimulated calpain activity at 1.0 microM Ca2+ concentration. Triton X-100, oxidized glutathione (GSSG), and calpain activator did not affect the Ca2+ requirement. Liposomes containing GD1a, Cerb, and m calpain also showed recognizable calpain activity at a significantly reduced Ca2+ concentration (0.4 microM), confirming the glycolipid-mediated enzyme modulation. These studies indicate that specific lipid mixtures can stimulate m calpain activity at an intracellular level of Ca2+.

Animals↗

PubMed1996

Role of calpain in spinal cord injury: increased calpain immunoreactivity in rat spinal cord after impact trauma.

Impact spinal cord injury (20 g-cm) was induced in rat by weight drop. The immunoreactivity of mcalpain was examined in the lesion and adjacent areas of the cord following trauma. Increased calpain immunoreactivity was evident in the lesion compared to control and the immunostaining intensity progressively increased after injury. The calpain immunoreactivity was also increased increased in tissue adjacent to the lesion. mCalpain immunoreactivity was significantly stronger in glial and endothelial cells, motor neurons and nerve fibers in the lesion. The calpain immunoreactivity also increased in astrocytes and microglial cells in the adjacent areas. Proliferation of microglia and astrocytes identified by GSA histochemical staining and GFAP immunostaining, respectively, was seen at one and three days after injury. Many motor neurons in the ventral horn showed increased calpain immunoreactivity and were shrunken in the lesion. These studies indicate a pivotal role for calpain and the involvement of glial cells in the tissue destruction in spinal cord injury.

Animals↗

PubMed1995

The localization of mcalpain in myelin: immunocytochemical evidence in different areas of rat brain and nerves.

A major part of brain mcalpain activity has been found associated with myelin, but its presence in the myelin sheath has not been clearly demonstrated by microscopic (morphological) means. Using myelin mcalpain antisera the localization of mcalpain has been investigated in tissue of rat CNS and PNS by immunohistochemical methods. These experiments also have been carried out by double labeling studies using antibodies to myelin basic protein (MBP) and neurofilament protein (NFP). Our results indicate calpain/MBP immunoreactivity in the myelin sheath surrounding the axon while NFP antibody stained inside the axon in spinal cord; pons, cerebellum, trigeminal nerve, and sciatic nerve. Patches of light immunoreactivity of calpain were also seen in the axonal cytoplasm. The calpain immunostaining of myelin was similar to that of MBP staining indicating the presence of calpain in myelin. This finding supports the view that calpain is a constituent of myelin, may be involved in the normal turnover of myelin proteins. In pathological situations such as in demyelinating and other brain degenerative diseases, myelin may be autodigestive.

Animals↗

PubMed1995

Calpain secreted by activated human lymphoid cells degrades myelin.

Calpain secreted by lymphoid (MOLT-3, M.R.) or monocytic (U-937, THP-1) cell lines activated with PMA and A23187 degraded myelin antigens. The degradative effect of enzymes released in the extracellular medium was tested on purified myelin basic protein and rat central nervous system myelin in vitro. The extent of protein degradation was determined by SDS-PAGE and densitometric analysis. Various proteinase inhibitors were used to determine to what extent protein degradation was mediated by calpain and/or other enzymes. Lysosomal and serine proteinase inhibitors inhibited 20-40% of the myelin-degradative activity found in the incubation media of cell lines, whereas the calcium chelator (EGTA), the calpain-specific inhibitor (calpastatin), and a monoclonal antibody to m calpain blocked myelin degradation by 60-80%. Since breakdown products of MBP generated by calpain may include fragments with antigenic epitopes, this enzyme may play an important role in the initiation of immune-mediated demyelination.

Animals↗

PubMed1995

Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain.

Blood↗

PubMed1994

Peptide bond specificity of calpain: proteolysis of human myelin basic protein.

In order to determine the peptide bond specificity of calpain, human myelin basic protein (HMBP) was treated with purified calpain of bovine brain. Upon incubation, HMBP component I (HMBP-I) was degraded into several peptides as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Component I was more susceptible to degradation than components II and III. HMBP degradation products were separated by high performance liquid chromatography (HPLC) and the cleavage sites in HMBP molecules were determined by peptide sequence analysis and by N- and C-terminal analyses. The major cleavage site was found to be 94Val-95Thr with several minor cleavages at 49Arg-50Gly, 18Ala-19Ser, 23His-24Ala, 27Gly-28Phe, 59Asp-60Ser, 70Gly-71Ser, 97Arg-98Thr, 110Ser-111Leu, 145Asp-146Ala, and 156Leu-157Gly. These results indicate that calpain is involved in the limited proteolysis of human myelin basic protein and prolonged incubation causes further digestion of the large peptides.

