Single chloride channels in endosomal vesicle preparations from rat kidney cortex

Summary Endocytotic vesicles from rat kidney cortex, isolated by differential centrifugation and enriched on a Percoll gradient, contain both an electrogenic H⁺ translocation system and a conductive chloride pathway. Using the dehydration/rehydration method, we fused vesicles of enriched endosomal vesicle preparations and thereby made them accessible to the patch-clamp technique. In the fused vesicles, we observed Cl^– channels with a single-channel conductance of 73±2 pS in symmetrical 140mm KCl solution (n=25). The current-voltage relationship was linear in the range of –60 to +80 mV, but channel kinetic properties dependended on the clamp potential. At positive potentials, two sublevels of conductance were discernible and the mean open time of the channel was 10–15 msec. At negative voltages, only one substate could be resolved and the mean open time decreased to 2–6 msec. Clamp voltages more negative than –50 mV caused reversible channel inactivation. The channel was selective for anions over cations. Ion substitution experiments revealed an anion permeability sequence of Cl^–=Br^–=I^–>SO ₄ ^2– F^–. Gluconate, methanesulfonate and cyclamate were impermeable. The anion channel blockers 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS, 1.0mm) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 0.1mm) totally inhibited channel activity. Comparisons with data obtained from radiolabeled Cl^–-flux measurements and studies on the H⁺ pump activity in endocytotic vesicle suspensions suggest that the channel described here is involved in maintenance of electroneutrality during ATP-driven H⁺ uptake into the endosomes.     

Fusion of liposomes and rat brain microsomes examined by two assays

Summary Liposomes are prepared from rat brain microsomal lipid and loaded with either Tb³⁺ or dipicolinic acid (DPA) to test fusion with the Tb-DPA assay. They are also loaded with octadecyl Rhodamine B chloride (R₁₈) to test fusion with the R₁₈ assay. The addition of either Ca²⁺ or Mg²⁺ to loaded liposomes develops fluorescence with both assays. The fluorescence elicited by Mg²⁺ is similar to that elicited by Ca²⁺ if assessed with R₁₈, but much higher if determined by Tb-DPA. The Ca²⁺-dependent fluorescence of the Tb-DPA complex is not suppressed by the addition of EDTA, and therefore it is internal to vesicles. The contrary is true for the Mg²⁺-dependent fluorescence. Rat brain microsomes can be disrupted by adding octylgucoside and reconstituted by removing it by dialysis. We use this procedure to load microsomes with DPA. This allows the use of the Tb-DPA assay for testing the fusion of rat brain microsomes. Reconstituted microsomes fuse with liposomes. This fusion has characteristics similar to those of liposome-liposome fusion. However, no microsome-microsome fusion could be detected with either method. The two methods give different results, owing to the chemical properties of the assays. Indeed Tb-DPA implies the retention of vesicle content, whereas this is not required by the R₁₈ assay.     

Fusion of cultured dog kidney (MDCK) cells: I. Technique,fate of plasma membranes and of cell nuclei

Summary The evaluation of the intracellular signal train and its regulatory function in controlling transepithelial transport with electrophysiological methods often requires intracellular measurements with microelectrodes. However, multiple impalements in epithelial cells are hampered by the small size of the cells. In an attempt to avoid these problems we fused cells of an established cell line, Madin Darby canine kidney cells, originally derived from dog kidney, to giant cells by applying a modified polyethylene glycol method. During trypsin-induced detachment from the ground of the petri dish, individual cells grown in a monolayer incorporate volume and mainly lose basolateral plasma membrane by extrusion. By isovolumetric cell-to-cell fusion, spherical giant cells are formed within 2 hr. During this process a major part of the individual cell plasma membranes is internalized. Over three weeks following cell plasma membrane fusion degradation of single cell nuclei and cell nuclear fusion occurs. We conclude that this experimental approach opens the possibility to investigate ion transport of epithelia in culture by somatic cell genetic techniques.     

