Intracellular binding of lead in the kidney: The partial isolation and characterization of postmitochondrial lead binding components

Gel chromatography of kidney postmitochondrial fractions from control rats 2 hr after injection of ²⁰³Pb or after in vitro incubation with ²⁰³Pb disclosed the presence of two fractionated Pb-binding components plus binding in the void volume and total volume regions. The binding of Pb to the two components, with molecular weights of 11,500 and 63,000 daltons, was markedly decreased in Pb-pretreated rats. Sodium dodecyl sulfate-gel electrophoresis and autoradiography showed the presence of one major ²⁰³Pb band with an estimated molecular weight of 60,000 daltons. The 11,500-dalton peak did not incorporate ¹⁴C-leucine nor did concomitant administration of cycloheximide with the ²⁰³Pb inhibit incorporation of ²⁰³Pb activity, suggesting that the component is a preformed constituent of the kidney. In vitro incubation of brain, liver and lung postmitochondrial supernatants with ²⁰³Pb disclosed that these two binding components were also present in brain but not in liver or lung, suggesting a target tissue-specific localization for these Pb-binding macromolecules.     

Determination of very-long-chain fatty acids in plasma by a simplified gas chromatographic—mass spectrometric procedure

The concentration of very-long-chain fatty acids (VLCFA) (straight chain, more than 22 carbon atoms) in plasma or in cultured fibroblasts is one of the most important diagnostic criteria for the diagnosis of the peroxisomal disorders. A sensitive method for VLCFA assay in plasma, using small sample volume and a simplified procedure, is described. After adequate extraction and derivatization, methyl esters of VLCFA are separated, identificated and quantified by gas chromatography—mass spectrometry (GC—MS). The method is sensitive, reproducible, accurate and relatively simple. GC—MS equipment used for routine organic acid analysis can be used.     

Satellite III DNA: Regions of extreme endonuclease resistance and interindividual polymorphism in the Mb size range

Southern analysis of within-gel digested and restricted human cells has revealed very large Satellite III restriction fragments which show clear inter-individual length polymorphism. The Mb and sub-Mb length of these fragments indicate that they arise from regions of heterochromatin which contain homogeneous Satellite III sequences of peculiar resistance to common endonucleases. Based on sequence alone, such regions would be little digested by endonuclease digestion of chromatin in metaphase, regardless of its method of preparation. Polymorphic regions such as these might be expected to stain as part of the C-banding seen in endonuclease treated metaphase chromosomes, and may in part account for inter-individual C-band heteromorphisms.     

Evidence that plasma membrane electrical potential is required for vesicular stomatitis virus infection of MDCK cells: A study using fluorescence measurements through polycarbonate supports

Summary We used fluorescence microscopy of Madin-Darby Canine Kidney (MDCK) cells grown on polycarbonate filters to study a possible link between plasma membrane electrical potential (_pm) and infectivity of vesicular stomatitis virus (VSV). Complete substitution of K⁺ for extracellular Na⁺blocks VSV infection of MDCK cells as well as baby hamster kidney (BHK) cells. When we independently perfused the apical and basal-lateral surfaces of high resistance monolayers, high K⁺ inhibited VSV infection of MDCK cells only when applied to the basal-lateral side; high K⁺ applied apically had no effect on VSV infection. This morphological specificity correlates with a large decrease in _pm of MDCK cells when high K⁺ buffer is perfused across the basal-lateral surface. Depolarization of the plasma membrane by 130 mm basal K⁺ causes a sustained increase of cytosol pH in MDCK cells from 7.3 to 7.5 as reported by the fluorescent dye BCECF. Depolarization also causes a transient increase of cytosol Ca²⁺ from 70 to 300 nm as reported by the dye Fura-2. Neither increase could explain the block of VSV infectivity by plasma membrane depolarization. One alternative hypothesis is that _pm facilitates membrane translocation of viral macromolecules as previously described for colicins, mitochondrial import proteins, and proteins secreted by Escherichia coli.We thank Kenneth Spring for many helpful discussions concerning fluorescence digitized imaging systems, James Russell for his collaboration in the design of our imaging system, Herbert Chase for suggestions on dye loading into MDCK cells, and Manfred Schubert and George Harmison for providing expertise on VSV.     

The interaction of the Escherichia coli mutD and mutT pathways in the prevention of A:T-->C:G transversions.

