Rabbit Thymus Research Articles

Ganglioside composition of rabbit thymus

Gangliosides of rabbit thymus were extracted and analyzed by the ganglioside-mapping procedure developed previously, 1 g of thymus contained 205.1 nmol of lipid-bound sialic acid and the relative amounts of monosialoganglioside and di- and trisialoganglioside fractions were 76.6 and 23.3%, respectively, as based on lipid-bound sialic acid. No ganglioside containing N-acetylgalactosamine was detected in rabbit thymus. The predominant component was N- glycoloylneuraminosyl lacto- N-norhexaosyl ceramide, NeuGc(α, 2–3)Gal(β, 1–4)GlcNAc(β, 1–3)Gal(β,1–4)-GlcNAc (β, 1–3)Gal(β, 1–4)Glc(β, 1–1) ceramide, which constituted 38.4% of the total gangliosides. The other major gangliosides were N-glycoloylneuraminosyl lacto O- N-neotetraosyl ceramide (31.3%), GD3 containing N- glycoloylneuraminic acid (11.8%), GM3 containing N-glycoloylneuraminic acid (10.6%) and GM3 containing N-acetylneuraminic acid (6.4%). C-18 sphingosine was the only long-chain base in all the gangliosides. Palmitic acid was the major fatty acid of thymus gangliosides and chromatographically more polar gangliosides contained higher proportions of palmitic acid: 46.3% in GM3, 47.5% in GD3, 60.2% in N-glycoloylneuraminosyl lacto- N-neotetraosyl ceramide and 89.6% in N-glycoloylneuraminosyl lacto- N-norhexaosyl ceramide.

Comparative study on ganglioside compositions of various rabbit tissues: Tissue-specificity in ganglioside molecular species of rabbit thymus

Ganglioside compositions of various organs of rabbit (NIBS strain, male, 10 months old) were studied. Organs examined contained lipid-bound sialic acid at various concentrations but the amounts in extraneural tissues were less than one-fifth of that in brain. The gangliosides of various tissues were analyzed by ganglioside-mapping and by isolating individual components and determining their structures chemically or enzymatically. According to their backbone asialocarbohydrate chain, the major gangliosides of various tissues were classified into three groups: (1) lactose and ganglio- N-triose (lung, stomach, liver, intestine, kidney, testis and muscle); (2) ganglio- N-tetraose (brain); (3) lacto- N-neotetraose (thymus). 70% of all thymus gangliosides had a lacto- N-neotetraose backbone, which was tissue-specific. In marked contrast to the case in other tissues, in thymus N-glycoloylneuraminic acid constituted 90% of the total lipid-bound sialic acid, and all molecular species of thymus gangliosides contained N-glycoloylneuraminic acid. Palmitic acid was a major fatty acid of thymus gangliosides. Distinct differences were found in the fatty acid compositions of gangliosides with longer carbohydrate chains in various tissues.

Fluorescence polarization studies and biochemical properties of membranes exfoliated from the cell surface of rabbit thymocytes in situ

In the course of our work on membrane phenomena related to the differentiation of lymphocytes in the rabbit thymus, we isolated membranous material from the extracellular compartment of this organ. With respect to their ultra-structural appearance, enzyme activity, lipid composition (cholesterol/phospholipid molar ratio, fatty acid composition of total phospholipids, phospholipid composition) and lipid fluidity, these membranes were shown to exhibit characteristics similar to those of purified plasma membranes isolated from disrupted thymocytes. Moreover, their antigenic specificity as determined in a cytotoxicity adsorption test was identical. From our experiments, we hypothesize that the extracellular membrane fragments found in the rabbit thymus are derived mainly from material shed by immature thymocytes.

Synthesis of histone model sequences for immobilization on a cross-linked polyacrylate matrix.

