Cytochalasin D and cationized ferritin as probes for the morphological investigation of blebbing in two human cell lines

Summary The potent fungal metabolite cytochalasin D (CD) and cationized ferritin (CF) are used in combination to test for negative charge distribution on blebs (knobs). Two established human epithelial cell lines, WISH and HeLa, that display blebs in various phases of the cell cycle or under certain culture conditions (37,46) are investigated. CD alone, applied at a low concentration (1.0 μg/ml) and for a short time period (3 min), causes blebs to appear as the prevalent surface feature. These are filled mainly with free ribosomes. Additionally, feltlike mats, presumed to be disorganized, compacted microfilaments, are formed directly beneath the cell membrane. These are especially evident in the cortical cytoplasm below the blebs or bleb clusters. CF (0.345 mg/ml), applied for a 5-min period after CD administration (1.0 μg/ml) for 3 min, appears along the surface of microvilli, at the base of blebs, and in vesicles beneath the bleb clusters. In some cases, microfilaments (6 nm in diameter) are closely related to the vesicles. CF does not preferentially bind to the apical cell membrane of blebs. Above areas of the subplasmalemmal microfilaments, CF membrane binding is apparent, even under circumstances where the filaments are disorganized by cytochalasin treatment. These results seem to show the following: (a) bleb membranes are different from the remainder of the cell and do exhibit a loss of negative charge and (b) surface charge may be dependent on the presence or structural integrity of membrane-related 6-nm microfilaments. The support of this research by a grant from the Baylor College of Dentistry and The Oklahoma College of Osteopathic Medicine and Surgery is gratefully acknowledged. The assistance of Dr. J. H. Martin, Department of Pathology, Baylor University Medical Center, is also greatly appreciated.     

日本血吸虫尾蚴的组织化学及扫描电镜观察

日本血吸虫尾蚴具有一个头腺、2对前钻腺及3对后钻腺,其解剖学位置、构造、化学成分及功能均有区别。钻腺分泌物含有多糖酶及蛋白酶。尾蚴的前端系特化的头器结构,它具有半月形嵴、钻腺开口及乳突。     

食蟹猴疟原虫晚期卵囊、成孢子细胞与子孢子扫描电镜观察

本研究对实验感染食蟹猴疟原虫后9、10和14天的斯氏按蚊阳性蚊胃,进行扫描电镜观察。晚期(分化的)卵囊表面呈现凹凸不平皱褶。成孢子细胞体常呈圆球形或椭圆形,表面光滑,子孢子芽从其表面长出。子孢子体细长,稍弯曲,体表光滑;虫体前部较细,顶端稍平,后部稍粗,末端钝圆。本文对成孢子细胞不同发育阶段的形态予以较详细描述,对子孢子出囊方式做了初步讨论。     

A comparison of techniques for isolation of the outer membrane proteins of Haemophilus influenzae type b

We compared several rapid techniques used for extraction of outer membrane proteins from gram-negative enteric bacteria to Haemophilus influenzae type b. After lysis of cells with a French press, the inner and outer membranes were separated by isopycnic centrifugation. Each membrane was identified by density, morphology, enzymatic activity, and susceptibility to solid-phase iodination of intact cells. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we identified 10 polypeptides which were enriched in the outer membrane band compared to the inner membrane band. Using these proteins, we compared the polypeptide pattern of outer membranes with that obtained by (1) selective solubilization with sodium dodecyl-beta-D-maltoside, octyl-beta-D-glucopyranoside, Triton X-100, sodium, or cholamidopropyl dimethylaminopropanesulfonate; (2) extraction with chaotropic agents and heat; and (3) differential centrifugation of vesicles shed during transition from log growth phase to stationary growth phase. There were definable differences between the polypeptide pattern of membranes obtained with each rapid technique compared to the polypeptide pattern of isolated outer membranes. The polypeptide pattern of lithium extracts and the Triton X-100 insoluble fractions of total membranes most closely approximated the polypeptide pattern of isopycnically isolated outer membranes. Depending on the outer membrane protein sought, one of these rapid techniques can be utilized when a rapid method of outer membrane protein isolation is required.     

Molecular motion underlying activation and inactivation of sodium channels in squid giant axons

Summary Measurements of the changes in birefringence associated with changes in membrane potential were made with internally perfused squid giant axons in low sodium solutions at 0–8°C. The time course of the birefringence changes share many properties of the gating (polarization) currents previously studied in this nerve. Both can be demonstrated as an asymmetry in the response to voltage pulses symmetrical about the resting potential which is not present about a hyperpolarized holding potential. Both have a rapid relaxation, which precedes the sodium permeability change. Both exhibit an initial delay or rising phase. Both are reversibly blocked by perfusion with 30mm colchicine; neither are altered by changes on sodium concentrations or 300nm tetrodotoxin. The birefringence response has a decrease in the amplitude of the rapid relaxation associated with the appearance of a slow relaxation. This is similar to the immobilization of fast gating charges which parallels sodium current inactivation.The amplitude of the birefringence and the gating current responses is consistent with a change in the alignment of several hundred peptide bonds per sodium channel.     

