Metabolic inhibitors and mitosis: II. Effects of dinitrophenol/deoxyglucose and nocodazole on the microtubule cytoskeleton

Summary To examine the effects exerted on the microtubule (MT) cytoskeleton by dinitrophenol/deoxyglucose (DNP/DOG) and nocodazole, live PtK₁ cells were treated with the drugs and then fixed and examined by immunofluorescence staining and electronmicroscopy. DNP/DOG had little effect on interphase MTs. In mitotic cells, kinetochore and some astral fibers were clearly shortened in metaphase figures by DNP/DOG. Nocodazole rapidly broke down spindle MTs (except those in the midbody), while interphase cells showed considerable variation in the susceptibility of their MTs. Nocodazole had little effect on MTs in energy-depleted (DNP/DOG-treated) cells. When cytoplasmic MTs had all been broken down by prolonged nocodazole treatment and the cells then released from the nocodazole block into DNP/DOG, some MT reassembly occurred in the ATP-depleted state. MTs in permeabilized, extracted cells were also examined with antitubulin staining; the well-preserved interphase and mitotic arrays of MTs showed no susceptibility to nocodazole. In contrast, MTs suffered considerable breakdown by ATP, GTP and ATPS; AMPPNP had little effect. This susceptibility of extracted MT cytoskeleton to nucleotide phosphates was highly variable; some interphase cells lost all MTs, most were severely affected, but some retained extensive MT networks; mitotic spindles were diminished but structurally coherent and more stable than most interphase MT arrays.We suggest that: 1. in the living cell, ATP or nucleotide triphosphates (NTPs) are necessary for normal and nocodazole-induced MT disassembly; 2. the NTP requirement may be for phosphorylation; 3. shortening of kinetochore fibers may be modulated by compression and require ATP; 4. many of these results cannot be accomodated by the dynamic equilibrium theory of MT assembly/disassembly; 5. the use and role of ATP on isolated spindles may have to be reevaluated due to the effects ATP has on the spindle cytoskeleton of permeabilized cells.     

Crystal-associated matrix components in rat incisor enamel

Summary Sections of glutaraldehyde-OsO₄-fixed, plastic-embedded rat incisor enamel were left untreated, stained, decalcifed (1% formic acid in 10% sodium citrate), or decalcified-stained. The presence of apatite crystals was monitored with electron diffraction. After brief decalcification and staining, apatite crystals and matrix components were visualized in the same field. The ghost was continuous with crystal fragments, and the coat appeared as a dense line next to crystals and ghosts. Position of ghosts and crystals at the ameloblast-enamel junction (AEJ) of the secretion zone suggested that there may be a lag of no more than 1/5 min between the elaboration of ghost and crystal. A major change in enamel morphology occurs between the AEJ and the deep enamel of the secretion zone. The ghost becomes thinner, the coat more pronounced, and the crystal enlarges. There is only little change from the deep secretion to the maturation zone enamel.     

The use of amphotericin B to detect inhibitors of cellular cholesterol biosynthesis

Pores formed in the membranes of animal cells by complexes of sterols and the polyene antibiotic amphotericin B can efficiently kill the cells. Thus, in the absence of exogenous sources of cholesterol, inhibitors of enzymes in the cholesterol biosynthetic pathway render cells resistant to amphotericin B. Preincubation of Chinese hamster ovary cells with compactin or 25-hydroxycholesterol, inhibitors of the synthesis of the key intermediate mevalonate, protected cells from amphotericin B killing and this protection was reversed by the addition of exogenous mevalonate. The ability of compactin to confer amphotericin B resistance on normal cells was abolished when cells were provided exogenous cholesterol by the receptor-mediated endocytosis of low density lipoprotein. Low density lipoprotein receptor-defective Chinese hamster ovary cells were not subject to this low density lipoprotein-dependent amphotericin B killing. Exogenous mevalonate did not prevent 4,4,10 beta-trimethyl-trans-decal-3 beta-ol, an inhibitor of mevalonate conversion to sterols, from protecting cells from amphotericin B. A simple two-step protocol in which cells are preincubated (15-24 h) with potential inhibitors and then treated (3-6 h) with amphotericin B was devised to provide a sensitive method for detecting direct (e.g., competitive) and regulatory inhibitors of cholesterol biosynthesis. This protocol may prove useful in detecting potential antihypercholesterolemia drugs and is currently being used to isolate mutants in receptor-mediated endocytosis.     

