Establishment and maintenance of nuclear position during zoospore formation in allomyces macrogynus: roles of the cytoskeleton

The involvement of the microtubule (MT) and actin microfilament (MF) cytoskeletons in establishing nuclear positions during zoosporogenesis in Allomyces macrogynus was assessed using selective cytoskeletal disrupting treatments and documented with light microscopy. These experiments were coupled with low-speed centrifugation studies to determine the degree to which cytoskeletal elements anchor nuclear position. At the onset of zoospore formation, nuclei were positioned only in cortical cytoplasmic regions of the zoosporangia (ZS). Immunofluorescence microscopy revealed that MTs primarily emanated from centrosomal regions into the surrounding cytoplasm at this stage. During delimitation of the cytoplasm into individual uninucleate zoospores, nuclei migrated from cortical regions to become distributed throughout the cytoplasm. Coincident with nuclear migrations, MTs were primarily organized at and emanated from nuclear surfaces, forming extensive perinuclear arrays. Nuclear migrations were suppressed in ZS induced to sporulate in the presence of cytochalasin D, an actin MF inhibiting compound. Disruption of MTs with nocodazole did not block nuclear migrations, although resultant nuclear spacing was irregular. Centrifugation treatments of control and drug-treated ZS demonstrated that nuclear positions were stabilized by perinuclear MT arrays. The results indicate that nuclear motility in ZS of A. macrogynus is the result of an actin-based system while perinuclear MTs arrays function to establish and fix nuclear position during zoospore formation. Copyright 1998 Academic Press.     

Cryopreservation of embryogenic tissue of sweet potato (Ipomoea batatas): use of sucrose and dehydration for cryoprotection

Embryogenic tissue of two sweet potato (Ipomoea batatas (L) LAM) genotypes, TIB 10 and Nemanete (Nem), was established from in vitro axillary meristems on Murashige and Skoog (1962) media supplemented with 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid respectively. Embryogenic aggregates of approximately 1.5–2.0 mm in diameter were subjected to a rapid or a two-step freezing protocol in liquid nitrogen following alginate encapsulation, sucrose preculture and varying degrees of dehydration. Up to 28% of encapsulated embryogenic aggregates of TIB 10 survived rapid freezing without dehydration. This was not enhanced by dehydration prior to freezing. However, survival after dehydration was enhanced up to 74% by incorporating an initial slow cooling step prior to plunging the tissue into liquid nitrogen. Following freezing, embryogenic tissue appeared to develop normally and retained its competence to produce mature embryos and plantlets. Similar results were obtained with Nem, although the survival percentages were much lower.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - MS Murashige and Skoog (1962) medium     

Gene-Based Sequencing Identifies Lipid-Influencing Variants with Ethnicity-Specific Effects in African Americans

Although a considerable proportion of serum lipids loci identified in European ancestry individuals (EA) replicate in African Americans (AA), interethnic differences in the distribution of serum lipids suggest that some genetic determinants differ by ethnicity. We conducted a comprehensive evaluation of five lipid candidate genes to identify variants with ethnicity-specific effects. We sequenced ABCA1, LCAT, LPL, PON1, and SERPINE1 in 48 AA individuals with extreme serum lipid concentrations (high HDLC/low TG or low HDLC/high TG). Identified variants were genotyped in the full population-based sample of AA (n = 1694) and tested for an association with serum lipids. rs328 (LPL) and correlated variants were associated with higher HDLC and lower TG. Interestingly, a stronger effect was observed on a “European” vs. “African” genetic background at this locus. To investigate this effect, we evaluated the region among West Africans (WA). For TG, the effect size among WA was the same in AA with only African local ancestry (2–3% lower TG), while the larger association among AA with local European ancestry matched previous reports in EA (10%). For HDLC, there was no association with rs328 in AA with only African local ancestry or in WA, while the association among AA with European local ancestry was much greater than what has been observed for EA (15 vs. ∼5 mg/dl), suggesting an interaction with an environmental or genetic factor that differs by ethnicity. Beyond this ancestry effect, the importance of African ancestry-focused, sequence-based work was also highlighted by serum lipid associations of variants that were in higher frequency (or present only) among those of African ancestry. By beginning our study with the sequence variation present in AA individuals, investigating local ancestry effects, and seeking replication in WA, we were able to comprehensively evaluate the role of a set of candidate genes in serum lipids in AA.     

