Mechanism of interaction of the cyanine dye DiS−C3-(5) with renal brush-border vesicles

Summary The equilibrium binding mechanism and kinetics of binding of diS–C₃-(5) (3,3-dipropylthiodicarbocyanine iodide) to rabbit renal brush-border membrane vesicles (BBMV) were examined using steady-state and time-resolved fluorescence, and fluorescence stopped-flow methods. In aqueous solution, diS–C₃-(5) exists as a monomer at concentrations <5 m with fluorescence emission peak at 670 nm (excitation 622 nm), anisotropyr=0.102, and lifetime =1.2 nsec (23°C). Upon addition of increasing BBMV (voltage clamped to 0 mV using K⁺/valinomycin), the 670 nm emission peak decreases, corresponding to formation of a nonfluorescent membrane dimer, and subsequently a new emission peak at 695 nm increases, corresponding to membrane monomer. Dynamic depolarization studies show that aqueous diS–C₃-(5) rotation is unhindered with a rotational rateR=0.57 nsec^–1 while membrane monomer is hindered with steady-state anisotropyr=0.190, lifetime =2.1 nsec,R=0.58 nsec^–1 and limiting anisotropyr =0.11. Based on equilibrium fluorescence titrations, the membrane monomer-dimer (M-D) dissociation constant,K _d=[M]²/[D][BBMV], is 0.0013, where BBMV is expressed as membrane phospholipid concentration. Three distinct kinetic processes are identified by stopped-flow experiments in which BBMV are mixed with diS–C₃-(5). There is rapid binding of diS–C₃-(5) to the membrane to form bound monomer with a 6-msec exponential time constant. The membrane monomer at the membrane outer surface then aggregates to form bound dimer at the outer surface with a concentration independent time constant of 30 msec. The overall dimerization reaction probably consists of a rate-limiting reorientation process (30 msec) followed by a rapid dimerization which occurs on a nanosecond time scale. Finally, there is a 0.8 to 1 sec translocation of membrane dimer between symmetric sites at the inner and outer membrane surfaces. The translocation reaction is the step which is probably sensitive to changes in transmembrane electrical potential.     

Quantitative gap junction alterations in mammalian heart cells quickly frozen or chemically fixed after electrical uncoupling

Summary The gap junction morphology was quantified in freeze-fracture replicas prepared from rat auricles that had been either quickly frozen at 6 K or chemically fixed by glutaraldehyde, in a state of normal cell-to-cell conduction or in a state of electrical uncoupling. The general appearance of the gap junctions was similar after both preparative procedures. A quantitative analysis of three gap junctional dimensions provided the following measurements in the quickly frozen conducting auricles (mean±sd): (a) P-face particles' diameter 8.27±0.74 nm (n =5709), (b) P-face particles' center-to-center distance 10.78±2.12 nm (n=4800), and (c) E-face pits' distance 9.99±2.19 nm (n=1600). Corresponding values obtained from chemically fixed tissues were decreased by about 3% for the particle's diameter and about 5% for the particles' and pits' distances. Electrical uncoupling by the action of either 1 mM 2–4-dinitrophenol (DNP), or 3.5 mMn-Heptan-1-ol (heptanol), induced a decrease of the particle's diameter, which amounted to –0.69±0.01 nm (mean ±se) in the quickly frozen preparations and –0.71±0.01 nm in the chemically fixed ones. The particles' distance was decreased by –0.96±0.04 nm in the quickly frozen samples and by –0.90 ±0.03 nm in the chemically fixed ones and the E-face pits' distance was similarly reduced. All differences were statistically significant (P<0.001 for all dimensions). Electrical recoupling after the heptanol effect promoted a return of these gap junctional dimensions towards normal values, which was about 50% complete within 20 min. It is concluded that very similar morphological alterations of the gap junctional structure are induced in the mammalian heart by different treatments promoting electrical uncoupling and that these conformational changes appear independently of the preparative procedure. The suggestion that the observed decrease of the particles' diameter is genuinely related to the closing mechanism of the unit cell-to-cell channel set in thei centers is thus confirmed.     

