Reserve substances inPaxillus involutus sclerotia

Summary The ectomycorrhizal fungus,Paxillus involutus, produces sclerotia in culture. These can be induced to form on agar medium by exposing mycelium grown at 25°C to various temperatures between6°C and 15°C. Sclerotia formed at 10°C and above were large and covered with drops of exudate, while those formed at 6°C or 8°C were very small and did not produce an exudate. Mature sclerotia were bounded by a compact rind and contained abundant storage reserves. Histochemistry of the larger sclerotia showed large quantities of protein stored as protein bodies in the cytoplasm, lipid present as small droplets, glycogen granules stored in the cytoplasm and polyphosphate present as small granules in the cytoplasm and in the protein bodies. Energy dispersive X-ray microanalysis confirmed the presence of phosphate in the granules and was used to map its distribution throughout the sclerotium. The smaller sclerotia induced at 8°C and below on the same medium had the same basic structure and composition, but lacked the complex phenolic cell network found in large sclerotia, and had abundant extracellular polysaccharides. The rind was not well developed and these small sclerotia are interpreted to have been arrested at an early stage of development.     

Beta beta homodimers exist in native rabbit skeletal muscle tropomyosin and increase after denaturation-renaturation.

Native tropomyosin from rabbit skeletal muscle (RSTm) consists mainly of alpha alpha and alpha beta coiled coils (alpha/beta approximately 3-4/1). In some extant studies, no beta beta molecules have been found. In this study, RSTm from several different preparations was disulfide cross-linked, both preparation and cross-linking being done under nondenaturing conditions. The cross-linked product was assayed for the presence of beta beta molecules cross-linked at both C36 and C190 (beta = beta). In such cross-linked RSTm, 3-8% beta = beta is detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, C4 reversed-phase high-performance liquid chromatography, and a free-solution capillary electrophoresis experiment. This percentage becomes approximately 4-10% beta beta when corrected for incomplete double cross-linking and is independent of protein concentration (0.1-10.0 mg/mL), indicating that the observed beta beta species are not artifacts due to intermolecular cross-linking. Upon denaturation and subsequent renaturation either by heating to 55 degrees C or by incubating at 45 degrees C followed by quenching to room temperature, or by guanidine hydrochloride exposure followed by phased renaturation by dialysis, the fraction of beta beta increases, indicating that the reassociation favors homodimer formation somewhat over random association. This result differs from the random association observed when the sulfhydryl on one of the chains is carboxyamidomethylated (Holtzer, M.E., Breiner, T., & Holtzer, A., 1984, Biopolymers 23, 1811-1833), and from the overwhelming heterodimer preferences reported for tropomyosins from other organisms (Lehrer, S.S., Qian, Y., & Hvidt, S., 1989, Science 246, 926-928; Lehrer, S.S. & Qian, Y., 1990, J. Biol. Chem. 265, 1134-1138).     

Ultrastructural visualization of exocytosis in the pig pineal gland

Summary The pinealocytes of the pig contain conspicuous dense bodies, the nature and role of which are not yet fully elucidated. The aim of this study was to demonstrate whether or not these structures are involved in the secretion process. The tannic acid-Ringer incubation (TARI)-method, which allows a clear-cut ultrastructural study of secretory discharge by exocytosis, has been used. The results indicate that pig pinealocytes release the content of the dense bodies with an amorphous inner structure into the extracellular space via exocytosis and that this secretion is quantitatively important. The secreted material is proteinaceous in nature; this indicates that polypeptides are released by the pineal.     

