Aldehyde pararosanilin (true aldehyde fuchsin) stains purified insulin, oxidized or not, following adequate fixation with either formalin or Bouin's fluid

Gomori reported that aldehyde fuchsin stained the granules of pancreatic islet beta cells selectively and without need of permanganate pretreatment. Others adopted permanganate oxidation because it makes staining faster though much less selective. All aldehyde fuchsins are not equivalent, being made from "basic fuchsin" whose composition may vary from pure pararosanilin to one of its methylated homologs, rosanilin or a mixture. Mowry et al. have shown that only aldehyde fuchsin made from pararosanilin stained unoxidized pancreatic beta cells (PBC). Aldehyde fuchsins made from methylated homologs of pararosanilin stain PBC cells only after oxidation, which induces basophilia of other cells as well; these are less selective for PBC. Is the staining of PBC by aldehyde fuchsins due to insulin? Others have been unable to stain pure insulin with aldehyde fuchsins except in polyacrylamide gels and only after oxidation with permanganate. They have concluded that insulin contributed to the staining of oxidized but not of unoxidized PBC. This view denies any inherent validity of the more selective staining of unoxidized PBC cells as an indication of their insulin content. We describe here indisputable staining of unoxidized pure insulins by aldehyde fuchsin made with pararosanilin. Dried spots of insulin dissolved in the stain unless fixed beforehand. Spots of dried insulin solution made on various support media and fixed in warm formalin vapor were colored strongly by the stain. Insulin soaked Gelfoam sponges were dried, fixed in formalin vapor and processed into paraffin. In unoxidized paraffin sections, presumed insulin inside gel spaces was stained strongly by aldehyde pararosanilin. Finally, the renal tubules of unoxidized paraffin sections of kidneys from insulin-injected mice fixed in either Bouin's fluid or formalin were loaded with material stained deeply by aldehyde pararosanilin. This material was absent in renal tubules of mice receiving no insulin. The material in the spaces of insulin-soaked gels and in the renal tubules of insulin-injected mice was proven to be insulin by specific immunostaining of duplicate sections. The same material was also stained by aldehyde pararosanilin used after permanganate. So, this dye stains oxidized or unoxidized insulin if fixed adequately.     

Patterns of Root Colonization in Epacridaceous Plants Collected from Different Sites

Root colonization was studied in ten species of the Epacridaceaeat three sites in Victoria by morphological and cross-inoculationexperiments. The sites and genera chosen were Cranbourne [Epacrisimpressa Labill. andLeucopogon ericoides(Smith) R. Br.] andRye [L. parviflorus(Andrews) Lindley] on the Mornington Peninsula,and the Grampians[Astroloma conostephioides(Sond.) Benth.,A.humifusum(Cav.) R. Br.,A pinifolium(R. Br.) Benth,Brachylomadaphnoides(Smith) Benth.,E. impressa, E. impressavar.grandifloraBenth.andStyphelia adscendensR. Br.] in western Victoria. For morphologicalstudies, samples of roots from each species at each site werecleared and stained and examined microscopically. For cross-inoculationstudies, cuttings from each site were struck in potting mediuminoculated with soil from the same and other sites. The ericoidmycorrhizae in the roots of plants found at or grown in Cranbourneand Rye soils were similar. Both were significantly differentfrom the internal hyphae found in the roots of plants foundat or grown in Grampians soils, which were three times largerin diameter and formed dense coils which filled the host celland invaded adjacent epidermal cells. This suggests that morethan one fungus is involved in the relationships, that the MorningtonPeninsula sites had a different fungus from the Grampians siteand that host specificity is low. Vesicular structures werealso found commonly on plants at the Grampians site, in contrastwith other sites. Epacridaceae; root; fungus; mycorrhiza; morphology; inoculation     

Effects of Water Stress on Photosynthesis and Carbon Partitioning in Soybean (Glycine max [L.] Merr.) Plants Grown in the Field at Different CO(2) Levels

The effects of water stress and CO₂ enrichment on photosynthesis, assimilate export, and sucrose-P synthase activity were examined in field grown soybean plants. In general, leaves of plants grown in CO₂-enriched atmospheres (300 microliters per liter above unenriched control, which was 349 ± 12 microliters per liter between 0500 and 1900 hours EST over the entire season) had higher carbon exchange rates (CER) compared to plants grown at ambient CO₂, but similar rates of export and similar activities of sucrose-P synthase. On most sample dates, essentially all of the extra carbon fixed as a result of CO₂ enrichment was partitioned into starch. CO₂-enriched plants had lower transpiration rates and therefore had a higher water use efficiency (milligrams CO₂ fixed per gram H₂O transpired) per unit leaf area compared to nonenriched plants. Water stress reduced CER in nonenriched plants to a greater extent than in CO₂-enriched plants. As CER declined, stomatal resistance increased, but this was not the primary cause of the decrease in assimilation because internal CO₂ concentration remained relatively constant. Export of assimilates was less affected by water stress than was CER. When CERs were low as a result of the imposed stress, export was supported by mobilization of reserves (mainly starch). Export rate and leaf sucrose concentration were related in a curvilinear manner. When sucrose concentration was above about 12 milligrams per square decimeter, obtained with nonstressed plants at high CO₂, there was no significant increase in export rate. Assimilate export rate was also correlated positively with SPS activity and the quantitative relationship varied with CER. Thus, export rate was a function of both CER and carbon partitioning.     

