Antigenic activity of oral whole-culture meningococcal serogroup B vaccine

The whole-culture vaccine preparation of Neisseria meningitidis, serogroup B, obtained by cultivation in a computer-controlled bioreactor, was studied. The preparation was shown to contain antigenically active polysaccharide, outer membrane proteins, as well as lipooligosaccharide, faintly pyrogenic and with low toxicity. After oral immunization of rabbits a multiple increase in the levels of IgG antibodies to these antigens in their blood serum was noted during the period of observation (303 days).     

Perspectives for development of polyvalent meningococcal vaccine

Experimental whole-culture oral polyvalent meningococcal vaccines against serogroups A, B and C consisting of three monovalent components in different proportions have been developed and evaluated. Kinetics of IgG response to meningococcal antigens (outer membrane proteins, polysaccharide, lypooligosaccharide of these serogroups) in sera of rabbits immunized orally with these preparations was measured. Sharp rise of IgG levels (on 2 - 3 orders) compared to baseline has been detected as well as persistence of high titers during the observation period (322 days).     

Experimental reconsideration of the utility of serum starvation as a method for synchronizing mammalian cells

Accurate cell-size determinations support the prediction that serum starvation and related whole-culture methods cannot synchronize cells. Theoretical considerations predict that whole-culture methods of synchronization cannot synchronize cells. Upon serum starvation, the fraction of cells with a G1-phase amount of DNA increased, but the cell-size distribution is not narrowed. In true synchronization, the cell-size distribution should be narrower than the cell-size distribution of the original culture. In contrast, cells produced by a selective (i.e. non-whole-culture) method have a specific DNA content, a narrow size distribution, and divide synchronously. The general theory leading to the conclusion that whole-culture methods for synchronization do not work implies that one can generalize these serum-starvation results to other cell lines and other whole-culture methods, to conclude that these methods do not synchronize cells.     

Rejoinder: whole-culture synchronization cannot, and does not, synchronize cells

There have been numerous proposals suggesting that whole-culture methods - in which all cells in a growing culture are treated identically - can synchronize cells. An explicit defense of these methods has been presented (Spellman and Sherlock, this issue, pp. 270-273, ). Here, this defense of whole-culture 'synchronization' is subjected to a critical evaluation leading to the conclusion that whole-culture synchronization cannot synchronize cells - at all. Whole-culture methods cannot produce a set of cells that reflects the size and genome composition of cells of any particular cell-cycle age during the normal cell cycle. Thus, in addition to the well-recognized problem of artifacts, it is proposed that experiments using whole-culture treatments (usually starvation or inhibition methods) are not suitable for cell-cycle analysis because these methods do not produce a synchronized culture.     

Spraydrying of yeast-lytic enzymes from Arthrobacter sp.

Summary Spray drying processes are widely used for large scale preservation of biological materials e.g milk, whey, yeast, egg white, etc. Nevertheless, there is an increasing tendency for drying of catalytic active proteins. The paper presents results from experiments which were carried out to test the applicability of spray drying for the preservation of several bacterial enzymes used for the lysis of yeast cell-walls (-1,3-glucanase, -mannanase, chitinase and amylase) and formed by an Arthrobacter species. Enzyme solutions were obtained by concentrating the cell free cultivation liquid by cross-flow ultrafiltration. Due to the low protein content of the liquid preparation, several substances were added as stabilizers or carriers (eg. sucrose, KCl, MgSO₄) and the effect was studied in further experiments. Spray drying was carried out at inlet air temperatures of 110 – 120°C and outlet air temperatures of 65 – 72°C. Best results were obtained by the addition of 10% KCl, the preparation having a crystalline consistency and a 60% yield in activity.     

Thymidine block does not synchronize L1210 mouse leukaemic cells: implications for cell cycle control, cell cycle analysis and whole-culture synchronization

Abstract.   It has been predicted that whole-culture methods of synchronization cannot synchronize cells. We have tested whether thymidine block, one type of whole-culture synchronization, can synchronize L1210 cells. We demonstrate experimentally that the thymidine block method cannot produce a synchronized culture. Although thymidine-treated cells are arrested primarily with an S-phase amount of DNA, there is no narrowing of the cell size distribution and there is no synchronized division pattern following release from the thymidine block. In contrast to a whole-culture synchronization method, cells produced by a selective (i.e. non-whole-culture) method not only have a specific DNA content, but also have a narrow size distribution and divide synchronously. Generalizing the results to other cell lines, we suggest that these conclusions call into question experimental measurements of gene expression during the division cycle based on thymidine inhibition synchronization.     

