Occluding junctions in a cultured transporting epithelium: Structural and functional heterogeneity

Summary MDCK cells (epithelioid of renal origin) form monolayers which are structurally and functionally similar to transporting epithelia. One of these similarities is the ability to form occluding junctions and act as permeability barriers. This article studies the junctions of MDCK monolayers formed on a permeable and transparent support (a disk of nylon cloth coated with collagen) by combining two different approaches: (i)Scanning of the electric field: the disk is mounted as a flat sheet between two Lucite chambers and pulses of 20–50 A cm^–2 are passed across. The apical surface of the monolayer is then scanned with a microelectrode to detect those points where the current is flowing. This shows that the occluding junctions of this preparation are not homogeneous, but contain long segments of high resistance, intercalated with sites of high conductance. (ii)Freeze fracture electron microscopy: the junctions are composed of regions of eight to ten strands intercalated with others where the strands are reduced to one or two ridges. The sites of high conductance may correspond to those segments where the number of junctional strands is reduced to 1 or 2. It is concluded that the occluding junctions of MDCK monolayers are functionally and morphologically heterogeneous, with tight regions intermixed with leaky ones.     

Mercury contamination in Lake Xolotlán (Managua)

Total mercury was measured in different compartments of Lake Xolotlán's (Managua) ecosystemviz., sediments, water, fish and men. Sediments from 18 localities at 5 depths inside the sediment (5, 10, 15, 20 and 25 cm) contained an average concentration of 0.62 g Hg.g^–1±0.46 at the surface, with extreme values of 0.16 and 1.8 g.g^–1. The highest concentration was observed at 25 cm depth in front of the chlor-alkaly factory (ELPESA). This maximum is associated with the period of highest production of this factory. The highest mercury concentrations in water were also measured close to the discharge of ELPESA,viz. 787 g.Hg^–1 in January and 506 g.g^–1 in April. The mean mercury concentrations measured in the muscles of the most consumed fish were 0.63 g.g^–1±0.22 (extreme values 0.22 and 1.45) inCichlasoma managuense, and 0.07 g.g^–1±0.14 (extreme values 0.004 and 0.63) inC. citrinellum. The concentration in the liver was 0.79 g.g^–1±1.29 inC. managuense and 0.62 g.g^–1±0.44 inC. citrinellum. Human hairs (n=98) of fishermen and their families contained 5.03 g.g^–1±6.2 (extreme values 0.02 and 38.22). The mean concentration measured in men was 6.22 g.g^–1±6.34 (n=58), and in women 3.39 g.g^–1±5.7 (n=40). The average mercury concentration of hairs of workers of ELPESA was 91.24 g.g^–1±156.9 (extreme values 0.46 and 724.53; n=32). We conclude that total mercury levels in the various ecosystem compartments are very high and mercury contamination in the lake may be considered as dangerous for human health.     

Meiotic analysis ofElymus caucasicus,E. longearistatus,and their interspecific hybrids with twenty-threeElymus species (Triticeae,Poaceae)

Interspecific hybridizations were carried out between the two tetraploidsElymus caucasicus andE. longearistatus, and 23 tetraploids and hexaploids ofElymus containing SH, SY, SYH, and SYW genomes and representing various geographical regions. Meiotic pairing was studied in the two target species and their hybrids. It is concluded from this study that (i) interspecific hybridization is fairly easy to perform although strong reproductive barriers exist between the species; (ii)Elymus caucasicus andE. longearistatus are allotetraploids, and share the diverged SY genomes; (iii) the divergence of SY genomes is correlated with the geographic distance between theElymus spp. studied.     

