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.     

How does inactivation change timing of replication in the human X chromosome?

Summary The kinetics of replication of the inactive (late replicating) X chromosome (LRX) were studied in karyotypically normal lymphocytes and human amniotic fluid cells. Both cell types were successively pulse labeled with 1-h or 1/2-h thymidine pulses in an otherwise BrdU-substituted S phase after partial synchronization of the cultures at G₁/S. For the first time with this technique, the entire sequence of replication was analyzed for the LRX from the beginning to the end of the S phase, with special reference to mid S (R-band to G-band transition replication). The inactive X is the last chromosome of the metaphase to start replication, with a delay of 1 or 2h, after which time a thymidine pulse results in R-type patterns. In mid S, the inactive X is the first chromosome to switch to G-type replication (without overlapping of both types and without any detectable replication pause). Until the end of S, a thymidine pulse results in G-type patterns. To rule out artifacts that might arise by the synchronization of cultures in these experiments, controls were carried out with BrdU pulses and the BrdU antibody technique without synchronization. In the course of replication, no fundamental difference was seen between the two different cell types examined. In contrast to studies using continuos labeling, this study did not reveal an interindividual difference of replication kinetics in the LRXs of the seven individuals studied; thus it is concluded that the inactive X chromosome shows only one characteristic course of replication.     

Epidemiological analysis of acute respiratory diseases (ARD) of viral etiology in the Czech Socialist Republic (CSR] and German Democratic Republic (GDR) over the period 1979 to 1984

An analysis is made of the ARD reported in CSR and the GDR over the period July 1st, 1979 to June 30th, 1984. During that time, there were 27,810,000 cases reported in CSR in the framework of ARD epidemiological surveillance, representing 2.67 cases per one inhabitant, whereas in the GDR, the total number of reported ARD was 28,900,000 yielding 1.73 cases per person. However, the GDR reported higher morbidity per one child of preschool age. The authors believe that the differences in the reported incidence of ARD between the two countries are due to differences in the reporting systems and medical officers' activity during an epidemic and in the interim period. Approximately one third of ARD reported annually in the two countries falls to the period of influenza epidemics. The authors also analyze the etiology of the influenza epidemics which affected the two countries in 1980, 1981, 1982, 1983 and 1984. In most seasons, the causative agents and morbidity excesses were different in the two countries. The drift variant B/USSR/100/83, which caused a major epidemy in CSR in 1984, has not to date been implicated in the DGR in the etiology of ARD. The cyclic epidemic due to Mycoplasma pneumoniae occurred in the GDR already in 1979-80, while CSR experienced it a year later. There was a temporal and territorial correlation between the course of A(H1N1) influenza epidemic in the two countries in 1984.     

Scavenging of Hydrogen Peroxide in the Endosperm of Ricinus communis by Ascorbate Peroxidase

The fat-storing endosperm of Ricinus communis L. was found tocontain an ascorbate peroxidase (EC 1.11.1.11 [EC] ), which is nearlyas active as catalase (EC 1.11.1.6 [EC] ) in degradation of hydrogenperoxide (H₂O₂) at its physiological concentrations. This ascorbateperoxidase probably functions together with monodehydroascorbatereductase (EC 1.6.5.4 [EC] ) or dehydroascorbate reductase (EC 1.8.5.1 [EC] )and glutathione reductase (EC 1.6.4.2 [EC] ) to remove the H₂O₂ producedduring the transformation of fat to carbohydrate in the glyoxysomes.The activities of these enzymes as well as the content of ascorbateand glutathione increase parallel to the activities of glyoxysomalmarker enzymes during the course of germination. Inhibitionof catalase by aminotriazole results in increases of the ascorbateperoxidase activity and of the glutathione content. All fourenzymes are predominantly localized in the cytosol of the Ricinusendosperm with low activities found in the plastids and themitochondria. The results suggest, that the ascorbate-dependentH₂O₂ scavenging pathway, which has been shown to be responsiblefor the reduction of photosynthetically derived H₂O₂ in thechloroplasts, operates also in the Ricinus endosperm. (Received June 5, 1990; Accepted July 31, 1990)     

