A protein of 175,000 daltons associated with striated rootlets in ciliated epithelia, as revealed by a monoclonal antibody

Basal bodies from laying quail oviduct were semipurified and used as immunogen to produce monoclonal antibodies. On 38 clones obtained and among those staining the apical pole of the ciliated cell, CC-310 was chosen because it labeled the apical region with a punctuated aspect, suggesting a staining of basal bodies or of basal body-associated structures; the basal pole was also labeled. The ultrastructural localization performed by the immunogold technique showed that the labeling was mainly associated with the striated rootlets. The basal feet, the side of the basal bodies, and the basal poles of the demembranated cells were also decorated. The identification of the antigen performed by immunoblots of deciliated cortices revealed two proteins of 175,000 and 40,000, whereas immunoblots of basal bodies showed only the 175,000-mw protein. The possibility of these two proteins sharing the same epitope, located at both poles of the cell, is discussed. Immunofluorescence ascertained that CC-310 decorated the striated rootlets in ciliated epithelia from other species: mussel, frog, and human tissue. Finally, when tested on cultured cell lines, CC-310 labeled the centrosome and its associated rootlets on PtK2 during interphase. During mitosis the poles of the mitotic spindle were stained without any apparent rootlet-like structure.     

Myosin at the apical pole of ciliated epithelial cells as revealed by a monoclonal antibody

A monoclonal antibody (CC-212), obtained in a fusion experiment in which basal bodies from quail oviduct were used as immunogen, has been shown to label the apical pole of ciliated cells and to react with a 200-kD protein. This monoclonal antibody was demonstrated to be an anti-myosin from smooth muscle or from nonmuscular cells using the following criteria: On Western blots it reacted with the myosin heavy chains from gizzard and platelet extracts and from cultured cell line extracts, but did not react with striated muscle myosin heavy chains. By immunofluorescence it decorated the stress fibers of well-spread cells with a characteristic striated pattern, while it did not react with myotubes containing organized myofibrils. On native ciliated cells as well as on Triton-extracted ciliated cortices from quail oviduct, this monoclonal antibody decorated the apical pole with a stronger labeling of the periphery of the apical area. Ultrastructural localization was attempted using the immunogold technique on the same preparation. Myosin was associated with a filamentous material present between striated rootlets and the proximal extremities of the basal bodies. No labeling of the basal body itself or of axoneme was observed.     

Immunocytochemical staining of breast carcinoma with the monoclonal antibody NCRC 11: a new prognostic indicator.

The staining of breast cancer with a new monoclonal antibody, NCRC 11, was studied in a series of 126 women with primary breast carcinoma. Tumour samples embedded in paraffin were tested, and the minimum duration of follow up was five years or to death. Altogether 119 tumours stained positively. There was a strong relation between the intensity of staining, divided on a four point scale, and patient survival. Patients whose tumours exhibited intense staining had an improved survival compared with those with less intensely staining tumours (p less than 0.0001). Staining related weakly to histological grade but not significantly to oestrogen receptor state or the pathological stage of lymph node disease. Mathematical analysis showed the relation to survival to be independent of the other known prognostic factors. Inclusion of intensity of staining with other factors in a prognostic index might permit a more accurate estimation of prognosis in patients with breast cancer.     

The anatomy of the nervous system of the hydrozoan jellyfish,Polyorchis penicillatus,as revealed by a monoclonal antibody

Dissociated cells from the margin and tentacles of the hydromedusa Polyorchis penicillatus were centrifuged in a Percoll gradient to remove cnidocytes. The resulting formaldehyde-fixed cells were used to inoculate mice to produce monoclonal antibodies. One of the hybridomas, which secreted antibodies against all neurons, was cloned and designated as mAb 5C6. Immunohistochemical labelling with mAb 5C6 of whole-mount preparations and paraffin sections provided a far more complete picture of the organisation of the hydromedusan nervous system than was previously available when using neuronal labelling techniques that restrict labelling to certain neuronal types. Besides confirming anatomical features described in earlier studies these techniques allowed us to discover a number of new structures and to determine connections that were only suspected. Such findings included: 1.The discovery of an arch-like connection between the swimming motor neuron network at the apices of the subumbrellar muscle sheets 2.An orthogonal network connecting each pair of radial nerves in each radius 3.Continuity of a central branch of the radial nerve with the radial innervation of the manubrium 4.Details of the sensory neuronal contribution to the microanatomy of the ocelli and cnidocyte batteries 5.Presence of specialised receptor cells in the margin at the bases of tentacles 6.Neurons apparently innervating the radial muscles of the velum 7.Isolated neurons in the peduncle and gonads Electronic Publication     

