STABILITY OF ISOLATED MITOTIC APPARATUS DURING STORAGE*

Mitotic apparatus (MA) were isolated from sea urchin zygotes using various isolation procedures, and various properties of the isolated MA were studied and compared. MA isolated using hexylene glycol had birefringences which depended on the pH of the isolation medium, the lower the pH the higher the MA birefringence. The stability of the MA Birefringence also depended on the pH of the hexylene glycol isolation medium (the lower the pH the slower the rate of decay of birefringence), as did the final birefringence reached after prolonged storage. MA isolated using glycerol-dimethylsulphoxide (MTME) had much more stable birefringence than MA isolated using hexylene glycol; their birefringence decay rates were about 1000 times slower than those of MA isolated using hexylene glycol. Birefringence which remained after extraction of MA with H₂O, or 0.5 M KC1 was also studied; the results depended on the MA isolation medium, on the medium the MA were stored in, and on the amount of time the MA were stored after isolation, as described in detail in the text. These results are discussed, and it is suggested that several components (including, perhaps, oriented ribosomes) contribute to birefringence of isolated MA.     

A possible bio-assay for chromosome movement in isolated mitotic apparatus

We isolated mitotic apparatus (MA) from sea urchin zygotes using several methods. The isolated MA were injected into nucleated frog eggs or into enucleated frog eggs, and 24 h later we determined whether or not the eggs had undergone normal cleavage. Normal cleavage occurred in about 50% of the cases when MA isolated in glycerol-dimethyl sulphoxide were injected into either nucleated or enucleated frog eggs. Likewise for MA isolated initially in hexylene glycol and transferred immediately into glycerol-dimethyl sulphoxide. No cleavage occurred when MA isolated in hexylene glycol (and stored in hexylene glycol) were injected into frog eggs. We discuss two possible interpretations of the results. In one interpretation cleavage of the frog eggs is a bioassay, measuring the ability of the isolated MA to support chromosome movement. In the other interpretation the isolated MA contribute only nuclear material and cleavage initiation factors, and there is no chromosome movement in the isolated MA per se.     

Peripheral proteins of human erythrocytes

Water soluble, nonglycosylated proteins have been extracted from human erythrocyte membranes by two different methods and characterized immunochemically and by PAGE. The spectrin peripheral protein complex (PAGE bands 1 + 2) has been equated with two antigens of intermediate mobility in immunoelectrophoretic analysis of crude spectrin developed with antiserum to bands 1 + 2 purified by elution from gels. Nonspectrin proteins, including catalase, remain in close association with isolated membranes, and display solubility properties similar to those of spectrin. Along with spectrin, they may also function in the intact cell as peripheral proteins.     

Acylation of cell-associated IL-1 by palmitic acid

To determine whether membrane-associated IL-1 is palmitylated, we labeled LPS-activated human monocytes with [3H]palmitic acid. The plasma membranes were isolated, and the membrane proteins extracted and analyzed simultaneously by SDS-PAGE-autoradiography and Western blot analysis from the same gel. When the monocytes were labeled with [3H]palmitate, 23- and 31-kDa bands were visualized, for membrane-associated IL-1 and its precursor, respectively. The 31- and 23-kDa bands were excised from several gels and rehydrated and analyzed again by SDS-PAGE, autoradiography, and Western blot analysis. The 23- and 31-kDa bands appeared again by both methods. To further investigate membrane-associated IL-1 acylation, human monocytes were labeled with [3H]palmitate, the plasma membranes isolated, and the membrane proteins extracted by CHAPS detergent. Immunoprecipitation of isolated membrane proteins using anti-IL-1 antibodies revealed two bands of 23 and 31 kDa after autoradiography. The studies demonstrate that both membrane-associated IL-1 and the IL-1 precursor are acylated with palmitic acid.     

