Characterisation of 24-kD proteins from rat testes using polyclonal sera reactive to human sperm antigens

A group of antigens of 24-kD Mr from rat testes were characterised biochemically. These antigens were part of a larger molecule of approximately 200 kD. On treatment with disulfide bond reducing agent, the 200-kD molecule was reduced to subunits. Immunoreactivity was confined to a doublet of approximately 24 kD and a single band of approximately 50 kD Mr after the reduction. Glycoprotein in nature, this antigen shared immunoreactive epitopes with a 40-kD antigen on human spermatozoa. Antiserum raised in rabbits against the 24-kD antigen from rat testes reacted with antigens on the acrosome of human spermatozoa. Agglutination of sperm could be induced by the antiserum. The carbohydrate residue could be removed by mannosidase digestion. Chemical deglycosylation studies showed a slight decrease in molecular weight. Immunoreactivity was however not completely lost after chemical deglycosylation. Isoelectric focusing of the antigen identified nine isoelectric species. Two relatively minor species showed immunoreactivity. Acrosome-reacted spermatozoa showed loss of antigens from acrosome.     

Proteomic analysis of endogenous nitrotryptophan-containing proteins in rat hippocampus and cerebellum

Nitration of tryptophan residues is a novel post-translational modification. In the present study, we examined whether NO₂Trp (nitrotryptophan)-containing proteins are produced in the hippocampus and cerebellum of the adult rat under physiological conditions in vivo. Using Western blot analysis with anti-6-NO₂Trp-specific antibody, we found many similar immunoreactive spots in the protein extracts from both regions. These spots were subsequently subjected to trypsin digestion and LC-ESI-MS/MS (LC-electrospray ionization-tandem MS) analysis. We identified several cytoskeletal proteins and glycolytic enzymes as NO₂Trp-containing proteins and determined the position of nitrated tryptophan residues with significant ion score levels (P<0.05) in several proteins in both regions. We also observed that the total amount of NO₂Trp-containing proteins in the cerebellum was significantly greater than that in the hippocampus (P<0.05). Moreover, IP (immunoprecipitation) assays using anti-aldolase C antibody showed that the relative intensity of immunostaining for NO₂Trp over aldolase C was much higher in cerebellum than in hippocampus. The amounts of nNOS (neuronal nitric oxide synthase) and eNOS (endothelial nitric oxide synthase) were much greater in cerebellum than in hippocampus. This is the first evidence of several specific sites of nitrated tryptophan in proteins under physiological conditions in vivo.     

Analysis of the cuticular proteins of Hyalophora cecropia with polyclonal antibodies

The cuticular proteins from different anatomical regions and metamorphic stages of Hyalophora cecropia were analyzed with polyclonal antibodies raised against cuticular protein extracts from each stage. Western blots of 2D gels coupled with detection of antibody-antigen binding with avidin-biotinylated-horseradish peroxidase complexes (ABC method) proved to be extremely sensitive. Reactions of polyclonal antisera with blots of extracts of different cuticular regions revealed the following: (1) glycosylated cuticular proteins were highly antigenic; (2) there was less cross-reaction between rigid and flexible cuticles from the same metamorphic stage than among cuticles with similar mechanical properties from different stages; (3) proteins with identical molecular weights and isoelectric points were antigenically indistinguishable.     

Characterisation of monoclonal and polyclonal antibodies to 19-linked conjugates of testosterone with corresponding radioligands