The synthesis of three peptide sequences which are useful as histone models, [Lys]5, [Lys]5-Pro and the sequence 17-27 of the lysine-rich histone from rabbit thymus Pro-Ala-Lys-Lys-Lys-Lys-Ala-Ala-Lys-Lys-Pro is described. Three different methods for the synthesis are applied. [Lys] 5 is synthesized from the amino end beginning with N alpha-acryloyl-N epsilon-Cbo-lysine by successive coupling of Cbo-lysine to yield the pentalysine monomer CH2=CH-CO-[Lys(Cbo)]5 which can be used directly for radical copolymerization. [Lys]5-Pro is synthesized according to the conventional peptide synthesis using the carbobenzoxy group for N alpha-protection and the tert,-butyl group for side chain protection. The carboxyl function is protected as tert.-butyl ester. The undecapeptide is synthesized in a similar manner. Instead of purifying the intermediate peptides by extraction, chromatography is used exclusively. The isolation of the unprotected peptides Lys-Pro, [Lys]3-Pro, [Lys]4-Pro, [Lys]5-Pro and Pro-Ala-[Lys]4-[Ala]2-[Lys]2-Pro and their characterization using amino acid analysis, electrophoreses and field desorption mass spectrometry is also reported.

Regional, cellular, and subcellular distribution of Thy-1 antigen in rat nervous tissues.

The regional, cellular, and subcellular distribution of Thy-1 antigen in rat nervous tissues was investigated with rabbit and anti-rat thymus antiserum. Thy-1 antigen was found most abundantly in the cerebrum, including the cerebral cortical layer, caudate nucleus, thalamus, and hypothalamus, and in the midbrain. It was found in lower amounts in the pons, medulla oblongata, cerebellum, and spinal cord. The content of Thy-1 antigen, however, was low in the retina, cervical sympathetic ganglion, and sciatic nerve, and there was little in the pineal body and adrenal medulla. Thy-1 antigen was present in the neuronal cell-enriched fraction, whereas the glial cell-enriched fraction lacked Thy-1 antigen. In a subcellular fractionation study, Thy-1 antigen was found to occur mainly in the synaptosomal membrane and microsomes and was low in the highly purified myelin fraction. The amount of Thy-1 antigen in the cerebral synaptosomal fraction was much higher than those in the cerebellar synaptosomal fraction. The distribution of Thy-1 antigen among the respective areas within the cerebrum was not significantly different, and it is suggested that Thy-1 antigen may be present evenly in the neuronal membranes of the cerebrum. Based on these results, Thy-1 antigen is considered to represent a useful marker of cerebral neuronal membranes, especially in the synaptic region, regardless of the kind of neurotransmitters.

The amino acid sequence of residues 1-104 of CTL-1, a bovine H1 histone.

The amino acid sequence of the first 104 residues of calf thymus H1 histone subfraction (CTL-1), one of the H1 histones from bovine thymus, was determined by use of peptides derived from chymotryptic and staphylococcus protease digestion. The first 35 residues differ from the corresponding regions of a rabbit thymus H1 (RTL-3) and trout H1 by 30 and 39%, respectively, while in the region between residues 36 and 104 CTL-1 differs from the rabbit histone by only 4% and from the trout histone by only 16%. Although the differences between CTL-1 and sea urchin H1 are much greater, it is still evident that the sequence between residue 36 and residue 104 has been conserved much more than that between residues 1 and 35.

Identification of the immunogenically active components of the Sm and RNP antigens.

The spectrum of cellular targets in the autoimmune diseases is both large and varied and includes among the nuclear components the so-called Sm and RNP antigens associated with systemic lupus erythematosus. The use of immunoaffinity chromatography with dual specificity for the Sm and RNP antigens has allowed for their substantial purification from rabbit thymus in parallel and in quantity. In lieu of a functional assay, the use of a counterimmunoelectrophoresis assay provided a sensitive and rapid means of monitoring the distribution of the two antigens during purification and ensured the isolation of complexes containing the components required for antigenicity. The resulting purified complex consisted of nine polypeptides having molecular weights of approximately 9000 to 44,000 and two small RNAs of similar size. However, limited proteolysis of the isolated complex suggested that most of these polypeptides were not actually required for antigenic activity. Unlike Sm in crude thymus extracts, purified Sm was RNase sensitive. Thus, one of the major diagnostic criteria used to distinguish Sm and RNP antigens in crude extracts was shown to be invalid for purified material, suggesting that both antigens from rabbit thymus are actually ribonucleoprotein complexes.