Hippocampal AMPA-type receptor complexes containing GluR3 and GluR4 are paralleling training in the Multiple T-Maze

Although it is well-known that AMPA receptors are involved in spatial learning and memory, published data on GluR3 and GluR4 are limited. Moreover, there is no information about GluR3 and GluR4 receptor complex levels in spatial memory training. It was therefore the aim of the study to determine the above-mentioned receptor levels following training in the Multiple T-Maze (MTM). Results from the MTM and hippocampal membrane proteins from C57BL/6J mice were taken from an own previous study and GluR3 and GluR4 receptor complexes were run on blue native gel electrophoresis followed by immunoblotting and quantification of bands. Subsequently, GluR3 and GluR4 were identified under denaturing conditions from two-dimensional gels by mass spectrometry (nano-LC-ESI-MS/MS). Hippocampal levels of GluR3 containing complexes (apparent molecular weight between 480 and 720) were decreased while GluR4 containing complexes (apparent molecular weight between 480 and 720) were increased. GluR4 complex levels in trained mice were correlating with latency and speed. Mass spectrometry unambiguously identified the two receptor subunits. The findings show that GluR3 and GluR4 may have different functions in the processes of spatial memory training in the MTM and indeed, different neurobiological functions of the two receptor subunits have been already reported. GluR3 and GluR4 receptor complex rather than subunit levels are paralleling training in the MTM and GluR4 complex levels were even linked to memory training, which may be of relevance for understanding molecular memory processes, interpretation of previous work or for design of future AMPA receptor studies.     

Specific dimerization of the light chains of human immunoglobulin

1. The light chains of human immunoglobulin were allowed to dimerize in vitro on removal of the dispersing agents acetic acid or urea. 2. On electrophoresis in polyacrylamide gel at pH8.8 the dimers yielded up to nine regularly spaced bands. This approximates to the number of electrophoretic components known to occur among the monomers. 3. Single electrophoretic components of the dimers were isolated from the gel, dissociated into monomers, and subjected as such to electrophoresis in urea-containing gels. Each gave two adjacent bands. 4. Similarly, after all the light chains as monomers had been subjected to electrophoresis in urea-containing gels, single electrophoretic components were isolated and allowed to dimerize. When examined now as dimers in the absence of urea, each component gave two adjacent bands. 5. These findings are explicable on the following basis. (a) The dimerization of the light chains is specific, at least inasmuch as it occurs between monomers of the same electrophoretic mobilities. (b) With the buffer constant, different light chains undergo different changes in net charge on being transferred from urea-containing to urea-free solution; in this way two different chains of the same initial charge can acquire a charge difference of 1. 6. Experiments with Bence-Jones proteins and other homogeneous light chains gave results substantiating the conclusions (a) and (b).     

Augmentation and dispersion of thein vivo mitotic apparatus of living marine eggs

Summary The effects of a number of organic reagents on the structure of the mitotic apparatus (MA) of several invertebrate eggs were investigated. In general, the agents induced either a large increase in the volume and retardation of the MA (hexylene glycol, dimethylsulfoxide, etc.) or caused it to partially or completely disappear (formamide, dithiodiglycol, dimethylacetamide, etc.). The immediate effects of the agents on the MA were reversible, but reversibility of embryonic development was only obtained if the eggs did not remain in the reagent for too long a time. The analysis of the specific effects of these agents suggests that the eggs utilized possess a pool of unoriented (or unpolymerized) MA material which may be reversibly affected by the agents studied. In the case of augmenting agents, the material is oriented into the MA or into aster-like bodies, in the case of dispersing agents, the material returns to the pool. Although no decision could be made as to whether the agents act directly or indirectly on the MA, the totality of the observations support the latter possibility.     

Changes in tubulin heterogeneity during postnatal development of rat brain.

Tubulin isolated from rat brain at various stages of postnatal development was subjected to isoelectric focusing on polyacrylamide gels. Multiple bands, indicative of the heterogeneity of the protein, were apparent at all developmental ages. When isoelectric focusing patterns of tubulin from brains of increasing developmental age were compared, changes in the distribution and relative intensities of the bands were observed. These changes were most pronounced between 8–12 days of age and were seen whether the tubulin was isolated by DEAE-cellulose chromatography or by successive cycles of assembly-disassembly. The isoelectric focusing pattern of tubulin isolated from the 22-day-old animal was indistinguishable from that of the protein obtained from 30-day-old rat brain. These developmental changes in tubulin heterogeneity may relate to changes in the assembly properties of the microtubule protein or may reflect age-dependent changes in the relative contributions of mitotic spindles, axons, dendrites, and glia to the total pool of tubulin in brain.     

