Histamine-sensitizing Factor, Mouse-protective Antigens, and Other Antigens of Some Members of the Genus Bordetella

The three species of the genus Bordetella-B. pertussis, B. parapertussis, and B. bronchiseptica-have many antigens in common. Studies on representative strains of these species have shown that there are only a few specific antigens in each species. Whole-cell vaccines and extracts from B. pertussis contained specific mouse-protective antigen and a histamine-sensitizing factor. In addition, whole-cell vaccines and some saline extracts protected mice against intracranial challenge with B. bronchiseptica. Cells and a saline extract of B. parapertussis also protected against B. bronchiseptica but not against B. pertussis. Whole cells of B. bronchiseptica protected against B. bronchiseptica, but only one of three saline extracts protected against this challenge. Neither whole cells nor saline extracts from B. bronchiseptica protected against B. pertussis. The antigen in B. pertussis responsible for cross-protection against B. bronchiseptica was less resistant to heat than the protective antigen in B. bronchiseptica. Since histamine-sensitizing factor was not detected in B. bronchiseptica or B. parapertussis cells or extracts, this factor is not required to protect mice against B. bronchiseptica challenge. Whether B. pertussis vaccines protected against B. bronchiseptica by a nonspecific mechanism was not established, but it is clear that the specific antigen responsible for protection against B. pertussis was found only in B. pertussis and not in B. bronchiseptica or B. parapertussis.     

Initiation of simian virus 40 DNA replication in vitro.

Exogenously added simian virus 40 (SV40) DNA can be replicated semiconservatively in vitro by a mixture of a soluble extract of HeLa cell nuclei and the cytoplasm from SV40-infected CosI cells. When cloned DNA was used as a template, the clone containing the SV40 origin of DNA replication was active, but a clone lacking the SV40 origin was inactive. The major products of the in vitro reaction were form I and form II SV40 DNAs and a small amount of form III. DNA synthesis in extracts began at or near the in vivo origin of SV40 DNA synthesis and proceeded bidirectionally. The reaction was inhibited by the addition of anti-large T hamster serum, aphidicolin, or RNase but not by ddNTP. Furthermore, this system was partially reconstituted between HeLa nuclear extract and the semipurified SV40 T antigen instead of the CosI cytoplasm. It is clear from these two systems that the proteins containing SV40 T antigen change the nonspecific repair reaction performed by HeLa nuclear extract alone to the specific semiconservative DNA replication reaction. These results show that these in vitro systems closely resemble SV40 DNA replication in vivo and provide an assay that should be useful for the purification and subsequent characterization of viral and cellular proteins involved in DNA replication.     

Quantitation of M antigen in Lancefield extracts of group A streptococci type 12 using electro-immuno assay

Anti-type 12 serum incorporated in agarose-polyethylene glycol gel in a concentration of 1.5% (vol/vol) was found to enable a distinct "rocket" precipitate in electro-immuno assay using hot hydrochloric acid extract of type 12 group A streptococci. This precipitate was removed by trypsin treatment of the extract and on addition of anti-M12 typing serum but not of five other typing sera to the extract. The streptococcal component responsible for this precipitate was eluted from a CM-cellulose ion exchange column at pH 6.5. These findings demonstrated that the precipitate was caused by the M12 antigen. Crossed immuno-electrophoresis of hot hydrochloric acid extracts of three different type 12 group A streptococci showed that the electrophoretic mobility of the M12 antigens was similar in the three extracts. A linear correlation was obtained between the concentration of the M12-antigen and the height of the precipitate obtained in the electro-immuno assay using different dilutions of a standard type 12 extract. M12 antigen could thus be quantitated by the electro-immuno assay. In quantitation experiments, uniformly prepared extracts of five randomly selected, freshly-isolated type 12 strains were found to contain from 130 to 1850% of M12 antigen, respectively (expressed in % of the content of the standard type 12 extract).     