Synthese vernetzter lysin‐ und lysinpeptidgele

AbstractThe syntheses of models of histones are described. The lysine derivative Nε‐Boc‐lysine methylester, the protected peptides (Lys(Z))5,(Lys(Boc))5‐Pro‐OtBu and the protected sequence 17–27 of the histone H1 of rabbit thymus, Pro‐Ala‐(Lys(Boc))4‐Ala‐Ala‐(Lys(Boc))2‐Pro‐OMe, were transferred via three different ways into insoluble gels. The first synthetic way started with acrylamides, obtained by the reaction of the active benzotriazol‐1‐yl acrylate (3) with Nα‐amino groups of the lysine derivative and of derivatives of lysine peptides. The acrylic peptides were copolymerized with tetramethylene dimethacrylate and thus transferred into insoluble gels. In a second way partially protected peptides were coupled with carboxylic groups of cross‐linked polyacrylic gels. The third way followed the principle of the solid phase peptide synthesis. Cleavage of the protecting groups resulted in lysine and lysine peptide gels, which resemble different models of histones. The peptide gels can be used both for column chromatographic investigations of the peptide‐nucleotide interaction and for affinity‐chromatography.

The detection and identification of antibodies to saline extractable nuclear antigens by counterimmunoelectrophoresis

AbstractSera containing antibodies to the saline extractable nuclear antigens RNP, Sm, and Ha can be identified with counterimmunoelectrophoresis (CIE) by matching their precipitin reactions to those of reference sera. The CIE reaction of Sm is unusual in that two preciptin lines are formed, one of which shows partial immunologic identity with the reaction to RNP. Sera were identified with the reaction to RNP. Sera were identified by CIE which reacted with additional acidic antigens in rabbit thymus extract, some of which had the same enzyme sensitivity profiles as RNP and Ha but were immunologically not identical and could not be detected by hemagglutination with red cells coated with calf thymus nuclear extract. Although the incidence of reactivity to RNP, Sm, and Ha was similar to previous reports, review of the clinical manifestations of 128 patients with systemic lupus erythematosus (SLE) did not provide statistically significant confirmation of previously to reactivity and particular patterns of disease expression. With due attention paid to technical artifacts, CIE is a sensitive and specific method for identifying sera reactive with acidic nuclear antigens that has practical advantages over alternative methods.

Differences among subfractions of H1 histone in retention of linear and superhelical DNA on filters.

Four kinds of rabbit thymus H1 histone differ among themselves in their ability to retain DNA on nitrocellulose filters. This is true for linear, or superhelical DNA, but the order of effectiveness of the different H1 histones depends on the physical conformation of the DNA. For linear DNA the binding efficiencies of the H1 histones are: RTL2 = RTL3 greater than RTL4 greater than RTL1. This order of effectiveness parallels the effectiveness of the H1 histones previously found for the condensation of linear DNA as observed by circular dichroism and viscosity. The binding efficiencies of the various histones toward superhelical DNA were: RTL4 greater than RTL3 greater than RTL1 greater than RTL2. The variation in amino acid sequence between different rabbit thymus H1 histones might thus introduce structural variations in nucleohistone fibers and perhaps in chromatin.

Nuclear antigens: Characterization of the acidic nuclear ribonucleoprotein-Sm-antigen complex by analytical and preparative isoelectric focusing

The soluble ribonucleoprotein nuclear antigen (RNP), associated with the Sm antigen and present in extracts of rabbit thymus aceton powder, was purified by ion-exchange chromatography. Analytical isoelectric focussing (IEF) showed two bands at pH 5.2 and two bands at pH 5.4. After treatment by RNAse these 4 bands disappeared, and a new band with a pI of 6.1, representing the intact protein moiety, appeared. During preparative IEF in granulated gels the antigenicity of RNP was lost. Only the Sm antigen could be detected by counter-immunoelectrophoresis. Fractions with Sm antigenicity refocussed as two intense bands (pI 6.8 and 7.2) and one or two fainter bands (pI 6.1). On reducing SDS-PAGE the SM antigen revealed two bands of apparent molecular weights of about 110, 000 and 28, 500, indicating a total molecular weight of 138, 500.