Short repetitive sequences in green algal mitochondrial genomes: potential roles in mitochondrial genome evolution

Current data on green algal mitochondrial genomes suggest an unexpected dichotomy within the group with respect to genome structure, organization, and sequence affiliations. The present study suggests that there is a correlation between this dichotomy on one hand and the differences in the abundance, base composition, and distribution of short repetitive sequences we observed among green algal mitochondrial genomes on the other. It is conceivable that the accumulation of GC- rich short repeated sequences in the Chlamydomonas-like but not Prototheca-like mitochondrial genomes might have triggered evolutionary events responsible for the distinct series of evolutionary changes undergone by the two green algal mitochondrial lineages. The similarity in base composition, nucleotide sequence, abundance, and mode of organization we observed between the short repetitive sequences present in Chlamydomonas-like mitochondrial genomes on one hand and fungal and vertebrate homologs on the other might extend to some of the roles that the short repetitive sequences have been shown to have in the latter. Potential involvements we propose for the short repetitive sequences in the evolution of Chlamydomonas-like mitochondrial genomes include fragmentation and scrambling of the ribosomal-RNA-coding regions, extensive gene rearrangements, coding-region deletions, surrogate origins of replication, and chromosomal linearization.      

Accomplishments of the Trustees and laboratory staff of the Biological Stain Commission, 2002–2013

During the 12 years from 2002 to 2013, the Trustees and laboratory personnel of the Biological Stain Commission (BSC) can claim many accomplishments. These accomplishments are itemized under 11 categories: continuous publication of the official journal, Biotechnic & Histochemistry; production of four special issues of Biotechnic & Histochemistry devoted to specific dyes or stains; standardization of staining and dye purity; mechanisms of staining and prediction of dye behavior; publication of books or book chapters; effects of fixation and processing on staining; cancer research; immunohistochemistry; BSC Laboratory activities; miscellaneous publications; and administrative accomplishments.     

Glyoxal fixation: how it works and why it only occasionally needs antigen retrieval

Over the past 13 years, glyoxal has become the leading alternative to formaldehyde as a histological fixative because of its low inhalation risk, faster reaction rate and selective control over crosslinking. The latter attribute is especially important, because most of the difficulties relating to use of formaldehyde-fixed specimens for immunohistochemistry stem from its aggressive crosslinking behavior. With suitable catalysts or other reaction accelerators, glyoxal forms 2-carbon adducts with nearly all end groups in proteins and carbohydrates, leaving most of them unimpaired for subsequent immunohistochemical demonstration. Only arginine is seriously impaired by the formation of imidazoles, which is the basis for the well known arginine blockade method using glyoxal. A special glyoxal-specific antigen retrieval method using high pH and high temperature effectively reverses the blockade and restores immunoreactivity. Other methods for antigen retrieval are rarely beneficial and in most cases damage the specimen. Special stains work well, except silver methods for Helicobacter pylori. Routine hematoxylin and eosin preparations exhibit clarity and cellular detail rarely seen with formaldehyde.     

News from the Biological Stain Commission No. 15

In the 15^th issue of News from the Biological Stain Commission (BSC), under the heading of Regulatory affairs, the Biological Stain Commission’s International Affairs Committee presents information from the plenary meetings of the International Standards Organization ISO/TC 212 Clinical laboratory testing and in vitro diagnostic test systems held on August 22–24, 2012 in Berlin, Germany. An additional discussion of the use of food dyes in India also is included.     

Systematic sequencing of cDNA clones using the transposon Tn5

In parallel with the production of genomic sequence data, attention is being focused on the generation of comprehensive cDNA-sequence resources. Such efforts are increasingly emphasizing the production of high-accuracy sequence corresponding to the entire insert of cDNA clones, especially those presumed to reflect the full-length mRNA. The complete sequencing of cDNA clones on a large scale presents unique challenges because of the generally small, yet heterogeneous, sizes of the cloned inserts. We have developed a strategy for high-throughput sequencing of cDNA clones using the transposon Tn5. This approach has been tailored for implementation within an existing large-scale ‘shotgun-style’ sequencing program, although it could be readily adapted for use in virtually any sequencing environment. In addition, we have developed a modified version of our strategy that can be applied to cDNA clones with large cloning vectors, thereby overcoming a potential limitation of transposon-based approaches. Here we describe the details of our cDNA-sequencing pipeline, including a summary of the experience in sequencing more than 4200 cDNA clones to produce more than 8 million base pairs of high-accuracy cDNA sequence. These data provide both convincing evidence that the insertion of Tn5 into cDNA clones is sufficiently random for its effective use in large-scale cDNA sequencing as well as interesting insight about the sequence context preferred for insertion by Tn5.