Immunohistochemical localization of cholinergic nerve terminals

Summary Most of the published light-microscopic methods for the localization of cholinergic nerve pathways present various difficulties of interpretation. The production and characterization of an antiserum that binds specifically to cholinergic terminals is described. The antiserum was raised to small synaptosomes prepared from the purely cholinergic electric organ of Torpedo marmorata. It was shown to lyse cholinergic synaptosomes in a mixed population derived from guinea-pig cortex. After partial purification by adsorption onto nonspecific antigens, it was used to label nerve endings in several tissues of Torpedo, rats and guinea pigs using indirect immunofluorescence histochemistry. The antiserum appears to provide a highly specific means of localizing cholinergic nerve endings in these tissues.     

The localization and rate of disappearance of a synaptic vesicle antigen following denervation

Summary A proteoglycan-specific antiserum has been used to monitor the effects of denervation in the electric organ of Torpedo marmorata. The antiserum was produced by injecting a highly purified synaptic vesicle fraction prepared from the electric organs of Torpedo marmorata. Following absorption the serum appears to be specific towards synaptic vesicles. The ultrastructural localization of the antigen determined by immuno-electron microscopy confirmed the specificity of the antiserum and showed that it did not crossreact with the proteoglycans of the basal lamina. The rate of disappearance of the vesicle proteoglycans following denervation was evaluated by means of the antiserum and was compared to the rate of disappearance of other vesicular and nerve terminal-associated markers. The results suggest that degeneration affects the vesicular constituents at varying rates resulting in a progressive disappearance of the entire functional capacity of the synaptic vesicles.     

Further evidence that glycosaminoglycan specific to cholinergic synaptic vesicles recycles during electrical stimulation of the electric organ of Torpedo marmorata

Summary Semiquantitative immunohistochemical methods were used to demonstrate that at least some of the glycosaminoglycan contained within cholinergic synaptic vesicles is recycled during successive electrical stimulations of the electric organ of Torpedo marmorata.     

Membrane proteins of synaptic vesicles and cytoskeletal specializations at the node of Ranvier in electric ray and rat

Summary Binding sites for antibodies against membrane proteins of synaptic vesicles have been shown to be enhanced at nodes of Ranvier in electromotor axons of the electric ray Torpedo marmorata and sciatic nerve axons of the rat, using indirect immunofluorescence and monoclonal antibodies against the synaptic vesicle transmembrane proteins SV2 and synaptophysin (rat) or SV2 (Torpedo). In the electric lobe of Torpedo, vesicle-membrane constituents occurred at higher density in the proximal axon segments covered by oligodendroglia cells than in the distal axon segments where myelin is formed by Schwann cells. Antibody binding sites were enhanced at nodes forming the borderline of the central and peripheral nervous systems. Filamentous actin was present in the Schwann-cell processes covering both the nodal and the paranodal axon segments as suggested by the pattern of phalloidin labelling. Furthermore, in rat sciatic nerve, Schmidt-Lanterman incisures were intensely labelled by phalloidin. A similar nodal distribution was found for binding sites of antibodies against actin and myosin. Binding of antibodies to tubulin was enhanced at nodes in Torpedo electromotor axons. The apparent nodal accumulation of constituents of synaptic vesicle membranes and the presence of filamentous actin and of myosin are discussed in relation to the substantial constriction of the axoplasm at nodes of Ranvier.     

An immunohistochemical study of synaptogenesis in the electric organ of Torpedo marmorata by use of antisera to vesicular and presynaptic plasma membrane components

Summary Synaptogenesis has been studied in the electric organ of embryonic Torpedo marmorata by use of two antisera directed against components of synaptic vesicles (anti-SV) and presynaptic plasma membranes (ap-anti-TSM), respectively. The anti-SV serum was previously shown to recognize a proteoglycan specific for synaptic vesicles. The ap-anti-TSM serum was raised to plasma membranes of synaptosomes derived from the electromotor nerve terminals and affinity-purified on electric-organ gangliosides. The vesicular antigen was first detectable at the 81-mm stage of development, which is 1–2 weeks earlier than the formation of morphologically mature presynaptic terminals, but is coincident with a rise in choline acetyltransferase levels and the ability of the electric organ to generate discharges. The gangliosidic antigen recognized by the ap-anti-TSM was first detectable on the ventral electrocyte surface at the 93-mm stage of development. This indicates that specific carbohydrate epitopes, not present on the growth cones, are expressed during maturation of the nerve terminal. The nerve terminal components recognized by these sera arose pari passu with neurite coverage of the ventral surface of the electrocyte, reaching a maximum in the adult. In contrast, postsynaptic aggregates of acetylcholine receptor, rendered visible with rhodamine-labeled -bungarotoxin, arose previous to the presynaptic antigens, reaching a maximum surface density at 110 mm and then declining in the adult.     

