Horseradish peroxidase: a reliable or a misleading tool for the investigations on the origin of the proteins of the aqueous humor?

The concept of the blood-aqueous barrier is largely based on the use of horseradish peroxidase (HRP). The present investigation was designed to check its reliability as a macromolecular tracer, especially with regard to the transport of plasma proteins. Rabbits were killed 5 min to 24 h after being intravenously injected with HRP. The tracer diffused rapidly, reaching the aqueous humor of the eye in 3 min or less and was detected at high concentration in the narrow space between the outer epithelial layer of the ciliary epithelium and the wall of the pervious capillaries in the stroma of the processes. HRP appeared to migrate from the blood to the posterior chamber, permeating the tight junctions, viz., the anatomical basis of the blood-aqueous barrier. It was detected at higher concentration at the anterior surface of the iris, at short time intervals; this was interpreted as penetration of the tracer from the aqueous humor of the anterior chamber. The choroid was also labeled in continuation with the reaction in the stroma of the pars plana of the ciliary body which, in turn, sometimes reached the iris root. Therefore, the pervious blood vessels of the choroid could be a source of macromolecules for the iris root. HRP also induced the formation of lysosomes in the ciliary epithelium. This can hardly be accepted as the way in which plasma proteins are physiologically transported to the aqueous humor. However, the pathway of HRP migration over short time intervals seems to be in agreement with previous research indicating that the entrance of serum albumin into the posterior chamber is the first step of its incorporation into the aqueous humor. Received: 7 June 1996 / Accepted: 15 January 1997     

Evolutionary history of Scinax treefrogs on land‐bridge islands in south‐eastern Brazil

Aim We investigated how Pleistocene refugia and recent (c. 12,000 years ago) sea level incursions shaped genetic differentiation in mainland and island populations of the Scinax perpusillus treefrog group. Location Brazilian Atlantic Forest, São Paulo state, south‐eastern Brazil. Methods Using mitochondrial and microsatellite loci, we examined population structure and genetic diversity in three species from the S. perpusillus group, sampled from three land‐bridge islands and five mainland populations, in order to understand the roles of Pleistocene forest fragmentation and sea level incursions on genetic differentiation. We calculated metrics of relatedness and genetic diversity to assess whether island populations exhibit signatures of genetic drift and isolation. Two of the three island populations in this study have previously been described as new species based on a combination of distinct morphological and behavioural characters, thus we used the molecular datasets to determine whether phenotypic change is consistent with genetic differentiation. Results Our analyses recovered three distinct lineages or demes composed of northern mainland São Paulo populations, southern mainland São Paulo populations, and one divergent island population. The two remaining island populations clustered with samples from adjacent mainland populations. Estimates of allelic richness were significantly lower, and estimates of relatedness were significantly higher, in island populations relative to their mainland counterparts. Main conclusions Fine‐scale genetic structure across mainland populations indicates the possible existence of local refugia within São Paulo state, underscoring the small geographic scale at which populations diverge in this species‐rich region of the Atlantic Coastal Forest. Variation in genetic signatures across the three islands indicates that the populations experienced different demographic processes after marine incursions fragmented the distribution of the S. perpusillus group. Genetic signatures of inbreeding and drift in some island populations indicate that small population sizes, coupled with strong ecological selection, may be important evolutionary forces driving speciation on land‐bridge islands.     

Wnt Pathway Activation Increases Hypoxia Tolerance during Development

Adaptation to hypoxia, defined as a condition of inadequate oxygen supply, has enabled humans to successfully colonize high altitude regions. The mechanisms attempted by organisms to cope with short-term hypoxia include increased ATP production via anaerobic respiration and stabilization of Hypoxia Inducible Factor 1α (HIF-1α). However, less is known about the means through which populations adapt to chronic hypoxia during the process of development within a life time or over generations. Here we show that signaling via the highly conserved Wnt pathway impacts the ability of Drosophila melanogaster to complete its life cycle under hypoxia. We identify this pathway through analyses of genome sequencing and gene expression of a Drosophila melanogaster population adapted over >180 generations to tolerate a concentration of 3.5–4% O₂ in air. We then show that genetic activation of the Wnt canonical pathway leads to increased rates of adult eclosion in low O₂. Our results indicate that a previously unsuspected major developmental pathway, Wnt, plays a significant role in hypoxia tolerance.     

Fine structure of the intersegmental membrane glands of the sixth abdominal sternum of female Nomia melanderi (Hymenoptera,Apoidea)

The fine structure of the intersegmental glands of the sixth abdominal sternum in 1-week old females of Nomia melanderi is presented. The plasma membrane of the secretory cell is unfolded in many places and is covered by a basement membrane. The microvillous surface is invaginated to form a rather long sinuous cavity. The endoplasm is almost entirely filled by secretory granules. Many secretory granules are located close to the inner surface of the invaginated plasma membrane. The invagination contains a porous ductule, apparently of cuticulin origin, that is connected directly with the inner layer of the transport duct of the duct-forming cell. This type of arrangement allows the direct flow of the secretory substance to the outside in a continuous way. The cylindrical duct-forming cell, besides having typical cell organelles, contains a cuticular transport duct. This duct is composed of a thin cuticulin layer surrounded by a rather thick epicuticular one. The results suggest that the secretory cell has two secretory cycles. The first occurs while the gland is differentiating (at the pupal stage) and is involved in secretion of the cuticulin that forms the porous ductule. The second cycle, which starts by the beginning of nesting, is involved in the secretion of a substance that is carried to the outside via the transport duct of the duct-forming cell.     

