Lack of evidence for the involvement of a β-D-galactosyl-specific lectin in the fusion of chick myoblasts

The role of a β-D-galactosyl-specific lectin, first reported by Teichberg et al., in the fusion of myoblasts in vitro was investigated. The concentration of this lectin in embryonic chick skeletal muscle was found to reach maximal levels at the time of myoblast fusion in vivo. β-D-Galactosyl-β-thiogalactopyranoside and lactose are potent inhibitors of agglutination of trypsinized rabbit erythrocytes caused by the lectin. However, at concentrations of 50 mM these compounds had no effect on either nonsynchronous fusion of myoblasts or on the release of synchronized myoblast cultures from EGTA fusion block. The presence of the agglutinin in the external membranes of chick myoblasts and myotubes could not be demonstrated. It is, therefore, concluded that the involvement of the lectin in the fusion of chick myoblasts remains questionable.     

Toxicological implications of a commercial formulation of deltamethrin (Decis®) in developing chick embryo

A deltamethrin containing insecticide formulation (Decis®) was evaluated for its toxic potential in developing chick embryos. For the present study, three water emulsified concentrations of Decis® (12.5 mg L^?1, 25 mg L^?1, and 50 mg L^?1) were used. Fertilized eggs of Gallus domesticus were immersed in these three concentrations of the insecticide for 60 min at 37°C on day 0 of incubation and kept for incubation till embryonic day 7. Recovered embryos were evaluated for teratogenic and biochemical changes. The results revealed that administration of Decis® at its lower concentrations (12.5 mg L^?1 and 25 mg L^?1) did not show any significant teratological changes but the significant number of abnormal survivors was observed at 50 mg L^?1 of dose concentration when compared with vehicle-treated control. Among biochemical changes, total glycogen and RNA contents of embryos was significantly decreased at 25 mg L^?1 and 50 mg L^?1 of Decis® concentrations. Similarly, significant alteration (p ≤ .05) was observed in alanine transaminase activity at 50 mg L^?1 concentration of Decis®. Thus, the present study concluded that the no-effect-level for developmental toxicity for Decis® is below the concentration of 25 mg L^?1 under standard laboratory conditions.     

Are egg yolk lipoproteins metabolized by the chick embryo in the same manner as plasma lipoproteins are in the adult?

• 1.1. Egg yolk very low density lipoproteins were found to contain proteins with cofactor activity for lipoprotein lipase.
• 2.2. Lipoprotein lipase activity in adipose tissue of chick embryos increased several-fold on days 16 and 17, coinciding with the time when utilization of yolk lipoproteins by the embryo becomes rapid.
• 3.3. Embryonic blood plasma was found to contain cholesterol esterifying activity.

     

Amino acid uptake in the developing chick embryo heart. The effect of insulin on α-aminoisobutyric acid accumulation

1. The uptake of (14)C-labelled alpha-aminoisobutyric acid by 5-day-old chick embryo hearts was investigated in vitro, together with the effect of insulin thereon. 2. At equilibrium the distribution ratio of this amino acid analogue between intracellular and extracellular water attained values greater than unity. Insulin enhanced the rate of alpha-aminoisobutyric acid accumulation and increased the value of its final concentration in the cell water. 3. The rate of alpha-aminoisobutyric acid accumulation and the effect of insulin on it were independent of the presence of glucose in the incubation medium. Bovine and chicken insulin were equally effective, and the action of the hormone was specifically prevented by an anti-insulin serum but not by puromycin. 4. A linear relationship was observed between the intracellular accumulation of the analogue and the logarithm of the insulin concentration in the range 50muunits-100m-units/ml. of incubation medium. 5. Evidence was obtained for the occurrence of two different transport processes for alpha-aminoisobutyric acid in the chick embryo heart: one subject to saturation and one that was not saturated by reasonable concentrations of the analogue. Insulin increased the effectiveness of the saturable component, increasing the maximal velocity of transport without altering the concentration for half-maximal velocity of transport, and decreased the contribution of the non-saturable component.     