Profile of a 24 kDa Mycobacterium tuberculosis antigen in the urine samples of tuberculosis patients under therapy regime

Secretion of a low molecular weight (24 kDa) Mycobacterium tuberculosis-specific antigen was analysed in the urine samples of tuberculosis patients under antimycobacterial therapy regime. The urine samples of sputum-positive and culture-positive tuberculosis patients under therapy regime were collected after 2, 3, 4, 5 and 6 months of antimycobacterial therapy. After concentration of the samples by ultrafiltration the proteins were resolved by SDS–PAGE. The antibodies raised in rabbits against M. tuberculosis strain H₃₇Ra were used to check specificity of the bands by Western blotting. It was found that the tuberculosis patients secreted a 24 kDa M. tuberculosis-specific antigen in their urine. This band was present in the samples taken upto 5 months of drug therapy but was absent in the samples taken after 6 months of antimycobacterial therapy. This study gives evidence in support of the continuation of chemotherapy of tuberculosis patients for at least 6 months. Also, the 24 kDa excreted/secreted antigen can be used as a marker for monitoring the drug therapy as well as for the diagnosis of tuberculosis patients. Moreover, the urine sample taken in the study is a non-invasive and safer sampling method as compared to sampling blood or sputum which is the usual procedure in these patients.     

Immunohistochemical localization of laminin,neural cell adhesion molecule,collagen type IV and T-61 antigen in the embryonic retina of the Japanese quail by in vivo injection of antibodies

Summary Antibodies against laminin (LN), fibronectin (FN), collagen type IV (Col IV), neural cell adhesion molecule (N-CAM), T-61 antigen, actin, tubulin and neurofilament protein were injected into the eyes of quail embryos (Coturnix coturnix japonica) of different ages. Twenty h after injection, the heads of the embryos were fixed and the antibodies visualized in sections with the use of fluorescein-isothiocyanate (FITC) or peroxidase-labeled second antibodies by light- and electron microscopy. Antibodies against cell surface molecules, such as N-CAM, LN, Col IV and T 61, labeled matrix and membrane components of the retinal cells in different antigen-specific patterns. Antibodies against intracellular antigens, such as actin, tubulin and neurofilament protein labeled nonspecifically the vitreous body and the inner basal lamina of the retina, but resulted in only a very weak and diffuse labeling of retinal cells. N-CAM was detected in high concentration in the optic fiber layer on the surface of axons and on the membranes of all retinal cells. Col IV, LN and T 61 antigen were found predominantly in the optic fiber layer. LN and Col IV were located on the surface of axons and the endfeet of ventricular (neuroepithelial) cells in a patchy distribution. The T-61 antigen was found in early stages in the cell-free space of the optic fiber layer, on the surface of ventricular cells and axons, and at later stages also in high-density patches between nerve fibers. The distribution of LN and T-61 antigen together with data from in vitro experiments suggests a crucial role of these proteins in axon extension in the avian retina during early development of the optic fiber layer.     

家蚕蜕皮液羧肽酶A的表征及免疫荧光定位分析

蜕皮是许多变态发育昆虫的一种重要生理现象,昆虫通过蜕皮液中的酶对新旧表皮进行分离。已有相关蛋白组学的研究证明,家蚕蜕皮液中具有一种含量丰富的羧肽酶A(Bombyx mori-carboxypeptidase A, Bm-CPA),目前对其作用功能尚不清楚。为了更好地了解Bm-CPA在家蚕蜕皮发育过程的作用,本研究通过生物信息学分析、实时荧光定量PCR、抗体制备、免疫荧光染色和毕赤酵母表达等方法对Bm-CPA进行了研究。结果显示,Bm-CPA具有保守的M14锌羧肽酶结构域和糖基化位点,并且受蜕皮激素(20-hydroxyecdysone, 20E)调控,在眠期和上簇期的表皮中大量表达;免疫荧光染色显示Bm-CPA在眠期的表皮中富集,Bm-CPA抑制剂会导致幼虫因无法蜕皮而死亡;通过毕赤酵母表达系统在体外成功获得大量的重组Bm-CPA蛋白。这些结果为深入了解家蚕蜕皮发育过程提供了一定的参考。     