Only aldehyde fuchsin made from pararosanilin stains pancreatic B cell granules and elastic fibers in unoxidized microsections: problems caused by mislabelling of certain basic fuchsins

The most distinctive property of aldehyde fuchsin is its staining of certain nonionic proteins and peptides in unoxidized cells and tissues. These substances include granules of pancreatic islet B cells, elastic fibers and hepatitis B surface antigen. Aldehyde fuchsin made from two different basic fuchsins, each certified by the Biological Stain Commission and labelled C.I. (Colour Index) No. 42500 (pararosanilin), did not stain pancreatic B cells at all. Stain Commission's records and retesting showed that each of the "faulty" basic fuchsins was not pararosanilin, but rosanilin, whose Colour Index number is 42510. These basic fuchsins were labelled with the wrong Colour Index number when packaged. Additional basic fuchsins were coded by V.M.E. and tested by R.W.M. for their capacity to make satisfactory aldehyde fuchsins. Only certain of these aldehyde fuchsins stained unoxidized pancreatic islet B cells. The same aldehyde fuchsins stained elastic fibers strongly. Each basic fuchsin whose aldehyde fuchsin was judged satisfactory proved to be pararosanilin. Aldehyde fuchsin solutions made from other basic fuchsins stained elastic fibers only weakly and did not stain pancreatic B cells at all in unoxidized sections. Each basic fuchsin whose aldehyde fuchsin was unsatisfactory proved to be rosanilin. It appears that only aldehyde fuchsin made from pararosanilin stains unoxidized pancreatic B cell granules dependably. We found that basic fuchsins from additional lots of Commission-certified pararosanilin and rosanilin were also labelled with incorrect Colour Index numbers when packaged. Steps were taken to prevent recurrences of such mislabelling which has made it difficult until now to correlate differences in the properties of pararosanilin and rosanilin. A table is provided of all basic fuchsins that have been certified by the Biological Stain Commission since 1963 when they began the practice of subdesignating basic fuchsins according to whether they are pararosanilins or nonpararosanilins. The consumer can readily determine from the certification number on the label the correct subdesignation of any Commission-certified basic fuchsin listed here. Until now, mislabelling of some lots of pararosanilin as rosanilin and vice-versa has confused and frustrated the users of basic fuchsins in other applications such as the carbol fuchsin staining of tubercle bacilli and certain cytochemical tests, e.g. esterase and acid phosphatase, that utilize hexazotized pararosanilin as a coupling reagent. Consumers experiencing trouble with any Commission-certified dye should look to the Biological Stain Commission for help. This is an important reason for purchasing, whenever possible, only Biological Stain Commission certified dyes.     

Conserved and clustered RNA recognition sequences are a critical feature of signals directing RNA localization in Xenopus oocytes

Although it is widely regarded that the targeting of RNA molecules to subcellular destinations depends upon the recognition of cis-elements found within their 3' untranslated regions (UTR), relatively little is known about the specific features of these cis-sequences that underlie their function. Interaction between specific repeated motifs within the 3' UTR and RNA-binding proteins has been proposed as a critical step in the localization of Vg1 RNA to the vegetal pole of Xenopus oocytes. To understand the relative contributions of repeated localization element (LE) sequences, we used comparative functional analysis of Vg1 LEs from two frog species, Xenopus laevis and Xenopus borealis. We show that clusters of repeated VM1 and E2 motifs are required for efficient localization. However, groups of either site alone are not sufficient for localization. In addition, we present evidence that the X. borealis Vg1 LE is recognized by the same set of RNA-binding proteins as the X. laevis Vg1 LE and is capable of productive interactions with the X. laevis transport machinery as it is sufficient to direct vegetal localization in X. laevis oocytes. These results suggest that clustered sets of cis-acting sites within the LE direct vegetal transport through specific interactions with the localization machinery.     

Ultrasound imaging in an experimental model of fatty liver disease and cirrhosis in rats

Background

Atypical scrapie was first identified in Norwegian sheep in 1998 and has subsequently been identified in many countries. Retrospective studies have identified cases predating the initial identification of this form of scrapie, and epidemiological studies have indicated that it does not conform to the behaviour of an infectious disease, giving rise to the hypothesis that it represents spontaneous disease. However, atypical scrapie isolates have been shown to be infectious experimentally, through intracerebral inoculation in transgenic mice and sheep. The first successful challenge of a sheep with 'field' atypical scrapie from an homologous donor sheep was reported in 2007.

Results

This study demonstrates that atypical scrapie has distinct clinical, pathological and biochemical characteristics which are maintained on transmission and sub-passage, and which are distinct from other strains of transmissible spongiform encephalopathies in the same host genotype.

Conclusions

Atypical scrapie is consistently transmissible within AHQ homozygous sheep, and the disease phenotype is preserved on sub-passage.     

Mapping of citrullinated fibrinogen B-cell epitopes in rheumatoid arthritis by imaging surface plasmon resonance

Introduction  

Rheumatoid arthritis (RA) frequently involves the loss of tolerance to citrullinated antigens, which may play a role in pathogenicity. Citrullinated fibrinogen is commonly found in inflamed synovial tissue and is a frequent target of autoantibodies in RA patients. To obtain insight into the B-cell response to citrullinated fibrinogen in RA, its autoepitopes were systematically mapped using a new methodology.