Molecular evolution of enzyme structure: Construction of a hybrid hamster/Escherichia coli aspartate transcarbamoylase

Summary Aspartate transcarbamoylase (ATCase, EC 2.1.3.2) is the first unique enzyme common to de novo pyrimidine biosynthesis and is involved in a variety of structural patterns in different organisms. InEscherichia coli, ATCase is a functionally independent, oligomeric enzyme; in hamster, it is part of a trifunctional protein complex, designated CAD, that includes the preceding and subsequent enzymes of the biosynthetic pathway (carbamoyl phosphate synthetase and dihydroorotase). The complete complementary DNA (cDNA) nucleotide sequence of the ATCase-encoding portion of the hamster CAD gene is reported here. A comparison of the deduced amino acid sequences of the hamster andE. coli catalytic peptides revealed an overall 44% amino acid similarity, substantial conservation of predicted secondary structure, and complete conservation of all the amino acids implicated in the active site of theE. coli enzyme. These observations led to the construction of a functional hybrid ATCase formed by intragenic fusion based on the known tertiary structure of the bacterial enzyme. In this fusion, the amino terminal half (the “polar domain”) of the fusion protein was provided by a hamster ATCase cDNA subclone, and the carboxyl terminal portion (the “equatorial domain”) was derived from a clonedpyrBI operon ofE. coli K-12. The recombinant plasmid bearing the hybrid ATCase was shown to satisfy growth requirements of transformedE. coli pyrB ⁻ cells. The functionality of thisE. coli-hamster hybrid enzyme confirms conservation of essential structure-function relationships between evolutionarily distant and structurally divergent ATCases.     

下肢长骨骨重的非对称性

本文测量82副中国成年下股长骨即股骨,胫骨和腓骨的重量,在两侧骨重相差>1％时,则股骨、胫骨和腓骨及其三骨总重均以非对称性为多,均具有非常显著性差异(P<0.001)。除腓骨重具有明显侧别差异外,其余无显著性侧别差异,对腓骨侧差解释为:①腓骨主要机能是肌的附着,而下肢对踢和足趾取物具有明显的侧别差异;②以两侧骨重相差>1％时为非对称性的标准,由于腓骨绝对重量较小,在两侧相差<0.5克时即多属非对称性。     

Morphological and molecular characterization of fertile tetraploid somatic hybrids produced by protoplast electrofusion and PEG-induced fusion between Lycopersicon esculentum Mill. and Lycopersicon peruvianum Mill.

Summary Mesophyl protoplasts of two genotypes of cultivated tomato (Lycopersicon esculentum Mill.) and one of its wild relative species (Lycopersicon peruvianum Mill.) were fused by using electrofusion and polyethyleneglycol-induced fusion. Forty-three fertile tetraploid somatic hybrid plants, each deriving from separate calli, were recovered from both fusion procedures. Electrofusion appeared more efficient than chemical fusion for the production of somatic hybrids. These plants appeared morphologically similar, whatever the fusion procedure and tomato genotype. They had intermediate leaf, inflorescence, and flower morphology. After self-pollination, the hybrids set fruit of intermediate size and color. The hybrid nature of these plants was confirmed by isoelectric focusing of the Rubisco small subunits used as nuclear markers. L. esculentum and L. peruvianum were distinguished by means of two chloroplast markers: CF1-ATPase subunit as analyzed by isoelectro-focusing and ct DNA restriction patterns. All hybrids displayed both ct markers of only one parent with no biased transmission. Mitochondrial (mt) DNAs were prepared from flower buds by using miniaturized CsCl gradients. Preliminary analysis indicated that mt genomes from the hybrids all differed from those of both parents. mt DNA Sall restriction enzyme analysis revealed that all but two hybrids contained one novel fragment of 13.5 kb. Gene mapping experiments showed that the mt apocytochrome b and ATPase subunit 9 homologies in the somatic hybrid mt DNA resembled L. esculentum and L. peruvianum, respectively; the mt nad5 probe distinguished at least four distinct patterns in the hybrids. These results indicated that mt DNA rearrangements involving intergenomic recombinations occurred through protoplast fusion. A greater mt DNA polymorphism was induced with chemical fusion than with electrofusion.     