Amino Acid Sequence↗

PubMed1993

Calcium-activated neutral proteinase (calpain) in rat brain during development: compartmentation and role in myelination.

The activity of both forms (microM and mM Ca(2+)-sensitive) of calcium-activated neutral proteinase (calpain) was determined in developing rat brain. Triton X-100 did not affect mcalpain activity at the earlier ages (1-5 days postpartum) whereas mcalpain activity significantly increased at 16 days and older. The mcalpain activity in brain was negligible at earlier ages (1-7 days) and the peak activity occurred between 16 and 30 days after birth. The peak activity of mcalpain in myelin was found between 16 and 30 days of age and myelin from rats older than 30 days contained 40-50% of the brain mcalpain activity. In contrast, 70-80% of the brain mcalpain activity was in cytosol at younger ages (1-10 days) and decreased to 30% with increasing age (90 days). On the other hand, mu calpain was found mainly (65-75%) associated with a membrane fraction (microsomes) before 10 days and the majority of the activity was found in cytosol (68%) between 16 and 30 days. Immunoblot studies revealed mcalpain in both myelin and cytosol from developing rat brain. These results indicate that mcalpain is present in myelin and suggest that it may be involved in the formation of myelin sheath.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1993

Differential distribution of calpain in human lymphoid cells.

Calpain, a calcium-activated neutral proteinase, is ubiquitously present in human tissues. To determine if lymphoid cells implicated in pathogenesis of demyelination may harbor calpain in a functionally active form, we determined both muCalpain and mCalpain activities in human lymphoid cell lines. DEAE-cellulose and phenylsepharose column chromatography were used to isolate the enzyme from the natural inhibitor, calpastatin. Lymphocytic lines (CCRF-CEM, MOLT-3, MOLT-4, M.R.) showed predominance of muCalpain (55-80%) whereas the monocytic line (U-937) showed predominance of mCalpain (77%). Proportion and subcellular distribution of both isoforms varied among cell lines. Calpains isolated from U-937 cells degraded myelin basic protein. These results indicate that human lymphoid cells harbor functionally active calpain that can degrade myelin components in vitro. The study suggests a degradative role for calpain in demyelinating diseases.

Calcium-Binding Proteins↗

PubMed1992

Calcium-activated neutral proteinase (calpain) activity in C6 cell line: compartmentation of mu and m calpain.

Calcium-activated neutral proteinase (calpain) activity was determined, including in cytosol and membrane fractions, in rat glioma C6 cell line. The mu and m forms of calpain were separated by DEAE and phenylsepharose column chromatography and with removal of the endogenous inhibitor calpastatin. C6 cells contained more mcalpain than the mu isoform. More than 70% of mcalpain activity was membrane-associated and 20% was cytosolic. Isolated plasma membrane also contained 69% of the mcalpain activity. In contrast, approximately 80% of mucalpain activity was cytosolic and 16% was membranous. Half-maximal activity for mu and mcalpain was obtained at 1 microM and 0.2 mM CaCl2, respectively. Trypsin dissociation of cells reduced activity. Triton X-100 stimulated mcalpain activity of the whole homogenate and the membrane pellet but not of the cytosol. Activity of the myelin marker enzyme adenosine 2'3'-cyclic nucleotide 3'-phosphohydrolase (CNPase), was also found in C6 cells. The identification of calpain and CNPase in C6 cells is in keeping with an interpretation that C6 differentiation resembles, at least in part, that of the myelin-forming oligodendroglial cells.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1992

Susceptibility of myelin proteins to a neutral endoproteinase: the degradation of myelin basic protein (MBP) and P2 protein by purified bovine brain multicatalytic proteinase complex (MPC).

Multicatalytic proteinase complex (MPC) was isolated from bovine brain and the susceptibility of myelin basic protein (MBP) and P2 protein of bovine central and peripheral nervous system was examined. SDS-polyacrylamide electrophoretic analysis of purified MPC revealed protein bands of molecular weight ranging from 22-35 kDa. The enzyme is activated by SDS at a concentration less than 0.01%. Upon incubation with MPC, purified MBP and P2 proteins were degraded into smaller fragments. There was a 57% and 100% loss of MBP at 2 and 6 hours of incubation. The P2 protein which is not susceptible to any endogenous non-lysosomal enzyme thus far studied was digested into small peptide fragments only in the presence of SDS (0.01%) and not in its absence. These results indicate that MPC which is active at physiological conditions may have a role in the turnover of myelin proteins and in demyelinating diseases.

