Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly committed intermediate progenitor subtypes. Each of these subtypes expresses a set of specific molecular markers that, together with specific morphological criteria, can be used for their identification. Typically, immunofluorescent techniques are applied involving subtype-specific antibodies in combination with exo- or endogenous proliferation markers. We herein describe immunolabeling methods for the detection and quantification of all stages of adult hippocampal neurogenesis. These comprise the application of thymidine analogs, transcardial perfusion, tissue processing, heat-induced epitope retrieval, ABC immunohistochemistry, multiple indirect immunofluorescence, confocal microscopy and cell quantification. Furthermore we present a sequential multiple immunofluorescence protocol which circumvents problems usually arising from the need of using primary antibodies raised in the same host species. It allows an accurate identification of all hippocampal progenitor subtypes together with a proliferation marker within a single section. These techniques are a powerful tool to study the regulation of different progenitor subtypes in parallel, their involvement in brain pathologies and their role in specific brain functions.     

Complement Requirement of Neutralizing Antibodies in Different Classes of Immunoglobulin Appearing in Rabbits and Guinea Pigs after Primary and Booster Immunizations with Herpes Simplex Virus

Rabbits and guinea pigs were immunized with herpes simplex virus and bled periodically. The sera were fractionated into slow IgG, fast IgG and IgM by DEAE-cellulose column chromatography, and complement-requiring (CRN) and nonrequiring neutralizing (N) antibody activities were estimated. In early sera of rabbits, the two IgG and IgM fractions possessed about equal CRN activities, although some animals showed a slightly lower activity in fast IgG. In guinea pigs, the early CRN activity resided mainly in slow IgG (7 S γ₂). The early IgG antibody of guinea pigs differed from that of rabbits in that it resembled IgM in resistances to heating at 70 C and to 2-mercaptoethanol. The level of CRN IgM antibody in rabbits declined following a peak reached in 2 to 3 weeks, whereas such a decline was never observed in guinea pigs. N IgG antibody was developed a few weeks after the first immunization in rabbits and much retarded in guinea pigs. In both species, booster immunization quickly evoked N antibody in the two IgG fractions and also CRN IgM antibody, but in the case of rabbits the IgM antibody disappeared soon. It is concluded that IgG plays an important role in humoral immunity from the initial stage of the immunization course.     

Enzymatic Activities of Allergen Extracts from Three Species of Dust Mites and Cockroaches Commonly Found in Korean Home

Allergen extracts from dust mites and cockroaches commonly found in Korean homes were used to evaluate their enzymatic activity as they are believed to influence allergenicity. Allergen extracts were prepared from 3 dust mite species (Dermatophagoides farinae, D. pteronyssinus, and Tyrophagus putrescentiae) and 3 cockroach species (Blattella germanica, Periplaneta americana, and P. fuliginosa) maintained in the Korea National Arthropods of Medical Importance Resource Bank. Proteins were extracted in PBS after homogenization using liquid nitrogen. The activities of various enzymes were investigated using the API Zym system. No significant difference in phosphatase, lipase, or glycosidase activity was observed among the 6 allergen extracts, but much difference was observed in protease activity. Protease activity was assessed in more detail by gelatin zymography and the EnzChek assay. Extract from T. putrescentiae showed the highest protease activity, followed by those of the cockroach extracts. Extracts from D. farinae and D. pteronyssinus showed only weak protease activity. Gelatinolytic activity was detected mainly in a 30-kDa protein in D. farinae, a 28-kDa protein in D. pteronyssinus, a > 26-kDa protein in T. putrescentiae, a > 20-kDa protein in B. germanica, and a > 23-kDa protein in P. americana and P. fuliginosa. The information on various enzymatic activities obtained in this study may be useful for future studies. In particular, the strong protease activity found in cockroach extracts could contribute to sensitization to cockroach allergens, which is known to be associated with the development of asthma.     

Enzymatic synthesis of (+)- and (-)-bisdechlorogeodin with sulochrin oxidase from Penicillium frequentans and Oospora sulphurea-ochracea

Sulochrin oxidase is a blue copper-containing glycoenzyme that catalyzes a stereospecific formation of bisdechlorogeodin from sulochrin. The enzyme has been isolated from Penicillium frequentans and Oospora sulphureaochracea which catalyzes the formation of (+)-form and (-)-form of bisdechlorogeodin respectively. The Penicillium enzyme has a molecular weight of 157,000 and contains 19.5% of carbohydrates. Amino acid and carbohydrate compositions are given. The enzyme has probably a dimeric structure containing 6 Cu-atoms. Apparent K _m-values of various substrates are presented. The Oospora enzyme has a molecular weight of 128,000 and except for its stereospecificity its properties are very similar to those of the Penicillium enzyme.     