An immunological study of rabbit tonsil lymphocytes with anti-thymocyte serum (author's transl)

A study of tonsillar immunological function was performed by investigating how rabbit tonsil antibody-producing lymphocytes after sensitization against sheep red blood cells (SRBC) were affected by anti-thymocyte serum (ATS).Antiserum was obtained by sensitizing goats with mixture of rabbit thymus cells and Freund's adjuvant and by absorbing serum with rabbit red blood cells. In vitro the sensitization by Marbrook's method and in vivo the sensitization by intravenous injection were performed. Plaque forming cell (PFC) assay was carried out according to the method of Cunningham & Szenberg in tonsils, lymph nodes and spleens.Results were as follows;1) In vitro anti-SRBC response of lymphocytes treated by ATS and complement remarkably suppressed.2) When lymphocytes sensitized in vivo were treated with ATS and complement, survival rate of PFC was significantly higher than that of total cells.3) Tonsil cells indicated higher sensitivity for ATS than lymphnode cells and spleen cells did.4) The number of direct or indirect PFC per a million of the lymphocytes from tonsils was always less than that from lymph nodes and spleens.5) Judging from the number of total indirect PFC after in vivo sensitization, the amount of antibodies produced in the cervical lymphnode was 46 times and that in the spleen was 258 times more than that in the tonsil.The author discussed these results and considered that the tonsil was peripheral lymphatic organ but its functions were, different from that of lymphnode and spleen, obtaining, folding and transmissing the antigenic information rather than producing antibody.

Antigen-induced in vitro lymphocyte blastogenesis in the rabbit: A T-cell response

In vitro lymphocyte stimulation of sensitized rabbit lymphocytes to specific antigen (ovalbumin) was found to depend on thymic-dependent lymphocytes. This conclusion is based on an enhanced response upon enrichment with T lymphocytes by passage of lymphocytes through nylon wool, and on the elimination of the response after treatment of lymphocytes with complement and an antiserum to rabbit thymus cells prepared in a goat. Specificity of the antiserum was demonstrated by elimination of in vitro T-cell function and retention of in vitro B-cell functions.

Regulatory Influences on the Response of Rabbit T Cells to Concanavalin A and Phytohaemagglutinin

The proliferative response, induced in rabbit spleen cells by concanavalin A (Con A) and phytohaemagglutinin (PHA), is abolished when T cells are killed with antibody against rabbit thymus lymphocyte antigen (RTLA) in the presence of complement. The response was examined with purified spleen T cells, to which various helper cell fractions were added; it could be shown that B cells help the responding T cells. The helper effect in the response to PHA is abolished and the response to Con A is reduced by any manoeuvre which destroys or removes B cells. Help by B cells is given when helper cells have lost proliferative capacity as a consequence of mitomycin-C treatment. Spleen cells adhering to the walls of culture tubes help suspended T spleen cells in their response to Con A. This help could be abolished by complement mediated cell kill with antibody to rabbit bursal equivalent lymphocyte antigen (RABELA). On the other hand, the helper effect in Con A response was increased when T cells were removed. Thus the response of T cells to Con A is regulated by helper B cells and suppressor T cells.

Two subpopulations of cortisone-resistant lymphoid cells in the rabbit thymus.

The final product of lymphoid cell differentiation in the thymus along common or separate (1) maturation pathways is the small lymphocyte. A minor, cortisone-resistant (CR), part of small thymic lymphocytes contributes to the generation of immunocompetent peripheral T cells, a high, cortisone-sensitive (CS), proportion of incompetent small cells appears to die within the thymus (2,3). Candidate (precursor) cells for the functionally various classes of T cells are to be found within the category of CR small thymic lymphocytes, which, in the present study, was analysed with physical and morphological methods. Evidence for a possible separation and identification of two subpopulations of CR small thymic lymphocytes will be presented.

A study of the T cell differentiation in the rabbit thymus using a special staining and immuno-ferritine technique

A study of the T cell differentiation in the rabbit thymus using a special staining and immuno-ferritine technique

Analysis of the high mobility group proteins associated with salt-soluble nucleosomes.