Antibody response to a solubilized tumor-associated membrane antigen (TAMA) from the murine lewis lung tumor

A tumor-associated membrane antigen (TAMA) has been solubilized from the C57B1/6 murine Lewis lung tumor using the detergent Triton X100. Xenoantiserum prepared in rabbits was used to identify this tumor antigen in Triton extracts by double immunodiffusion in agar. The antigen was partially purified using a combination of DEAE and Sephadex G200 chromatography followed by sucrose gradient isoelectric focusing. Isoelectric focusing also showed the antigen to have a pI at pH 4.7. Sodium dodecylsulfate polyacrylamide gel electrophoresis indicated the antigen to be of 37,000 molecular weight. The 125I protein A antibody-binding assay was used to further characterize the specificity of the antigen using rabbit antisera to the Triton extract. The same assay system also detected a specific antitumor humoral response in syngeneic mice immunized with the Triton extract.     

Immunological studies of canine ovarian antigens

Studies were conducted to determine the feasibility of producing canine ovary antiserum in the rabbit and to characterize the antigenic composition of the canine ovary. Ovaries from dogs were obtained and corpora lutea (CL) macroscopically removed. Following homogenization of ovaries, adult male rabbits were immunized by injecting the ovarian preparation. Unabsorbed antiserum cross-reacted with canine ovarian extract and with other reproductive and non-reproductive organ extracts to form precipitin bands. Species cross-reactivity was also observed by testing the unabsorbed antiserum with extracts from organs of the cat and rat. Absorption of antiserum with canine liver and lung extracts removed antibodies not specific to the canine ovary. One band was observed when such absorbed antiserum was allowed to react with the canine ovarian extract. The absorbed antiserum produced no bands with extracts from other canine reproductive and non-reproductive organs tested. These experiments suggested that the canine ovary contained at least 1 organ-specific antigen. This organ-specific antigen was located in the isolated canine ovarian cells by the immunofluorescence technique.     

Stability and stabilization of potential feed additive enzymes in rumen fluid*

Four commercial preparations of fibrolytic enzymes, from Irpex lacteus, Trichoderma viride, Aspergillus niger, and a mixture designed to be similar to the I. lacteus extract, were incubated in vitro with digesta taken from the rumen of sheep receiving a grass hay/concentrate diet, and the survival of major enzyme activities was measured. Some activities, including the beta-1,4-endoglucanase and xylanase from the extract derived from Aspergillus niger, were stable for at least 6 h in rumen fluid. The same activities in the other extracts also retained substantial activity for several hours. beta-Glucosidase and beta-xylosidase activities were much more labile, most being almost completely destroyed after 1 h, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that most proteins in the extracts were digested extensively after up to 7 h of incubation. Adding bovine serum albumin (0.5 g/l) to the incubation increased the half-life of Trichoderma viride beta-glucosidase activity from less than 0.5 h to 3 h. Proteins extracted from plant materials, particularly the soybean 7S globulin fraction, also conferred protection from proteolytic breakdown, but none was as effective as bovine serum albumin. It was concluded that the stability of most fibrolytic enzymes in rumen fluid is not likely to be a limiting factor in the use of enzymes as feed additives for ruminants; but if the enzymes are not stable, means can be found for their stabilization.     

渔游蛇雄性生殖周期及其肾性节的生殖生理学研究:Ⅱ.肾性节对精子的体外效应及生化分析

通过精子体外短期孵育表明：渔游蛇（Ｎａｔｒｉｘ ｐｉｓｃａｔｏｒ）肾性节具有营养精子的功能。聚丙烯酰 胺凝胶制备性电泳分离得到了肾性节特异性蛋白Ａ、Ｂ、Ｃ三因子。其中Ａ因子能显著提高精子在 体外的存活率，由Ａ１Ａ２两个亚基构成，分子量分别为６００００和３２０００。而Ｂ、Ｃ因子对精子存活 率无明显的影响。     

Antibodies to saline extractable tissue antigen in sera of patients with renal diseases