Purification of ribulose 1,5-bisphosphate carboxylase/oxygenase and of carboxysomes from Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa (Montagu)

The bacterial symbionts of many marine invertebrates contain ribulose 1,5-bisphosphate (RuBP) carboxylase but apparently no carboxysomes, polyhedral bodies containing RuBP carboxylase. In the few cases where polyhedral bodies have been observed they have not been characterised enzymatically. Polyhedral bodies, 50–90 nm in diameter, were observed in thin cell sections of Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa and RuBP carboxylase activity was detected in both soluble and particulate fractions after centrifugation of cell-free extracts. RuBP carboxylase purified 90-fold from the soluble fraction was of high molecular weight and consisted of large and small subunits, with molecular weights of 53,110 and 11,100 respectively. Particulate RuBP carboxylase activity was associated with polyhedral bodies 50–100 nm in diameter, as revealed by density gradient centrifugation and electron microscopy. Therefore, the polyhedral bodies were inferred to be carboxysomes. Native electrophoresis of isolated carboxysomes demonstrated a major band which comigrated with the purified RuBP carboxylase and three minor bands of lower molecular weight. Sodium dodecyl-sulphate (SDS) gel electrophoresis of SDS-dissociated carboxysomes demonstrated nine major polypeptides two of which were the large and small subunits of RuBP carboxylase. The RuBP carboxylase subunits represented 21% of the total carboxysomal protein. The most abundant polypeptide had a molecular weight of 40,500. Knowledge of carboxysome composition is necessary to provide an understanding of carboxysome function.Abbreviations FPLC fast performance liquid chromatography - IB isolation buffer - PAGE polyacrylamide gel electrophoresis - RuBP carboxylase - ribulose 1,5-bisphosphate carboxylase/oxygenase - SDS sodium dodecyl-sulphate     

一例硬皮病人自发抗核仁抗体的免疫荧光及其与银染的比较研究

从硬皮病人血清中筛选出一例含有自发抗核仁抗体的血清,利用这个血清对核仁抗原的性质及其在细胞中随分裂周期不同产生的分布变化做了初步研究,并把结果与核仁嗜银蛋白做了比较。间接免疫荧光染色及细胞化学分析表明,这种核仁抗原的性质是蛋白质,其分布与嗜银蛋白相似,在间期,抗原呈颗粒状簇集在核仁中,而在分裂中期,抗原颗粒与染色体NORs部位接合,但有证据指出,这种抗原蛋白与核仁嗜银蛋白有所不同,同时还发现,经长时间秋水仙素处理诱导产生微核化的多核细胞中尽管微核的数目远多于细胞中NORs的数目,免疫荧光染色和银染都显示出每个微核中类核仁小体的存在。这说明(1)类核仁小体也是由核仁物质构成;(2)某些类核仁小体的产生可能与NORs无关。对这个现象的意义进行了讨论。     

Improved preservation of the form and contents of wall vesicles and the golgi apparatus in freeze substituted hyphae ofSaprolegnia

Summary Secretory vesicles involved in cell wall synthesis (wall vesicles) and the Golgi apparatus have been compared in conventionally fixed and freeze substituted hyphae of the oomycete fungusSaprolegnia ferax. Wall vesicles freeze substituted in various fluids range from spherical to tubular and contain an intensely staining, phosphorous rich matrix. In contrast diverse conventional fixations cause artefactual constrictions in most tubular vesicles and loss of their intensely staining contents. These data are interpreted to show the existence of an intravesicular skeletal system, with cellular regulation, to determine vesicle morphology and intravesicular synthesis of a hypothetical phosphorylated glycolipid cell wall precursor. Whilst freeze substitution gives superior preservation of wall vesicle morphology, it does not demonstrate any preferential association between wall vesicles and microtubules thus suggesting that microtubules are only indirectly involved in wall vesicle transport. Freeze substitution is superior to conventional fixation for analysis of the Golgi apparatus because it uniquely reveals both differentiation of a specific single cisterna in each Golgi body and greater differences in membrane thicknesses throughout the endomembrane system.     