Biological effects of interferon,produced by recombinant bacteria of the probiotic preparation subalin

The present review deals with the analysis of biological and functional activities of recombinant bacteria Bacillus subtilis IF-alpha 2335 are producing a human interferon. The interferon-producing bacteria are constructed on a basis commercial probiotic strain B.subtilis 2335, carrying a recombinant plasmid pMBM 105 with the gene of human alpha-2 interferon. The implementation of the recombinant strain in the preparation probiotic, received a designation "Subalin", necessitates to verify a number of immunologic activities and to perform successive protective effects. Interferon, synthesized by recombinant bacteria shows the activity on macroorganism at oral and rectal application of preparation. Subalin was shown antivirus and antitumor activity and preservation by recombinat bacteria of antagonistic properties. The mechanisms of the positive effect of subalin were considered: this effect was shown to be due to the action of interferon excreted by recombinant bacteria into the mucous of different biotopes of host.     

Lack ofPer os toxicity or pathogenicity in rats fed the fungusHirsutella thompsonii

Symptoms of toxicity or pathogenicity were not observed in white rats fed either whole-culture broth or mycelia of the parasitic fungusHirsutella thompsonii. This fungus is presently under consideration as a microbial control agent of the citrus rust mite,Phyllocoptruta oleivora.     

A murine colitis model developed using a combination of dextran sulfate sodium and <Emphasis Type="Italic">Citrobacter rodentium</Emphasis>

Adult mice were treated with dextran sulfate sodium (DSS) and infected with Citrobacter rodentium for developing a novel murine colitis model. C57BL/6N mice (7-week-old) were divided into four groups. Each group composed of control, dextran sodium sulfate-treated (DSS), C. rodentium-infected (CT), and DSS-treated and C. rodentium-infected (DSS-CT) mice. The DSS group was administered 1% DSS in drinking water for 7 days. The CT group was supplied with normal drinking water for 7 days and subsequently infected with C. rodentium via oral gavage. The DSS-CT group was supplied with 1% DSS in drinking water for 7 days and subsequently infected with C. rodentium via oral gavage. The mice were sacrificed 10 days after the induction of C. rodentium infection. The DSS-CT group displayed significantly shorter colon length, higher spleen to body weight ratio, and higher histopathological score compared to the other three groups. The mRNA expression levels of tumor necrosis factor (TNF)-α and interferon (INF)-γ were significantly upregulated; however, those of interleukin (IL)-6 and IL-10 were significantly downregulated in the DSS-CT group than in the control group. These results demonstrated that a combination of low DSS concentration (1%) and C. rodentium infection could effectively induce inflammatory bowel disease (IBD) in mice. This may potentially be used as a novel IBD model, in which colitis is induced in mice by the combination of a chemical and a pathogen.     

Quantitative immuno-gold labelling and ultrastructural preservation after cryofixation (combined with different freeze-substitution and embedding protocols) and after chemical fixation and cryosectioning. Analysis of the secretory organelle matrix of Paramecium trichocysts.

Among the variety of parameters affecting immuno-gold labelling efficiency, mainly the effects of different preparative protocols were tested. Preservation of ultrastructure and of antigenicity are the salient features of this study. We have labelled insoluble components of the secretory matrix of Paramecium trichocysts with specific antisera, using 10 nm colloidal gold particles. The highest labelling efficiency was obtained with fast freezing (cryofixation, either by sandwich or spray-freezing), freeze-substitution in methanol (without added fixatives) and hydrophilic Lowicryls, particularly when applied at low temperatures (K11M at 193 K). The presence of different chemical fixatives always reduced the labelling density and some recommendations from the literature do not appear advisable. Methods commencing with fixation at greater than or equal to 0 degree C, such as "progressive lowering of temperature" (PLT) or preparation of cryostat sections, i.e. with chemical pretreatments, always resulted in lower labelling density. Our data appear, therefore, relevant for optimal immuno-gold labelling of insoluble antigens and emphasize the potential of cryofixation as a primary preparation step. In addition, ultrastructural preservation was also superior after cryofixation.     