Binding of the hydrophilic beta-adrenergic antagonist [3H]CGP-12177 to cardiac tissue slices: characterization and ontogenetic studies in dogs

A new technique was developed to characterize the binding of a hydrophilic beta-adrenergic antagonist, [3H]CGP-12177, to 1-mm thick slices of canine cardiac tissue. This technique was used to quantify the density (Bmax) and the affinity (Kd) of these receptors in the right ventricular conus (RVC) and the left ventricle (LV) at day 1 to 6 weeks of age, and in the adult. Binding was found to be reversible, saturable, stereospecific, of high affinity, and thermolabile. There was an increase in the density of beta-adrenergic receptors between day 1 (Bmax = 2.2 +/- 0.3 fmol/mg tissue in RVC and 2.9 +/- 0.8 fmol/mg tissue in the LV) and 2 weeks of age postnatally, after which it remained constant until 6 weeks of age (Bmax = 7.5 +/- 0.4 and 6.8 +/- 0.9 fmol/mg tissue in RVC and LV, respectively); however, by 6 weeks of age it had not reached adult levels (10.3 +/- 1.0 fmol/mg tissue). The affinity of these receptors did not change between early neonatal life (Kd = 1.3 +/- 0.4 nM) and adulthood (Kd = 1.4 +/- 0.2 nM). The density of beta-adrenergic receptors in the RVC was similar to that in the LV. This new method of quantifying beta-adrenergic receptors in cardiac tissue is simple and fast, and requires minimal tissue handling. It proved to be useful in studying the development of cardiac beta-adrenergic receptors with age.     

Production of ethanol by alginate-entrapped Saccharomyces cerevisiae strain "14-12"

Cells of S. cerevisiae strain "14-12" of different ages were immobilized in sodium alginate and used for conversion of glucose to ethanol. Immobilized cells of 48 hr old were the most potential. Employment of high counts of alginate-entrapped cells shortened the period required for production of the maximal alcohol yield. However, the percentage surviving cells decreased with increasing initial cell counts. Maximal accumulation of ethanol (4.18 g/100 ml) was obtained after 4 days of static fermentation with 1.8 X 10(8) immobilized yeast cells. The residual viable cell count was found to represent 3-fold the surviving percentage in a control experiment using an inoculum of the free yeast cells. Immobilized yeast cells could convert about 85% of the available sugars to ethanol over 28 days of the repeated-batch fermentation. The immobilized cells retained 50% of their viability for 16 days. After 48 days of repeated fermentation only 6% of the yeast cells were viable, and on the 52nd day no viable cells could be detected.     

Ethanol tolerance ofSaccharomyces cerevisiae and its relationship to lipid content and composition

Saccharomyces cerevisiae strain 14-12 is a highly ethanol-tolerant organism. It can grow in the presence of 13% ethanol but growth is completely prevented at 14% ethanol. A relationship was detected between yeast lipids and ethanol tolerance. A gradual decrease of lipid content was recorded as the concentration of supplemented ethanol increased. Moreover, free fatty acids were comparatively decreased in these lipid extracts. When separately added to media with 14% ethanol different lipids produced varied stimulatory effects on yeast growth. Maximum yield of yeast growth was obtained at 14% ethanol in the presence of lecithin, palmitic acid and cholesterol. Yeast lipids produced in the presence of these fractions are characterized by a relatively high percentage of free fatty acids. The change in the percentage of free fatty acids was shown to be the controlling factor in ethanol tolerance.     

In vivo development and pathogenicity of Ascosphaera proliperda (Ascosphaeraceae) to the alfalfa leafcutting bee, Megachile rotundata

First instar larvae of the leafcutting bee, Megachile rotundata, were fed on either artificial or natural provisions containing spores of Ascosphaera proliperda. Two isolates were used as a source of inocula: one originated from in vitro isolates obtained while culturing what was thought to be pure spores of A. aggregata, the second originated from in vitro cultures from Denmark. Histological and scanning electron microscopy studies revealed that the spores germinated in the gut lumen and the developing hyphae invaded all tissues, after which they penetrated through larval integument and began the sexual phase of the life cycle aerially. Virtually all fungus-exposed larvae developed symptoms of disease regardless of source of inoculum, type of provision, and spore dose (1.5 × 10³ to 3 × 10⁶) per insect. It was concluded that the fungus was pathogenic to the alfalfa leafcutting bee under laboratory conditions and future studies should be conducted to determine its etiology, cross infectivity, and natural distribution in other bee taxa.