Monoclonal antibodies against chicken type V collagen: production, specificity, and use for immunocytochemical localization in embryonic cornea and other organs

Two monoclonal antibodies have been produced against chick type V collagen and shown to be highly specific for separate, conformational dependent determinants within this molecule. When used for immunocytochemical tissue localization, these antibodies show that a major site for the in situ deposition of type V is within the extracellular matrices of many dense connective tissues. In these, however, it is largely in a form unavailable to the antibodies, thus requiring a specific “unmasking” treatment to obtain successful immunocytochemical staining. The specificity of these two IgG antibodies was determined by inhibition ELISA, in which only type V and no other known collagen shows inhibition. In ELISA, mixtures of the two antibodies give an additive binding reaction to the collagen, suggesting that each is against a different antigenic determinant. That both antigenic determinants are conformational dependent, being either in, or closely associated with, the collagen helix is demonstrated by the loss of antibody binding to molecules that have been thermally denatured. The temperature at which this occurs, as assayed by inhibition ELISA, is very similar to that at which the collagen helix melts, as determined by optical rotation. This gives strong additional evidence that the antibodies are directed against the collagen. The antibodies were used for indirect immunofluorescence analyses of cryostat sections of corneas and other organs from 17 to 18-day-old chick embryos. Of all tissues examined only Bowman’s membrane gave a strong staining reaction with cryostat sections of unfixed material. Staining in other areas of the cornea and in other tissues was very light or nonexistent. When, however, sections were pretreated with pepsin dissolved in dilute HAc or, surprisingly, with the dilute HAc itself dramatic new staining by the antibodies was observed in most tissues examined. The staining, which was specific for the anti-type V collagen antibodies, was largely confined to extracellular matrices of dense connective tissues. Experiments using protease inhibitors suggested that the “unmasking” did not involve proteolysis. We do not yet know the mechanism of this unmasking; however, one possibility is that the dilute acid causes swelling or conformational changes in a type-V collagen-containing supramolecular structure. Further studies should allow us to determine whether this is the case.     

Biomass and annual production of the freshwater mussel Elliptio complanata in an oligotrophic softwater lake

SUMMARY. An extensive survey of Mirror Lake, New Hampshire, was carried out by divers with SCUBA to assess the importance of the freshwater mussel Elliptio complanata in this softwater lake ecosystem. Density (0.032 adults m⁻²), biomass (52 mg m⁻² as dry organic matter) and annual production (6.4 mg m⁻² as dry organic matter) of the mussel population are low when compared with results from other studies, corresponding with the general observation that mussels are scarce in soft, oligotrophic waters. We reject the traditional view that the low mussel density is a result of low calcium concentrations in Mirror Lake, and propose that mussel populations may be regulated by a scarcity of appropriate fish hosts in unproductive lakes. Elliptio complanata is probably not important in the metabolism or biochemistry of the Mirror Lake ecosystem.     

Influence of day length and temperature on number of main stem leaves and time to flowering in lupin

A growth chamber experiment was carried out to investigate the influence of day length and temperature on the development of flowering in eight varieties of the three grain lupin species Lupinus albus (Wat and C3396), L. angustifolius (Gungurru, Polonez and W26) and L. luteus, (Juno, Radames and Teo). The plants were grown at two temperatures, 10°C and 18°C, in combination with five daylength regimes: 10, 14, 18, 24 h day at full light intensity and 10 h full light extended with 8 h low intensity light. Increased daylength decreased days from sowing to flowering in all varieties, but had little effect on thermal time to flowering in most varieties. However, C3396, W26 and Radames had a significantly longer thermal time to flowering at high, non‐vernalising temperature (18°C) at short daylengths. Low light intensity daylength extension did not significantly influence thermal time to flowering. For flower initiation, measured as number of leaves on the main stem three types of response were found. All varieties formed fewer leaves on the main stem at 10°C than at 18°C, although the two thermo‐neutral varieties of L. luteus, Juno and Teo, gave only a small response to temperature and daylength. In Polonez, Gungurru and Wat, low temperature decreased leaf number, but there was only a small response to changes in daylength. Three varieties, C3396, W26 and Radames, showed longer thermal time to flowering at 18°C with short daylengths. This could be explained by a greater number of main stem leaves formed at short daylength at non‐vernalising temperatures. Increased daylength decreased leaf number in these varieties, but never to a smaller number than for plants grown at 10°C. In these varieties, low intensity extension of the daylength had a similar (W26, Radames) or decreased (C3396) effect compared to full light extension. The hastening of time to flowering by long days could be separated into two effects: a high light energy effect hastened development by increasing the rate of leaf appearance in all varieties, while low light energy in thermo‐sensitive varieties was able to substitute for vernalisation by decreasing leaf number.     