Immunohistochemical localization and radioenzymatic measurements of serotonin (5-hydroxytryptamine) in hearts of Aplysia and several bivalve mollusks

Summary Serotonin immunoreactivity was localized in hearts of the opisthobranch gastropod, Aplysia californica (sca hare) and several species of bivalve mollusks, the heterodonts, Mercenaria mercenaria (quahog or cherry stone clam), Protothaca staminea (little neck clam), and the pteriomorphs, Hinnites multirugosus (rock scallop), Crassostrea virginica (eastern oyster), Mytilus edulis (eastern mussel), and Geukensia demissa (ribbed mussel). In addition, serotonin was assayed in the ventricles, auricles and heart-associated tissues in A. californica, M. mercenaria, H. multirugosus, and G. demissa with a sensitive radioenzymatic assay. Serotonin concentrations and the density of innervation were significantly higher in members of the subclass Heterodonta compared to the subclass Pteriomorpha. Serotonin immunoreactivity was observed in all species surveyed except G. demissa, which also contained relatively low concentrations of serotonin. Varicose fibers presumably corresponding to release sites were localized in the ventricles, auricles, and the auricular-ventricular valves. We hypothesize that in the species where serotonin-immunoreactive fibers are present, serotonin serves to modulate cardiac myogenic activity. The significance of the observed distribution and concentration of serotonin to the physiological effects of serotonin on cardiac function in these species is discussed.     

Changes in axonal transport in neurones of Asterias vulgaris and Asterias forbesei produced by colchicine and dimethyl sulfoxide

Summary Gas flow analysis, autoradiography, and electron microscopy were performed on radial nerve cords of sea stars injected intragastrically with [¹⁴C] leucine tracer, a precursor of axonal proteins. Neurotubules, neurofilaments, and axonal transport were demonstrated for the first time in sea stars. This transport occurred at two rates (fast phase: 240–480 mm/da, slow phase: 20–40 mm/da) moving in both the cellulifugal and centripetal directions. The rate in the centripetal direction was slightly slower. Displacement of peaks of radioactivity appeared cyclic.Transneuronal transport apparently occurred between several groups of neurones suggesting the possibility of metabolic communication, perhaps across the synapse, between groups of neurones or even areas of the nervous system. Regenerating nerve cords demonstrated an increase in the rate (400–600 mm/da) and quantity (6–11 times as much) of label transported to cord termini.The relationship between colchicine and the disruption of neurotubular integrity, resulting in disturbances in fast phase transport, was confirmed. More significantly, the association between nerve injury and site or concentration of treatment is emphasized. Treatment of nerve cords with dimethyl sulfoxide (DMSO) produced more subtle effects than did colchicine, reversible 4–6 hours following the local treatment of the involved nerves. Axon bundles swelled and the organization of axons within the bundles was distorted. Stereochemical structure was suggested from this work as playing a key role in effecting fast phase transport.The authors are indebted to Dr. B. Kiefer, Department of Biology, Wesleyan University, Dr. L. Ross, Department of Anatomy, Medical College of Pennsylvania, and Dr. M. Shelanski, Department of Molecular Biology, Harvard University for their help and constructive criticism.     

Heterogeneity of auxin-accumulating membrane vesicles from Cucurbita and Zea: a possible reflection of cell polarity

When microsomes from hypocotyls of Cucurbita pepo L. or coleoptiles of Zea mays L. were centrifuged on dextran-sucrose gradients a heterogeneity of auxin-accumulating vesicles was observed. Vesicles from the top part of the gradient showed saturable, specific accumulation of indole-3-acetic acid with only a small stimulation by phytotropins, and with very few binding sites for 1-N-naphthylphthalamic acid. In the vesicles from the lower part of the gradient, net accumulation of indole-3-acetic acid could be strongly increased by addition of phytotropins; binding of 1-N-naphthylphthalamic acid was high in this region. After two-phase partitioning, both kinds of vesicles were found in the upper-phase membrane fraction considered to be purified plasma membrane. The hypothesis is discussed that vesicles can be separated from the apical and basal parts of the cell's plasmalemma.Abbreviations CCO cytochrome-c oxidase - CCR KCN-insensitive NADH-dependent cytochrome-c reductase - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IDPase inosine 5-diphosphatase - ION3 ionophore mixture of carbonylcyanide-3-chlorophenylhydrazone, nigericin and valinomycin - 1-NAA 1-naphthaleneacetic acid - NPA 1-N-naphthylphthalamic acid - PBA 2-(1-pyrenoyl)benzoic acid - UDPG uridine diphosphoglucose     

