Recognition of 29 kDa surface-associated adhesive molecule of Entamoeba histolytica by monoclonal antibodies

Abstract Monoclonal antibodies have been developed and used as specific probe to locate and identify a 29-kDa molecule of axenic Entamoeba histolytica trophozoites. Monoclonal antibody produced by clone C8 (MoAb C8) strongly agglutinated the amoebic trophozoites. THe immunofluorescence of live E. histolytica trophozoites and surface fluorescence of acetone-fixed trophozoites by MoAb C8 indicated existence of a 29-kDa molecule on surface-associated plasma membrane of E. histolytica . The monoclonal antibody belonged to IgG₁ isotype. The prior treatment of E. histolytica trophozoites with MoAb C8 resulted in significant ( P < 0.01) reduction in adherence of amoebic trophozoites to cultured Chinese Hamster Ovary cells and significant ( P < 0.01) reduction in cytotoxicity to cultured Baby Hamster Kidney cells. Pretreatment of amoebic trophozoites with MoAb C8 prior to cultivation in TPS-1 medium resulted in significant ( P < 0.01) reduction in growth of the parasite. Thus, the data suggested that the surface-exposed 29-kDa molecule may be one of the receptors involved in E. histolytica host cell interactions and may possibly modulate amoebic disease processes.     

Immunocytochemical markers revealing retinal and pineal but not hypothalamic photoreceptor systems in the Japanese quail

Summary The retinal proteins opsin,-transducin, S-antigen and interstitial retinol-binding protein (IRBP) are essential for the processes of vision. By use of immunocyto-chemistry we have employed antibodies directed against these photoreceptor proteins in an attempt to identify the photoreceptor systems (retina, pineal and deep brain) of the Japanese quail. Opsin immunostaining was identified within many outer (basal portion) and inner segments of retinal photoreceptor cells and limited numbers of photoreceptor perikarya. Opsin immunostaining was also demonstrated in limited numbers of pinealocytes with all parts of these cells being immunoreactive. These results differ from previous observations. In contrast to the results obtained with the antibody against opsin, S-antigen and-transducin immunostaining was seen throughout the entire outer segments and many photoreceptor perikarya of the retina. In the pineal organ immunostaining was seen in numerous pinealocytes in all follicles. These results conform to previous findings in birds. In addition, IRBP has been demonstrated for the first time in the avian retina and pineal organ. These findings underline the structural and functional similarities between the retina and pineal organ and provide additional support for a photoreceptive role of the avian pineal. No specific staining was detected in any other region of the brain in the Japanese quail; the hypothalamic photoreceptors of birds remain unidentified.     

An 83-kDa embryonic-type nuclear antigen is detected within the germinal vesicles of oocytes of the ascidianHalocynthia roretzi

Summary Monoclonal antibodies were raised against germinal vesicles which were isolated from fully grown oocytes of the ascidianHalocynthia roretzi. Immunoblot analyses revealed that one of the antibodies, designated Hgv-2, recognized a single band with a molecular weight of about 83 kDa. The antibody, visualized by indirect immunohistochemistry, reacted only with the germinal vesicles of oocytes and did not react with test cells, follicle cells, and other somatic cells of the gonad. During embryogenesis the antigenicity was found in interphase nuclei of all embryonic cells. The antibody did not react with chromosomes or the mitotic apparatus. The antigenicity was retained by interphase nuclei of larval cells, but it disappeared from nuclei of juveniles about 7 days after metamorphosis.     

Immobilization of enzymes on alumina by means of pyridoxal 5′-phosphate

A number of proteases have been immobilized on alumina in a two-step procedure: the first step converted them into semisynthetic phosphoproteins which, in the second step, spontaneously bonded to alumina through their phosphate function. The immobilized enzymes thus obtained showed the physical properties typical of the inorganic carrier and a high activity on low molecular weight substrates.     

DNA hybridization analysis of mycobacterial DNA using the 18-kDa protein gene of Mycobacterium leprae

Abstract DNA hybridization studies using a 611-base pair (bp) probe, encoding the entire 18-kDa protein of Mycobacterium leprae , demonstrated that M. simiae, M. intracellulare, M. kansasii, M. terrae , ADM-2, M. avium, M. scrofulaceum, M. gordonae and M. chelonei appear to posses DNA sequences homologous to the 18-kDa protein gene of M. leprae . RFLP analysis revealed that the restriction sites in the M. leprae 18-kDa gene were not conserved in the putative gene homologs of M. simiae and M. intracellulare . The restriction patterns observed with the 611-bp probe were useful in differentiating M. intracellulare, M. simiae , and M. leprae from each other, as well as in distinguishing strains of M. simiae serovar 1. Finally, the presence of homologous sequences in various mycobacteria did not affect the specificity of a previously described PCR test for detection of M. leprae , based on the M. leprae 18-kDa protein gene.     