Monoclonal antibodies to testosterone T were produced using testosterone 19-O-carboxymethyl ether (T19C) and testosterone 19-hemisuccinate (T19H) immunogens. All antibodies were characterised with iodinated derivatives of both T19C and T19H. Monoclonal antibodies derived from the T19C immunogen had similar titres and assay sensitivities with both T19-tracers. In contrast antibodies derived from the T19H immunogen bound the homologous but not the heterologous tracer. Individual antibodies showed a wide variation in cross-reactivity with 5 alpha-dihydrotestosterone, DHT (4.4-100%), androstenedione AN (0.5-100%) and progesterone, Po (0.08-5.4%). One antibody 3F11 derived from a T19C immunogen gave 50% displacement of tracer with 180 pgT/tube and low cross-reactivity of 12% with DHT, 3.0% with AN and 1.1% with Po. In general, assay sensitivity and antibody specificity were poorer with an [125I]-histamine conjugate of T-3-carboxymethyloxime than with T19 tracers. Radioimmunoassays for T in extracted human serum were developed with [125I]T19C as tracer and monoclonal antibody 3F11 (T19C immunogen) and rabbit antiserum T19H3R1 (T19H immunogen). Sensitivities of the extracted assays were 43 and 20 pg/tube respectively and results correlated well with those obtained after chromatographic separation of testosterone (r = 0.97 for both antibodies). We conclude that 19-linked derivatives of T are highly immunogenic for the production of specific testosterone antibodies. Selection of the appropriate iodinated tracer is essential to achieve optimal titre, assay sensitivity and specificity, since these characteristics vary widely with individual monoclonal antibodies, and classical bridge recognition is not observed.     

Immunophotochemical analysis of mineralocortin by polyclonal antibodies against the native receptor from rat kidney

We have obtained a polyclonal antiserum by immunizing fawn Burgundy rabbits with the mineralocorticoid receptor (MCR) purified biochemically from rat kidneys. High titers of anti-MCR activity were obtained in radioimmunoassays within 3 weeks and increased with a booster shot. In Western blot analysis, the antibody revealed a major band of 94–98 kDa in renal cytosol from rat and beef kidneys. We also developed a fluorographic procedure where the MCR linked covalently to tritiated R-5020, following ultraviolet irradiation, gave imprints superimposable on the Western blot profile. The fluorographic pattern was specific since it was largely abolished in the presence of cold RU 26752 that is specific to MCR, or mineralocortin. The immune IgG precipitated rat renal MCR-[³H]RU 26752 complexes in a dose-dependent manner and also recognized MCR bound to the natural hormone aldosterone. During gel permeation chromatography on Sephacryl, the elution profile of [³H]RU 26752 shifted to high-molecular-weight regions in the presence of immune IgG. The receptor protein could be immunolocalized primarily to the principal cells of the collecting duct in rat kidney but the intercalated cells and glomeruli were not labeled, contrary to beef kidney where a uniform pattern of immunostaining was evident. These should permit large-scale purification of the MCR for detailed physicochemical studies and for screening of the MCR-positive tissues during various pathophysiological syndromes.     

Differences of antioxidant systems in the cerebellum and hippocampus

The effect of portacaval shunting on the antioxidant status of the cerebellum and hippocampus has been studied in rats using standard methods of enzymatic analysis. Endogenous ammonia levels and activities of eight antioxidant enzymes were shown to be unequal in these two brain regions and to respond differently to intrahepatic portosystemic shunt surgery.     

Changes of mitochondrial membrane proteins in rat cerebellum during aging

Qualitative and quantitative changes of mitochondrial membrane proteind during aging were investigated. Free (non-synaptic) mitochondria were purified from rat cerebellum at different ages (4, 8, 12, 16, 20, and 24 months). Mitochondrial outer membrane (OM), inner membrane (IM) and matrix (MX) were separated and the proteins were extracted and analyzed by gel-electrophoresis.After staining, the gels were scanned densitometrically to quantify the proteins. No significant changes in the quantity of OM or MX protein subunits were observed, while serveral statistically significant quantitative changes in IM proteins with age were found. These age-dependent modifications of inner membrane mitochondrial proteins may play an important role in energy transduction, transport systems and regulatory enzymatic activities in mitochondria.     

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.     

