A Selective Stain for Renal Basement Membranes

Paraffin sections from human kidneys fixed in Carnoy's fluid No. 2 were treated consecutively with periodic acid-sodium bisulfite and stained with resorcin-fuchsin. Basement membranes were colored black in cross sections, dark gray in tangential sections. Cytoplasm, nuclei, reticulin and collagen fibers remained unstained or were only lightly colored, depending on duration of fixation. Elastic fibers were colored black. In sections counterstained with Kernechtrot, the sharp black coloration of basement membranes and the pink staining of nuclei facilitated the study of glomerular lesions. After counterstaining with Van Gieson's picro-fuchsin, the black basement membranes contrasted well with the red reticulin and collagen fibers. Because this method does not require differentiation, it gave uniform results in the hands of different users. No fading was observed in section stored for 3 yr.     

Continental-scale distribution and diversity of Ceratobasidium orchid mycorrhizal fungi in Australia

Background and AimsMycorrhizal fungi are a critical component of the ecological niche of most plants and can potentially constrain their geographical range. Unlike other types of mycorrhizal fungi, the distributions of orchid mycorrhizal fungi (OMF) at large spatial scales are not well understood. Here, we investigate the distribution and diversity of Ceratobasidium OMF in orchids and soils across the Australian continent.MethodsWe sampled 217 Ceratobasidium isolates from 111 orchid species across southern Australia and combined these with 311 Ceratobasidium sequences from GenBank. To estimate the taxonomic diversity of Ceratobasidium associating with orchids, phylogenetic analysis of the ITS sequence locus was undertaken. Sequence data from the continent-wide Australian Microbiome Initiative were used to determine the geographical range of operational taxonomic units (OTUs) detected in orchids, with the distribution and climatic correlates of the two most frequently detected OTUs modelled using MaxEnt.Key ResultsWe identified 23 Ceratobasidium OTUs associating with Australian orchids, primarily from the orchid genera Pterostylis, Prasophyllum, Rhizanthella and Sarcochilus. OTUs isolated from orchids were closely related to, but distinct from, known pathogenic fungi. Data from soils and orchids revealed that ten of these OTUs occur on both east and west sides of the continent, while 13 OTUs were recorded at three locations or fewer. MaxEnt models suggested that the distributions of two widespread OTUs are correlated with temperature and soil moisture of the wettest quarter and far exceeded the distributions of their host orchid species.ConclusionsCeratobasidium OMF with cross-continental distributions are common in Australian soils and frequently have geographical ranges that exceed that of their host orchid species, suggesting these fungi are not limiting the distributions of their host orchids at large spatial scales. Most OTUs were distributed within southern Australia, although several OTUs had distributions extending into central and northern parts of the continent, illustrating their tolerance of an extraordinarily wide range of environmental conditions.     

Three metal ions participate in the reaction catalyzed by T5 flap endonuclease

Protein nucleases and RNA enzymes depend on divalent metal ions to catalyze the rapid hydrolysis of phosphate diester linkages of nucleic acids during DNA replication, DNA repair, RNA processing, and RNA degradation. These enzymes are widely proposed to catalyze phosphate diester hydrolysis using a "two-metal-ion mechanism." Yet, analyses of flap endonuclease (FEN) family members, which occur in all domains of life and act in DNA replication and repair, exemplify controversies regarding the classical two-metal-ion mechanism for phosphate diester hydrolysis. Whereas substrate-free structures of FENs identify two active site metal ions, their typical separation of > 4 A appears incompatible with this mechanism. To clarify the roles played by FEN metal ions, we report here a detailed evaluation of the magnesium ion response of T5FEN. Kinetic investigations reveal that overall the T5FEN-catalyzed reaction requires at least three magnesium ions, implying that an additional metal ion is bound. The presence of at least two ions bound with differing affinity is required to catalyze phosphate diester hydrolysis. Analysis of the inhibition of reactions by calcium ions is consistent with a requirement for two viable cofactors (Mg2+ or Mn2+). The apparent substrate association constant is maximized by binding two magnesium ions. This may reflect a metal-dependent unpairing of duplex substrate required to position the scissile phosphate in contact with metal ion(s). The combined results suggest that T5FEN primarily uses a two-metal-ion mechanism for chemical catalysis, but that its overall metallobiochemistry is more complex and requires three ions.     