Change in the heterogeneous distribution of tubulin isotypes in mitotic microtubules of the sea urchin egg by treatment with microtubule depolymerizing or stabilizing drugs.

In the mitotic sea urchin egg, the spindle microtubules were composed of different tubulin isotypes from those of astral microtubules using monoclonal antibodies [Oka et al. (1990) Cell Motil. Cytoskeleton, 16, 239-250]. Three of the antibodies, D2D6, DM1B, and YL1/2, were specific for spindle microtubules, astral microtubules and reactive with both microtubules, respectively. The mitotic sea urchin egg was treated with microtubule depolymerizing (colcemid and nocodazole) and stabilizing (hexylene glycol) drugs and change in the heterogeneous distribution of the tubulin isotypes was investigated by the immunofluorescence procedure using these three monoclonal anti-tubulin antibodies. We observed that: (1) the microtubule depolymerizing drugs caused quick depolymerization of most mitotic microtubules, and a small number of spindle microtubules remaining were stained with all three antibodies; (2) hexylene glycol induced many microtubules in the mitotic apparatus, which was stained with D2D6 but was not stained with DM1B; (3) hexylene glycol also induced a great number of miniasters in the cytoplasm, and they were stained with three antibodies. These results suggest that these drugs altered the distribution of tubulin isotypes in the mitotic microtubules during depolymerization or polymerization within a short time.     

Serial analysis of zein by isoelectric focusing and sodium dodecyl sulfate gel electrophoresis

Zein, the major storage protein of maize (Zea mays L.) endosperm, was extracted from a number of inbreds with alcohol plus a reducing agent. Isoelectric focusing (IEF) separated total zeins into 41 components, while sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separated total zeins into about 15 components. Each procedure gave characteristic patterns of zein bands for a number of maize inbreds. IEF and SDS-PAGE were used serially so that each band separated by IEF could be assayed as an individual SDS-PAGE sample. Some IEF bands revealed only a single band after SDS-PAGE, while others revealed two or more bands. A nomenclature system is presented which integrates the two separation systems with information about chromosome locations of zein genes, maize mutations which affect zein synthesis, and inbred sources for different zeins. SDS-PAGE of zein gives apparent molecular masses which vary widely according to the standards used and the properties of the gels, therefore an artificial nomenclature for identifying zein bands after SDS-PAGE is presented. The new nomenclature provides a flexible system which is useful and can be conveniently used in different laboratories.     

The second peptidoglycan hydrolase of Streptococcus faecium ATCC 9790 covalently binds penicillin.

A second peptidoglycan hydrolase (muramidase-2) of Streptococcus faecium ATCC 9790 (Enterococcus hirae) has been purified to apparent homogeneity. The enzyme has been shown to be a beta-1,4-N-acetylmuramoylhydrolase (muramidase; EC 3.2.1.17) and to differ in substrate specificity from a previously isolated muramidase. Purified enzyme appears as two protein staining bands with molecular masses of 125 and 75 kilodaltons (kDa) on polyacrylamide gels after sodium dodecyl sulfate electrophoresis. Elution and renaturation of protein bands from sodium dodecyl sulfate-polyacrylamide gels showed that both proteins have muramidase-2 activity. Both proteins have been shown to bind radioactive benzylpenicillin and have the same electrophoretic mobilities as penicillin-binding proteins 1 and 5 present in membrane preparations of this organism, respectively. Incubation of a [14C]penicillin G-labeled 125-kDa form of the enzyme with crude alkaline extracts from S. faecium (which did not contain added proteinase inhibitors) showed the endogenous conversion of the radiolabeled 125-kDa form to the radiolabeled 75-kDa form of the enzyme.     

