Biomedical subjects

P Ghosh

Publications and source records attributed to P Ghosh.

At least 19 recordsLinked to original sources

PubMed1997

Crystal structure of colicin Ia.

The ion-channel forming colicins A, B, E1, Ia, Ib and N all kill bacterial cells selectively by co-opting bacterial active-transport pathways and forming voltage-gated ion conducting channels across the plasma membrane of the target bacterium. The crystal structure of colicin Ia reveals a molecule 210 A long with three distinct functional domains arranged along a backbone of two extraordinarily long alpha-helices. A central domain at the bend of the hairpin-like structure mediates specific recognition and binding to an outer-membrane receptor. A second domain mediates translocation across the outer membrane via the TonB transport pathway; the TonB-box recognition element of colicin Ia is on one side of three 80 A-long helices arranged as a helical sheet. A third domain is made up of 10 alpha-helices which form a voltage-activated and voltage-gated ion conducting channel across the plasma membrane of the target cell. The two 160 A-long alpha-helices that link the receptor-binding domain to the other domains enable the colicin Ia molecule to span the periplasmic space and contact both the outer and plasma membranes simultaneously during function.

Biological Transport↗

PubMed1997

Variation in proteoglycan metabolism by articular chondrocytes in different joint regions is determined by post-natal mechanical loading.

In this study we investigated the hypothesis that cartilage from defined regions of ovine stifle joints, which were subjected to differing mechanical stresses, contained phenotypically distinct chondrocyte populations. Chondrocyte phenotypes were identified by the relative biosynthesis of the proteoglycans (PGs) aggrecan, biglycan and decorin. Articular cartilage (AC) from adult and neonatal ovine stifle joints were examined. Cells were cultured as both full-depth AC explants and in alginate beads after their isolation from the AC matrix. When chondrocytes from the various topographical regions of adult ovine knee joints were cultured as explants they demonstrated a consistent difference with regard to the metabolism of aggrecan and decorin. Significantly, this topographically-dependent phenotypic expression of PGs was preserved when the chondrocytes were cultured in alginate beads. In adult joints, chondrocytes from the central region of the tibial plateau not covered by the meniscus, which is subjected to high mechanical loads in-vivo, synthesized less aggrecan but more decorin than cells from regions covered by the meniscus. When chondrocytes from identical AC regions of neonatal ovine joints were cultured as explants, no topographical difference in aggrecan nor decorin metabolism could be detected. The results of this study, in association with the existing literature, lead us to propose that post-natal mechanical loading of AC could select for chondrocyte clones or induce a lasting modulation of chondrocyte phenotypic expression in different joint regions. Such cellular changes could result in the synthesis of PG populations that confer properties to AC most suited to resist the variable mechanical stresses in the different joint regions. This study serves to emphasize the importance of using cartilage from identical joint areas when examining PG metabolism by chondrocytes. Further investigation into the relationship between mechanical loading, regional chondrocyte phenotype selection and the response of these cells to anabolic and catabolic factors may provide important insights into the focal nature of AC degeneration in osteoarthritis.

Aggrecans↗

PubMed1996

Assembly, specific binding, and crystallization of a human TCR-alphabeta with an antigenic Tax peptide from human T lymphotropic virus type 1 and the class I MHC molecule HLA-A2.

T lymphocytes use TCR-alphabeta to bind and to recognize complexes of antigenic peptides bound to MHC proteins located at the surface of APCs. We have assembled and crystallized this intercellular complex of TCR/peptide/MHC from soluble human TCR-alphabeta and soluble peptide/HLA-A2 complexes. The soluble TCR-alphabeta binds specifically to its in vivo ligand, the complex of HLA-A2, and a peptide from the Tax protein of human T lymphotropic virus type 1. The soluble TCR also binds in vitro to an altered peptide ligand, which appears to be a partial agonist in T cell assays as determined by its ability to elicit different cytolytic and lymphokine secretion responses. Heterodimerization and the antigenic specificity of the TCR do not require its interchain disulfide bond, transmembrane segments, or glycosylations. Crystals of the TCR/peptide/HLA-A2 complex diffract x-rays, providing the means to study in atomic detail the mechanism of Ag-specific cell-cell recognition between T cells and target cells.