Fluorescent and Ferritin Labelling of Cuticle Surface Carbohydrates of Caenorhabditis elegans and Panagrellus redivivus

Caenorhabditis elegans and Panagrellus redivivus were investigated for surface carbohydrates using fluorescent-labelled and ferritin-labelled lectins. Rhodamine-labelled Concanavalin A was specifically located in the cephalic region of both species. Rhodamine-labelled wheat germ agglutinin was located over the entire cuticle of P. redivivus but was absent on C. elegans. Rhodamine-labelled peanut agglutinin and Limax flavus agglutinin did not label nematodes of either species. Galactose and sialic acid were not detected on either species, whereas mannose-glucose residues were specifically localized in the head areas of both species. No detectable N-acetylglucosamine occurred on C. elegans, but it was evenly distributed over the cuticle surface of P. redivivus.     

Aerenchyma and lenticel formation in pine seedlings: A possible avoidance mechanism to anaerobic growth conditions

Seedlings of pond pine ( Pinus serotina Michx.), sand pine [ P. clausa (Engelm.) Sarg.], and loblolly pine ( P. taeda L., wet-site and drought-hardy seed sources) were grown in hydroponic solution culture using a non-circulating, continuously flowing design under anaerobic or aerobic conditions to determine whether flooding tolerance was correlated with enhanced internal root aeration. Transport of atmospheric O₂ from the shoot to the root of anaerobically grown loblolly and pond pine seedlings was demonstrated via rhizosphere oxidation, using both reduced indigo-carmine solution and a polarographic, ensheathing Pt-electrode. Stem and root collar lenticels were the major sites of atmospheric O₂ entry for submerged roots in these seedlings. No O₂ leakage was detected from roots of aerobically grown pine seedlings. Longitudinal and radial pathways for gaseous diffusion via intercellular air spaces in the pericycle and between ray parenchyma cells, respectively, were demonstrated histo-logically in anaerobically grown loblolly and pond pines. Rhizosphere oxidation, and lenticel and aerenchyma development in roots of flood-intolerant sand pine seedlings grown in anaerobic solutions were minimal. Only 15 days of anaerobic growth conditions were necessary to increase internal root porosities of loblolly and pond pine seedlings – although not to the extent found in seedlings treated for 30 or 75 days. Histological results indicated that root tissue in the secondary stage of growth was capable of forming intercellular air spaces, demonstrating a degree of internal plasticity – at least in the more flood-tolerant loblolly and pond pine seedlings.     

荧光显微术在当代植物细胞生物学研究中的应用

自从八十年前第一次在显微镜下观察到生物组织经紫外线照射后发射荧光的现象以来,荧光显微术不断获得进步,现已发展成细胞生物学中一个重要的研究手段。高度的灵敏性和专一性、制样与观察程序的简便、尤其是适宜于活细胞研究等特点,是它所具有的独特长处。荧光显微术特别是免疫荧光技术在现代医学生物学研究中的应用是一个     

Sperm-oocyte membrane fusion in the human during monospermic fertilization

Sperm-oocyte membrane fusion has been observed during monospermic fertilization of a human oocyte in vitro. Women were stimulated with both clomiphene citrate and human menopausal gonadotropin and were given human chorionic gonadotropin before a LH-surge. Twelve oocytes, collected at laparoscopy from six women who became pregnant by IVF, were allowed to mature for 7–14 hours in vitro and inseminated with preincubated sperm, fixed between 1–3 hours after insemination, and examined by transmission electron microscopy. Membrane fusion had occurred in one ovum 2 hours after insemination, and the oocyte had resumed maturation and was at anaphase II of meiosis. Cortical granules had been exocytosed, and some of their contents were visible at the surface close to the oolemma all around the oocyte. The sperm that fused with this oocyte was acrosome-reacted and had been partly incorporated into the ooplasm, while the anterior two-thirds of its head was phagocytosed by a tongue of cortical ooplasm. Membrane fusion had occurred between the oolemma and the plasma membrane overlying the postacrosomal segment of the sperm head, posterior to the equatorial vestige. Sperm chromatin had not decondensed, and serial sections revealed a midpiece attached to the basal plate and a tail located deeper in the ooplasm, all devoid of plasma membrane. Supplementary sperm penetrating the inner zona, approaching the perivitelline space, had undergone the acrosome reaction but had a persistent vestige of the equatorial segment of the acrosome with intact plasma membrane. Evidence of sperm chromatin decondensation was seen in other oocytes, 3 hours after insemination, which were at telophase II of meiosis. Eight oocytes penetrated by sperm were monospermic, while four were unfertilized. The general pattern of sperm fusion and incorporation appears to conform to that seen in most other mammals. The study also reveals that sperm have to complete the acrosome reaction before fusing with the egg.     

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.