Spontane Mutation von einer Monoauxotrophie zu einer anderen in einem Schritt (Auxotrophiesprungmutation)

Summary Serratia strain CV produces spontaneously about 0,5% auxotrophic mutants of different types. By means of a special auxanographic technique, among about 60 monoauxotrophs, 5 were found which frequently and spontaneously mutate to a certain other auxotrophic state loosing the first one (arg 1 ^– nia ^–;arg 2 ^– his ^–,ad, hyx ^–;thr ^– met ^–;prol ^– his ^–;leu ^– ad, hyx ^–). It could be shown that this auxotrophic leap happens in ohne step without a prototrophic intermediate step. By backmutation experiments with the mutationsleu ^– ad ^– it was shown that the second auxotrophy acts as a suppressor mutation to the first auxotrophy. Since the biochemical reaction sequence blocked by the first mutation seems not to be related to the chain blocked by the second mutation, and since the second mutants do not feed the first mutants syntrophically, it is not probable that the suppression of the first auxotrophy is caused by a substance accumulated as an effect of the second mutation, removing the first auxotrophy by intracellular feeding. Rather, the second auxotrophy-gene (in its mutated, suppressing allelic state, e. g.ad ^–) seems to contribute information to the production of the enzyme determined usually by the first auxotrophy-gene, which information was lost by the first mutation (e. g. leu ^–). Thus, the information on the specificity of an enzyme would be stored not only in one single gene but in several genes.     

Über Chromatin und DNS-Synthese im Nucleolus

Zusammenfassung Im Nucleolus der Leberzellen von Ratten ist elektronenmikroskopisch — mit Formalinfixierung — kontrastreiches Chromatin und autoradiographisch — mit H³-Thymidin — eine DNS-Synthese nachweisbar. Das Chromatin ist nicht in die netzigen Anteile der Nucleolarsubstanz (Nucleolonema), sondern in die dazwischen liegenden Aufhellungen eingelassen, doch sind nur einige dieser Räume und auch diese oft nur unvollständig von Karyoplasma (= Chromosomensubstanz) ausgefüllt. Die einzelnen Chromatin-Einschlüsse erreichen normalerweise die lichtmikroskopischesichtbarkeitsgrenzenicht. Lediglich in hepatozellulärenTumornucleolen sind bereits in gewöhnlichen Präparaten feulgenpositive Strukturen festzustellen.Aus den Befunden wird gefolgert: der Nucleolus der Somazellen ist generell von — meist sublichtmikroskopischen — Anteilen aufgelockerter und aufgesplitterter Chromosomen durchzogen. Dabei handelt es sich um die nucleolusorganizer-Region der Nucleolarchromosomen, denen der organisierte Nucleolus auch in der Intermitose verhaftet bleibt.     

Regeneration of plantlets through organogenesis in the Himalayan yellow poppy,Meconopsis paniculata

Plantlet formation through organogenesis in callus cultures of Himalayan yellow poppy,Meconopsis paniculata D.Don (Prain), a threatened taxon of ornamental value, is described. Hypocotyl segments from 3-month-old laboratory-raised seedlings produced callus on agar-solidified Murashige and Skoog medium (MS) containing 10 M -naphthaleneacetic acid and 1 M kinetin. Shoots differentiated best from callus on MS containing 10 M indolebutyric acid (IBA) and 1 M 6-benzyladenine. The regenerated shoots rooted best on MS medium containing 10 M IBA. From seed germination to differentiation of plantlets through the two-step organogenesis process required 28–29 weeks.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - FAA formalin-acetic acid-alcohol - BA 6-benzyladenine - IAA indole-acetic acid - IBA indolebutyric acid - GA₃ gibberellic acid - NAA -naphthaleneacetic acid - RH relative humidity     

Modeling of Alternative Pathways of Electron Transport to Photosystem I in Isolated Thylakoids

