Light and electron microscopical postembedding lectin histochemistry for WGA-binding sites in the renal cortex of the mouse embedded in polyhydroxy aromatic resins LR-White and LR-Gold

Summary This work describes a technique which permits study of the postembedding lectin histochemistry for WGA-binding sites at the light and electron microscopical level on the same resin embedded tissue without removing or etching of the resin. Unfixed kidney pieces or kidney pieces fixed in 4% formaldehyde were embedded in the hydrophilic polyhydroxy aromatic resins LR-Gold and LR-White, following dehydration in up to 70% ethanol, 90% ethanol or 100% ethanol. LR-Gold was cryopolymerised at –25° C using the light sensitive initiator benzil, whereas LR-White was heat-cured at +50° C. The localisation of WGA-binding sites at the light microscopical level was investigated using FITC-labelled WGA. The ultrastructural localisation of WGA-binding sites was performed using 15 nm gold-labelled WGA. The best fluorescence staining results were obtained on fixed or unfixed tissue dehydrated in up to 70% ethanol and embedded in LR-Gold. At the ultrastructural level, the best staining results for WGA-binding sites were seen on tissue sections, dehydrated in up to 90% ethanol prior to embedding in LR-Gold.     

The possible roles of residues 79 and 80 of the Trp repressor from Escherichia coli K-12 in trp operator recognition

We constructed mutants of the Trp repressor from Escherichia coli K-12 with all possible single amino acid exchanges at positions 79 and 80 (residues 1 and 2 of the recognition helix). We tested these mutants in vivo by measuring the repression of synthesis of β-galactosidase with symmetric variants of α- and β-centered trp operators, which replace the lac operator in a synthetic lac system. The Trp repressor carrying a substitution of isoleucine 79 by lysine, showed a marked specificity change with respect to base pair 7 of the α-centered trp operator. Gel retardation experiments confirmed this result. Trp repressor mutant IR79 specifically recognizes a trp operator variant with substitutions in positions 7 and 8. Another mutant, with glycine in position 79, exhibited loss of contact at base pair 7. We speculate that the side chain of Ile79 interacts with the AT base pairs 7 and 8 of the α-centered trp operator, possibly with the methyl groups of thymines. Replacement of thymine in position 7 or 8 by uracil confirms the involvement of the methyl group of thymine 8 in repressor binding. Several Trp repressor mutants in position 80 (i.e. AI80, AL80, AM80 and AP80) broaden the specificity of the Trp repressor for α-centered trp operator variants with exchanges in positions 3, 4 and 5.     

G2 phase cell cycle disturbance as a manifestation of genetic cell damage

The predominant cell cycle change induced by X-rays and clastogens in peripheral blood mononuclear cells is the accumulation of cells in the G2 phase of the cell cycle. We show that this accumulation consists of cells that are either delayed or arrested within the G2 phase. Since both X-rays and DNA crosslinking chemicals are known to damage DNA, the G2 phase inhibition caused by these agents is thought to be one of the primary manifestations of (unrepaired) DNA damage. This interpretation is supported by two additional findings. (1) Older individuals have elevated baseline levels of mononuclear blood cells that are delayed and/or arrested in the G2 phase of the cell cycle. This coincides with the increased chromosomal breakage rates reported for older individuals. (2) Irrespective of their age, individuals with inherited genetic instability syndromes (such as Fanconi anemia and Bloom syndrome) exhibit elevated G2 phase cell fractions. We show that the method used to detect such induced or spontaneous cell cycle changes, viz. BrdU-Hoechst flow cytometry, is a rapid and highly sensitive technique for the assessment of genetic cell damage.Dedicated to Professor Ulrich Wolf on the occasion of his 60th birthday     

Carbon isotope composition of N2-fixing and N-fertilized legumes along an elevational gradient