Motility-dependence of the heterogenous staining of culture cells by a monoclonal anti-tropomyosin antibody

A monoclonal antibody (CG1) which recognizes tropomyosin isoforms 1 and 3 of chicken embryo fibroblasts was used to detect what is a motility-dependent change in the availability of the antigenic determinant in tropomyosin molecules along microfilaments. Immunofluorescence microscopy with this antibody revealed a heterogenous staining pattern among chicken embryo fibroblasts cells such that a population (17%) of cells showed only background staining. Stress fibers in about half the population of the cells stained weakly with this antibody, while the stress fibers in another population of cells (35%) showed very strong staining. After glycerination or cytochalasin B treatment, all of the cells became positive in reaction to CG1 antibody, suggesting that the antigenic determinant was present in every cell. On the other hand, all of the cells after brief nonionic detergent treatment became negative to CG1 antibody. The CG1 staining pattern was not significantly changed in cells at different stages after release from colcemid blockage, nor was a brief treatment of cells with buffer containing 2 M urea, mild trypsin, chymotrypsin, or V.8 protease effective in changing the reactivity. However, most of the cells with a morphology typical of movement, and all of the contracted, glycerinated cells were strongly positive to CG1 antibody. These results suggest that the unmasking of the CG1 determinant may be motility-dependent. Immunoblot analysis showed that forced modification on the cysteine residue of tropomyosin molecules, caused either by performic acid oxidation or by disulfide cross-linking with the chemical 5,5'-dithiobis (2-nitrobenzoate), results in drastic changes in the reactivity of the different isoforms to CG1 antibody. These results indicate that the cysteine residue is involved in the CG1 determinant. The motility-dependent unmasking of this determinant may suggest an important role for nonmuscle tropomyosin in regulating cell motility.     

Novel polysaccharide antigen of Orientia tsutsugamushi revealed by a monoclonal antibody

Orientia tsutsugamushi , the causative agent of scrub typhus, is an obligate intracellular bacterium that replicates in the cytosol of host cells. Although several protein antigens have been characterized and cloned, little information exists regarding the polysaccharide antigen of this bacterium. In this study, we identified and characterized a novel antigen defined by a monoclonal antibody (MAb), NT19, against O. tsutsugamushi . Immunofluorescence microscopic studies showed that the NT19 antigen is released from the bacteria in the cytosol of host cells forming aggregates with bacteria. Immunoblot analysis showed that MAb NT19 recognized a strong band with a molecular mass of 20 kDa that was resistant to proteinase K digestion and sensitive to periodate oxidation, suggesting that the NT19 antigen is a polysaccharide. The function of this polysaccharide is not known, but considering its distribution within a bacterial microcolony, it is suspected to be involved in forming a biofilm-like structure within host cells.     

Specific distribution of an epididymal 50 kDa protein revealed by an anti-Mos protein monoclonal antibody.

An anti-Mos protein monoclonal antibody, 4A6, was used to investigate the distribution of the antigen in the epididymis, in which the c-mos gene is reportedly expressed. The 4A6-reactive antigen was found on the basement membrane and luminal surface of the epithelial cells in the caput epididymis of BALB/c male mice as well as in the proximal corpus epididymis, the cauda epididymis, and the vas deferens. The 4A6 antigen was also found on the luminal surface of the epithelial cells in the epididymis of male germ cell-deficient C57BL/6J-Wv/Wv mice. This confirmed that the 4A6 antigen does not derive entirely from the testicular c-Mos protein but is synthesized in the epididymis. Western blot analysis revealed that the molecular weight of the epididymal 4A6 antigen was 50 kDa, which is unusually high for the c-Mos protein. With its specific distribution in the epididymis, the protein should play a specific role in functions of the epididymis.     