Multiple serum samples originating from 110 renal allograft recipients were examined against saline extract of normal human kidney by means of double diffusion gel precipitation. Eleven recipients were found to be positive; 99 of 106 sera from these patients were positive. Pretransplantation sera were available from 7 of these recipients and 6 patients were found "positive." The precipitation reaction was composed of one line. Identity reactions were formed between the lines produced by sera from all patients except 1. Sera of patients from end-stage renal disease produced similar reaction; however, only 3 of 234 sera from patients with nonrenal diseases precipitated the kidney extract. None of 154 normal sera were positive. Several positive sera also were positive in complement fixation tests with human kidney extract. Evidence was presented that the antibodies under study combined with a nonorgan-specific but species-restricted tissue antigen. The hypothesis was advanced that these antibodies are autoantibodies formed in response to a sequestered antigen released as a result of tissue damage. Apparently, the antigen is released frequently in immunogenic form from injury to kidney but infrequently from injury to other organs.     

Delayed hypersensitivity to crude and partially purified murine tumor-specific transplantation antigens and alloantigens utilizing the footpad swelling assay

Antigen preparations extracted from C3H/HeJ methylcholanthrene-induced fibrosarcomas by the 3M KCl extraction procedure were assessed for tumor-specific and allospecific antigenicity. Specificity of crude tumor antigen preparations and of fractions from preparative isoelectric focusing was investigated by evocation of footpad swelling (FPS) in syngeneic mice immunized with irradiated fibrosarcoma cells. Tumor immune mice displayed delayed hypersensitivity as positive FPS responses to challenge with 3M KCl extract and with fraction (Fr) 15 (pH 5.7 to 6.0) from preparative isoelectrically focused 3M KCl extract. Crude extracts and Fr 15 exhibited immunoprotective activity in vivo. Immune mice demonstrated a specific FPS response only to crude antigen preparations of Fr 15 from immunizing tumors, not to materials from a noncross-reactive neoplasm. DBA/2J mice immunized with C3H/HeJ spleen cells displayed FPS to challenge with crude antigen preparations, but not with the tumor-specific Fr 15. Alloantigen activity, however, was detected by a positive FPS response in C3H-immune DBA mice in fractions from the pH range 5.1 to 5.5. These experiments demonstrated that the FPS assay provides the setting for detection of specific delayed hypersensitivity responses to crude and fractionated tumor antigen preparations and for delineation of tumor-specific and histocompatibility antigen activities in fractions from crude extracts.     

Simian virus 40 origin- and T-antigen-dependent DNA replication with Drosophila factors in vitro.

DNA replication of double-stranded simian virus 40 (SV40) origin-containing plasmids, which has been previously thought to be a species-specific process that occurs only with factors derived from primate cells, is catalyzed with an extract derived from embryos of the fruit fly Drosophila melanogaster. This reaction is dependent upon both large T antigen, the SV40-encoded replication initiator protein and DNA helicase, and a functional T-antigen binding site at the origin of DNA replication. The efficiency of replication with extracts derived from Drosophila embryos is approximately 10% of that observed with extracts prepared from human 293 cells. This activity is not a unique property of embryonic extracts, as cytoplasmic extracts from Drosophila tissue culture cells also support T-antigen-mediated replication of SV40 DNA. By using highly purified proteins, DNA synthesis is initiated by Drosophila polymerase alpha-primase in a T-antigen-dependent manner in the presence of Drosophila replication protein A (RP-A; also known as single-stranded DNA-binding protein), but neither human RP-A nor Escherichia coli single-stranded DNA-binding protein could substitute for Drosophila RP-A. In reciprocal experiments, however, Drosophila RP-A was able to substitute for human RP-A in reactions carried out with human polymerase alpha-primase. These results collectively indicate that many of the specific functional interactions among T antigen, polymerase alpha-primase, and RP-A are conserved from primates to Drosophila species. Moreover, the observation that SV40 DNA replication can be performed with Drosophila factors provides a useful assay for the study of bidirectional DNA replication in Drosophila species in the context of a complete replication reaction.     