Dinitrogen-fixing legumes are frequently assumed to be less water-use efficient than plants utilizing soil mineral N, because of the high respiratory requirements for driving N₂ fixation. However, since respiration is assumed not to discriminate against ¹³C, any differences in water-use efficiency exclusively due to respiration should not be apparent in carbon isotope discrimination () values. Our objective was to determine if the source of N (N₂ fixation versus soil N) had any effect on of field-grown grain legumes grown at different elevations. Four legume species, Glycine max, Phaseolus lunatus, P. vulgaris, and Vigna unguiculata, were grown on five field sites spanning a 633 m elevational gradient on the island of Maui, Hawaii. The legumes were either inoculated with a mixture of three effective strains of rhizobia or fertilized weekly with urea at 100 kg N ha^-1 in an attempt to completely suppress symbiotic N₂-fixing activity. In 14 of 20 analyses of stover and 12 of 15 analyses of seed values were significantly higher (p=0.10) in the inoculated plants than the N-fertilized plants. Nitrogen concentrations were generally higher in the fertilized treatments than the inoculated treatments. The different values obtained depending on N-source may have implications in using as an indicator of water-use efficiency or yield potential of legumes.     

Location of antibiotic resistance determinants, copy control, and replication functions on the double-selective streptococcal cloning vector pGB301

Summary The physical map of the streptococcal cloning vector pGB301 (9.8 kb) has been extended by localizing the cleavage sites of restriction endonucleases AvaII, AvaI, BclI, BstEII, and PvuII. The latter four enzymes cleaved pGB301 at unique sites. Insertion of chromosomal DNA from Staphylococcus aureus strain 3Ar^–m^– into the single BstEII site of pGB301 led to inactivation of the plasmid's chloramphenicol resistance determinant. Twelve deletion derivatives of pGB301 were isolated either by in vitro manipulation of pGB301 or as spontaneous deletion mutants following transformation of Challis by mixtures of recombinant plasmids. The overlapping deletions which spanned a continuous sequence of 7.7 kb ranged in size from 0.3 kb to 4.1 kb and allowed to localize the chloramphenicol and MLS-resistance determinants, the copy control function, and the replication region on the physical map of pGB301. Plasmid pGB301 together with its deletion mutants constitutes a valuable tool for further molecular cloning experiments in streptococci.     

Successful bone marrow transplantation in a patient with DNA ligase IV deficiency and bone marrow failure

Cirrhotic cardiomyopathy is the term used to describe a constellation of features indicative of abnormal heart structure and function in patients with cirrhosis. These include systolic and diastolic dysfunction, electrophysiological changes, and macroscopic and microscopic structural changes. The prevalence of cirrhotic cardiomyopathy remains unknown at present, mostly because the disease is generally latent and shows itself when the patient is subjected to stress such as exercise, drugs, hemorrhage and surgery. The main clinical features of cirrhotic cardiomyopathy include baseline increased cardiac output, attenuated systolic contraction or diastolic relaxation in response to physiologic, pharmacologic and surgical stress, and electrical conductance abnormalities (prolonged QT interval). In the majority of cases, diastolic dysfunction precedes systolic dysfunction, which tends to manifest only under conditions of stress. Generally, cirrhotic cardiomyopathy with overt severe heart failure is rare. Major stresses on the cardiovascular system such as liver transplantation, infections and insertion of transjugular intrahepatic portosystemic stent-shunts (TIPS) can unmask the presence of cirrhotic cardiomyopathy and thereby convert latent to overt heart failure. Cirrhotic cardiomyopathy may also contribute to the pathogenesis of hepatorenal syndrome. Pathogenic mechanisms of cirrhotic cardiomyopathy are multiple and include abnormal membrane biophysical characteristics, impaired β-adrenergic receptor signal transduction and increased activity of negative-inotropic pathways mediated by cGMP. Diagnosis and differential diagnosis require a careful assessment of patient history probing for excessive alcohol, physical examination for signs of hypertension such as retinal vascular changes, and appropriate diagnostic tests such as exercise stress electrocardiography, nuclear heart scans and coronary angiography. Current management recommendations include empirical, nonspecific and mainly supportive measures. The exact prognosis remains unclear. The extent of cirrhotic cardiomyopathy generally correlates to the degree of liver insufficiency. Reversibility is possible (either pharmacological or after liver transplantation), but further studies are needed.     