Effect of Na+, K+, ouabain,amiloride and ethacrynic acid on the transepithelial potential across Malpighian tubules of Locusta

A transepithelial potential of +8.74±0.29 mV (n = 85) has been recorded across the Malpighian tubules of Locusta. The effect of varying the Na⁺ and K⁺ concentration in the bathing medium on the transepithelial potential has been determined. The data show that the transepithelial potential does not obey the Nernst equation for K⁺. Ouabain, ethacrynic acid and amiloride all inhibit the transepithelial potential. The results are discussed in relation to the nature of the mechanisms of cation transport across the Malpighian tubules.     

Entamoeba histolytica. I. Aerobic metabolism

The respiration of intact trophozoites harvested from axenic cultures of Entamoeba histolytica was studied with the polarographic technique utilizing the Clark oxygen electrode. A typical Q_o2 value for the freshly harvested amebae was 1 μatom oxygen/mg protein/hr.It was conclusively demonstrated that this parasite, a putative anaerobe, not only consumes oxygen when provided, but has a high affinity for the gas.Added glucose, galactose, and ethanol increased the respiratory rates, whereas other carbohydrates were without effect on the endogenous respiration. Intermediates of the tricarboxylic acid cycle, amino and fatty acids did not stimulate the respiration of E. histolytica.Inhibitors of the mammalian respiratory chain (cyanide, antimycin) as well as agents that inhibit enzymes catalyzing the tricarboxylic acid cycle (malonate, fluoropyruvate, fluoroacetate, fluorocitrate) had little effect on the endogenous or glucose-supported respiration. Alkylating agents (iodoacetamide, iodoacetate), cinnamate, and N-ethylymaleimide strongly inhibited the oxygen consumption of E. histolytica. The chemotherapeutic agents, Paromomycin, Emetine and Metronidazole, at concentrations that inhibit growth in vitro, did not restrict the respiration.Storage of the trophozoites at 4 C led to progressive deterioraion of the parasites and loss of endogenous and glucose-supported respiration. The deterioration was paralled by loss of SH-materials from the amebae. Likewise, sonication or lysis with detergents abolished both the endogenous respiration and response to glucose.Exogenous NADH or NADPH evoked only marginal increases in oxygen consumption of the freshly harvested amebae, but were effective respiratory substrates with stored or sonicated organisms. Addition of vitamin K₃ greatly enhanced the endogenous and glucose-supported respiration of the intact amebae, as well as enhancing the response of stored or sonicated amebae to reduced pyridine nucleotides.     

The secretory nature of the excretory gland cells of Strephanurus dentatus. 3. Proteinase inhibitors

A proteinase inhibitor(s) was found in extracts of the excretory gland cells, intestines, esophagi, reproductive organs, and body walls from Stephanurus dentatus adults. The specific activity of the inhibitor(s) in the excretory gland cell extract was 45–175 times greater than in the other tissues. It is heat stable at pH 5.0 and inhibits the esterolytic activity of trypsin and chymotrypsin using p-toluenesulfonyl-l-arginine methyl ester hydrochloride (TAME) and benzoyl-l-tyrosine ethyl ester (BTEE) as the substrates, respectively, and also the proteolytic activity of both chymotrypsin and trypsin using casein as the substrate. S. dentatus adults maintained in NCTC 109 medium, secreted a trypsin inhibitor.     

Comparison of ordinations and classifications of vegetation data

The methodology of comparing the results of multivariate community studies (resemblance matrices, ordinations, hierarchical and nonhierarchical classifications) is reviewed from two viewpoints: basic strategy and measure employed. The basic strategy is determined by 7 choices concerning the type of results, consensus methods or resemblance measures, hypothesis testing or exploratory analysis, lack or presence of reference basis, data set congruence or algorithmic effects, number of factors responsible for differences among results, and the number of properties considered in the comparison. Included is a brief summary of methods applicable to vegetation studies. Examples from a grassland survey demonstrate the utility of comparisons in evaluating the effects of plot size, data type, standardization, taxonomic level and number of species on classifications and ordinations.Abbreviations OUC = Operational Unit of Comparison - PCA = Principal Components Analysis - PCoA = Principal Coordinates Analysis - SSA = Incremental Sum of Squares Agglomeration     