Neuroblastoma × Glioma Hybrid Cells Synthesize Enkephalin-Like Opioid Peptides

Partially purified extracts from neuroblastoma X glioma hybrid cells 108CC15 inhibit, like opioids, the prostaglandin E1-evoked formation of cyclic AMP in a dose-dependent manner in the same hybrid cells. The inhibition is prevented by the opioid antagonist naloxone. In addition, the same extract competes with [3H]naloxone and [3H]Leu-enkephalin for binding to opioid receptors of hybrid cell membranes and to a specific antiserum, respectively. The opioid activity in the extracts is destroyed by carboxypeptidase A and leucine aminopeptidase, but not by trypsin. Further purification of the extracts by HPLC, TLC, or high-voltage paper electrophoresis reveals in each case two active fractions which behave like Met- and Leu-enkephalin. The Met-enkephalin-like, but not the Leu-enkephalin-like, fraction is inactivated by treatment with BrCN. Dimethylaminonaphtylsulfonyl (dansyl) derivatives of Met- and Leu-enkephalin correspond to [3H]dansyl derivatives of Met-like substances from hybrid cells. Three to four times as much Met-enkephalin-like as Leu-enkephalin-like material is present in the extract. The overall concentration of opioid peptides in the hybrid cells varies between 0.03 and 1.0 pmol Leu-enkephalin equivalents per mg protein. The amount of opioids in the hybrid cells is strongly dependent on the cell density. The findings suggest that neuroblastoma X glioma hybrid cells contain opioid peptides that are very similar, if not identical, to Met- and Leu-enkephalin. Opioid activity can also be detected in other neuronal cell lines and even in glioma cells.     

BANANA GEL

The conditions for the formation of gels from banana extracts were studied. Gels were obtained with extracts more alkaline than pH 7.0 with very small quantities of calcium, strontium, and barium salts, the gel formation with these salts decreasing in the indicated order. In solutions more acid than pH 6.0, no gels were obtained with these salts. Magnesium, lithium, and sodium salts did not cause gel formation either in acid or alkaline solutions. Pancreatine gave a gel on incubation with banana extract at pH 5.0. The gel-forming property of banana extracts was destroyed on boiling.     

Existence of 28R-antigen in a certain strain of group F streptococcus.

An inter-Group common antigen was detected between Group A type 28 (Small)- and Group F (21/58/O'Mahoney, Colindale)-streptococcal cells by the T-typing agglutination reaction. The characteristics of this antigen coincide with those of the 28R-antigen, which was first detected in the Group A type 28 (Small) cells by Lancefield in 1943, in the following points: 1) It can be extracted from the cells with HC1 at pH 2.0 at 100 C in a stable state; 2) It can be kept in a stable state by heating in an alkaline solution at pH 7.8; 3) The antigen on the heat-killed cells was not affected by trypsin digestion at pH 7.8 but was destroyed by pepsin digestion at pH 2.0.     

Evaluation of anti-Plasmodium falciparum antibodies in Senegalese adults using different types of crude extracts from various strains of parasite

To date, no consensus exists on the type of crude Plasmodium falciparum Ags to be used in a standard assay for the evaluation of the overall anti-blood-stage immune response in humans. Comparison of the dose-dependent reactivity of using a pool of hyper-immune Senegalese sera to saponin and water schizont extracts of the Senegalese 07/03 isolate indicated similar reactivity on both types of antigen preparations. Water schizont extracts from three different strains of P. falciparum adapted to in vitro culture probed with a panel of specific mouse antisera and monoclonal antibodies reacting with conserved antigens showed similar antigenic content. Seroreactivity of immune individuals living in three different areas of endemicity was assessed in parallel on water crude extracts. The individual IgG, IgM and IgG subclass antibody responses to the various schizont preparations correlated positively. The specific IgM response was higher on the Senegalese schizont extract than on the FCR3 extract and was highest in Dielmo villagers. The IgG response was similar in all three locations and was strain independent. These results indicate that monitoring IgG antibody levels to the widely distributed FCR3 strain using an easily prepared crude lysate might represent a valuable reference ELISA allowing homogenisation and comparison of data from different laboratories.     