THE MITOTIC APPARATUS : Structural Changes after Isolation

The fibrous structure of the mitotic apparatus (MA) isolated from dividing sea urchin eggs undergoes no changes visible in phase contrast during extended storage, but the solubility of the MA rapidly decreases after isolation. Polarization microscopy shows that a decrease in the birefringence of the MA also occurs after isolation and is correlated with the loss of solubility. This loss of birefringence indicates that some structural change takes place during this period, and such a change was demonstrated by means of electron microscopy. The tubular filaments which form the spindle of the intracellular MA and of the freshly isolated MA were found to break down during storage to rows of dense granules, this loss of continuity presumably accounting for the loss of birefringence. The interrelations of the observed changes and the significance of these observations for investigations on the isolated MA are discussed.     

Differences in the surface properties of the mating types of Physarum polycephalum

In the slime mold Physarum polycephalum, formation of a diploid plasmodium occurs when compatible haploid amoebae fuse. To study cell surface changes associated with the fusion process, a non-destructive method known as aqueous, two-phase partitioning was employed. Using a two-phase system of dextran and polyethylene glycol, we observed that the two mating types (RSD4 and MA185) have different partition coefficients and hence different surface properties. Based on their partitioning behavior, MA185 cells appear to have a more hydrophobic surface than RSD4 amoeba. The partition coefficient of both cell types decreased with time. If amoebae were maintained in culture until they encysted, differences in their surface were not detectable.     

Active uptake of sucrose by plant plasma membrane vesicles: determination of some important physical and energetical parameters

Changes in general protease activity and the levels of the zein storage proteins were monitored during germination and early seedling growth of maize ( Zea mays L . inbred A636). General endosperm endoprotease activity, measured in vitro using azocasein as a substrate, increased continuously to day 5 and remained high thereafter. The increase was in parallel with the loss of zein protein as determined by immunoblot analysis, with a total loss of detectable zein by 10 days after inhibition of the seeds. A method was developed for the specific in vitro assay of zein degrading activity by monitoring the release of soluble radioactivity from the immobilized storage protein. The in vitro pH optimum for both general protease and zein degrading activities in homogenates prepared from isolated endosperm was 4.0. However, the curve for general protease activity was asymmetrical suggesting the presence of more than 1 protease. This was verified by activity staining of gelatin-containing polyacrylamide gels, which suggested the presence of 3 major and 3 minor protease activities. Both general protease and zein-specific activities were enhanced more than 2-fold when 2-mercaptoethanol (ME) was included in the assays, and both were inhibited by fhiol-protease directed compounds. Most of the general protease activity but none of the zein-specific protease activity bound to con A-Sepharose. Both the con A-binding and nonbinding fractions were analyzed using gelatin activity gels. The 3 minor activity bands were present in the fraction which was specifically eluted from the con A column by a-methyl-mannoside, while the 3 major activity bands, corresponding to the zein-specific protease activity, did not bind to the immobilized lectin.     

The Freshwater Mussel (Elliptio complanata) as a Sentinel Species: Vitellogenin and Steroid Receptors

Freshwater mussels, Elliptio complanata were collected froma reference and pollutant-impacted pond on Cape Cod, MA. Glutathione-S-transferase(GST) activity was measured in gill, hepatopancreas and foot.In addition, content of seven heavy metals were measured inwhole bodies. GST activity was significantly elevated in hepatopancreasand foot, as was whole body cadmium level in animals from thecontaminated site suggesting that these animals have been exposedto organic and inorganic contaminants. Sodium dodecyl acrylamidegel electrophoresis (SDS-PAGE) analysis showed putative vitellogeninswith molecular weight 180 and 205 kDa bands only in the ovary.In non-denatured gel electrophoresis ovarian extracts revealedtwo higher molecular weight bands at 550 and 700 kDa, whichwere reproductive stage specific. Western blotting of SDS-PAGEand non-denatured gels using the anti-scallop yolk-protein antibodyconfirmed the presence of cross-reacting bands of the same molecularweights in the ovary but not other tissues. Although severalexperiments involving steroid hormone exposure were done, nosignificant changes in vitellogenin protein levels were observed.However, using an anti-human ERß antibody, ERßpositive bands were observed both in female foot, and the ovary.No cross reactivity with the antibody was observed in hepatopancreas.Additional studies are required to resolve questions of vitellogeninregulation and the role of (xeno)estrogens in bivalve molluscs.     