Reduction of Photoautotrophic Productivity in the Cyanobacterium Synechocystis sp. Strain PCC 6803 by Phycobilisome Antenna Truncation

Truncation of the algal light-harvesting antenna is expected to enhance photosynthetic productivity. The wild type and three mutant strains of Synechocystis sp. strain 6803 with a progressively smaller phycobilisome antenna were examined under different light and CO(2) conditions. Surprisingly, such antenna truncation resulted in decreased whole-culture productivity for this cyanobacterium.     

Preservation of alveolar type II pneumocyte lamellar bodies for electron microscopic studies.

Simultaneous fixation with glutaraldehyde and osmium tetroxide, followed by an uranyl acetate (UA) treatment before dehydration and embedding (Hirsch and Fedorko 1968) ensures a very good preservation of lamellar bodies (LB's) as well as of the cellular membranes in type II pneumocyte. The uranyl acetate treatment appeared to be the most efficient step of the procedure. The morphological aspect of lamellar bodies after such a preparation was similar to that observed after freeze-etching of lipid retaining methods. Moreover, the Hirsch-Fedorko procedure is very simple and can easily be used for routine ultrastructural and radioautographic studies. On the other hand, it appeared that the uranyl acetate phospholipid "complex" is very sensitive to the pH of chemical solutions used after sectioning. The "complex" is variously dissolved by alkaline solutions, photographic developers or stains. The best preservation of ultrastructure was obtained with neutral or acidic developers and acidic stains.     

A new integrated concept for the improved preparation of sections of fresh or frozen tissue for light microscope histochemistry

In order to obtain thin sections of plant tissues which combined good morphological preservation and the preservation of the substances and enzyme activities in the tissues, a concept of section preparation by external stabilization was developed. The main components are as follows: appropriate supporting medium; surface coating before each sectioning process, the coating being either non-permanent, permanent, or semi-permanent; suitable techniques for affixing the coated sections to the slides using either pressure-sensitive adhesive or solvent-based adhesive; and mounting media with defined refractive indices (preferably UV-curable, water-soluble monomers). By this approach, sections exhibiting excellent morphological and physiological preservation were obtained using either a cryostat at -30 degrees C or a rotary microtome at room temperature.     

Synthesis of a naphthylvinylpyridine derivative and its use for affinity chromatography of choline acetyltransferase

N-(10-carboxy)decamethylene-4(1-naphthylvinyl)pyridinium chloride, a derivative of the choline acetyltransferase (CAT) inhibitor naphthylvinylpyridine (NVP) was synthesized and used as a ligand for affinity chromatography of choline acetyltransferase. The preparation of this inhibitor included the quaternization of naphthylvinylpyridine with 11-Br-undecanoic acid methyl ester to obtain N-(10-carbomethoxy)decamethylene-4-(1-naphthylvinyl)pyridinium bromide, followed by hydrolysis to free the carboxylic group. This inhibitor (C11-NVP+) had a potency comparable to that of N-methyl-4(1-naphthylvinyl) pyridinium iodide (C1-NVP+) which is the most potent derivative of NVP but which lacks a functional group for conjugation to Sepharose. The C11-NVP+ was then bound through the carboxylic group to aminoalkyl Sepharose by a carbodiimide promoted condensation reaction. Interaction of CAT with the inhibitor retarded its elution from a column of Sepharose-C11-NVP+ and permitted the purification of the enzyme to electrophoretic homogeneity starting from a preparation in which CAT represented about 20% of the total proteins. Conventional procedures of protein purification had previously been unsuccessful in isolating the enzyme in pure form. Inhibition studies showed that CAT could exhibit either a "high" or a "low" sensitivity to inhibition by naphthylvinylpyridine and its derivatives (I50 with C1-NVP+ = 0.57 microM or 5.2 microM). A direct relationship existed between the sensitivity of CAT to these inhibitors and the retention of the enzyme by the affinity column.     

Short-term preservation of mouse oocytes at 5 degrees C.

The temporary preservation of oocytes without freezing would be useful for some experiments. ICR mouse oocytes were kept in a preservation medium under mineral oil for 1, 2, 3, 4 or 7 days at 5 degrees C, and 1 or 2 days at 37 degrees C. In vitro fertilization was attempted on oocytes rinsed with TYH medium after preservation. More than 70% of morphologically normal oocytes were recovered from each preservation group. Fertilization rates of oocytes preserved for 1, 2, 3, 4 or 7 days at 5 degrees C were 69.9, 66.5, 45.3, 26.7 and 8.8% respectively. Fertilization rates of oocytes preserved for 1 or 2 days at 37 degrees C were 9.6 and 1.6%, respectively. Preservation of oocytes at 5 degrees C has some capability as a method of short-term storage without freezing.     