Structure and composition of influenza virus. A small-angle neutron scattering study

A detailed analysis is presented of the small-angle neutron scattering curves of homogeneous solutions of influenza B virus, both intact and after treatment with bromelain, which removes the external glycoprotein spikes. The two sets of data are consistent with the following low-resolution structure: the virus particles are spherical, about 1200 A in diameter and of Mr about 180 X 10(6). The lipid bilayer is centred at a radius of 425 A, is 40 A to 50 A thick and constitutes 25% to 28% of the virus mass. The surface glycoproteins, predominantly haemagglutinin, contribute 40% to 46% of the total mass. Surprisingly little protein is found in the interior of the virus. It is suggested that the reason for this is that many particles do not contain the full complement of ribonucleoprotein complexes. These results are in good agreement with recent scanning transmission electron microscopic measurements of molecular mass and cryo-electron microscopic observations of the same preparations. Appendix 1 describes a new method of deriving spherical shell models from contrast variation neutron scattering data on viruses, in which scattering curves from all measured contrasts are used simultaneously. There is also a discussion of the assumptions and limitations implicit in the structural interpretation of such models, with emphasis on viruses containing lipid bilayers. Appendix 2 examines the effect on the scattering curves of various arrangements of the surface glycoproteins.     

Improved Method for Isolating Synaptosomes from 11 Regions of One Rat Brain: Electron Microscopic and Biochemical Characterization and Use in the Study of Drug Effects on Nerve Terminal γ-Aminobutyric Acid in Vivo

A procedure is described for the rapid preparation of nerve ending particles (synaptosomes) from 11 regions of one rat brain. The synaptosomal fractions have been characterized by electron microscopy and determination of four marker enzymes, i.e., glutamate decarboxylase (GAD), acetylcholinesterase, succinate dehydrogenase, and glycerol 3-phosphate dehydrogenase. Comparison with a much lengthier standard (Ficoll-sucrose) preparation showed that the synaptosomal yield of the new procedure was substantially better as judged by both morphological evaluation and protein recovery. The improved synaptosome preparation was used for determination of regional gamma-aminobutyric acid (GABA) levels in synaptosomal fractions. The postmortem increase in GABA level during removal and dissection of brain tissue and homogenization and fractionation procedures could be minimized by rapid processing of the tissue at low temperatures and inclusion of the GAD inhibitor 3-mercaptopropionic acid (3-MP; 1 mM) in the homogenizing medium. The addition of GABA (0.2 mM) to the homogenizing medium did not alter the GABA levels in the synaptosomes, indicating that no significant redistribution of GABA occurred during subcellular fractionation in sodium-free media. Synaptosomal GABA levels determined in the 11 rat brain areas showed the same regional distribution as the GABA-synthesizing enzyme GAD. On the basis of these findings, it was suggested that the synaptosome preparation could be used to evaluate the in vivo effects of drugs on nerve terminal GABA. Treatment of rats with a convulsant dose of 3-MP (50 mg/kg i.p.) 3 min before decapitation significantly lowered synaptosomal GABA levels in olfactory bulb, hippocampus, thalamus, tectum, and cerebellum. The 3-MP-induced seizures and reduction of GABA levels could be prevented by administration of valproic acid (200 mg/kg i.p.) 15 min before the 3-MP injection. The data indicate that the improved synaptosome preparation offers a convenient method of preparing highly purified synaptosomes from a large number of small tissue samples and can provide useful information on the in vivo effects of drugs on regional GABA levels in nerve terminals.     

Quantitative ultrastructural analysis of barley sperm

Summary Complete serial ultrathin sections of seven sperm pairs, computer-assisted measurements of cell, nuclear and organelle surface areas and volumes, and three-dimensional imagery were used to demonstrate that a process of cytoplasm and organelle elimination occurs during sperm maturation in barley. The number of mitochondria per sperm cell is reduced by 50%; sperm cell surface area and volume are reduced by 30% and 51% respectively. Mean volume and surface area per mitochondrion are significantly less in mature sperms. No examples of mitochondrial fusion or degeneration were observed within sperm cells. These data, along with observations of plasma membrane apposition and vesiculation within cytoplasmic extensions containing mitochondria, support the proposition that cytoplasm and organelle loss results primarily from the formation of cytoplasmic projections that are subsequently discarded from the sperm cell body. Comparisons of the quantitative data, including the number of mitochondria, indicate that differences between sperm cells of a pair are absent to very slight. Spatial organization within the pollen grain is such that the mature sperms, as well as the sperms and vegetative nucleus, are not in close proximity.     

Cytoskeletal changes visualized by fluorescence microscopy during amoeba-to-flagellate and flagellate-to-amoeba transformations inPhysarum polycephalum

Summary Changes in the intracellular distribution of microtubules and microfilaments during amoeba-to-flagellate and flagellate-to-amoeba transformations inPhysarum polycephalum were examined by fluorescence microscopy using anti-tubulin antibody and NBD-phallacidin, respectively. Amoebae contained an extensive microtubular cytoskeleton, which was converted to a flagellar cone structure during transformation to flagellates in liquid medium. When flagellates reverted back to amoebae, this conical structure disintegrated prior to flagella resorption. Amoebae showed some microfilament-enriched domains along the periphery, from which numerous filamentous extrusions, probably pseudopods and filopods, emanated. Flagellates contained a ridge, a sheet-like structure, along their dorsal axis, especially in the earlier stages of flagellation. Another microfilament-enriched thick filamentous structure ran along the dorsal axis, starting from the anterior tip of the cell. This structure apparently coincided spatially with one of the bundles of microtubules. During the reversion to amoebae, other localized microfilaments were transiently observed at the posterior end. A model of cytoskeletal changes in the transformations between these two cell types was proposed.