Immunochemical and Enzymatic Comparisons of the Tryptophan Synthase α Subunits from Five Species of Enterobacteriaceae

The reactive surface structures of alpha subunits of tryptophan synthase from Escherichia coli, Shigella dysenteriae, Salmonella typhimurium, Aerobacter aerogenes, and Serratia marcescens were compared by measuring (i) their reactivities in micro-complement-fixation assays with antibodies directed specifically to E. coli wild-type alpha subunit, (ii) their reactivities in enzyme neutralization assays with the same antibodies, and (iii) their binding affinities for tryptophan synthase beta(2) subunits. The enzymes from the four heterologous species cross-reacted in the microcomplement-fixation assays with the anti-E. coli alpha subunit antibodies, each to a different degree. However, neutralization titers of the antibodies reacting with the various alpha subunits were comparatively similar, and the beta(2) subunit-binding and -stimulating abilities of the alpha subunits were even more closely alike. The results suggested that the tertiary structure of the beta(2) subunit-binding site of the alpha subunit has been conserved, relative to the rest of the molecule, during the evolutionary divergence of the species of Enterobacteriaceae.     

Monoclonal Antibodies to Rat Brain Acetylcholinesterase: Comparative Affinity for Soluble and Membrane-Associated Enzyme and for Enzyme from Different Vertebrate Species

Seven unique monoclonal antibodies were generated to rat brain acetylcholinesterase. Upon density gradient ultracentrifugation, immunoglobulin complexes with the monomeric enzyme appeared as single peaks of acetylcholinesterase activity with a sedimentation coefficient approximately 3S greater than that of the free enzyme. This behavior is consistent with the assumption of one binding site per enzyme molecule. Apparent dissociation constants of these antibodies for rat brain acetylcholinesterase calculated on the basis of this assumption ranged from about 10 nM to more than 1,000 nM. Some of the antibodies were less able to bind the membrane-associated enzyme that required detergent for solubilization than the naturally soluble acetylcholinesterase of detergent-free brain extracts. Species cross-reactivity was investigated with crude brain extracts from mammals (human, mouse, rabbit, guinea pig, cow, and cat) and from other vertebrates (chicken, frog, and electric eel). Three antibodies bound rat acetylcholinesterase exclusively; one had nearly the same affinity for all mammalian acetylcholinesterases investigated; the remaining three showed irregular binding patterns. None of the antibodies recognized frog and electric eel enzyme. Pooled antibody was found to be suitable for specific immunofluorescence staining of large neurons in the ventral horn of the rat spinal cord and smaller cells in the caudate nucleus. Other potential applications of these antibodies are discussed.     

Population Density of Chimpanzees and Gorillas in the Petit Loango Reserve,Gabon: Employing a New Method to Distinguish Between Nests of the Two Species

We studied the vegetation and population density of chimpanzees (Pan troglodytes troglodytes) and gorillas (Gorilla gorilla gorilla) in the Petit Loango Reserve, Gabon. The study site consists mainly of primary and secondary forest, and it also includes patches of savanna, inundated forest, and coastal scrub. The density of herbaceous plants of Marantaceae and Zingiberaceae, which serve as important foods for great apes at other study sites, is extremely low. It was very difficult to distinguish between the nests of chimpanzees and gorillas because most gorilla nests were made in trees. We developed a new method to identify the builder of nests via a combination of field observation of nests and fecal samples, and examination of hair samples under a scanning electron microscope. Nests of chimpanzees were mainly in primary forest, and the estimated density (0.78 individual per km ² ) is higher than the average density in Gabon and comparable to the nest density in the Lopé Reserve, Gabon. Nests of gorillas were mainly in secondary forest, and their estimated density (0.21) is similar to the average next density in Gabon but lower than the gorilla nest density in the Lopé Reserve. The low density of herbaceous foods seemed to influence the density of gorillas but not the density of chimpanzees.     

菊花不同部位及根际土壤水浸液对其扦插苗生长的自毒效应

以自毒性较强的菊花品种 '高压太子' 插穗为材料,调查了菊花枯落物、叶、茎、根系和根际土壤水浸液处理对其扦插苗生长和生理指标的影响,以探讨菊花自毒作用机制.结果表明,菊花不同部位水浸液处理对其扦插生根的根长、鲜重及根数均有抑制作用,并以叶和枯落物水浸液对根长的抑制作用最强,且均达到极显著水平;随水浸液浓度的升高,其对扦插苗根长、鲜重及根数的抑制作用越明显.水浸液处理导致扦插苗叶片可溶性糖、可溶性蛋白和叶绿素含量显著下降,显著抑制根系脱氢酶与叶片硝酸还原酶活性.可见,菊花地上部和根际土水浸液可能通过抑制其扦插苗根系脱氢酶与叶片硝酸还原酶活性,降低叶片叶绿素、可溶性糖和可溶性蛋白的含量,在营养水平上抑制不定根的形成而产生明显的自毒效应,且该自毒作用表现出明显的浓度梯度效应和部位差异.     