Amino Acid Sequence↗

PubMed1996

Association of human fas (CD95) with a ubiquitin-conjugating enzyme (UBC-FAP).

A novel human ubiquitin conjugating enzyme (UBC) was found to associate with Fas (CD95). The mRNA for this UBC Fas-associated protein (FAP) was widely expressed in human tissues, and the protein was identified in several mammalian cell lines. UBC-FAP shows strong homology to two recently identified UBCs, Hus5 and Ubc9, which control yeast cell cycle progression. UBC-FAP, but not an active site mutant, complemented ubc9-1(ts) mutants. This suggests that UBC-FAP is a human homologue of Ubc9, possesses ubiquitin conjugating activity, and may play an important role in mammalian cell cycle regulation. A single amino acid substitution in the death domain of Fas that abolishes Fas-mediated apoptosis also abolished Fas association with UBC-FAP, suggesting that UBC-FAP may play a role in Fas signal transduction. The sequence of UBC-FAP is identical to that of HsUbc9, a UBC recently shown to interact with Rad51.

Animals↗

PubMed1996

Structure of the complex between human T-cell receptor, viral peptide and HLA-A2.

Recognition by a T-cell antigen receptor (TCR) of peptide complexed with a major histocompatibility complex (MHC) molecule occurs through variable loops in the TCR structure which bury almost all the available peptide and a much larger area of the MHC molecule. The TCR fits diagonally across the MHC peptide-binding site in a surface feature common to all class I and class II MHC molecules, providing evidence that the nature of binding is general. A broadly applicable binding mode has implications for the mechanism of repertoire selection and the magnitude of alloreactions.

Crystallography, X-Ray↗

PubMed1996

Retinoic acid-induced transcriptional modulation of the human interferon-gamma promoter.

Disregulation of vitamin A metabolism is able to generate different immunological effects, including altered response to infection, reduced IgG production, and differential regulation of cytokine gene expression (including interleukin-2 and -4 and interferon-gamma (IFN-gamma)). In particular, IFN-gamma gene expression is significantly affected by vitamin A and/or its derivatives (e.g. retinoic acid (RA)). Here, we analyze the effect of retinoic acid on IFN-gamma transcription. Transient transfection assays in the human T lymphoblastoid cell line Jurkat demonstrated that the activation of the IFN-gamma promoter was significantly down-regulated in the presence of RA. Surprisingly, two different AP-1/CREB-ATF-binding elements situated in the initial 108 base pairs of the IFN-gamma promoter and previously shown to be critical for transcriptional activity were unaffected by RA. Utilizing promoter deletions and electrophoretic mobility shift analysis, we identified a USF/EGR-1-binding element cooperating in the modulation of IFN-gamma promoter activity by RA. This element was found to be situated in a position of the IFN-gamma promoter close to a silencer element previously identified in our laboratory. These results suggest that direct modulation of IFN-gamma promoter activity is one of the possible mechanisms involved in the inhibitory effect of retinoids on IFN-gamma gene expression.

Humans↗

PubMed1996

Using antibodies to perturb the coordination sphere of a transition metal complex.

Metal ions in the active sites of many metalloenzymes exhibit distinctive spectral and chemical features which are different from those of small inorganic complexes. These features are the result of the unusual geometric and electronic constraints that are imposed on the metal ion within the protein environment. Much effort has been invested to try to mimic this feature of metalloenzymes in synthetic systems, but this remains a formidable task. Here we show that one of the key lessons learned from the science of catalytic antibodies--that binding energy can be converted into chemical energy--can be exploited to 'fine-tune' the physicochemical properties of a metal complex. We show that an antibody's binding site can reversibly perturb the coordination geometry of a metal ion, and can stabilize a high-energy coordinated species. Specifically, antibodies designed to bind the organosilicon compound 1 also bind the geometrically similar Cu(I) complex 2. However, the antibody binds a slightly compressed form of 2, which is closer in size to 1. This distortion is manifested by a spectral shift--an 'immunochromic' effect.

Animals↗

PubMed1996

Activation of nuclear factor of activated T cells in a cyclosporin A-resistant pathway.