Kinetics of dark decay of absorbance changes at 830 nm (₈₃₀) was examined in thylakoids isolated from leaves of pea seedlings at various concentrations of exogenous NADPH or NADH. Absorbance changes were induced by far-red light to avoid electron donation from photosystem II. In the presence of either biological reductant, the kinetics of ₈₃₀ decay reflecting dark reduction of 700⁺, the primary electron donor of photosystem I, was fitted by a single exponential term. The rate of 700⁺ reduction increased with the rise in the concentration of both NADPH and NADH. The values of K _M and V _max for 700⁺ reduction estimated from concentration dependences were 105 ± 21 M and 0.32/s for NADPH or 21 ± 8 M and 0.12/s for NADH. The rate of P700⁺ reduction by either NADPH or NADH significantly increased in the presence of rotenone, a specific inhibitor of chloroplast reductase. The value of V _max was changed only in the presence of rotenone, whereas K _m was practically unaffected. Unlike the chloroplasts of intact leaves, the only enzyme mediating the input of reducing equivalents from NADPH or NADH to the electron transport chain was concluded to be present in thylakoids.     

The North Sea: source or sink for nitrogen and phosphorus to the Atlantic Ocean?

Annual nitrogen and phosphorus budgets for the whole North Sea taking into account the most recent data available were established. The area considered has a total surface of approximately 700,000km² and corresponds to the definition by OSPARCOM (Oslo and Paris Commission) with the exclusion of the Skagerrak and Kattegat areas. Input and output fluxes were determined at the marine, atmospheric, sediment and continental boundaries, and riverine inputs based on river flows and nutrient concentrations at the river–estuary interface were corrected for possible estuarine retention. The results showed that the North Sea is an extremely complex system subjected to large inter-annual variability of marine water circulation and freshwater land run-off. Consequently, resulting total N (TN) and P (TP) fluxes are extremely variable from 1 year to another and this has an important influence on the budget of these elements. Total inputs to the North Sea are 8870±4860kTNyear^–1 and 494±279kTPyear^–1. Denitrification is responsible for the loss of 23±7% of the TN inputs while sediment burial is responsible for the retention of only of 2±2% of the TP input. For TN, due to the large variability on marine and estuarine fluxes, and to the uncertainty related to the denitrification rate, it was concluded that the North Sea could either be a source (1930kTNyear^–1) or a sink (1700kTNyear^–1) for the waters of the North Atlantic Ocean. For TP it was concluded that the North Sea is mostly a source (–4 to 52kTPyear^–1) for the waters of the North Atlantic Ocean.     

Comparative systematic study on “Spirillum” 5175, Campylobacter and Wolinella species

Physiological tests, redetermination of G+C values with HPLC and DNA-DNA hybridization were used to determine the taxonomic affiliation of Spirillum 5175. This facultatively sulfur-reducing bacterium was compared to the type strains of the phenotypically most similar species Wolinella succinogenes and Campylobacter sputorum biovar bubulus. In addition to morphology, the following physiological properties were in common: use of elemental sulfur, nitrate, nitrite, aspartate, fumarate or malate as electron acceptor for growth with hydrogen or formate under anoxic conditions; microaerobic growth with 2% (v/v) oxygen. The G+C content of Wolinella succinogenes (51.8 mol%) and Campylobacter sputorum biovar bubulus (30.4 mol%) differs about 10 mol% from the G+C content of Spirillum 5175 (40.6 mol%). No significant DNA homology could be detected between the three strains. These differences excluded affiliation of Spirillum 5175 with the genera Wolinella or Campylobacter despite phenotypic similarities. On the basis of our results and DNA-rRNA hybridization studies by other authors, we established the new genus Sulfurospirillum for the freeliving Campylobacter-like bacteria Spirillum 5175 and Campylobacter spec. DSM 806. Strain Spirillum 5175 is described as the type strain of the new genus and species Sulfurospirillum deleyianum.Dedicated to R. S. Wolfe on the occasion of his 70th birthday     