Hypomorphic mutations in the gene encoding a key Fanconi anemia protein, FANCD2, sustain a significant group of FA-D2 patients with severe phenotype

FANCD2 is an evolutionarily conserved Fanconi anemia (FA) gene that plays a key role in DNA double-strand-type damage responses. Using complementation assays and immunoblotting, a consortium of American and European groups assigned 29 patients with FA from 23 families and 4 additional unrelated patients to complementation group FA-D2. This amounts to 3%-6% of FA-affected patients registered in various data sets. Malformations are frequent in FA-D2 patients, and hematological manifestations appear earlier and progress more rapidly when compared with all other patients combined (FA-non-D2) in the International Fanconi Anemia Registry. FANCD2 is flanked by two pseudogenes. Mutation analysis revealed the expected total of 66 mutated alleles, 34 of which result in aberrant splicing patterns. Many mutations are recurrent and have ethnic associations and shared allelic haplotypes. There were no biallelic null mutations; residual FANCD2 protein of both isotypes was observed in all available patient cell lines. These analyses suggest that, unlike the knockout mouse model, total absence of FANCD2 does not exist in FA-D2 patients, because of constraints on viable combinations of FANCD2 mutations. Although hypomorphic mutations arie involved, clinically, these patients have a relatively severe form of FA.     

Amazonian Anthrosols Support Similar Microbial Communities that Differ Distinctly from Those Extant in Adjacent, Unmodified Soils of the Same Mineralogy

We compared the microbial community composition in soils from the Brazilian Amazon with two contrasting histories; anthrosols and their adjacent non-anthrosol soils of the same mineralogy. The anthrosols, also known as the Amazonian Dark Earths or terra preta, were managed by the indigenous pre-Colombian Indians between 500 and 8,700 years before present and are characterized by unusually high cation exchange capacity, phosphorus (P), and calcium (Ca) contents, and soil carbon pools that contain a high proportion of incompletely combusted biomass as biochar or black carbon (BC). We sampled paired anthrosol and unmodified soils from four locations in the Manaus, Brazil, region that differed in their current land use and soil type. Community DNA was extracted from sampled soils and characterized by use of denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism. DNA bands of interest from Bacteria and Archaea DGGE gels were cloned and sequenced. In cluster analyses of the DNA fingerprints, microbial communities from the anthrosols grouped together regardless of current land use or soil type and were distinct from those in their respective, paired adjacent soils. For the Archaea, the anthrosol communities diverged from the adjacent soils by over 90%. A greater overall richness was observed for Bacteria sequences as compared with those of the Archaea. Most of the sequences obtained were novel and matched those in databases at less than 98% similarity. Several sequences obtained only from the anthrosols grouped at 93% similarity with the Verrucomicrobia, a genus commonly found in rice paddies in the tropics. Sequences closely related to Proteobacteria and Cyanobacteria sp. were recovered only from adjacent soil samples. Sequences related to Pseudomonas, Acidobacteria, and Flexibacter sp. were recovered from both anthrosols and adjacent soils. The strong similarities among the microbial communities present in the anthrosols for both the Bacteria and Archaea suggests that the microbial community composition in these soils is controlled more strongly by their historical soil management than by soil type or current land use. The anthrosols had consistently higher concentrations of incompletely combusted organic black carbon material (BC), higher soil pH, and higher concentrations of P and Ca compared to their respective adjacent soils. Such characteristics may help to explain the longevity and distinctiveness of the anthrosols in the Amazonian landscape and guide us in recreating soils with sustained high fertility in otherwise nutrient-poor soils in modern times.