The mechanism of action of the immunosuppressive drug cyclosporin A (CsA) is the inactivation of the Ca2+/calmodulin-dependent serine-threonine phosphatase calcineurin by the drug-immunophilin complex. Inactive calcineurin is unable to activate the nuclear factor of activated T cells (NFAT), a transcription factor required for expression of the interleukin 2 (IL-2) gene. IL-2 production by CsA-treated cells is therefore dramatically reduced. We demonstrate here, however, that NFAT can be activated, and significant levels of IL-2 can be produced by the CsA-resistant CD28-signaling pathway. In transient transfection assays, both multicopy NFAT- and IL-2 promoter-beta-galactosidase reporter gene constructs could be activated by phorbol 12-myristate 13-acetate (PMA)/alpha-CD28 stimulation, and this activation was resistant to CsA. Electrophoretic mobility shift assay showed the induction of a CsA-resistant NFAT complex in the nuclear extracts of peripheral blood T cells stimulated with PMA plus alphaCD28. Peripheral blood T cells stimulated with PMA/alphaCD28 produced IL-2 in the presence of CsA. Collectively, these data suggest that NFAT can be activated and IL-2 can be produced in a calcineurin independent manner.

Base Sequence↗

PubMed1996

Biotinylated hyaluronan: a versatile and highly sensitive probe capable of detecting nanogram levels of hyaluronan binding proteins (hyaladherins) on electroblots by a novel affinity detection procedure.

Hyaluronan influences cellular proliferation and migration in developing, regenerating and remodelling tissues and in tissues undergoing malignant tumour-cell invasion. The widespread occurrence of hyaluronan-binding proteins indicates that the recognition of hyaluronan is important to tissue organisation and the control of cellular behaviour. A number of extracellular matrix and cellular proteins, which have been termed the hyaladherins, have specific affinities for hyaluronan. These include cartilage link-protein, hyaluronectin, neurocan, versican and aggrecan, which all bind to HA within the extracellular matrix. Cellular receptors for hyaluronan such as CD44 and RHAMM (receptor for hyaluronate-mediated motility) have also been identified. In the present study biotinylated hyaluronan (bHA) was prepared by reacting adipic dihydrazide with a 170 kDa hyaluronan sample using the bifunctional reagent 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide. The resultant free amine moeity of the hydrazido-hyaluronan was then reacted with biotin succinimidyl ester (sulfo-NHS-biotin) to prepare the bHA. After 4-20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotting to nitrocellulose membranes, bHA and avidin alkaline phosphatase conjugate could be used in conjunction with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrates to specifically visualise with high sensitivity (> or = 2 ng), bovine nasal cartilage link-protein, aggrecan hyaluronan binding region, and human fibroblast hyaluronan receptors such as CD-44. Conventional Western blotting using specific monoclonal antibodies to these proteins was also used to confirm the identities of these proteins.

Aggrecans↗

PubMed1996

Biotinylated trypsin and its application as a sensitive, versatile probe for the detection and characterisation of an ovine chondrocyte serine proteinase inhibitor using Western blotting.

Biotinylated trypsin (bT) was used as a probe on Western blots of 10-20% polyacrylamide gradient Tris-Tricine gels for the detection of serine proteinase inhibitors (SPIs) isolated from extracts of ovine articular cartilage and from chondrocyte conditioned culture medium. The major cartilage SPI, a 58 kDa glycoprotein, was purified by sequential Sephacryl S--300 gel permeation, concanavalin A affinity, anion exchange and Superose 12 fast protein liquid chromatography (FPLC). A low molecular weight SPI of approximately 6 kDa was also detectable in cartilage extracts after prolonged storage at 4 degrees C and following affinity chromatography on immobilised chymotrypsin, suggesting that the 6 kDa inhibitor may have arisen from proteolytic modification of the 58 kDa SPI. Analysis of chondrocyte conditioned culture medium using the bT detection system revealed that the major SPI present was the 6 kDa species although a small amount of the 58 kDa SPI was also detectable. The large molecular weight 'native' cartilage SPI was distinct from ovine alpha1-proteinase inhibitor (alpha1-PI) when examined by native polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF). The 6 kDa cartilage SPI was indistinguishable from basic pancreatic trypsin inhibitor (BPTI, aprotinin) following sodium dodecyl sulfate (SDS)-PAGE under reduced and nonreduced conditions. Amino terminal sequencing of the 6 kDa cartilage SPI revealed a strong (90%) homology with BPTL.