Glutamate-like immunoreactivity revealed in rat olfactory bulb, hippocampus and cerebellum by monoclonal antibody and sensitive staining method

Although there is good evidence favoring L-glutamate as a major excitatory amino acid transmitter, relatively little is known about the distribution of nerve terminals using this substance. A method visualizing glutamate-like immunoreactivity at the light microscopic level by means of a monoclonal antibody, mAb 2D7, is described. --The antigen used for immunization was a glutaraldehyde-linked glutamate-BSA conjugate, and hybridomas were differentially screened by ELISA for production of antibodies recognizing glutamate- but not aspartate-BSA. The crossreactivity of 'anti-glutamate' mAb 2D7 as estimated in absorption tests was low even with conjugates closely related to glutamate-BSA.--Semithin sections from rapidly perfusion-fixed, plastic-embedded rat brain tissues were etched and stained by a combination of the peroxidase-antiperoxidase method and silver enhancement of the diaminobenzidine reaction product. Only this amongst several other immunohistochemical methods tried produced labeling patterns which showed terminal-like elements in brain regions such as olfactory bulb, hippocampus and cerebellum, and which were mostly consistent with already available information on systems using glutamate as neurotransmitter. Particularly striking was the staining of elements reminiscent of mossy fiber terminals in hippocampus and cerebellum as well as of cerebellar parallel fiber terminals.     

Glutamate-like immunoreactivity revealed in rat olfactory bulb,hippocampus and cerebellum by monoclonal antibody and sensitive staining method

Summary Although there is good evidence favoring l-glutamate as a major excitatory amino acid transmitter, relatively little is known about the distribution of nerve terminals using this substance. A method visualizing glutamate-like immunoreactivity at the light microscopic level by means of a monoclonal antibody, mAb 2D7, is described. — The antigen used for immunization was a glutaraldehyde-linked glutamate-BSA conjugate, and hybridomas were differentially screened by ELISA for production of antibodies recognizing glutamate- but not aspartate-BSA. The cross-reactivity of anti-glutamate mAb 2D7 as estimated in absorption tests was low even with conjugates closely related to glutamate-BSA. — Semithin sections from rapidly perfusion-fixed, plastic-embedded rat brain tissues were etched and stained by a combination of the peroxidase-antiperoxidase method and silver enhancement of the diaminobenzidine reaction product. Only this amongst several other immunohistochemical methods tried produced labeling patterns which showed terminal-like elements in brain regions such as olfactory bulb, hippocampus and cerebellum, and which were mostly consistent with already available information on systems using glutamate as neurotransmitter. Particularly striking was the staining of elements reminiscent of mossy fiber terminals in hippocampus and cerebellum as well as of cerebellar parallel fiber terminals.     

Detection of adenocarcinoma in peritoneal washings by staining with monoclonal antibody B72.3

Peritoneal washings are routinely performed in the staging evaluation of carcinomas of the ovary and in "second-look" explorations; major problems in the evaluation of these specimens continue to be the distinction between atypical mesothelium and adenocarcinoma and the identification in an otherwise inflammatory specimen of rare cells of adenocarcinoma, which may be undetected by the most trained individual. Monoclonal antibody (MAb) B72.3, reactive with an oncofetal, tumor-associated glycoprotein (termed TAG-72; MW greater than 1000 kd) expressed in a variety of epithelial malignancies but not generally expressed in benign or malignant mesothelium, was reacted with sections from the paraffin-embedded cell blocks of 185 peritoneal washings from 180 patients with extant cancer or a prior history of malignancy. One hundred four of the washings were initially interpreted as atypical mesothelium, with no evidence of malignancy; when reacted with MAb B72.3, 6 of these specimens demonstrated groups of metastatic adenocarcinoma cells not appreciated by the usual cytologic criteria. Of the 81 washings interpreted as showing cells of adenocarcinoma, 73 demonstrated expression of TAG-72 from both gynecologic and nongynecologic malignancies. In the remaining 49 cases without an associated malignant process, MAb B72.3 did not stain atypical mesothelium, but did react, however, with benign endometrial cells and müllerian inclusions in two cases. MAb B72.3 may be used as a diagnostic adjunct to the routine cytologic evaluation of malignancy in peritoneal washings; reactivity with MAb B72.3 may indicate the need for further evaluation to define the presence of a malignancy or an advanced cancer.     