Size and number of tandem repeat arrays can determine somatic homologous pairing of transgene loci mediated by epigenetic modifications in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> nuclei

The chromosomal arrangement of different transgenic repeat arrays inserted at various chromosomal positions was tested by FISH in Arabidopsis 2C leaf and root nuclei. Large lacO (∼10 kb) but not tetO (4.8 kb) or small lacO (∼2 kb) arrays were, in general, more often spatially associated with heterochromatic chromocenters (CC) than flanking regions (that either overlap the array insert position or are between 5 and 163 kb apart from the insert site). Allelic and ectopic pairing frequencies of lacO arrays were significantly increased only in nuclei of lines with two large lacO arrays inserted at different positions on the same chromosome arm. Within the same lines, root nuclei showed a significantly lower increase of pairing frequencies at the insert position compared to leaf nuclei but still a higher frequency than in the wild-type situation. Thus, the frequencies of homologous pairing and association with heterochromatin of transgenic repeats may differ with the construct, the chromosomal insertion position, the cell type and with the number and repetitiveness of inserts. Strong CpG methylation is correlated with a high frequency of homologous pairing at large repeat array loci in somatic cells but has no impact on their association with CCs. These results show that single low-copy arrays apparently do not alter interphase chromatin architecture and are more suitable for chromatin tagging than multiple high copy arrays.     

The Exotic Legume Tree Species Acacia holosericea Alters Microbial Soil Functionalities and the Structure of the Arbuscular Mycorrhizal Community

The response of microbial functional diversity as well as its resistance to stress or disturbances caused by the introduction of an exotic tree species, Acacia holosericea, ectomycorrhized or not with Pisolithus albus, was examined. The results show that this ectomycorrhizal fungus promotes drastically the growth of this fast-growing tree species in field conditions after 7 years of plantation. Compared to the crop soil surrounding the A. holosericea plantation, this exotic tree species, associated or not with the ectomycorrhizal symbiont, induced strong modifications in soil microbial functionalities (assessed by measuring the patterns of in situ catabolic potential of microbial communities) and reduced soil resistance in response to increasing stress or disturbance (salinity, temperature, and freeze-thaw and wet-dry cycles). In addition, A. holosericea strongly modified the structure of arbuscular mycorrhizal fungus communities. These results show clearly that exotic plants may be responsible for important changes in soil microbiota affecting the structure and functions of microbial communities.     

Dose-related effects of red wine and alcohol on hemodynamics, sympathetic nerve activity, and arterial diameter

The cardiovascular benefits of light to moderate red wine consumption often have been attributed to its polyphenol constituents. However, the acute dose-related hemodynamic, vasodilator, and sympathetic neural effects of ethanol and red wine have not been characterized and compared in the same individual. We sought to test the hypotheses that responses to one and two alcoholic drinks differ and that red wine with high polyphenol content elicits a greater effect than ethanol alone. Thirteen volunteers (24-47 yr; 7 men, 6 women) drank wine, ethanol, and water in a randomized, single-blind trial on three occasions 2 wk apart. One drink of wine and ethanol increased blood alcohol to 38 +/- 2 and 39 +/- 2 mg/dl, respectively, and two drinks to 72 +/- 4 and 83 +/- 3 mg/dl, respectively. Wine quadrupled plasma resveratrol (P < 0.001) and increased catechin (P < 0.03). No intervention affected blood pressure. One drink had no heart rate effect, but two drinks of wine increased heart rate by 5.7 +/- 1.6 beats/min; P < 0.001). Cardiac output fell 0.8 +/- 0.3 l/min after one drink of ethanol and wine (both P < 0.02) but increased after two drinks of ethanol (+0.8 +/- 0.3 l/min) and wine (+1.2 +/- 0.3 l/min) (P < 0.01). One alcoholic drink did not alter muscle sympathetic nerve activity (MSNA), while two drinks increased MSNA by 9-10 bursts/min (P < 0.001). Brachial artery diameter increased after both one and two alcoholic drinks (P < 0.001). No beverage augmented, and the second wine dose attenuated (P = 0.02), flow-mediated vasodilation. One drink of ethanol dilates the brachial artery without activating sympathetic outflow, whereas two drinks increase MSNA, heart rate, and cardiac output. These acute effects, which exhibit a narrow dose response, are not modified by red wine polyphenols.     