The effect of β-bungarotoxin, or geniculate ganglion lesion on taste bud development in the chick embryo

Chick taste bud (gemmal) primordia normally appear on embryonic day (E) 16 and incipient immature, spherical-shaped buds at E17. In ovo injection of β-bungarotoxin at E12 resulted in a complete absence of taste buds in lower beak and palatal epithelium at developmental ages E17 and E21. However, putative gemmal primordia (solitary clear cells; small, cell groupings) remained, lying adjacent to salivary gland duct openings as seen in normal chick gemmal development. Oral epithelium was immunonegative to neural cell adhesion molecule (NCAM) suggesting gemmal primordia are nerve-independent. Some NCAM immunoreactivity was evident in autonomic ganglion-like cells and nerve fibers in connective tissue. After unilateral geniculate ganglion/otocyst excision on E2.5, at developmental ages E18 and posthatching day 1, ∼12% of surviving ipsilateral geniculate ganglion cells sustained ∼54% of the unoperated gemmal counts. After E18, proportional stages of differentiation in surviving developing buds probably reflect their degree of innervation, as well as rate of differentiation. Irrespective of the degree of geniculate ganglion damage, the proportion of surviving buds can be sustained at the same differentiated bud stage as on the unoperated side, or may differentiate to a later bud stage, consistent with the thesis that bud maturation, maintenance, and survival are nerve-dependent.     

Deficiencies of the müllerian ducts induced by norethindrone in the female chick embryo

Norethindrone produces two effects on müllerian ducts (MD) of female chick embryos. It induces the loss of the lower end of both ducts, as a result of a stop in their development, before 8 days. After 12 days NET causes regressions of the upper part of the MD particularly of the oviduct. NET like estrogens are the only known substances which present these both properties.     

Changes in expression of two endogenous β-galactoside-binding isolectins in the dermis of chick embryonic skin during development in ovo and in vitro

In order to elucidate the roles of metal-independent animal lectins, we systematically investigated changes in expression of 2 kinds of -galactoside-binding isolectins (MW 14 and 16 kDa) in the dermis of chick embryonic tarsometatarsal skin during the course of development. These lectins were immunohistochemically located at different stages of development both in ovo and in vitro by light and electron microscopy. Light-microscopic observation showed that while positive staining for the 14-kDa lectin was weak at days 8 and 10 it became intense after day 13. In contrast, staining for the 16-kDa lectin was intense at days 8, 10, and 13, but it became weak after day 17 when keratinization of the epidermis was completed. Immuno-electron-microscopic observation revealed that both the 14 and 16-kDa lectins were located on the basement membrane, in the extracellular matrix, and in both the cytoplasm and the nucleus of dermal fibroblasts. Distribution of the 2 isolectins was also examined in cultured skin explants in vitro. The results were almost the same as those obtained in ovo when the skin explant was keratinized in the presence of hydrocortisone. However, in the skin explant where keratinization was prevented and mucous metaplasia was induced by the addition of vitamin A, the distribution of the 14-kDa lectin in the epidermis was significantly affected. These results indicate that (1) the expression of the 2 isolectins is differently regulated in both the dermis and epidermis, (2) the 16-kDa lectin is involved in the early stage of the formation of the dermis and the basement membrane and is replaced by the 14-kDa lectin as keratinization of the epidermis occurs, and (3) the expression of the 2 isolectins in the dermis is not significantly affected by the induction of mucous metaplasia, in contrast to their drastic changes in the epidermis.     

Study of the residues involved in the binding of β1 to β3 subunits in the sodium channel

The voltage-gated sodium channel (VGSC) is a complex, which is composed of one pore-forming α subunit and at least one β subunit. Up to now, five β subunits are known: β1/β1A, β1B, β2, β3, and β4, encoded by four genes (SCN1B∼SCN4B). It is critical to have a deep understanding of the interaction between β1 and β3 subunits, two subunits which frequently appear in many diseases concurrently. In this study, we had screened out the new template of β1 subunit for homology modelling, which shares higher similarity to β3. Docking studies of the β1 and β3 homology model were conducted, and likely β1 and β3 binding loci were investigated. The results revealed that β1–β3 is more likely to form a di-polymer than β1–β1 based on molecular interaction analysis, including potential energy analysis, Van der Waals (VDW) energy analysis and electrostatic energy analysis, and in addition, consideration of the hydrogen bonds and hydrophobic contacts that are involved. Based on these analyses, the residues His122 and Lys140 of β1 and Glu 66, Asn 131, Asp 118, Glu 120, Glu133, Asn135, Ser 137 of β3 were predicted to play a functional role.     