Massive ribonucleoprotein bodies in gametophyte vegetative cells of the mossTimmiella barbuloides (Brid.) Moenk

Summary Vegetative cells of the gametophyte phase of the mossTimmiella barbuloides (Pottiales) are characterized by large cytoplasmic bodies of spherical shape (SBs) whose ribonucleoprotein composition is cytochemically demonstrated. SBs seem to be derived from massive aggregation of cytoplasmic ribosomes, with possible participation by rough endoplasmic reticulum elements. SBs have been found in stereids, parenchymatous cells and young hydroids of the gametophyte stem, and in euricysts of the leaf nerve. The SBs develop early in the course of cell differentiation and, once formed, persist until advanced stages of cell senescence.     

Fine structure and cytochemistry of the nuclei of the primitive ciliateTracheloraphis crassus (Karyorelictida)

Summary The nuclei ofTracheloraphis crassus were studied using light and electron microscopy combined with Bernhard's RNP staining and pronase digestion. The nuclear apparatus of this species consists of a longitudinal row of 11–43 macronuclei and 4–16 micronuclei. Like in all karyorelictids, the macronuclei are unable to divide and become segregated during cytokinesis; their number is supplemented in every cell cycle by differentiation of several new macronuclei from micronuclei.Each adult macronucleus contains a single compact endonuclear aggregate of several large chromocenters, readily destained with EDTA, and several RNP containing nucleoli. There is continuity between the material of the chromocenters and the decondensed DNP fibrils in the nuclear matrix. The nucleoli contain NORs in the form of fibrillar centers. The endonuclear aggregate includes also groups of RNP granules which are especially resistant to EDTA destaining. A microfibrillar sphere, usually localized at the periphery of the aggregate, contacts one or several nucleoli. The sphere is not bleached with EDTA, and only its periphery becomes digested with pronase. The macronuclear matrix consists of both protein fibrils and pronase-resistant fibrils, the latter being localized at the nuclear periphery.Developing macronuclear primordia contain loose strands of decondensed chromatin; only later they form chromocenters and nucleoli.The micronuclei reproduce by mitosis with typical chromosomes (2n=66). During interphase, they are filled with condensed chromatin which can be bleached with EDTA; they form no nucleoli. Ring-like lamellae, existing in the cavities of the chromatin mass, stain for RNA (after Bernhard) and are pronase-sensitive. These lamellae resemble the kinetochore material conserved during interphase in another karyorelictid ciliate,Trachelocerca geopetiti.     

Mesenchymal control of branching pattern in the fetal mouse lung

The effect of mesenchyme on specialization of respiratory epithelium in the fetal mouse was tested in organ cultures. Heterologous combinations were made between respiratory and non-respiratory lung epithelia and the corresponding mesenchymes. Isolated terminal respiratory buds of fetal mouse lungs were recombined with mesenchyme from chick lung parabronchi, mouse trachea or from the avascular, non-respiratory air sacs of chick lungs. Isolated non-branching chick air sacs were combined with mouse terminal bud mesenchyme or mesenchyme from the respiratory branches of chick lungs. Air sac epithelia branched in a pattern characteristic of the chick lung when combined with chick respiratory mesenchyme and in a pattern characteristic of mouse lung when combined with mouse terminal bud mesenchyme. Mouse terminal bud epithelia did not branch with either mouse tracheal mesenchyme or chick air sac mesenchyme but branched in a chick pattern with chick parabronchial mesenchyme. Electron microscopic examination of the cultures showed that all chick air sac epithelial cultures failed to produce surfactant (lamellar bodies) even when they branched. Control cultures of mouse terminal buds contained large numbers of lamellar bodies; mesenchyme which suppressed branching reduced the number of lamellar bodies to only a few in a small proportion of the cells. Culture medium supplemented with growth factors and hormones increased the number of lamellar bodies in heterologous mouse combinations but did not bring the number to control levels. Supplemented medium had no effect on lamellar body production by chick air sac epithelium. The results indicate that branching pattern is determined by the mesenchyme surrounding the epithelial primordium. However, the capacity to synthesize surfactant is determined by the source of the epithelium; mesenchyme may control the degree of expression but not the absolute presence or absence of the differentiated condition.