Functional and spectral characterization of the respiratory chain of Chloroflexus aurantiacus grown in the dark under oxygen-saturated conditions

In this paper we attempt a functional and spectral characterization of the membrane-bound cytochromes involved in respiratory electron transport by membranes from cells of Chloroflexus aurantiacus grown in the dark under oxygen saturated conditions. We conclude that the NADH-dependent respiration is carried out by a branched respiratory chain leading to two oxidases which differ in sensitivity to CN^- and CO. The two routes also show a different sensitivity to the ubiquinone analogue, HQNO, the pathway through the cytochrome c oxidase being fully blocked by 5 M HQNO, whereas the alternative one is insensitive to this inhibitor. The cytochrome c oxidase containing branch is composed by at least two c-type haems with E _m 7.0 of +130 and +270 mV ( bands at 550/553 nm and 549 nm, respectively), plus a b-type cytochrome with E _m 7.0 of +50 mV ( band at 561 nm). From this, and previous work, we conclude that respiratory and photosynthetic electron transport components are assembled together and function on a single undifferentiated plasma membrane.Abbreviations HQNO heptylhydroxy-quinoline-N-oxide - UHDBT undecyl-hydroxydioxobenthiazole - Q/b-c ubiquinol/cytochrome c oxidoreductase complex - BChl bacteriochlorophyll     

An improved method of measuring photosynthetic electron transport in Euglena under in vivo conditions

A method is described to measure photochemical activity in intact cells of Euglena under in vivo conditions. The method employs a cell wall digesting enzyme (cellulysin) to induce enough permeability in the cell walls and membranes in order to allow dyes, commonly used to investigate light-dependent electron transport reactions to enter, but without inducing a concomittant efflux of metabolites. Between 1 and 2 h of incubation in 5% (w/v) cellulysin provided conditions which allowed measurement of photosystem I-, II- and I+II-dependent electron transport with rates up to 600% higher than in control cells; whereas other cell wall degrading enzymes (cellulase and pectinase) still did not increase the entry of the dyes. Cellulysin up to 2 h of incubation had little or no effect on whole cell respiration, photosynthetic O₂ evolution, or the export of potassium and (¹⁴C) labeled compounds out of cells; therefore cellulysin obviously did not change the normal habit or physiology of Euglena. Cellulysin (4 h digestion), cellulase and pectinase (2–4 h of incubation) on the other hand led to a lowering of respiration and light-dependent O₂ evolution, and increased the efflux of K⁺, but apparently decreased that of (¹⁴C)labeled fixation products.Abbreviations DBMIB dibromothymoquinone - DCPIP 2,6-dichlorophenol-indophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMMIB 2,3-dimethyl-5,6-methylenedioxy-p-benzoquinone - MV methylviologen - PSI photosystem I - PS II photosystem II     

An ultrastructural analysis of electromotor cell death in Torpedo marmorata and its counterpart in vitro

Summary Naturally occurring neuronal cell death has been investigated in the electromotor system of Torpedo marmorata and compared with neuronal death seen in expiant cultures of tissues from T. marmorata electric lobe. One objective of the study was to determine whether cell death in vitro was morphologically the same as cell death in vivo and to substantiate the validity of using in vitro models for studying naturally occurring cell death. Sequences of degeneration in vitro and in vivo have been established and compared: a single morphological sequence best represents this form of cell death in both conditions. In vivo, dying neurons are seen at all depths of the electric lobe indicating the involvement of different generations. Retrograde degeneration appears to be the first sign of cell death.     