Interactions between anion exchange and other membrane proteins in rabbit kidney medullary collecting duct cells

Summary In separated outer medullary collecting duct (MCD) cells, the time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate), to the MCD cell analog of band 3, the red blood cell (rbc) anion exchange protein, can be measured by the stopped-flow method and the reaction time constant, _DBDS, can be used to report on the conformational state of the band 3 analog. In order to validate the method we have now shown that the ID_50,DBDS,MCD (0.5±0.1 m) for the H₂-DIDS (4,4-diisothiocyano-2,2-dihydrostilbene disulfonate) inhibition of _DBDS is in agreement with the ID_50,Cl ^–,_MCD (0.94±0.07 m) for H₂-DIDS inhibition of MCD cell Cl^– flux, thus relating _DBDS directly to anion exchange. The specific cardiac glycoside cation transport inhibitor, ouabain, not only modulates DBDS binding kinetics, but also increases the time constant for Cl^– exchange by a factor of two, from _Cl=0.30±0.02 sec to 0.56±0.06 sec (30mm NaHCO₃). The ID_50,DBDS,MCD for the ouabain effect on DBDS binding kinetics is 0.003±0.001 m, so that binding is about an order of magnitude tighter than that for inhibition of rbc K⁺ flux (K _I,K ⁺,_rbc=0.017 m). These experiments indicate that the Na⁺,K^–-ATPase, required to maintain cation gradients across the MCD cell membrane, is close enough to the band 3 analog that conformational information can be exchanged. Cytochalasin E (CE), which binds to the spectrin/actin complex in rbc and other cells, modulates DBDS binding kinetics with a physiological ID_50,DBDS,MCD (0.076±0.005 m); 2 m CE also more than doubles the Cl^– exchange time constant from 0.20±0.04 sec to 0.50±0.08 sec (30mm NaHCO₃). These experiments indicate that conformational information can also be exchanged between the MCD cell band 3 analog and the MCD cell cytoskeleton.     

Characterization of inositol 1,4,5-trisphosphate-sensitive (IsCaP) and-insensitive (IisCaP) nonmitochondrial Ca2+ pools in rat pancreatic acinar cells

Summary We have measured Ca²⁺ uptake and Ca²⁺ release in isolated permeabilized pancreatic acinar cells and in isolated membrane vesicles of endoplasmic reticulum prepared from these cells. Ca²⁺ uptake into cells was monitored with a Ca²⁺ electrode, whereas Ca²⁺ uptake into membrane vesicles was measured with⁴⁵Ca²⁺. Using inhibitors of known action, such as the H⁺ ATPase inhibitors NBD-Cl and NEM, the Ca²⁺ ATPase inhibitor vanadate as well as the second messenger inositol 1,4,5-trisphosphate (IP₃) and its analog inositol 1,4,5-trisphosphorothioate (IPS₃), we could functionally differentiate two non-mitochondrial Ca²⁺ pools. Ca²⁺ uptake into the IP₃-sensitive Ca²⁺ pool (IsCaP) occurs by a MgATP-dependent Ca²⁺ uptake mechanism that exchanges Ca²⁺ for H⁺ ions. In the absence of ATP Ca²⁺ uptake can occur to some extent at the expense of an H⁺ gradient that is established by a vacuolar-type MgATP-dependent H⁺ pump present in the same organelle. The other Ca²⁺ pool takes up Ca²⁺ by a vanadate-sensitive Ca²⁺ ATPase and is insensitive to IP₃ (IisCaP). The IsCaP is filled at higher Ca²⁺ concentrations (10^–6 mol/liter) which may occur during stimulation. The low steady-state [Ca²⁺] of 10^–7 mol/liter is adjusted by the IisCaP.It is speculated that both Ca²⁺ pools can communicate with each other, the possible mechanism of which, however, is at present unknown.     

猕猴(Macaca mulatta)生命表研究

本文研究了猕猴雌性生命表。南湾猕猴各年度全群平均存活率高于93％,特定年龄组逐年存活率直到13岁仍达0.81以上,种群内禀增长率为0.1327,年周增长率为1.1419。用莱斯利矩阵对种群发展作预测,将预测值与1985-1987年的实测值作卡方适应性检验,两者无显著性差异。进一步分析发现该方法预测南湾猕猴种群动态,在7年内是有效的。