Perisynaptic Schwann cells at neuromuscular junctions revealed by a novel monoclonal antibody

This study aimed to generate a probe for perisynaptic Schwann cells (PSCs) to investigate the emerging role of these synapse-associated glial cells in the formation and maintenance of the neuromuscular junction (NMJ). We have obtained a novel monoclonal antibody, 2A12, which labels the external surface of PSC membranes at the frog NMJ. The antibody reveals PSC fine processes or “fingers” that are interposed between nerve terminal and muscle membrane, interdigitating with bands of acetylcholine receptors. This antibody also labels PSCs at the avian neuromuscular junction and recognizes a 200 kDa protein in Torpedo electric organs. In frog muscles, axotomy induces sprouting of PSC processes beyond clusters of acetylcholine receptors and acetylcholinesterase at denervated junctional branches. PSC branches often extend across several muscle fibers. At some junctions, PSC sprouts join the tips of neighboring branches. The average length of PSC sprouts is approximately 156 µ at 3-week denervated NMJs. PSC sprouting is accompanied by a significant increase in the number of Schwann cell bodies per NMJ. Following nerve regeneration, nerve terminals reinnervate the junction along the PSC processes. In vivo observations of normal frog muscles also show PSC processes longer than nerve terminals at some junctional branches. The results suggest that nerve injury induces profuse PSC sprouting that may play a role in guiding nerve terminal regeneration at frog NMJs. In addition, antibody 2A12 reveals the fine morphology of PSCs in relation to other synaptic elements and is a useful probe in elucidating the function of these synapse-associated glial cells in vivo.     

Domain unfolding of monoclonal antibody fragments revealed by non-reducing SDS-PAGE

Monoclonal antibodies and derived fragments are used extensively both experimentally and therapeutically. Thorough characterization of such antibodies is necessary and includes assessment of their thermal and storage stabilities. Thus, assessment of the underlying conformational stabilities of the antibodies is also important. We recently documented that non-reducing SDS-PAGE can be used to assess both monoclonal and polyclonal IgG domain thermal unfolding in SDS. Utilizing this same h2E2 anti-cocaine mAb, in this study we generated and analyzed various mAb antibody fragments to delineate the structural domains of the antibody responsible for the observed discrete bands following various heating protocols and analysis by non-reducing SDS-PAGE. Previously, these domain unfolding transitions and gel bands were hypothesized to stem from known mAb structural domains based on the relative thermal stability of those CH2, CH3, and Fab domains in the absence of SDS, as measured by differential scanning calorimetry. In this study, we generated and analyzed F(ab’)₂, Fab, and Fc fragments, as well as a mAb consisting of only heavy chains, and examined the thermally induced domain unfolding in each of these fragments by non-reducing SDS-PAGE. The results were interpreted and integrated to generate an improved model of thermal unfolding for the mAb IgG in SDS. These results and the model presented should be generally applicable to many monoclonal and polyclonal antibodies and allow novel comparisons of conformational stabilities between chemically or genetically modified versions of a given antibody. Such modified antibodies and antibody drug conjugates are commonly utilized and important for experimental and therapeutic applications.     

The sulfate-binding site structure of the human eosinophil cationic protein as revealed by a new crystal form

The human eosinophil cationic protein (ECP), also known as RNase 3, is an eosinophil secretion protein that is involved in innate immunity and displays antipathogen and proinflammatory activities. ECP has a high binding affinity for heterosaccharides, such as bacterial lipopolysaccharides and heparan sulfate found in the glycocalix of eukaryotic cells. We have crystallized ECP in complex with sulfate anions in a new monoclinic crystal form. In this form, the active site groove is exposed, providing an alternative model for ligand binding studies. By exploring the protein-sulfate complex, we have defined the sulfate binding site architecture. Three main sites (S1-S3) are located in the protein active site; S1 and S2 overlap with the phosphate binding sites involved in RNase nucleotide recognition. A new site (S3) that is unique to ECP is one of the key anchoring points for sulfated ligands. Arg 1 and Arg 7 in S3, together with Arg 34 and Arg 36 in S1, form the main basic clusters that assist in the recognition of ligand anionic groups. The location of additional sulfate bound molecules, some of which contribute to the crystal packing, may mimic the binding to extended anionic polymers. In conclusion, the structural data define a binding pattern for the recognition of sulfated molecules that can modulate the role of ECP in innate immunity. The results reveal the structural basis for the high affinity of ECP for glycosaminoglycans and can assist in structure-based drug design of inhibitors of the protein cytotoxicity to host tissues during inflammation.