Simian virus 40 origin auxiliary sequences weakly facilitate T-antigen binding but strongly facilitate DNA unwinding.

The complete simian virus 40 (SV40) origin of DNA replication (ori) consists of a required core sequence flanked by two auxiliary sequences that together increase the rate of DNA replication in monkey cells about 25-fold. Using an extract of SV40-infected monkey cells that reproduced the effects of ori-auxiliary sequences on DNA replication, we examined the ability of ori-auxiliary sequences to facilitate binding of replication factors and to promote DNA unwinding. Although the replicationally active form of T antigen in these extracts had a strong affinity for ori-core, it had only a weak but specific affinity for ori-auxiliary sequences. Deletion of ori-auxiliary sequences reduced the affinity of ori-core for active T antigen by only 1.6-fold, consistent with the fact that saturating concentrations of T antigen in the cell extract did not reduce the stimulatory role of ori-auxiliary sequences in replication. In contrast, deletion of ori-auxiliary sequences reduced the efficiency of ori-specific, T-antigen-dependent DNA unwinding in cell extracts at least 15-fold. With only purified T antigen in the presence of topoisomerase I to unwind purified DNA, ori-auxiliary sequences strongly facilitated T-antigen-dependent DNA conformational changes consistent with melting the first 50 base pairs. Under these conditions, ori-auxiliary sequences had little effect on the binding of T antigen to DNA. Therefore, a primary role of ori-auxiliary sequences in DNA replication is to facilitate T-antigen-dependent DNA unwinding after the T-antigen preinitiation complex is bound to ori-core.     

Altered immunochemical reactivity of Saccharomyces cerevisiae a-cells after alpha-factor-induced morphogenesis.

Antibodies were raised against Saccharomyces cerevisiae a-cells that had been exposed to the sex pheromone, alpha-factor. After adsorption of the antiserum with diploid cells, antibodies remained that reacted specifically with the mannan from haploid cells. The characteristic determinant was observed in mannan from pheromone-treated a-cells, in mannan from untreated alpha-cells, and at a much lower concentration, in mannan from control a-cells. The antigens from these three mannans appeared to be identical. The determinant was destroyed by mild-acid hydrolysis or periodate oxidation, but not by proteolysis or digestion with exo-alpha-mannanase. Mutants with altered mannan were unable to express the antigen. Complete acid hydrolysates mannan from alpha-factor-treated a-cells contained mannose, glucose, and N-acetylglucosamine. Partial acid hydrolysis, under conditions that destroyed the antigenic determinant, released only mannose and mannobiose. The mannose fraction was labeled to high specific activity during response of a-cells to alpha-factor if radioactive glucose was the carbon source. Neither alpha- not beta-D-mannopyranosyl phosphate was a hapten. The results are consistent with the presence of a haploid-specific antigen containing an acid-labile mannose determinant and show that the amount of this antigen in a-cell mannan is increased in response to alpha-factor.     

Biocatalysis of silica gel-immobilized chlorophyllase in a ternary micellar system

A partially purified enzymic extract from Phaeodactylum tricornutum was immobilized on silica gel and the specific activity of chlorophyllase in its free and immobilized states were compared in a ternary micellar system. The storage stability of the free and immobilized chlorophyllase extracts, maintained at temperatures ranging from 4 to 35°C for a period of 0–20 h, was temperature-dependent. The results also showed that the specific activity of the free and immobilized chlorophyllase extracts was highest at 30°C for long-term incubation, using chlorophyll and pheophytin as substrates and that a three-fold increase in the specific activity of the immobilized chlorophyllase was observed in comparison to that obtained with the free counterpart. The findings indicated that when free and immobilized chlorophyllase extracts were recovered and reused with both substrates, the immobilized chlorophyllase extract could be recycled for longer periods of time, while the free enzyme extract showed no activity after the first cycle.     