Immunohistochemical localization of aldehyde and xanthine oxidase in rat tissues using polyclonal antibodies

Tissues from male Wistar rats, fixed with 4% paraformaldehyde and embedded in paraffin, were studied with immunoperoxidase techniques using polyclonal antibodies raised against aldehyde oxidase or xanthine oxidase purified from rat liver. Immunohistochemical studies demonstrated that aldehyde oxidase-bearing cells were strongly stained in renal tubules, esophageal, gastric, intestinal and bronchial epithelium as well as liver cytoplasm. Weak but positive immunoreactivity was observed on the pulmonary alveolar epithelial cells, gastric glands and intestinal goblet cells. In contrast, it was demonstrated that cells with xanthine oxidase were strongly stained in renal tubules, esophageal, gastric, and small and large intestinal and bronchial epithelia etc. Positive immunostaining was also found in adrenal gland, skeletal muscle, spleen and cerebral hippocampus. Immunoreactivity againt aldehyde oxidase was not found in adrenal gland, spleen, mesentery or aorta, while immunoreactivity against xanthine oxidase was not found in mesentery or aorta. Although the significance of this ubiquitous and similar localization of aldehyde and xanthine oxidase seems unclear at present, these results may provide a clue as to the full understanding of the pathophysiological role of these oxidases in tissues.     

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.     

Regional expression of sulfatides in rat kidney: immunohistochemical staining by use of monospecific polyclonal antibodies

 Sulfatides¹ are quantitatively prominent glycosphingolipids of rat kidney. In order to gain insight into their possible physiological significance in this organ, and their possible role in Heymann’s nephritis, the main sulfatide components were localized immunohistochemically. The antibodies used recognized the sulfatides Sga1l, Stri1, Stri2, and Stet2a. Stri1 epitopes were expressed in the brush border of proximal tubuli, whereas Stri2-specific immuno staining was observed in cortical and medullar collecting ducts. Both Stri1 and Stri2 were also expressed by interstitial cells of the inner medulla. Stet2a was visualized in epithelia of the distal tubules of Henle’s loop and the juxtaglomerular apparatus, including the macula densa. The mAb Sulph-I, that recognizes the SO₃ ^(–)-3Galβ< epitope of Sgal1, seminolipid, and Slac1, prominently stained the cortical and medullar collecting ducts. No sulfatide was immuno-stained in the glomerular region. For this reason, the possibility of a direct involvement of anti-Stri1 and Stri2 antibodies in the etiology of Heymann’s nephritis appears to be less likely. A strict correlation between the local sulfatide content, as analyzed by chemical estimation, and the sulfatide expression, as observed by immunohistochemical methods, may not be possible. Accepted: 15 October 1998     

Morphological differentiation of nerve cells in thin organotypic cultures derived from rat hippocampus and cerebellum

Explants of cerebellum and hippocampus were cultured by means of the roller-tube technique. Large nerve cells such as Purkinje cells and pyramidal cells were injected with the fluorescent dye lucifer yellow. The results demonstrate that cultured neurons grow dendritic arborizations in a pattern that is reminiscent of their in situ counterparts. This finding supports the view that under the described culturing conditions nerve cells show a high degree of differentiation despite an altered biochemical and topographical environment.     

Post-translational changes of chromosomal proteins in rat cerebellum during postnatal development

Acetylation, phosphorylation and methylation of nuclear proteins in rat cerebellum at 10 and 30 days of age were investigated in vitro. Isolated nuclei were incubated in the presence of [1-14C]acetyl CoA, S-adenosyl [methyl-3H]methionine and [gamma-32P]ATP and then separated into histones and non histone proteins (NHP), which were further fractionated by polyacrylamide gel electrophoresis. The results obtained indicate that acetylation, phosphorylation and methylation of both basic and acidic proteins decrease from 10 to 30 days of age. Electrophoretic analysis of histones shows that the decrease mainly concerns H1, H3, and H2b fractions. The H3 fraction is always more labeled than the other fractions and shows the major changes during postnatal development. Phosphorylation of H2a and H4 fractions increases from 10 to 30 days of age, whereas acetylation and methylation of these fractions do not show significant changes from 10 to 30 days. The densitometric and radioactive patterns of NHP show considerable changes between 10 and 30 days, especially in the high molecular weight region. The incorporation of 14C-acetyl and 3H-methyl groups and of 32P phosphate appears to be generalized throughout the molecular weight range and decreases from 10 to 30 days of age. The methylation of an as yet unidentified protein with a molecular weight of approximately 110,000 daltons occurred at both ages.     