The mature mesonephric nephron of the rabbit embryo

Summary In freeze-fracture replicas of the entire cross-fractured mesonephros of 18 day rabbit embryos the basolateral and luminal cell faces of the different nephron segments were studied and compared with their metanephric counterparts. In the proximal tubule, the shallow zonula occludens exhibited only 1–2 strands and resembled the corresponding metanephric zonula, a very leaky type, which was found with a considerable paracellular flow component in sites of isotonic reabsorption. Gap junctions were restricted to the proximal tubule and were seen more frequently in its terminal segment. The distal tubule harboured two types of tight junctions. The most common type, a band of 5–8 closely parallel strands, matched the zonula occludens of the metanephric straight distal tubule. The observed particle density of the basolateral membrane (2,500±328/m²) was less than that of the proximal tubule (2,642±306). In addition, the collecting tubule exhibited a zonula occludens of the tight variety similar to that which occurred in the metanephric collecting duct. Rod-shaped particles of the luminal membrane were mainly concentrated in some of the intercalated cells but also had developed on principal cells, and occasionally, in the distal tubule. The Wolffian duct, with a deep tight zonula occludens, had an obviously rather inactive epithelium with no conspicuous transport-linked membrane specializations.     

Conservation status of parrot populations in an Atlantic rainforest area of southeastern Brazil

A census of four species of syntopic parrots was carried out using distance sampling methods on São Sebastião island, SE Brazil. Most of the 33593 ha island is covered by mature and secondary Atlantic rainforest. Almost 80% of these forests are within the Ilhabela Park. Although the species counted have marked differences in size and weight, density (individuals/km²) and estimated population size in 23500 ha of well-preserved forests were similar: Amazona farinosa (13.82±5.94; 3247±1395), Pionus maximiliani (15.79±7.04; 3712±1654), Brotogeris tirica (15.05±4.87; 3537±1143) and Pyrrhura frontalis (13.06±5.53; 3068±1298). Encounter rates of Forpus crassirostris and Pionopsitta pileata were very low, which suggests that there is only a small population of these species on the island. The São Sebastião forests still support healthy populations of parrots. Although woodpecker population estimates on the island are large enough to provide nesting sites for parrots, competition for holes with other secondary cavity nesters such as toucans, flycatchers and tytiras, and the selective cutting of dead trees for canoe construction, which is a common practice on the island, may limit hole availability for parrots.     

The Use of the [13C]/[12C] Ratio for the Assay of the Microbial Oxidation of Hydrocarbons

The study deals with a comparative analysis of the relative abundances of the carbon isotopes ¹²C and ¹³C in the metabolites and biomass of the Burkholderia sp. BS3702 and Pseudomonas putida BS202-p strains capable of utilizing aliphatic (n-hexadecane) and aromatic (naphthalene) hydrocarbons as sources of carbon and energy. The isotope compositions of the carbon dioxide, biomass, and exometabolites produced during the growth of Burkholderia sp. BS3702 on n-hexadecane (¹³C = –44.6 ± 0.2) were characterized by the values of ¹³C_{CO 2} = –50.2 ± 0.4, ¹³C_biom = –46.6 ± 0.4, and ¹³C_exo = –41.5 ± 0.4, respectively. The isotope compositions of the carbon dioxide, biomass, and exometabolites produced during the growth of the same bacterial strain on naphthalene (¹³C = –21 ± 0.4) were characterized by the isotope effects ¹³C_{CO 2} = –24.1 ± 0.4, ¹³C_biom = –19.2 ± 0.4, and ¹³C_exo = –19.1 ± 0.4, respectively. The possibility of using the isotope composition of metabolic carbon dioxide for the rapid monitoring of the microbial degradation of petroleum hydrocarbons in the environment is discussed.     