Preendocytotic alterations in cumulus cell gap junctions precede meiotic resumption in the rat cumulus-oocyte complex

Cumulus cells in the mammalian ovary are normally connected to each other and to their enclosed oocyte by an extensive network of gap junctions (GJs). We have shown that the loss of cumulus cell GJs is correlated temporally with meiotic resumption in the intact preovulatory rat follicle (Larsen et al., 1986). Here we describe morphological changes in GJ particle packing patterns (PPPs) that occur prior to GJ loss and meiotic resumption in hormonally stimulated rat cumulus-oocyte complexes (COCs). In the PMSG-primed rat, 89% of the cumulus cell GJ area detected by freeze-fracture electron microscopy consists of tightly packed junctional particles: 4% exhibit loose PPPs of randomly dispersed particles; and 7% contain a mixture of both tight and loose PPPs. One to 2 hr after stimulation with hCG, the area of GJs containing tight PPPs drops by 50%-60%, while junctions exhibiting loosely organized and mixed patterns increase concomitantly. These shifts in PPPs are accompanied by the appearance of unusual particle-free areas of puckered or ruffled nonjunctional membrane at the GJ periphery. Cumulus cell GJs from isolated COCs incubated in FSH-containing medium demonstrate a similar shift in PPPs prior to meiotic resumption. The appearance of fusing areas of particle-free nonjunctional membrane at the GJ periphery in vitro is correlated with GJ loss and is not seen in COCs treated with dihydrocytochalasin B to inhibit endocytotic removal of cumulus GJs. The structural and temporal nature of these morphological observations supports the hypothesis that interruption of junctional communication plays a role in meiotic maturation of the preovulatory oocyte.     

Molecular analysis of the extent of asymmetry in two asymmetric somatic hybrids of tomato

Summary Two somatic hybrid plants generated from a single fusion event between Lycopersicon esculentum and irradiated L. pennellii protoplasts have been analyzed at the molecular level. Over 30 loci have been analyzed using isozymes and RFLPs. All loci tested on chromosomes 2–10 were heterozygous, while those loci on chromosome 12 were homozygous L. pennellii in both somatic hybrids. In one of the somatic hybrids, 2850, loci on chromosome 1 were also homozygous L. pennellii. The other somatic hybrid, 28F5, was heterozygous at all chromosome 1 loci tested, but exhibited altered stoichiometry of parental bands as compared to the sexual hybrid. Loci on chromosome 2 from both somatic hybrids have altered stoichiometry, with L. pennellii alleles being four times more abundant than expected. Both somatic hybrids contain the L. esculentum chloroplast genome, while only L. pennellii polymorphisms have been detected in the mitochondrial genome.     

Paleohistology of Paget's disease in two medieval skeletons.

Paget's disease has been ascribed several times to specimens of archeological bone but, in the absence of microscopic examination, the evidence remains insubstantial. Suspected metabolic bone disease is described here in the archeological remains of a skeleton from a 16th century burial ground at Wells Cathedral, England and from a single medieval sacrum recovered from a large deposit of disarticulated bones from a churchyard at Barton-on-Humber, England. Radiographs showed apparent structural abnormality in one femoral shaft and calcaneus and in the isolated sacrum. Histomorphometry on undecalcified bone cores confirmed the regions of abnormality and showed not only increased trabecular width but also areas of "mosaic" woven bone together with extensive resorption cavities; these features contrasted with the normal structure and organized lamellar bone from sites elsewhere. Despite post-interment changes in surrounding tissues, the morphological stability of some of the osteocytes was remarkable. Preservation of the histology was sufficient to permit the assignment of a metabolic bone disorder and the nature of the sclerosis was consistent with Paget's disease.