Differential expression of metastasis-associated cell surface glycoproteins and mRNA in a murine large cell lymphoma

A metastatic variant cell subline of the Abelson virus-transformed murine large lymphoma/lymphosarcoma RAW117 has been selected in vivo ten times for liver colonization. Highly metastatic subline RAW117-H10 forms greater than 200 times as many gross surface liver tumor nodules as the parental line RAW117-P. Analysis of cellular proteins and glycoproteins indicates reduced expression of murine Moloney leukemia virus-associated p15, p30, and gp70, and increased expression of a sialoglycoprotein, gp150, in the highly metastatic H10 cells. Northern analyses of oncogene expression suggested that mRNA of various oncogenes was expressed equally or not expressed in the RAW117 cells of differing metastatic potential. Differential gene expression was examined using a cDNA library of 17,600 clones established from poly A+ mRNA isolated from H10 cells. The cDNA library was screened by the colony hybridization technique using probes made from both RAW117-P and -H10 cells. Approximately 99.5% of these cDNA clones were expressed identically in P and H10 cells. Of the few differentially expressed cDNA clones (approx. 150/17,600), one-half of these were identified as Moloney leukemia virus sequences in a separate probing with a radiolabeled Moloney leukemia virus probe. The remainder of the differentially expressed mRNA detected by colony hybridization of the cDNA library were expressed at higher levels (approx. 1/6) or lower levels (approx. 1/3) in the highly metastatic H10 cells.     

Haemagglutinin activity in the haemolymph of individual Acrididae (grasshopper) specimens

Twenty-four individual grasshopper specimens representing four Melanoplus spp. contained similar broad-spectrum haemolymphatic haemagglutinin. The agglutinin activity showed highest titre toward human ABO and rabbit cells among nine types of erythrocytes tested. Titre values differed between individual insects but agglutination specificity toward different erythrocytes was similar. Agglutination of type-O red cells by individual grasshopper haemolymph was inhibited by 34 of 41 tested carbohydrates, carbohydrate derivatives, alcohols and chelating agents. Individual insects showed similar patterns of haemagglutination inhibition. Non-inhibitory compounds were mannose and mannose derivatives (excepting N-acetylneuraminate), several glucose derivatives, amino sugars and ethanol. The observations indicated that haemolymph from an individual grasshopper contained complex heteroagglutinin activity similar to that found in haemolymph pooled from several insects. Determination of minimal effective inhibitor concentrations confirmed the presence of heteroagglutinin activity primarily directed toward galactose and glucose and related α-linked glycosidic derivatives.     

Biosynthesis of lysosomal endopeptidases

Despite the clear differences between the amino acid sequence and enzymatic specificity of aspartic and cysteine endopeptidases, the biosynthetic processing of lysosomal members of these two families is very similar. With in vitro translation and pulse-chase analysis in tissue culture cells, the biosynthesis of cathepsin D, a aspartic protease, and cathepsins B, H and L, cysteine proteases, are compared. Both aspartic and cysteine endopeptidases undergo cotranslational cleavage of an amino-terminal signal peptide that mediates transport across the endoplasmic reticulum (ER) membrane. Addition of high-mannose carbohydrate also occurs cotranslationally in the lumen of the ER. Proteases of both enzyme classes are initially synthesized as inactive proenzymes possessing amino-terminal activation peptides. Removal of the propeptide generates an active single-chain enzyme. Whether the single-chain enzyme undergoes asymmetric cleavage into a light and a heavy chain appears to be cell type specific. Finally, late during their biosynthesis both classes of enzymes undergo amino acid trimming, losing a few amino acid residues at the cleavage site between the light and heavy chains and/or at their carboxyltermini. During biosynthesis these enzymes are also secreted to some extent. In most cells the secreted enzyme is the proenzyme bearing some complex carbohydrate. Under certain physiological conditions the inactive secreted enzymes may become activated as a result of a conformational change that may or may not result in autolysis. Analysis of the biochemical nature of the various processing steps helps define the cellular pathway followed by newly synthesized proteases targeted to the lysosome.     

Isolation and Biochemical Characterization of a Neural Proteoglycan Expressing the L5 Carbohydrate Epitope

The monoclonal L5 antibody reacts with an N-glycosidically linked carbohydrate structure which is present on the neural cell adhesion molecule L1, neural chondroitin sulfate proteoglycans, and other not yet identified glycosylated proteins. Using this antibody, we isolated and characterized proteoglycans from adult mouse brain and cultured astrocytes biosynthetically labeled with Na2 35SO4 and a 3H-amino acid mixture. Our data suggest that the L5 proteoglycans of both sources are identical in their biochemical properties. The apparent molecular mass of the L5 proteoglycan is approximately 500 kDa. Digestion of the iodinated L5 proteoglycan from mouse brain and of the [35S]methionine-labeled L5 proteoglycan from cultured astrocytes with proteinase-free chondroitinases ABC and AC revealed three major core proteins with apparent molecular masses of approximately 380, 360, and 260 kDa. These represent molecularly distinct protein cores.     