Measurement of15N-13C J couplings in staphylococcal nuclease

Summary ¹⁵N-C and¹⁵N-C J couplings were measured for the backbone of staphylococcal nuclease, uniformly enriched with¹⁵N and¹³C. It is found that the^IJ_C'N coupling is similar for -sheet, J=14.8 ± 0.5 and for -helix, J = 14.8 ± 0.4 but tends to be larger for the unstructured N- and C-terminal ends of the protein (J=15.6 ± 0.5). On average,¹J_NC are smaller for -helical residues (J=9.6 ± 0.3 Hz) compared to -sheet (J=10.9 ± 0.8 Hz) and a substantial difference is observed for²J_NC in -helices (J=6.4 ± 0.4 Hz) and -sheets (J=8.3 ± 0.8 Hz).Dedicated to the memory of Professor V.F. Bystrov     

Laser kinetic spectroscopy studies of the bimolecular oxygenation of alpha- and beta-subunits within the R-state of human hemoglobin

Bimolecular oxygenation of tri-liganded R-state human hemoglobin (HbA) is described by bi-exponential kinetics with association rate constants k = 27.2 ± 1.3 (M·sec)^-1 and k = 62.9 ± 1.6 (M·sec)^-1. Both the observed processes have been assigned to the bimolecular oxygenation of - and -subunits of the native tetrameric protein by molecular oxygen. The quantum yields of photodissociation within the completely oxygenated R-state HbA are = 0.0120 ± 0.0017 and = 0.044 ± 0.005 for - and -subunits, respectively. The oxygenation reactions of isolated ^PCMB- and ^PCMB-hemoglobin chains are described by mono-exponential kinetics with the association rate constants k = 44 ± 2 (M·sec)^-1 and k = 51 ± 1 (M·sec)^-1, respectively. The quantum yields of photodissociation of isolated ^PCMB- and ^PCMB-chains (0.056 ± 0.006 and 0.065 ± 0.006, respectively) are greater than that observed for appropriate subunits within the R-state of oxygenated HbA.     

Degradation of Soluble Amyloid β-Peptides 1–40, 1–42, and the Dutch Variant 1–40Q by Insulin Degrading Enzyme from Alzheimer Disease and Control Brains

Insulin degrading enzyme (IDE) is a metalloprotease that has been involved in amyloid peptide (A) degradation in the brain. We analyzed the ability of human brain soluble fraction to degrade A analogs 1–40, 1–42 and the Dutch variant 1–40Q at physiological concentrations (1 nM). The rate of synthetic ¹²⁵I-A degradation was similar among the A analogs, as demonstrated by trichloroacetic acid precipitation and SDS-PAGE. A 110 kDa protein, corresponding to the molecular mass of IDE, was affinity labeled with either ¹²⁵I-insulin, ¹²⁵I-A 1–40 or ¹²⁵I-A 1–42 and both A degradation and cross-linking were specifically inhibited by an excess of each peptide. Sensitivity to inhibitors was consistent with the reported inhibitor profile of IDE. Taken together, these results suggested that the degradation of A analogs was due to IDE or a closely related protease. The apparent Km, as determined using partially purified IDE from rat liver, were 2.2 ± 0.4, 2.0 ± 0.1 and 2.3 ± 0.3 M for A 1–40, A 1–42 and A 1–40Q, respectively. Comparison of IDE activity from seven AD brain cytosolic fractions and six age-matched controls revealed a significant decrease in A degrading activity in the first group, supporting the hypothesis that a reduced IDE activity may contribute to A accumulation in the brain.     

Polar bears make little use of terrestrial food webs: evidence from stable-carbon isotope analysis

Summary The mean stable-carbon isotope ratios (¹³C) for polar bear (Ursus maritimus) tissues (bone collagen –15.7, muscle –17.7, fat –24.7) were close to those of the same tissues from ringed seals (Phoca hispida) (–16.2, –18.1, and –26.1, respectively), which feed exclusively from the marine food chain. The ¹³C values for 4 species of fruits to which polar bears have access when on land in summer ranged from –27.8 to –26.2, typical of terrestrial plants in the Arctic. An animal's ¹³C signature reflects closely the ¹³C signature of it's food. Accordingly, the amount of food that polar bears consume from terrestrial food webs appears negligible, even though some bears spend 1/3 or more of each year on land during the seasons of greatest primary productivity.     