Amino Acid Sequence↗

PubMed1996

Topographic variation in biglycan and decorin synthesis by articular cartilage in the early stages of osteoarthritis: an experimental study in sheep.

Articular cartilage contains large molecular weight proteoglycans that aggregate with hyaluronic acid (aggrecan) and small species, particularly biglycan (dermatan sulphate proteoglycan-1) and decorin (dermatan sulphate proteoglycan-2), that do not. Mechanical stresses have been shown to profoundly influence the metabolism of aggrecan by articular chondrocytes; however, there are limited corresponding data on the metabolism of dermatan sulphate proteoglycans 1 and 2. The objective of this study was to examine the metabolism of aggrecan, biglycan, and decorin in articular cartilage from different weight-bearing areas of normal ovine stifle joints and in joints 6 months after menisectomy, a procedure that has been shown to induce early osteorthritic changes. [35S]proteoglycans synthesised by cartilage explants from eight different weight-bearing regions of unoperated and meniscectomised ovine stifle joints during 48 hours of culture were separated by size-exclusion chromatography, hydrophobic chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were quantitated by phosphor-screen autoradiography. The synthesis and degradation of the proteoglycans were expressed relative to the DNA content of the explants. In control joints, the cartilage exposed to high contact stress synthesised significantly less proteoglycan overall and more decorin than joint regions bearing less stress. Explants from high stress regions also released significantly greater amounts of resident proteoglycans (dimethylmethylene blue positive) into media during culture. After lateral meniscectomy, the lateral tibial and femoral cartilages showed elevated biosynthesis of both 35S-dermatan sulphate proteoglycans 1 and 2. This chondrocyte biosynthetic response was accompanied by increased catabolism of aggrecan and the release of its degradation products into culture media. These experiments revealed, in normal joints, a topographic variation in proteoglycan synthesis by articular cartilage that was related to the mechanical stress to which the tissues were subjected in vivo. This biosynthetic pattern changed when the load distribution of the joint was altered by unilateral meniscectomy. These data suggest that an altered chondrocyte phenotypic expression of proteoglycans in response to abnormal mechanical loading is an early event in osteoarthritis.

Aggrecans↗

PubMed1996

Increased synthesis of matrix metalloproteinases by aortic smooth muscle cells is implicated in the etiopathogenesis of abdominal aortic aneurysms.

PURPOSE: The objective of this study was to identify the metalloproteinases elaborated by medial smooth muscle cells (SMCs) isolated from abdominal aortic aneurysm (AAA) and control arterial tissues and to ascertain if the levels produced by AAA SMCs were elevated. METHODS: SMC monolayers cultured from the outgrowth cells of tunica media explants were established, and their identity was determined by fluorescent microscopy by using a fluorescein isothiocyanate conjugated anti-SMC alpha-actin antibody. Matrix metalloproteinases (MMPs) produced by SMC monolayers in serum-free culture were examined by gelatin zymography and Western blotting with monoclonal antibodies to MMP-2, 3, and 9. RESULTS: Serum-free media from AAA SMCs contained metal-dependent elastolytic activity that cleaved the synthetic substrate succinyl trialanyl 4-nitroanilide (pH optima 7.2) and also 14C-insoluble elastin. The level of proteolytic activity found in these cultures was significantly greater than from control SMC media. Zymography established that AAA SMC media samples contained metal-dependent gelatinases of 50 to 64 and 92 kDa, which were identified respectively as MMP-2 and 9 by Western blotting by using monoclonal antibodies to these proteases. CONCLUSION: Medial SMCs isolated from AAA tissue produce significantly higher levels of MMP-9 and 2 than SMCs from control arterial tissues. These proteinases have the capacity to degrade elastin and a range of extracellular matrix proteins. From these data, we suggest SMCs may be involved in the abnormal degradation of the aortic wall in AAA through the excessive metalloproteinase activity produced by SMCs.

Adult↗

PubMed1996

Interactions of pentosan polysulfate with cartilage matrix proteins and synovial fibroblasts derived from patients with osteoarthritis.