The Escherichia coli mutT mutator allele produces high frequencies of exclusively A:T-->C:G transversions. This is thought to be caused by a failure to prevent or remove A:G mispairs during DNA replication. The mutD5 mutator allele maps to the dnaQ locus which encodes the epsilon subunit of the DNA polymerase III holoenzyme. This subunit provides 3'-->5' exonuclease, proofreading, activity for removing mispaired nucleotides at the 3' end of the newly synthesized DNA strand. mutD5 has an altered epsilon resulting in reduced levels of proofreading and subsequent high mutation frequencies for all base-pair substitutions. We have analyzed the interaction between mutD5 and mutT-induced A:T-->C:G transversions by measuring reversion frequencies in mutD5 and mutT single mutator strains and mutD5mutT double mutator strains using the well-characterized trpA58 and trpA88 alleles. We find that the double mutator strains produce more A:T-->C:G substitutions than would be expected from simple additivity of the single mutator strains. We interpret this to mean that the two systems, at least in part, do act together to prevent the same mutational intermediate from producing A:T-->C:G transversions. It is estimated that over 90% of the mutT-induced A:G mispairs are corrected by proofreading at the trpA58 site while only about 30% are corrected at trpA88. Reversion frequencies in the mutD5mutT double mutator strains indicate A:G misincorporations occur about 100 x more frequently at trpA58 than at the trpA88 site. Using these and other data we also provide estimations of the fidelity contributions for mutT editing, proofreading and methyl-directed mismatch repair at the two trpA sites for both transversions and the transition that could be scored. In the case of A:T-->C:G transversions, both mutT editing and proofreading make major contributions in error reduction with mismatch repair playing a small or no role at all. For the A:T-->G:C transition, proofreading and mismatch repair were both important in preventing mutations while no contribution was observed for mutT editing.     

Freeze fracture immunocytochemistry of light-harvesting pigment complexes in a cryptophyte

Summary Immunocytochemical techniques using colloidal gold as the marker have been used to examine the location of the two light harvesting pigment-protein complexes in cryptophyte chloroplasts. A comparison of post-embedding thin section labelling and freeze fracture labelling has been carried out onRhodomonas salina using polyclonal antibodies to a chlorophylla/c ₂ light-harvesting complex, phycoerythrin and the -subunit of phycoerythrin. The effect of different fixation procedures on the intensity of labelling and ac curacy of antigen location have been examined and the effectiveness of uranyl acetate and tannic acid in improving both the preservation of thylakoid structure and labelling density of phycoerythrin has been demonstrated. Freeze fracture labelling gives better spatial res olution of the different antigens than post-embedding labelling, as well as better definition of thylakoid membranes. It confirms the location of phycoerythrin in the thylakoid lumen and the location of the chlorophylla/c ₂ LHC in both appressed and unappressed thylakoid membranes.Abbreviations PE phycoerythrin - chl chlorophyll - LHC light-har-vesting complex     

Ovarian follicular dynamics in the llama

Ovarian follicular dynamics were determined in adult llamas by ultrasonography and palpation per rectum and hormone analysis (estradiol-17 beta and estrogen conjugates) of plasma and urine. The relationship of gonadotropin secretion to follicular development was determined by the analysis of plasma FSH and LH concentrations. Progesterone analysis of plasma was used to verify or deny the presence of CL. Final follicular development (from 3 mm) averaged 4.8 days, while the duration of the mature follicle (8-12 mm) averaged 5.0 days; regression of the follicle occurred over about 4 days. The development of a subsequent dominant follicle usually began within 2-3 days after onset of regression of the dominant follicle. While several follicles were present at the time of the demise of the dominant follicle, only one follicle continued to develop. The interval between ovarian follicle waves averaged 11.1 days. Dominant follicle activity alternated between ovaries in 81% of the cycles. The occurrence of dominant follicles was evenly distributed between ovaries. While plasma estradiol and estrogen conjugate concentrations were positively associated (p less than 0.05) with follicular activity, urinary estrogen conjugate concentrations best reflected ovarian follicular dynamics (p less than 0.001). Daily FSH concentrations in plasma were not correlated with follicular activity. LH concentrations in plasma were low in all animals throughout the study, indicating estrogen from developing ovarian follicles does not induce the release of LH. Progesterone values were low during the study, indicating that the llama does not spontaneously ovulate, at least under the conditions of this study. In summary, llamas have overlapping ovarian follicle waves that occur at about 11-day intervals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transsulphuration and methylation of homocysteine in control and mutant human fibroblasts

The ability of human skin-fibroblasts in monolayer culture to carry out transsulphuration and remethylation of homocysteine has been tested. The conversion of homocyst(e)ine to cyst(e)ine and methionine was studied in control and mutant cells by incubation for 16 h with l-[³⁵S]homocystine. Labelled cysteic acid and methionine sulphone were found in hydrolysates of oxidized cell proteins. The quantities found were dependent on the time of incubation and were used as a measure of cyst(e)ine and methionine formation, respectively. In control cells, labelled cyst(e)ine and labelled methionine were found. In cystathionine β-synthase-deficient cell lines, labelled cyst(e)ine formation was reduced, while labelled methionine formed was similar to that of controls, indicating the role of transsulphuration in the formation of cyst(e)ine observed in control cells. In a 5,10-methylenetetrahydrofolate reductase-deficient cell line, labelled methionine formation was reduced, indicating the role of N-5-methyltetrahydrofolate-requiring methylation of homocysteine in the formation of methionine observed in control cells.