Role of Glu445 in the substrate binding of β-glucosidase

A previously uncharacterized gene in Neosartorya fischeri was cloned and expressed in Escherichia coli. It was found to encode a β-glucosidase (NfBGL1) distinguishable from other BGLs by its high turnover of p-nitrophenyl β-d-glucopyranoside (pNPG). Molecular determinants for the substrate recognition of NfBGL1 were studied through an initial screening of residues by sequence alignment, a second screening by homology modeling and subsequent site-directed mutagenesis to alter individual screened residues. A conserved amino acid, E445, in the substrate binding pocket of wild-type NfBGL1 was identified as an important residue affecting substrate affinity. Replacement of E445 with amino acids other than aspartate significantly decreased the catalytic efficiency (k_cat/K_m) of NfBGL1 towards pNPG, mainly through decreased binding affinity. This was likely due to the disruption of hydrogen bonding between the substrate and the carboxylate oxygen of the residue at position 445. Density functional theory (DFT) based studies suggested that an acidic amino acid at position 445 raises the substrate affinity of NfBGL1 through hydrogen bonding. The residue E445 is completely conserved indicating that this position can be considered as a crucial determinant for the substrate binding among GHs tested.     

Immunohistochemical and in situ hybridization analysis of galectin-3, a β-galactoside binding lectin, in the urinary system of adult mice

Galectin is an animal lectin that has high affinity to β-galactoside of glycoconjugates. In the present study, cellular expression of galectin subtypes in the urinary system of adult mice was examined by in situ hybridization and immunohistochemistry. The major subtype expressed in the murine urinary system was galectin-3, which was expressed continuously from the kidney to the distal end of the urethra. The renal cortex expressed galectin-3 more intensely than the medulla. Renal galectin-3 immunoreactivity was strongest in the cortical collecting ducts, where principal cells were the sole cellular source. All cell layers of the transitional epithelium from the renal pelvis to the urethra strongly expressed galectin-3 at the mRNA and protein levels. An electron microscopic study demonstrated diffuse cytoplasmic localization of galectin-3 in principal cells of the collecting ducts and in the bladder epithelial cells. Urethral galectin-3 expression at the pars spongiosa decreased in intensity near the external urethral orifice, where the predominant subtype of galectin was substituted by galectin-7. The muscular layer of the ureter and urinary bladder contained significant signals for galectin-1. Taken together, the observations indicate that the adult urinary system shows intense and selective expression of galectin-3 in epithelia of the uretic bud- and cloaca-derivatives.     

Biosynthesis and secretion of fibronectin in the cultured mesenchymal cells of the developing chick müllerian duct

We have developed a method to separate and isolate the mesenchymal cells from the epithelial cells in the left Müllerian duct of the developing chick. We then cultured the mesenchymal cells in a serum-free medium. Through an enzyme-linked immunosorbent assay, we detected fibronectin synthesis and release into the medium at stages of Müllerian duct development. Our results demonstrate that the amount of fibronectin secreted by cultured cells gradually decreased in accordance with Müllerian duct differentiation. Similar observations found in the developing embryonic intestine indicate that the highest fibronectin synthesis occurs during early stages of development, when morphogenetic movement and mesenchymal-epithelial interaction are prominent features of embryonic organ differentiation and growth.     

Illuminating the binding interactions of galactonoamidines during the inhibition of β-galactosidase (E. coli)

Several galactonoamidines were previously identified as very potent competitive inhibitors that exhibit stabilizing hydrophobic interactions of the aglycon in the active site of β-galactosidase (Aspergillus oryzae). To elucidate the contributions of the glycon to the overall inhibition ability of the compounds, three glyconoamidine derivatives with alteration in the glycon at C-2 and C-4 were synthesized and evaluated herein. All amidines are competitive inhibitors of β-galactosidase (Escherichia coli) and show significantly reduced inhibition ability when compared to the parent. The results highlight strong hydrogen-bonding interactions between the hydroxyl group at C-2 of the amidine glycon and the active site of the enzyme. Slightly weaker H-bonds are promoted through the hydroxyl group at C-4. The inhibition constants were determined to be picomolar for the parent galactonoamidine, and nanomolar for the designed derivatives rendering all glyconoamidines very potent inhibitors of glycosidases albeit the derivatized amidines show up to 700-fold lower inhibition activity than the parent.     

Decrease in the secretion of anti-Müllerian hormone by the chick testis during development

At the end of embryonic life the chick embryonic testis possesses a low anti-Müllerian activity, as evidenced by the grafting method to female hosts. The percentage of grafted embryos presenting a Müllerian duct regression is not increased by administration of an anti-estrogenic drug (tamoxifen). This observation does not favour the hypothesis according to which the low percentage of regression could be due to a protection of Müllerian ducts by estrogens from the host ovary. It shows rather that the anti-Müllerian hormone secretion actually decreases during development.