Degradation of the 32-kilodalton thylakoid-membrane polypeptide of Chlamydomonas reinhardi Y-1

Isolated thylakoid membranes of Chlamydomonas reinhardi Y-1 with the 32-kDa polypeptide either radioactively labelled or unlabelled were incubated in vitro under various conditions in order to gain information about the degradation of the 32-kDa polypeptide. The degradation was higher at pH 6 compared with pH 7 and pH 8 and exhibited a temperature maximum between 20° C and 25° C (pH 6, pH 8). A light-dependent part of the total degradation was linearly dependent on white light of energy fluence rate between 1 and 20 mW·cm^-2 at 25° C and leveled out at higher fluence rates. The degradation in light was only slightly stimulated by ATP but was reduced by 3-(3-4-dichlorophenyl)-1,1-dimethylurea. Adenosine-5-diphosphate and heparin (2.7 mM and 200 g per 100 l, respectively) known to inhibit kinases, caused a 50% decrease in degradation indicating that a phosphorylation step is involved in degradating the 32-kDa polypeptide. Out of various inhibitors specific for different types of proteases, only those for thiol- and endoproteases showed intense effects. These results point to a proteolytic degradation of the 32-kDa polypetide by a thylakoid-membrane-bound thiol-endoprotease. Its activity yields soluble breakdown products with relative molecular masses (M_rs) of 23, 16.5, 11.3 and 10.7 kDa, and these are accumulated in the in-vitro system. Partial proteolytic digestion of thylakoids with Staphylococcus aureus V8 protease results in at least two labelled breakdown products (M_rs 23, and 16.5 kDa). It is assumed that cleaving at identical amino-acid residues of the 32-kDa polypeptide by the thylakoid-membrane-bound thiolendoprotease and the V8 protease results in these two breakdown products. They are derived from subsequent cleavage at amino-acid residues 60–242 and 60–189 according to the deduced protein sequence (Erickson et al. 1984, EMBO J. 3, 2753–2762).Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron) - LDS-PAGE lithiumdodecyl sulphate-polyacrylamide gel electrophoresis - M apparent molecular mass - PSII photosystem II - TCA trichloroacetic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Differential Expression of mRNA and Protein Encoding Retinal and Pineal S-Antigen During the Light/Dark Cycle

S-Antigen is a soluble cell protein unique to the retina and pineal gland. In the former, it is a well-characterized molecule that participates in light-induced signal transduction in photoreceptor cells. In the latter, the functional role is presently not known. The expression of S-antigen and its mRNA was examined in the rat retina and pineal gland throughout the diurnal cycle and with light interruption of the dark cycle. A cDNA for rat S-antigen was isolated from a pineal gland library to examine the mRNAs. A 1.7-kb mRNA for S-antigen was observed in both the pineal gland and the retina. Retinal S-antigen mRNA was expressed throughout the diurnal cycle and increased with light interruption of the dark cycle. In contrast, pineal gland S-antigen mRNA levels were detectable only during the dark and were absent preceding and during light. The phenotypic expression of immunoreactive S-antigen, identified with two S-antigen monoclonal antibodies (MAbs), MAb A9C6 and MAb C10C10, was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE) and isoelectric focusing (IEF) electrophoresis. Immunoblot analysis of gels after SDS-PAGE revealed a single 46-kDa protein in retina. In contrast, two bands of approximately 43 and 46 kDa were identified in the pineal gland. Immunoblots of the retinal extracts separated by IEF electrophoresis revealed five S-antigen isomers, which vary quantitatively throughout the diurnal cycle and when light interrupted the dark cycle. Immunoblots of the pineal gland samples separated by IEF electrophoresis indicated that the pineal gland possesses four pineal gland-specific forms of S-antigen in addition to the five forms present in the retina. The differences observed in the mRNA and protein analyses suggest tissue-specific structural components for S-antigen in the retina and pineal gland that are not regulated in the same manner.     

Postembryonic nongonadal cell lineages of the nematode Panagrellus redivivus: Description and comparison with those of Caenorhabditis elegans

The postembryonic nongonadal cell lineages of the nematode Panagrellus redivivus are described and compared with those of Caenorhabditis elegans. The newly hatched larvae of P. redivivus females and males and C. elegans hermaphrodites and males are very similar. An almost identical set of blast cells divides postembryonically in P. redivivus and C. elegans to produce similar changes in the neuronal, muscular, hypodermal, and digestive systems. Most of these cell lineages are invariant; however, there is substantial variability in the number of cell divisions in the relatively extensive lineages of the lateral hypodermis of P. redivivus. Typically, in P. redivivus females, 55 blast cells generate 635 surviving progeny and 29 cell deaths; in P. redivivus males, 59 blast cells generate 758 surviving progeny and 35 cell deaths. The lineages generating the cells of the male tails of P. redivivus and C. elegans are almost identical; thus, the grossly different characteristics of these structures must reflect differences in the morphogenesis of cells equivalent in lineage history. Laser ablation experiments demonstrate that the gonad induces vulva development and that cell-cell interactions are important in specifying the fates of hypodermal precursor cells. The lateral hypodermal lineages provide striking examples of the apparent construction of complex lineages from modular sublineages; one simple pattern of cell divisions and cell fates occurs 70 times in the P. redivivus female. The differences in cell lineage between P. redivivus and C. elegans are relatively minor, and many appear to have involved two types of evolutionary change: the replacement of sublineages, and the modification of sublineages by the four classes of lineage transformations previously proposed based on a comparison of P. redivivus and C. elegans gonadal cell lineages (Sternberg and Horvitz, 1981). These types of differences suggest that the genetic programming of cell lineage includes instructions specifying where and when a particular sublineage is utilized, and other instructions specifying the nature of that sublineage.