微卫星PCR产物变性与非变性PAGE-银染检测方法的比较

用鸡的微卫星引物对6个中国地方鸡种的两个微卫星位点进行PCR扩增。将扩增产物在变性与非变性聚丙烯酰胺凝胶（PAGE）上进行电泳,经银染,表明微卫星产物在二者上的电泳结果有明显的差异。在非变性聚丙烯酰胺凝胶中,表现为有较多的非特异带,而在变性胶中微卫星扩增产物条带清晰,易于鉴定。Abstract：The genomic DNAs from six chicken breeds in China were amplified using two microsatellite primers. The PCR products were detected by non-denatured and denatured PAGE gels respectively, and the gels were dyed by silver. There were distinct differences between the two kinds of gel. In non-denatured gels. There were many nonspecific bands while clear purposed bands were showed in denatured gels.     

Isolation and Characterization of Tubules and Plasma Membranes from Cytophaga columnaris

Tubular structures are released from cells of Cytophaga columnaris after lysis of the cells. To determine the nature of these tubules, they were purified and their composition was determined. Tubules were isolated after treating cell lysates with 1.0% sodium dodecyl sulfate at pH 8.1, which solubilizes all structural components except tubules. Plasma membranes from the same organism were isolated by discontinuous sucrose gradient centrifugation of lysed cells. Both tubules and membranes are composed of lipids and proteins. Lipids extracted from tubules and plasma membranes produced similar patterns when examined by thin-layer chromatography. Proteins solubilized from membranes were separated into 14 bands by polyacrylamide gel electrophoresis, whereas those solubilized from tubules separated into only 5 bands. The presence of lipids in tubules from C. columnaris supports the idea that they are derived from membranes of intact cells. In this respect they are similar to tubules produced by cells of Clostridium botulinum and different from other tubular structures ("rhapidosomes") found in cells of Saprospira grandis.     

Purification and some properties of myeloperoxidase and eosinophil peroxidase from human blood

Myeloperoxidase and eosinophil peroxidase have been isolated from outdated human blood. Peroxidase activity was extracted from washed leucocytes using 0.5 M-CaCl2 and the extract further purified by chromatography on concanavalin A--Sepharose, phenyl-Sepharose and finally by gel filtration. The final enzyme preparations were highly purified according to spectral and gel-electrophoretic criteria. Under reducing and denaturing conditions on polyacrylamide-gel electrophoresis myeloperoxidase gave rise to bands of Mr 57 000, 39 000 and 15 500, whereas the eosinophil enzyme yielded bands of Mr 50 000 and 15 500. Both enzymes were very resistant to denaturation either by the chaotropic agents urea and guanidinium chloride or by elevated temperatures. Spectral properties of the native and reduced forms of the enzymes are reported.     

The effect of ampholytes on ferritin isoelectric focussing patterns

Ferritin was subjected to isoelectric focussing (IEF) on agarose gels containing different commercial carrier ampholytes. In two gels protein staining revealed banded patterns which differed from one another, while a third gel yielded zones rather than discrete bands, indicating that the bands may be artefacts.The differences between banded patterns were studied by isolating bands from an IEF gel and refocussing these on gels containing either the original ampholyte or a different ampholyte preparation. Striking differences were noted.Chromatofocussing of ferritin resulted in the elution of broad peaks between the same pH limits as indicated by IEF patterns.     

Changes in the Electrophoretic Patterns of the Soluble Proteins of Winter Wheat and Rye following Cold Acclimation and Desiccation Stress

The degrees of freezing tolerance acquired by winter wheat (Triticum aestivium L.) and rye (Secale cereale L. cv Puma) were similar following a 4-week cold conditioning and a 24-hour desiccation stress. Soluble proteins were extracted from shoots of cold-conditioned or desiccation-stressed seedlings and electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. Quantitative changes in the electrophoretic patterns of the soluble proteins of the different cultivars grown in different environments were detected, but the changes were not equivalent following cold conditioning and desiccation stress. The abundance of two polypeptide bands showed a significant increase correlated to the degree of freezing tolerance and, hence, the polypeptides in these bands may play a role in the development of freezing tolerance.