Intrastriatal Administration of Methylmercury Increases In Vivo Dopamine Release

Mercury is a neurotoxin that exists in a number of physical and chemical forms, producing different effects in the brain. In the present work, we have studied the effects of intrastriatal administration of different doses (40 M, 400 M, and 4 mM) of organic mercury (methylmercury, MeHg) on the dopaminergic system of rat striatum, in conscious and freely-moving animals, using microdialysis coupled to Liquid Chromatography. In previous works, we have discussed the effects of chronic and acute administration of MeHg on striatal dopaminergic system assessing changes in both release and metabolism of striatal dopamine (DA). In the present study we report that the intrastriatal administration of different doses of MeHg (40 M, 400 M, and 4 mM) produced significant increases (907 ± 31%, 2324 ± 156%, and 9032 ± 70% of basal levels, respectively for the different doses) in DA release from rat striatal tissue associated with significant decreases in extracellular levels of its main metabolites dihydroxyphenylacetic acid (DOPAC) and homovallinic acid (HVA) using the dose of 4 mM MeHg (35 ± 3% and 48 ± 1%, respectively), whereas non-significant changes in metabolite levels were observed with the doses of 40 M and 400 M MeHg. We explain these effects as a result of stimulated DA release and/or decreased DA intraneuronal degradation.     

New expanded bed adsorbents for the recovery of DNA

A 20–40 m pellicular high density (3.7 g cm^–3) expanded bed material has been designed for the capture of DNA and other large macromolecules. Anion exchangers fashioned out of these supports exhibited dramatically enhanced DNA binding capacities over commercial anion exchange adsorbents (6 mg ml^–1, c.f. 50 g ml^–1 at 10% breakthrough), due to a combination of small particle and fuzzy surface architecture created through the coupling of polyethylene imine chains.     

Mercury budget of an upland-peatland watershed

Inputs, outputs, and pool sizes oftotal mercury (Hg) were measured in a forested 10 hawatershed consisting of a 7 ha hardwood-dominatedupland surrounding a 3 ha conifer-dominatedpeatland. Hydrologic inputs via throughfall andstemflow, 13±0.4 g m^–2 yr^–1over the entire watershed, were about doubleprecipitation inputs in the open and weresignificantly higher in the peatland than in theupland (19.6 vs. 9.8 g m^–2 yr^–1). Inputs of Hg via litterfall were 12.3±0.7g m^–2 yr^–1, not different in thepeatland and upland (11.7 vs. 12.5 g m^–2yr^–1). Hydrologic outputs via streamflow were2.8±0.3 g m^–2 yr^–1 and thecontribution from the peatland was higher despiteits smaller area. The sum of Hg inputs were lessthan that in the overstory trees, 33±3 gm^–2 above-ground, and much less than eitherthat in the upland soil, 5250±520 gm^–2, or in the peat, 3900±100 gm^–2 in the upper 50 cm. The annual flux of Hgmeasured in streamflow and the calculated annualaccumulation in the peatland are consistent withvalues reported by others. A sink for Hg of about20 g m^–2 yr^–1 apparently exists inthe upland, and could be due to either or bothstorage in the soil or volatilization.     

A novel technique for the study of bacterial cell mechanical properties

A micromanipulation method is described for measuring the bursting forces of bacteria and relating them to cell size. At a compression speed of 6.2 m s^–1, bursting forces of three samples of rapidly growing Staphylococcus epidermis from a batch culture varied from 3 to 34 N with an average value of 13.8 N (standard error 0.8 N). Escherichia coli grown in continuous culture at a specific growth rate of 0.5 h^–1 had bursting forces varying from 1 to 9 N with an average value of 3.6 N (standard error 0.4 N). In squeeze-hold experiments, force relaxation was observed, which was attributed to water loss from the cells, or viscoelasticity, or both. At high compression speed, such as 6.2 m s^–1, this relaxation could be neglected. Micromanipulation strength measurements might be used in studies of cell mechanical disruption and of the dependence of cell strength on cell physiology.