Ultrastructural localization of Lucifer Yellow and endocytosis in plant cells

Summary The fluorescent dye Lucifer Yellow CH (LYCH) was localized at the ultrastructural level with a precipitation method using barium chloride. Applying this technique, endocytosis of LYCH was examined in the nutrient absorptive trichomes of a carnivorous bromeliad. After a two hour incubation, the electron dense reaction product was localized in the membrane compartments of the endocytotic system. These structures included coated regions of the plasma membrane, coated and smooth vesicles, dictyosomes, partially coated reticulum, and smooth endoplasmic reticulum. This procedure demonstrates for the first time at the ultrastructural level endocytosis in whole plant cells, using a non-toxic compound.Abbreviations ER endoplasmic reticulum - BaCl₂ barium chloride - LYCH Lucifer Yellow CH - PCR partially coated reticulum     

Expression and characterization of a parasite-specific antigen on macrophages after infection withLeishmania donovani

A rabbit polyclonal antibody to crude soluble antigen ofLeishmania donovani promastigotes recognized a determinant expressed on the surface membrane of mouse peritoneal macrophages and human monocyte derived macrophages infectedin vitro. The determinant was recognized on infected macrophage surface only when F (ab)₂ fragments of anti-leishmanial antiserum was employed in immunofluorescence. F(ab)₂ fragments of human patient sera also could recognize the determinant. The expression of this antigen was not stage-specific for the parasite. Immunochemical analyses revealed this antigen to be of 51 kDa protein. Specific leaching of membrane proteins by trypsin showed three bands of expressed antigens of 26, 11 and 10 kDa, which in all likelihood might be arising from the 51 kDa antigen. The antigen was not expressed until 12 h of post infection, reached a maximum level at 24 h and thereafter attained a steady state level as studied upto 96 h of post infection. This typc of antigen might have a great potential in immunodiagnostics and site-specific drug targeting.     

Localization of substance P mRNA in cholinergic cells of the rat laterodorsal tegmental nucleus:In situ hybridization histochemistry and immunocytochemistry

1. In situ hybridization histochemical techniques in combination with immunocytochemistry and acetylcholinesterase (AChE) histochemistry were used to study the colocalization of messenger RNA (mRNA) encoding the neuropeptide substance P (SP) in cholinergic cells of the laterodorsal tegmental nucleus (LDT) of the rat pontine brain stem. 2. Alternate serial sections were hybridized with a 48-base, 35S-labeled synthetic oligonucleotide probe encoding SP using in situ hybridization histochemistry and processed either histochemically for AChE or immunocytochemically for choline acetyltransferase (ChAT). 3. In addition, serial section analysis was used to demonstrate the correlation between SP and SP mRNA in the same cells of the LDT. 4. These studies reveal that the cholinergic neurons of the LDT synthesize SP.     

Suitability of yeast- and Escherichia coli-expressed hepatitis B virus core antigen derivatives for detection of anti-HBc antibodies in human sera

Antibody to hepatitis B virus core antigen (anti-HBc) is one of the most important serological markers during hepatitis B virus (HBV) infection. The quality of the hepatitis B virus core antigen (HBcAg; diagnostic antigen) is crucial to the accuracy of anti-HBc detection. In an attempt to explore the suitability of recombinant HBcAg (rHBcAg) for diagnostic purposes, HBcAg was expressed in Escherichia coli (E. coli) and Pichia pastoris (P. pastoris) and evaluated for the detection of anti-HBc. The expression level of the recombinant protein satisfied the criteria for large-scale biologic production. P. pastoris- and E. coli-derived rHBcAg were purified with gel filtration followed by sucrose gradient (reagents A and C) or with a monoclonal anti-HBc antibody binding (reagents B and D) and were utilized to detect anti-HBc in competitive inhibition enzyme-linked immunosorbent assay (ELISA) format. The ELISA using P. pastoris-derived rHBcAg had a higher specificity and sensitivity than that using E.coli-derived rHBcAg to detect the anti-HBc standard panel. Serum specimens were collected from HBV-infected patients and healthy individuals (voluntary blood donors). Anti-HBc was detected in those specimens using P. pastoris- and E. coli-derived rHBcAg. The positive rate of anti-HBc detection in HBV-infected patients' sera was 100% with reagents A and B, 96.4% with reagent C, and 93.6% with reagent D. The negative rate in healthy control sera was 100% with reagents A and B, 97.0% with reagent C, and 99.7% with reagent D. These data indicate that P. pastoris-derived rHBcAg is superior to E.coli-derived rHBcAg for the detection of anti-HBc using the diagnostic ELISA.