Two methods have recently been described for the isolation of monomer nucleosomes enriched in transcribed sequences which depend on their solubility in 0.1 M NaCl (Levy, W.B. and Dixon (1978), Nucleic Acid Res., 5, 4155-4163) or solutions containing divalent metal ions (Bloom, K.S. and Anderson, J.N. (1978), Cell, 15, 141-150). Using these procedures the proteins associated with such nucleosomes from rabbit thymus, calf liver and hen oviduct nuclei were isolated and analysed. Increased amounts of proteins HMG14 AND HMG17 and small amounts of HMG1 and HMG2 were found associated with the four core histones H2A, H2B, H3 and H4 in these nucleosomes. HMG14 and HMG17 were found to be enriched 2 - 7 fold, suggesting an involvement of these two proteins with transcribed sequences. 0.1 M NaCl-soluble monomer nucleosomes prepared by the method of Levy and Dixon were analysed by polyacrylamide gel electrophoresis and found to be composed of principally two types of particle: 1. Core particles of 145 base pairs of DNA associated with the four core histones only. 2. Nucleosomes with 160 base pairs of DNA associated with the four core histones, increased amounts of HMG14 and 17, and no H1. Small amounts of HMG1 and HMG2 are also detected. These results suggest that HMG14 and HMG17 might be interacting with the 15 base pair linker DNA. A model is presented for the structure of transcriptionally active chromatin.

Studies on the association of the high mobility group non-histone chromatin proteins with isolated nucleosomes

Nucleosomes have been isolated from rabbit thymus by sucrose gradient centrifugation, and their high mobility group (HMG) protein content analysed by electrophoresis on polyacrylamide gels. The results suggest that proteins HMG 14 and HMG 17 are associated with the core particle of the nucleosome, and that there are two or more sub-populations of both HMG 1 and HMG 2 molecules. One sub-population appears to be fairly tightly bound to the nucleosome, while another is rapidly released from the chromatin by digestion with micrococcal nuclease. The latter fraction may participate in a higher order folding of the nucleosomes.

GM3 ganglioside in various tissues of rabbit. Tissue-specific distribution of N-glycolylneuraminic acid-containing GM31.

Molecular species and concentrations of GM3 ganglioside in rabbit tissues, brain, thymus, lung, liver, stomach, intestine, kidney, testis, muscle, and erythrocytes were determined. The highest concentration was found in lung (431.7 nmol/g wet tissue). With the exceptions of brain, thymus, and erythrocytes, GM3 was the dominant ganglioside and comprised more than 50% of the total gangliosides. In brain and thymus, GM3 composed 0.76% and 17.28% of the total gangliosides, respectively. The sialic acid composition of GM3 was determined by mild methanolysis and neuraminidase treatment combined with either permethylation or trimethylsilylation. N-Glycolylneuraminic acid-containing GM3 was found in thymus, lung, kidney, and intestine in addition to N-acetylneuraminic acid-containing GM3, but N-acetylneuraminic acid was the sole sialic acid of GM3 in the other tissues. Sixty-four percent of the thymus GM3 consisted of N-glycolylneuraminic acid. High concentrations of C-20 sphingosine and stearic acid were characteristic for brain GM3 and the major long chain base of the other tissues was C-18 spingosine. The fatty acid composition of GM3 varied in each tissue. The difference in mobility on a thin-layer plate was due to the difference in its molecular constitution, being derived from a combination of fatty acid, long chain base, and sialic acid.

Isolation and characterization of GD3 ganglioside having a novel disialosyl residue from rabbit thymus.

A major component of the di- and trisialoganglioside fraction of rabbit thymus was isolated. By neuraminidase treatment, methylation analysis, and CrO3 oxidation, it was identified as GD3 ganglioside with the novel disialosyl residue, NeuNGlyc(alpha, 2-8)NeuNAc(alpha, 2-3)Gal-(beta, 1-4)Glc(beta, 1-1)ceramide. Its concentration was 21.92 nmol/g of wet tissue (12.10% of the total gangliosides) and it contained 91.52% of the lipid-bound sialic acid in di- and trisialogangliosides of rabbit thymus.