Pentosan polysulfate (PPS) has been shown to improve symptoms of patients with osteoarthritis (OA) when studied under double-blinded conditions. Laboratory studies indicated that this drug exhibits multiple actions, including the preservation of articular cartilage (AC) proteoglycans in animal models of OA and the stimulation of hyaluronan synthesis by synovial fibroblasts in vitro and in vivo. As PPS is strongly anionic and has a molecular weight of approximately 5700 Da its ability to enter connective tissues rich in proteoglycans and interact with the resident cells has been questioned. In the present studies, experiments were undertaken to isolate and characterize proteins in human AC which have the potential to bind PPS. Thrombospondin was identified in 4.0 M GuHCl extracts of human AC as a PPS-binding protein. Furthermore, synovial fibroblasts derived from OA joints were shown to secrete thrombospondin and also bind PPS. Using bovine erythrocytes conjugated with PPS a rosetting of the synovial fibroblast could be demonstrated. The level of rosetting was not affected by pre-incubating cultures with thrombospondin antibody suggesting that PPS was interacting directly with the cells. Kinetic studies of 3H-PPS uptake by synovial fibroblasts showed saturation of binding sites within 30 min when cells were maintained at 4 degrees C but preservation of drug uptake for up to 120 min when cells were cultured at 37 degrees C. These data, together with the finding that cells labeled with drug at 37 degrees C showed higher incorporation, than at 4 degrees C after trypsin digestion suggests that PPS first binds to the cell membrane when at 37 degrees C is internalized, possibly by pinocytosis.

Aged↗

PubMed1996

Smooth muscle cell migration and proliferation is enhanced in abdominal aortic aneurysms.

BACKGROUND: The aetiology of abdominal aortic aneurysms (AAA) is as yet undetermined. Smooth muscle cells (SMC) have been implicated in the pathogenesis of AAA as a result of their ability to produce elastin degrading proteases. The present study was undertaken to examine AAA SMC and aortic occlusive disease (AOD) SMC in terms of their respective migration and proliferation in vitro, in order to identify intrinsic differences between these cells. METHODS: Five AAA specimens, four AOD and five inferior mesenteric artery (IMA) specimens were established in culture. The cultures were examined for the extent and the rate of SMC outgrowth and proliferation. Cells were counted following trypsinization using a haemocytometer. RESULTS: For the AAA explants, the cellular outgrowths were first seen at 6.7 days, after culture initiation, while the corresponding outgrowth in the AOD group required 8.8 days (P < 0.05) and the IMA group 11.4 days (P < 0.05). AAA cells reached confluency at a mean of 22.4 days while AOD SMC required 28.6 days (P < 0.05) and IMA 31 days (P < 0.05). In the first passage, the time for AAA SMC doubling was 5.3 days compared to 6.2 days for AOD (P < 0.05) and 8.1 days for the IMA group (P < 0.05). Greater than 98% of the cells, in both groups, stained positive to SMC alpha-actin. CONCLUSION: From these data it is clear that there are intrinsic differences in cellular kinetics between SMC from the two disease states, supporting the hypothesis that AAA are not the result of atherosclerosis.

Aged↗

PubMed1996

Variation in intervertebral disc serine proteinase inhibitory proteins with ageing in a chondrodystrophoid (beagle) and a non-chondrodystrophoid (greyhound) canine breed.

Intervertebral disc (IVD) proteoglycans (PGs) and trypsin inhibitory protein levels were assessed for groups of beagles and greyhounds aged between 4 and 80 months. A significant age-dependent decline in PGs and in trypsin inhibitory protein levels was evident in extracts of the beagle nucleus pulposus (NP). This was confirmed for the semi-purified trypsin inhibitor, using Western blotting. These age changes, however, were not evident in the greyhound NP. Furthermore, no significant ageing trends in PG or trypsin inhibitory protein levels were evident in extracts of the annulus fibrosus of either animal. The early onset of IVD degeneration in the beagle, a canine breed of the chondrodystrophoid (achondroplastic) classification, may be related to this decline in the anti-catabolic effects of the endogenous serine proteinase inhibitory proteins.

Aging↗

PubMed1996

Purification and characterisation of 6 and 58 kDa forms of the endogenous serine proteinase inhibitory proteins of ovine articular cartilage.