Biotransformations of Paralytic Shellfish Toxins by Bacteria Isolated from Bivalve Molluscs

Due to the possibility that bacteria could be involved in the clearance of paralytic shellfish toxins (PST) from bivalve molluscs, investigations into which, if any, bacteria were able to grow at the expense of PST focused on several common shellfish species. These species were blue mussels, oysters, razor fish, cockles, and queen and king scallops. Bacteria associated with these shellfish were isolated on marine agar 2216 and characterized by their carbon utilization profiles (BIOLOG). Selected isolates from groups demonstrating 90% similarity were screened for their ability to metabolize a range of PST (gonyautoxins 1 and 4 [GTX 1/4], GTX 2/3, GTX 5, saxitoxin, and neosaxitoxin) using a novel screening method and confirming its results by high-performance liquid chromatography. Results suggest that molluscan bacteria have different capacities to utilize and transform PST analogues. For example, isolates M12 and R65 were able to reductively transform GTX 1/4 with concomitant production of GTX 2/3, while isolate Q5 apparently degraded GTX 1/4 without the appearance of other GTXs. Other observed possible mechanisms of PST transformations include decarbamoylation by isolate M12 and sulfation of GTXs by isolates Q5, R65, M12, and C3. These findings raise questions as to the possible role of bacteria resident in the shellfish food transport system. Some researchers have suggested that the microflora play a role in supplying nutritional requirements of the host. This study demonstrates that bacteria may also be involved in PST transformation and elimination in molluscan species.     

MALDI-TOF mass spectrometry for the identification of freshwater snails from Senegal,including intermediate hosts of schistosomes

Freshwater snails of the genera Biomphalaria, Bulinus, and Oncomelania are intermediate hosts of schistosomes that cause human schistosomiasis, one of the most significant infectious neglected diseases in the world. Identification of freshwater snails is usually based on morphology and potentially DNA-based methods, but these have many drawbacks that hamper their use. MALDI-TOF MS has revolutionised clinical microbiology and has emerged in the medical entomology field. This study aims to evaluate MALDI-TOF MS profiling for the identification of both frozen and ethanol-stored snail species using protein extracts from different body parts. A total of 530 field specimens belonging to nine species (Biomphalaria pfeifferi, Bulinus forskalii, Bulinus senegalensis, Bulinus truncatus, Bulinus globosus, Bellamya unicolor, Cleopatra bulimoides, Lymnaea natalensis, Melanoides tuberculata) and 89 laboratory-reared specimens, including three species (Bi. pfeifferi, Bu. forskalii, Bu. truncatus) were used for this study. For frozen snails, the feet of 127 field and 74 laboratory-reared specimens were used to validate the optimised MALDI-TOF MS protocol. The spectral analysis yielded intra-species reproducibility and inter-species specificity which resulted in the correct identification of all the specimens in blind queries, with log-score values greater than 1.7. In a second step, we demonstrated that MALDI-TOF MS could also be used to identify ethanol-stored snails using proteins extracted from the foot using a specific database including a large number of ethanol preserved specimens. This study shows for the first time that MALDI-TOF MS is a reliable tool for the rapid identification of frozen and ethanol-stored freshwater snails without any malacological expertise.     

Plant N-glycan processing enzymes employ different targeting mechanisms for their spatial arrangement along the secretory pathway

The processing of N-linked oligosaccharides in the secretory pathway requires the sequential action of a number of glycosidases and glycosyltransferases. We studied the spatial distribution of several type II membrane-bound enzymes from Glycine max, Arabidopsis thaliana, and Nicotiana tabacum. Glucosidase I (GCSI) localized to the endoplasmic reticulum (ER), alpha-1,2 mannosidase I (ManI) and N-acetylglucosaminyltransferase I (GNTI) both targeted to the ER and Golgi, and beta-1,2 xylosyltransferase localized exclusively to Golgi stacks, corresponding to the order of expected function. ManI deletion constructs revealed that the ManI transmembrane domain (TMD) contains all necessary targeting information. Likewise, GNTI truncations showed that this could apply to other type II enzymes. A green fluorescent protein chimera with ManI TMD, lengthened by duplicating its last seven amino acids, localized exclusively to the Golgi and colocalized with a trans-Golgi marker (ST52-mRFP), suggesting roles for protein-lipid interactions in ManI targeting. However, the TMD lengths of other plant glycosylation enzymes indicate that this mechanism cannot apply to all enzymes in the pathway. In fact, removal of the first 11 amino acids of the GCSI cytoplasmic tail resulted in relocalization from the ER to the Golgi, suggesting a targeting mechanism relying on protein-protein interactions. We conclude that the localization of N-glycan processing enzymes corresponds to an assembly line in the early secretory pathway and depends on both TMD length and signals in the cytoplasmic tail.