Rapid preparation of a panel of polyclonal antibodies to Drosophila segmentation proteins

 We describe a method for rapidly raising a panel of high quality polyclonal antibodies from bacterially expressed proteins. Approximately 1²/₃ days of preparation is required per protein. One step that speeds up the procedure is the visualization of purified bands by precipitated sodium dodecyl sulfate (SDS). Antigenicity of the purified recombinant proteins may be increased by precipitation in double-distilled water. The results of using the serums obtained for fluorescent staining of Drosophila embryos are shown. Received: 2 February 1998 / Accepted: 19 February 1998     

High-level generation of polyclonal antibodies by genetic immunization

Antibodies are important tools for investigating the proteome, but current methods for producing them have become a rate-limiting step. A primary obstacle in most methods for generating antibodies or antibody-like molecules is the requirement for at least microgram quantities of purified protein. We have developed a technology for producing antibodies using genetic immunization. Genetic immunization-based antibody production offers several advantages, including high throughput and high specificity. Moreover, antibodies produced from genetically immunized animals are more likely to recognize the native protein. Here we show that a genetic immunization-based system can be used to efficiently raise useful antibodies to a wide range of antigens. We accomplished this by linking the antigen gene to various elements that enhance antigenicity and by codelivering plasmids encoding genetic adjuvants. Our system, which was tested by immunizing mice with >130 antigens, has shown a final success rate of 84%.     

Immunocytochemical localization of microtubule-associated protein 1 in rat cerebellum using monoclonal antibodies

Immunohistochemical staining with monoclonal antibodies showed that microtubule-associated protein 1 (MAP1) has a restricted cellular distribution in the rat cerebellum. Anti-MAP1 staining was found only in neurons, where it was much stronger in dendrites than in axons. There were striking variations in the apparent concentration of MAP1 in different classes of neurons. Purkinje cells were the most strongly labeled, while granule cell neurons gave a faint, threshold-level reaction with the antibody. The reaction of Golgi neurons was intermediate between these two extremes. Equivalent results were obtained using two different methods of tissue preparation. Thus MAP1 appears to be a neuron-specific protein that is highly concentrated in dendrites and occurs at markedly different levels in different types of neurons. These observations provide further indications of heterogeneity among brain microtubules.     

Lead-induced alterations of apoptosis and neurotrophic factor mRNA in the developing rat cortex, hippocampus, and cerebellum

Previous reports have recently shown the prototypic neurotoxicant, lead, to induce apoptosis in the brains of developing organisms. In the current study, timed-pregnant rats were exposed to lead acetate (0.2% in the drinking water) 24 h following birth at postnatal day 1 (PND 1). Dams and pups were continuously exposed to lead through the drinking water of the dam until PND 20. Postnatal exposure in the pups resulted in altered mRNA levels of the following apoptotic and neurotrophic factors: caspase 2 and 3, bax, bcl-x, brain-derived neurotrophic factor (BDNF). Ribonuclease protection assays were conducted to measure the factors simultaneously at the following postnatal time points: 9, 12, 15, 20, 25, days. Our results suggest a brain region- and time-specific response following lead acetate exposure. The region most vulnerable to alterations occurs in the hippocampus with alterations beginning at PND 12, in which caspase 3, bcl-x, BDNF increase with lead exposure. Significant treatment effects were not observed for both the cortex and cerebellum.     

Production and specificity of polyclonal antibodies against soluble proteins from the arbuscular mycorrhizal fungus Glomus intraradices

Rabbit polyclonal antibodies were produced against a soluble protein fraction from a vesicle and spore mixture of the arbuscular mycorrhizal fungus (AMF) Glomus intraradices. The protocol for isolation of vesicles and spores from plant roots was optimized to minimize debris contamination. Protein extract purification and preparation for immunization was adapted to increase protein content and immunogenicity. Active antisera were produced starting from the second boost immunization. Antibodies obtained were specific for surface antigens of AMF and revealed different patterns of soluble protein antigens in G. intraradices, G. constrictum and an unidentified Glomus species. Accepted: 6 December 2000