Amino Acid Sequence↗

PubMed1992

Effects of detergents on Ca(2+)-activated neural proteinase activity (calpain) in neural and non-neural tissue: a comparative study.

Calcium activated neutral proteinase (mcalpain) activity was determined in brain and other tissue of rat. More than 60% of the brain mcalpain activity was present in the particulate fraction while only 30% was in cytosol. In contrast, particulate fractions of liver, kidney, muscle, and heart contained about 8-12% of tissue mcalpain activity while 88% was present in cytosol. Removal of the endogenous inhibitor calpastatin increased the tissue mcalpain activity severalfold. Triton X-100 and deoxycholate (DOC) stimulated the neural calpain activity by ten-fold while activity in non-neural tissue was unaffected. Incubation with other detergents, e.g. Triton N-57 and thioglucopyranoside, stimulated brain calpain activity five-fold while Brij-35 did not have any effect. Sodiumdodecylsulphate (SDS), on the other hand, inhibited the enzyme activity. Brain contained the lowest calpain activity compared to non-neural tissue. The calpain activity in muscle, kidney and heart was three-fold greater than liver. Immunoblot identification of the enzyme revealed that calpain was predominantly in the particulate fraction and less in cytosol of brain while it was present mainly in cytosol and less in the pellet fractions of non-neural tissue.

Animals↗

PubMed1992

Pathogenesis of myelin breakdown in demyelinating diseases: role of proteolytic enzymes.

The mechanism by which the myelin sheath is degraded in demyelinating diseases is unknown. The demonstration of increased activities of both acid (cathepsins B, D, A) and neutral proteinases in tissue from experimental allergic encephalomyelitis (EAE) in animals and multiple sclerosis (MS, plaques) and the disappearance of myelin proteins implicate a role for proteolytic enzyme in myelin breakdown. The degradation of myelin basic protein (MBP) by proteinase yields encephalitogenic peptides and its loss has been found to cause structural alteration of the myelin sheath. This suggests that MBP degradation is an initial step in the breakdown of myelin in demyelinating diseases. A calcium-activated neutral proteinase (calpain), which degrades MBP, was found to increase in activity in MS tissue and cerebrospinal fluid (CSF), and its presence in myelin suggests that myelin may be autodigested in demyelinating disease. The source of increased proteinase activity has been indicated as macrophages, lymphocytes, and proliferative astrocytes (reactive cells). Increased proteinase activity is found in Schwann cells in Wallerian degeneration, and the presence of calpain in myelin-forming oligodendrocytes and Schwann cells suggests that these cells are likely sources of degradative enzymes. The involvement of proteolytic enzymes in the mechanism of myelin breakdown indicates the possible intervention with proteinase inhibitors for beneficial effect.

Animals↗

PubMed1991

Calcium-activated neutral proteinase (CANP; calpain) activity in Schwann cells: immunofluorescence localization and compartmentation of mu- and mCANP.

Calcium-activated neutral proteinase (CANP) activity was determined in cytosolic and membranous subcellular fractions of transformed Schwann cells (tSc). The muM and mM Ca(2+)-sensitive (mu- and mCANP) forms of CANP were separated by DEAE and phenyl Sepharose column chromatography, the latter step enabling removal of the endogenous inhibitor calpastatin. The tSc contained more muCANP than the mM isoform. More than 75% of mCANP activity was membrane-associated and 20% was cytosolic. In contrast, approximately 80% of muCANP was cytosolic and 15% was membranous. Triton X-100 stimulated activity of the whole homogenate and of the membrane pellet but did not stimulate CANP activity in the cytosolic fraction. Immunohistochemical distribution of mM enzyme was studied in both fixed and permeabilized tSc with cytosolic (anti-cyt-mCANP) and myelin (anti-my-mCANP) antibodies. Live cells (non-permeabilized) stained with anti-my-mCANP had a single filamentous ring circumscribing individual cells. Permeabilized cells treated with anti-my-mCANP had immunoreactive deposits throughout the intracellular space but sparing the perinuclear region. No immunohistochemical staining was detected when live cells were exposed to anti-cyt-mCANP whereas permeabilized cells had extensive intracellular staining with the most intense immunoreactivity in the perinuclear region. Our results indicate that both forms of CANP are present in tSc and that the activity of most of the muCANP is cytosolic while mCANP is particulate.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