Effects of extracts from sporoderm-broken spores of Ganoderma lucidum on HeLa cells

The effects of extracts from Ganoderma lucidum spores on the growth of human cervix uteri tumor HeLa cells as well as on the cell cycle and intracellular calcium level were investigated. Alcohol extracts were prepared from sporoderm-broken and sporoderm-nonbroken spores (termed extract I and extract II) of G. lucidum. Extract I was then subjected to silica gel chromatography to obtain extract III. Cytotoxicity was examined by means of trypan blue exclusion and MTT tests. It was found that extract I and extract III, but not extract II strongly inhibited the growth of HeLa cells, and that extract III was more effective than extract I. Moreover, extract III was shown to be capable of blocking the cell cycle at the transition from G₁ to S phase and inducing a marked decrease of intracellular calcium level, determined by flow cytometry and the specific fluorescent calcium probe Fura-2, respectively. These results imply that (1) the breaking of G. lucidum spores improves the release of cytotoxic activity and (2) the effective extract might influence the cell cycle and cellular signal transduction by altering the calcium transport system. This revised version was published online in July 2006 with corrections to the Cover Date.     

Protective effect of olive and juniper leaves extracts on nephrotoxicity induced by thioacetamide in male mice

This study, for the first time, evaluates the effect of olive and juniper leaves extracts and their combination on thioacetamide (TAA)-induced nephrotoxicity in male mice. The experimental mice were divided into eight groups. Group 1 was served as control. Group 2 was exposed to TAA. Group 3 was treated with TAA and olive leaves extract. Group 4 was subjected to TAA and juniper leaves extract. Group 5 was exposed to TAA and olive and juniper leaves extracts. Groups 6, 7 and 8 were treated with olive, juniper, and olive and juniper leaves extracts respectively. In mice treated with only TAA, significant increases of blood urea nitrogen and uric acid were observed after six weeks. Moreover, levels of serum creatinine, blood urea nitrogen and uric acid were statistically increased in mice administrated with only TAA for twelve weeks. Insignificant alterations in levels of these haematobiochemical parameters were noted in other treated groups after six and twelve weeks. Histopathological evaluations of renal sections from mice treated with only TAA for twelve weeks showed severe damage of the renal corpuscles. Furthermore, the renal sections from mice treated with TAA and olive leaves extract, TAA and juniper leaves extract, TAA and olive and juniper leaves extracts, olive leaves extract, juniper leaves extract, and olive and juniper leaves extracts showed normal structures. In addition, it is conceivable therefore, that these extracts exhibit protective influences against TAA-induced nephrotoxicity, probably mediated through the antioxidative pathway roles.     

Covalent affinity labeling by periodate-oxidized [alpha-32P]ATP of the large-T proteins of polyoma and SV40 viruses

Incubation of crude extracts from cells lytically infected with polyoma virus in the presence of periodate-oxidized [alpha-32P]ATP led to the radioactive labeling of one main polypeptide immunoprecipitated by anti-T antigen antibodies. It was absent from extracts of mock-infected cells and exhibited an apparent Mr value of 105,000, identical with that of the large-T viral protein. This polypeptide was unambiguously identified as large-T on the basis of the heat sensitivity of the in vitro labeling in extracts from cells infected with a tsa mutant. The amount of incorporated radioactivity was found to increase linearly with that of infected cell extract and with the incubation time. Labeling resulted from the specific binding of an oxidized ATP molecule to a nucleotide binding site of the large-T protein, since it was efficiently completed by unlabeled ATP. Study of the dependence on the concentration of oxidized ATP evidenced a first order kinetic for the labeling reaction. Similar results were obtained for the large-T protein of SV40 virus.