Molecular cloning of a gene encoding a Chlamydia psittaci 57-kDa protein that shares antigenic determinants with ca. 60-kDa proteins present in many Gram-negative bacteria

In order to develop reagents to study the immune response of guinea pigs to infection by Chlamydia psittaci guinea pig inclusion conjunctivitis strain (GPIC), we constructed a plasmid clone bank with C. psittaci DNA. One of the recombinant clones isolated produced large amounts of a 57-kilodalton (kDa) protein that was immunoreactive with sera from GPIC infected guinea pigs. While investigating this recombinant protein, we discovered that all the Gram-negative bacteria analyzed so far have immunoreactive proteins of similar size. This protein seems to be a 'common antigen' already described in various Gram-negative bacteria.     

Control of barley root respiration

Evidence from barley [ Hordeum distichum (L.) Lam. cv. Maris Mink], and from many other species, suggests that respiration is controlled by either supply of carbohydrate or demand for ATP. The relationship between root respiration rate (measured as O₂ consumption or CO₂ production) and ethanol-soluble carbohydrate content altered with time following selective pruning, and the change could not be accounted for by buffering of the cytoplasmic carbohydrate concentration by sugars in the vacuole. Exogenous sucrose supplied to the roots prevented any decline of the respiration rate in shoot-pruned plants, and if supplied for 24 h stimulated the respiration rate after any treatment. Root extension responded to sucrose in a similar manner. We suggest that respiration is under fine control by adenylates, but the capacity of the respiratory system is fixed by the supply of sucrose, possibly via coarse control of the respiratory machinery, or of the processes requiring metabolic energy.     

Differential Ultrastructural Localization of Myelin Basic Protein, Myelin/Oligodendroglial Glycoprotein, and 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase in the CNS of Adult Rats

In a light and electron microscopic immunocytochemical study we have examined the distribution of myelin basic protein (MBP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), and myelin/oligodendroglial glycoprotein (MOG) within CNS myelin sheaths and oligodendrocytes of adult Sprague-Dawley rats. Ultrastructural immunocytochemistry allowed quantitative analysis of antigen density in different myelin and oligodendrocyte zones: MBP was detectable in high density over the whole myelin sheath, but not in regions of loops, somata, or the oligodendrocyte plasma membrane. CNP reactivity was highest at the myelin/axon interface, and found in lower concentration over the outer lamellae of myelin sheaths, at the cytoplasmic face of oligodendrocyte membranes, and throughout the compact myelin. MOG was preferentially detected at the extracellular surface of myelin sheaths and oligodendrocytes and in only low amounts in the lamellae of compacted myelin and the myelin/axon border zone. Our studies, thus, indicate further the presence of different molecular domains in compact myelin, which may be functionally relevant for the integrity and maintenance of the myelin sheath.     

Confirmation of the Cholinergic Specificity of the Chol-1 Gangliosides in Mammalian Brain Using Affinity-Purified Antisera and Lesions Affecting the Cholinergic Input to the Hippocampus

An antiserum raised to Torpedo electromotor synaptosomal membranes (anti-TSM antiserum) induces a cholinergic-specific immune lysis of mammalian brain synaptosomes and recognizes a group of minor gangliosides appeared, therefore, to be specific to the cholinergic neuron and were designated Chol-1. To confirm the cholinergic specificity of the Chol-1 gangliosidic antigens, we have shown that not only does a mammalian ganglioside fraction that is enriched with respect to the Chol-1 gangliosides inhibit the cholinergic-specific immune lysis induced by the anti-TSM antiserum, but also it can be used to affinity-purify a subpopulation of immunoglobulins from the anti-TSM antiserum that also induce a cholinergic-specific lysis. Furthermore, we have demonstrated that fimbrial lesions, which cause a massive degeneration of cholinergic terminals in the ipsilateral hippocampus, lead to a loss of the Chol-1 gangliosides concomitant with that shown by choline acetyltransferase activity and that lesions to the entorhinal cortex, which cause a loss of mainly glutamergic synapses in the ipsilateral dentate gyrus leading to cholinergic sprouting from adjacent hippocampal areas and an increase in cholinergic markers in the dentate gyrus, produce concomitant increases in choline acetyltransferase activity and Chol-1 content. These results provide strong evidence in favour of the cholinergic specificity of the Chol-1 gangliosides.     

Protein synthesis and secretion in the human epididymis and immunoreactivity with sperm antibodies

The synthesis and secretion of proteins in the different regions of the human epididymis were studied in vitro. Epididymal tissues obtained from patients undergoing castration for prostatic carcinoma or from cadavers were incubated in the presence of [35S]methionine, and the resulting radiolabeled proteins were analysed on SDS-PAGE. The corpus region was found to be the most active segment in total protein synthesis. Significant qualitative and quantitative changes were observed in the pattern of proteins secreted from the different epididymal regions. To establish those epididymal proteins that interact with maturing sperm, the secreted products were immunoreacted with antibodies raised against a Triton X-100 extract of ejaculated human sperm heads. The antibodies react mainly with the head region of ejaculated spermatozoa as judged by indirect immunofluorescence. Protein A-gold labeling of freeze-fracture images showed gold particle distribution on the sperm plasma membrane. Western blot analysis of the secreted proteins revealed four bands (66, 37, 32, and 29 kDa) in the proximal regions and six additional bands (80, 76, 48, 27, 22, and 17 kDa) in the distal part of the epididymis. Immunoprecipitation of the secreted proteins with these antibodies revealed six radioactive bands of 170, 80, 76, 60, 48, and 37 kDa, which indicates that certain proteins of epididymal origin bind to the sperm plasma membrane.