检测梅毒螺旋体抗体的纤维膜条方法的建立及其在诊断中的应用

目的：建立以纤维膜为载体的检测梅毒螺旋体抗体的方法,检查病人血清中对梅毒螺旋体多种抗原的抗体,用于梅毒感染的诊断。方法：将基因工程表达及纯化的梅毒螺旋体蛋白tp15、tp17、tp42和tp47分别结合在纤维膜上,用载抗原的纤维膜条检查血清中的抗体,抗体阳性者在相应抗原位置显示出特异条带。结果：梅毒螺旋体感染者血清中存在特异性抗体,在检查的460份临床诊断的患者血清中,对tp15、tp17、tp42和tp47抗原的抗体检出率分别为41.3%、100%、98.7%和51.7%;134份献血员血清抗体阴性。结论：建立的检测梅毒螺旋体感染的方法可同时检查对多种抗原的抗体,以纤维膜条作为诊断条检测血清抗体方法简便,用于临床诊断更特异、更敏感。     

Characteristics of Polygalacturonate Lyase C from Bacillus subtilis 7-3-3 and Its Synergistic Action with PelA in Enzymatic Degumming

An alkaline polygalacturonate lyase (PGL) from Bacillus subtilis 7-3-3, PelC, with diverse depolymerization abilities for different pectin substrates was found. The PGL activity of PelC decreased with increasing degree of methyl esterification of the substrate. PelA and PelC displayed notable synergistic effects in the enzymatic degumming of ramie fibers. Gum loss rates increased by 62% when PelC was used to replace up to three-eighths of the PelA dose (PelC, 60 U g⁻¹ ramie fibers). To the best of our knowledge, this study is the first to report the synergistic action of members of polysaccharide lyase families 1 and 3, represented by PelA and PelC, respectively. The present paper provides new insights into the improvement and production of enzymes used in enzymatic degumming.     

新生大鼠肺成纤维细胞的原代培养改良法及细胞鉴定

探讨新生大鼠肺成纤维细胞原代培养的改良方法及细胞鉴定。用胰酶消化组织块结合的方法提取新生大鼠肺成纤维细胞,并纯化细胞,对肺成纤维细胞进行形态学观察,用HE染色及免疫组化染色法对细胞进行鉴定,并用MTT法测定细胞生长曲线。倒置相差显微镜下观察选用改良法获得的细胞,3 d后可见组织块周边有少许细胞,5 d后组织块周围有大量细胞爬出,生长迅速,10 d接近融合。经改良后的方法纯化细胞,细胞活性状态较好的为3~5代,5代以后的细胞增殖能力下降。对第3代肺成纤维细胞进行HE染色,镜下可见形态典型的成纤维细胞,免疫组化结果显示波形蛋白(Vi-mentin)阳性表达,细胞角蛋白(cytokeratin)阴性表达。MTT法检测第3代细胞于3~5 d处于对数生长期。胰酶消化组织块结合法是一种可靠快速的肺成纤维细胞分离纯化的培养方法,使用这种方法可得到具有典型形态特征且活性较好的肺成纤维细胞,初学者容易掌握。     

Purification of Glycogen Phosphorylase from Bovine Brain and Immunocytochemical Examination of Rat Glial Primary Cultures Using Monoclonal Antibodies Raised Against This Enzyme

The physiological function in brain of glycogen and the enzyme catalyzing the rate-limiting step in glycogenolysis, glycogen phosphorylase (EC 2.4.1.1), is unknown. As a first step toward elucidating such a function, we have purified bovine brain glycogen phosphorylase isozyme BB 1,700-fold to a specific activity of 24 units/mg protein. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent silver staining, a single major protein band corresponding to an apparent molecular mass of 97 kDa was observed. Mouse monoclonal antibodies raised against the enzyme were purified and shown to be monospecific as indicated by immunoblotting. Immunocytochemical examination of astroglia-rich primary cultures of rat brain cells revealed a colocalization of glycogen phosphorylase with the astroglial marker glial fibrillary acidic protein in many cells. The staining for the enzyme appeared at two levels of intensity. There were other cells in the culture showing no specific staining under the experimental conditions employed. Neurons in neuron-rich primary cultures did not show positive staining. The data suggest that glycogen phosphorylase may be predominantly an astroglial enzyme and that astroglia cells play an important role in the energy metabolism of the brain.     