Biosynthesis of digalactosyldiacylglycerol in plastids from 16:3 and 18:3 plants

Intact chloroplasts isolated from leaves of eight species of 16:3 and 18:3 plants and chromoplasts isolated from Narcissus pseudonarcissus L. flowers synthesize galactose-labeled mono-, di-, and trigalactosyldiacylglycerol (MGDG, DGDG, and TGDG) when incubated with UDP-[6-³H]galactose. In all plastids, galactolipid synthesis, and especially synthesis of DGDG and TGDG, is reduced by treatment of the organelles with the nonpenetrating protease thermolysin. Envelope membranes isolated from thermolysin-treated chloroplasts of Spinacia oleracea L. (16:3 plant) and Pisum sativum L. (18:3 plant) or membranes isolated from thermolysin-treated chromoplasts are strongly reduced in galactolipid:galactolipid galactosyltransferase activity, but not with regard to UDP-Gal:diacylglycerol galactosyltransferase. For the intact plastids, this indicates that thermolysin treatment specifically blocks DGDG (and TGDG) synthesis, whereas MGDG synthesis is not affected. Neither in chloroplast nor in chromoplast membranes is DGDG synthesis stimulated by UDP-Gal. DGDG synthesis in S. oleracea chloroplasts is not stimulated by nucleoside 5′-diphospho digalactosides. Therefore, galactolipid:galactolipid galactosyltransferase is so far the only detectable enzyme synthesizing DGDG. These results conclusively suggest that the latter enzyme is located in the outer envelope membrane of different types of plastids and has a general function in DGDG synthesis, both in 16:3 and 18:3 plants.     

Involvement of ferredoxin in desaturation of lipid-bound oleate in chloroplasts

Intact spinach (Spinacia oleracea) chloroplasts, pulse-labeled with [¹⁴C]acetate, desaturate newly formed fatty acids as ester groups of monogalactosyl diacylglycerol in a subsequent chase in the dark. Rupture of pulse-labeled chloroplasts by addition of a detergent solution 3-([3-cholamidopropyl]dimethylammonio)-1-propane sulfonate preserves part of this desaturation activity. Direct addition of different free fatty acids together with appropriate cofactors to detergent-ruptured chloroplasts results in fatty acid labeling of monogalactosyl diacylglycerol. During subsequent incubation these lipid-linked fatty acids are desaturated, i.e. 18:1 to 18:2 and 18:3 and to a small extent also 16:0 to 16:3. The formation of 18:2 was also observed after incorporation of 18:1 into sulfolipid and phosphatidyl choline. Density gradient centrifugation separated a membrane fraction from detergent-ruptured chloroplasts which in the presence of appropriate cofactors incorporated 18:1 and 18:2 into the above-mentioned lipids. In the light, desaturation was dependent on added ferredoxin, whereas in the dark, in addition to ferredoxin NAD(P)H was also required. Preliminary evidence for the involvement of membrane-bound ferredoxin:NADP oxidoreductase (FNR) as a third component of desaturation in the dark was obtained by inhibitor studies including antibodies against FNR. Desaturation of lipid-bound 18:1 and 18:2 resembles stearoyl-ACP desaturation with respect to its requirement of reduced ferredoxin and oxygen.     

Phytoplankton and periphyton responses to in situ experimental nutrient enrichment in a shallow subtropical lake

Experiments were performed in situ in shallow, subtropical LakeOkeechobee (Florida. USA) to quantify and compare the responsesof phytoplanklon (in 20 I clear polycarbonate carboys) and periphyton(on nutrient-diffusing clay substrates) to additions of nitrogenand/or phosphorus. During early and late summer. 1994, bothassemblages were nitrogen limited or co-limited by nitrogenand phosphorus, indicating the potential for competition betweenbenthic and planktonic communities. During late summer, therewas evidence that high phytoplankton biomass reduced light penetrationthrough the water column and may have suppressed periphytongrowth. The similar phytoplankton and periphyton taxonomic structures,both dominated by Lyngbya sp. and pennate diatoms, suggestedthat in shallow regions of this lake, resuspended meroplanktonmight account for a large portion of phytoplankton biomass.This phenomenon has been observed in other shallow, wind-drivenFlorida lakes.