Extracellular calcium protects cultured rat hepatocytes from injury caused by hypothermic preservation

Effects of various preservation solutions were compared in an experimental hypothermic preservation model using cultured rat hepatocytes. Hepatocytes prepared by the collagenase perfusion method were cultured for 48 hr, then the medium in each culture dish was exchanged for various preservation solutions, and the dishes were hypothermically (0-2 degrees C) stored in a refrigerator for 12-72 hr. After the preservation period, the hepatocytes were cultured again at 37 degrees C for 2 hr. Hepatocytes' viability after 18-hr preservation and reculture was greater when they were preserved in "intracellular" rather than "extracellular" solutions. Even with Euro-Collins solution (intracellular solution), hepatocyte viability decreased to approximately 20% after 24-hr preservation, and an increase in the cellular lipid peroxide content was observed. However, when this solution contained a submillimolar concentration of calcium, lipid peroxidation was significantly suppressed and hepatocyte viability was dramatically improved. Vitamin E was almost equally effective and a marked synergistic effect was observed with calcium. Calcium was found to be capable of maintaining the cellular glutathione level during cold storage, which seems to suppress lipid peroxidation and consequently improve hepatocyte survival.     

Synthesis of brassinolide and its biosynthetic precursors using methyl 3-hydroxy-2-methylpropionate

Formal synthesis of plant hormones that belong to the group of 24α-methylbrassinosteroids, including brassinolide and its biosynthetic precursors with one hydroxyl group in their side chain, was performed. Stereochemistry of a methyl group at the C24 atom was provided by the choice of the desired enanthiomer of methyl-3-hydroxy-2-methylpropionate and by the sequence of its conversions into the chiral intermediate that was necessary for the formation of the C23–C28 fragment of the side chain. The (22R,23R)-diol group was introduced by the Sharpless asymmetric dihydroxylation of the intermediate Δ²² -steroids, the products of consecutive reactions of attachment of a low-molecular sulfone to the steroid C22-aldehyde, acetylation, and reductive desulfurization of the intermediate β-acetoxysulfones. Reduction of 22-acetoxy-23,25-disulfones was shown to proceed with the preservation of the functional group at atom C22.     

Reply: whole-culture synchronization - effective tools for cell cycle studies

Studies of gene expression during the eukaryotic cell cycle in whole-culture synchronized cultures have been published using many methodologies. These procedures alter the state of the cell cycle for a population of cells, rather than purifying a population of cells that are in the same state. Criticism of these methods (e.g. see Cooper, this issue, pp. 266-269, ) suggests that these studies are flawed, and posits that such methodologies cannot be used to study the cell cycle because they alter the size and age distributions of the cultures. We believe that whole-culture cell cycle studies work even though they alter the size and age distributions: these cells still progress through the cell cycle and although we do not suggest that the methods are perfect, we will explain how these microarray studies have successfully identified cell cycle regulated genes and why these results are biologically meaningful.     

冷风干燥制备高活菌的恶臭假单胞菌菌粉

【目的】获得高活菌恶臭假单胞菌菌粉,提高菌体干燥及保藏存活率。【方法】选用冷风干燥法制备活菌粉,并优化吸附载体与保护剂。【结果】冷风干燥制备恶臭假单胞菌菌粉干燥存活率普遍达到65%以上,显著优于喷雾干燥(24%);对载体与保护剂进行正交试验优化,确定了载体为混合的硅藻土和碱处理玉米芯粉,混合比为1:2,保护剂(质量比)为甘露醇7%、谷氨酸钠5%、甘油1%,制得菌粉活菌数为1.03×1011 CFU/g,室温保藏30 d和4 °C保藏60 d存活率分别达到40.54%和71.67%。【结论】冷风干燥温度相对较低(10?40 °C),对菌体损伤小,碱处理玉米芯粉、甘露醇和谷氨酸钠是提高菌粉保藏存活率的重要因子,此法克服了革兰氏阴性菌菌粉不易制备和不耐保藏的瓶颈。