The major ovine articular cartilage (AC) serine proteinase inhibitory protein (SPI), a 58 kDa glycoprotein (SPI-58), was purified to homogeneity by sequential Sephacryl S-300 gel permeation, concanavalin A affinity, Mono Q anion exchange and Superose 12 FPLC. If precautions to prevent degradation of the native 58 kDa SPI were not undertaken during the early stages of its purification a SPI of approximately 6 kDa (SPI-6) was generated. SPI-6 could also be generated from SPI-58 by chymotrypsin affinity chromatography, suggesting that SPI-6 could be produced from SPI-58 in vivo by proteolytic processing within the tissue. SPI-6 was indistinguishable from the Kunitz inhibitor, bovine pancreatic trypsin inhibitor (BPTI) by SDS-PAGE under both reducing and non reducing conditions and showed a strong homology to BPTI in N-terminal sequence. These data suggest that the BPTI-like 6 kDa SPI constituted the inhibitory domain of the native 58 kDa SPI of ovine AC. Detection of [14C]-lysine-SPI-6 and SPI-58 in the serum free culture medium from ovine chondrocytes cultured in alginate beads in the presence of [14C]-lysine indicated that these SPIs were chondrocyte biosynthetic products. The inhibitory profiles of SPI-58 and SPI-6 differed somewhat suggesting that each may have an independent role in vivo.

Animals↗

PubMed1995

The structure of an intermediate in class II MHC maturation: CLIP bound to HLA-DR3.

A complex between HLA-DR3 and a fragment of invariant chain called CLIP was isolated from a human cell line defective in antigen presentation and its X-ray crystal structure determined. Previous data indicate that this complex is an intermediate in class II histocompatibility maturation, occurring between invariant chain-DR3 and antigenic peptide-DR3 complexes. The structure shows that the CLIP fragment binds to DR3 in a way almost identical to that in which antigenic peptides bind class II histocompatibility glycoproteins. The structure is the substrate for the loading of antigenic peptides by an exchange process catalysed by DM.

Amino Acid Sequence↗

PubMed1995

Metabotropic glutamate receptor mGluR5 subcellular distribution and developmental expression in hypothalamus.

The metabotropic glutamate receptor mGluR5 is a G-protein coupled receptor that plays a key role in release of Ca2+ from internal stores via inositol triphosphate mobilization. Western and Northern blot analyses revealed a greatly enhanced expression of mGluR5 in rats during early stages of hypothalamic development compared with the adult. This enhanced developmental expression provides an explanation for the dramatic physiological response of developing neurons to metabotropic glutamate receptor activation and supports the argument that metabotropic glutamate receptors may play an important role in hypothalamic development. During development, expression of the mGluR5 gene was reduced, not only in the hypothalamus but also in other regions of the brain. A differential decrease in mGluR5 protein was found in different brain regions with Western blot analysis. The hypothalamus showed a sixfold decrease in mGluR5 with development, whereas the cortex showed only a threefold decrease. Immunocytochemistry with an affinity-purified antibody against a peptide deduced from the cloned mGluR5 gene revealed selective expression in some regions in the adult hypothalamus. In the adult and developing (postnatal day 10) brain, immunoreactive neurons were found in the suprachiasmatic nucleus, preoptic area, lateral hypothalamus, and mammillary region, areas where the related metabotropic glutamate receptor mGluR1 is also found. In contrast, the ventromedial nucleus, an area critically involved in the regulation of food intake and metabolic balances, showed strong mGluR5 immunoreactivity but no mGluR1 immunoreactivity. Little or no mGluR5 staining was found in the neurosecretory neurons of the paraventricular, supraoptic, and arcuate nuclei. Ultrastructurally, mGluR5 was associated with the cytoplasmic face of the plasmalemma on hypothalamic dendrites, dendritic spines, and neuronal perikarya in the adult. The strongest immunoreactivity was found in patches on the membrane, sometimes associated with the postsynaptic side of synapses and sometimes associated with nonsynaptic dendritic or perikaryal membrane. Intense immunostaining was found on some astrocyte processes surrounding synaptic complexes containing asymmetrical synapses. These astrocytes would be in an ideal position to receive excitatory signals from glutamatergic axons. Unlike the punctate appearance of immunolabeling on neuronal membranes, astrocytes showed continuous staining along the plasma membrane.

Animals↗