Species complexes in Hericium (Russulales, Agaricomycota) and a new species - Hericium rajchenbergii - from southern South America

Species of the genus Hericium are generally recognized by macro morphology of their basidiomes, while they are very similar in the microscope. As a result, species boundaries are not always clear, and many collections are subsumed under incorrect names. We report on a thorough phylogenetic analysis based on ITS sequences to clarify the application of the most common names in the genus. H. alpestre, H. americanum, and H. erinaceus cannot readily be distinguished by their ITS sequences alone, while they are kept separate by characters from substrate preferences, geographical distribution, and macro morphology. A neotype is selected for H. alpestre. A well supported clade includes H. coralloides together with other, unnamed taxa. One of these, H. rajchenbergii from Argentina, is here described as new to science.     

Potassium channels from NG108-15 neuroblastoma-glioma hybrid cells. Primary structure and functional expression from cDNAs

The complete amino acid sequences of two potassium channel proteins from NG108-15 neuroblastoma-glioma hybrid cells have been deduced by cloning and sequencing the cDNAs. One of these proteins (NGK2) is structurally more closely related to the Drosophila Shaw gene product than to the Shaker and Shab gene products, whereas the other (NGK1) is identical with a rat brain potassium channel protein (BK2) which is more closely related to the Drosophila Shaker gene product. mRNAs derived from both the cloned cDNAs, when injected into Xenopus oocytes, direct the formation of functional potassium channels with properties of delayed rectifiers.     

A Simplified Method for Isolating Highly Purified Neurons, Oligodendrocytes, Astrocytes, and Microglia from the Same Human Fetal Brain Tissue

Elucidation of the underlying pathogenic mechanisms leading to apoptosis of neurons and oligodendrocytes and activation of microglia and astrocytes in different neurodegenerative and neuroinflammatory disorders remains a challenge in neuroscience. In order to overcome the challenge and find out therapeutic remedies, it is important to study live and death processes in each and every cell type of the brain. Here we present a protocol of isolating highly purified microglia, astrocytes, oligodendrocytes, and neurons, all four major cell types of the CNS, from the same human fetal brain tissue. As found in vivo, these primary neurons and oligodendroglia underwent apoptosis and cell death in response to neurodegenerative challenges. On the other hand, astroglia, and microglia, cells that do not die in neurodegenerative brains, became activated after inflammatory challenge. The availability of highly purified human brain cells will increase the possibility of developing therapies for different neurodegenerative disorders. M. Jana and A. Jana have equal contribution to the work.     

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver^1-3. Although the isolation methods of liver macrophages have been well-described^4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.After dissociation of the liver cells by two-step perfusion method^7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 10⁶ cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells⁹.In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.     

A Rapid and Sensitive Method for Determination of Relative Specificity of RuBisCO from Various Species by Anion-Exchange Chromatography

A rapid and sensitive method to determine the relative specificity() of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO)using anion-exchange chromatography is described. We employedthe open gas system for the reaction of RuBisCO to get the mostreliable CO₂ and O₂ concentrations in the reaction mixture.3-Phosphoglycerate and 2-phosphoglycolate which were formedin the RuBisCO reaction were completely separated and directlymeasured with anion-exchange chromatography without using radioisotopes.The determination of the value was accomplished in 3 h. The values of RuBisCO enzymes from higher land plants were between90 and 96, and those from bacteria including cyanobacteriumwere close to 45. These values were in agreement with previouslyreported values. The enzyme of the red macroalga Porphyra yezoensisexhibited a value of over 140, as expected from the reportedvalue of the enzyme from the red microalga Porphyridium cruenteum.RuBisCO from the green macroalga Ulva pertusa had a value closeto 70 and similar to that of the enzyme from the green microalgaChlamydomonas reinhardtii. ⁴On leave from Research and Development Center, Unitika Ltd.,23 Kozakura, Uji, Kyoto, 611 Japan. ⁵Present address: Nara Institute of Science and Technology,8916-5 Takayama-Cho, Ikoma, Nara, 630-01 Japan     

Comparative Electrophoretic Analyses of Soluble Proteins from Heterodera glycines Races 1-4 and Three Other Heterodera Species

Modified polyacrylamide gel and SDS-polyacrylamide gel electrophoretic systems using a low molarity tris-HCl buffer and equal pH of homogenizing buffer and stacking gel provided improved stacking for separation of soluble proteins from Heterodera schachtii, H. trifolii, H. lespedezae, and H. glycines races 1, 2, 3, and 4, compared with previous studies with cyst nematodes, The four Heterodera species were easily distinguished using the polyacrylamide gel system, but H. trifolii and H. lespedezae had similar protein patterns. H. glycines races were not separable by that system. The SDS-polyacrylamide gel system produced different protein patterns for all four Heterodera species although H. trifolii and H. lespedezae differed by only a single band, suggesting that these two may be subspecifically related. A protein band unique to H. glycines races 3 and 4 was not detected in SDS-polyacrylamide gel profiles from races 1 and 2. Molecular weight determinations were 55,000 for distinctive proteins in profiles of H. trifolii and 75,000 for H. glycines races 3 and 4.