The complementary effects of plant resistance and the choice of sowing and harvest times in reducing carrot fly (Psila rosae) damage to carrots

The benefits of combining a partially-resistant carrot cultivar with different sowing and lifting dates to reduce carrot fly, Psila rosae, damage were investigated at Wellesbourne in 1983 and 1984-85. The partially-resistant cv. Sytan was less damaged and supported fewer insects than the susceptible cv. Danvers on all lifting dates. The estimated reduction of carrot fly larvae on Sytan compared with Danvers ranged from 33 to 95%. Nine combinations of sowing and lifting dates provided more than 75% marketable roots of Sytan compared with only three combinations of dates for Danvers. An early June sowing of both cultivars provided roots of a marketable size with the least attack. More than 90% of Sytan roots were still marketable in December and fewer insects were produced by the end of the season on these roots than on those sown earlier. In addition, sowing in June decreased the number of pupae produced on cv. Danvers by 10 times compared with earlier sowings. Combining partial resistance with specific sowing and lifting times enabled satisfactory yields of marketable carrots to be obtained in a field infested by high populations of carrot fly.     

Relationship between the concentration of chlorogenic acid in carrot roots and the incidence of carrot fly larval damage

Chlorogenic acid (1·24-3·36 mg/g) was identified as the main phenolic component in the peel of carrots by hplc analysis. The higher the concentration of chlorogenic acid in different cultivars the greater the susceptibility to carrot fly larval damage. Increases in concentration were found both after carrot fly damage and after carrots had overwintered in the field. The presence and location of chlorogenic acid was confirmed in sections of carrot tissues, mounted in 0·05 M ammonia solution by viewing them using a u.v.-epifluorescent microscope. The importance of phenolic compounds and their function in the production of insect cuticle is discussed in relation to the different concentrations of chlorogenic acid and resistance to carrot fly in carrots.     

Contributions of Electron Microscopy to Fungal Classification

Such characters as surface ultrastructure of asexual and sexualpropagules, wall and septal ultrastructure, the fine structureof ascal tips, and ultrastructural aspects of nuclear divisionhave taxonomic significance for major groups of fungi. Informationderived from fine-structural analyses can be correlated withthat obtained from light-microscopic, chemical, and developmentalinvestigations. The versatility of electron-microscopic facilitiesmakes them powerful research tools in the hands of the innovativetaxonomist.     

HyLiTE: accurate and flexible analysis of gene expression in hybrid and allopolyploid species

Background

Forming a new species through the merger of two or more divergent parent species is increasingly seen as a key phenomenon in the evolution of many biological systems. However, little is known about how expression of parental gene copies (homeologs) responds following genome merger. High throughput RNA sequencing now makes this analysis technically feasible, but tools to determine homeolog expression are still in their infancy.

Results

Here we present HyLiTE – a single-step analysis to obtain tables of homeolog expression in a hybrid or allopolyploid and its parent species directly from raw mRNA sequence files. By implementing on-the-fly detection of diagnostic parental polymorphisms, HyLiTE can perform SNP calling and read classification simultaneously, thus allowing HyLiTE to be run as parallelized code. HyLiTE accommodates any number of parent species, multiple data sources (including genomic DNA reads to improve SNP detection), and implements a statistical framework optimized for genes with low to moderate expression.

Conclusions

HyLiTE is a flexible and easy-to-use program designed for bench biologists to explore patterns of gene expression following genome merger. HyLiTE offers practical advantages over manual methods and existing programs, has been designed to accommodate a wide range of genome merger systems, can identify SNPs that arose following genome merger, and offers accurate performance on non-model organisms.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0433-8) contains supplementary material, which is available to authorized users.     

Identification and characterization of the intracellular poly-3-hydroxybutyrate depolymerase enzyme PhaZ of Sinorhizobium meliloti

Background  

S. meliloti forms indeterminate nodules on the roots of its host plant alfalfa (Medicago sativa). Bacteroids of indeterminate nodules are terminally differentiated and, unlike their non-terminally differentiated counterparts in determinate nodules, do not accumulate large quantities of Poly-3-hydroxybutyrate (PHB) during symbiosis. PhaZ is in intracellular PHB depolymerase; it represents the first enzyme in the degradative arm of the PHB cycle in S. meliloti and is the only enzyme in this half of the PHB cycle that remains uncharacterized.