Glycoalkaloids and acclimation capacity of hybrids between <Emphasis Type="Italic">Solanum tuberosum</Emphasis> and the incongruent hardy species <Emphasis Type="Italic">Solanum commersonii</Emphasis>

F₁ and backcross hybrids between sexually incompatible species Solanum commersonii and Solanum tuberosum were characterized for glycoalkaloid content and capacity to cold acclimate. Glycoalkaloid (GA) analysis revealed that F₁ triploids and BC₁ pentaploids contained the glycoalkaloids of both parents. In BC₂ (near) tetraploids the situation was different, in that some hybrids produced the GAs of both parents, whereas others contained only the GAs of S. tuberosum. This suggested that the GAs from S. commersonii may be lost rapidly, and that they may have a simple genetic control. The total tuber GA content of BC₁ and BC₂ groups averaged quite acceptable levels (165.9 mg/kg in BC₁ and 192.8 mg/kg in BC₂), with six genotypes having a GA content <200 mg/kg fresh weight. The F₁ triploid hybrids expressed a capacity to cold acclimate similar to S. commersonii, whereas BC₁ and BC₂ genotypes generally displayed an acclimation capacity higher than the sensitive parent but lower than S. commersonii. However, one BC₁ and two BC₂ genotypes with an acclimation capacity as high as S. commersonii were identified. The polar lipid fatty acid composition in S. commersonii and its hybrid derivatives showed that, following acclimation, there was a significant increase in 18:3. Correlation analysis between the capacity to cold acclimate and the increase in 18:3 was significant, suggesting that the increase in 18:3 can be used as a biochemical marker for the assisted selection of cold-acclimating genotypes in segregating populations.Communicated by G. Wenzel     

Morbidity and mortality of red foxes (Vulpes vulpes) and gray foxes (Urocyon cinereoargenteus) admitted to the Wildlife Center of Virginia, 1993-2001

The medical records of 48 red foxes (Vulpes vulpes) and 35 gray foxes (Urocyon cinereoargenteus) examined at the Wildlife Center of Virginia (Waynesboro, Virginia, USA) from 1993 to 2001 were reviewed. The most common diagnosis in red foxes was orphaned (33%), followed by trauma (27%), undetermined diagnosis (23%), and sarcoptic mange (17%). Trauma (46%) was the most frequent cause of morbidity and mortality in gray foxes followed by orphaned (23%), undetermined (20%), toxoplasmosis (6%), presumptive canine distemper (3%), and rabies (3%). One gray fox had concurrent toxoplasmosis and presumptive canine distemper (3%). Similar diseases were detected in previous studies at a diagnostic laboratory; however in this study, trauma and orphaned animals were more common than infectious diseases. The lack of diagnostic information on some cases limited the usefulness of this study, and more emphasis should be placed on performing postmortem examinations of wildlife presented to wildlife rehabilitation centers.     

Purification and characterization of three beta-glycosidases from midgut of the sugar cane borer,Diatraea saccharalis

Three beta-glycosidases, named betaGly1, betaGly2 and betaGly3, were isolated from midgut tissues of the sugar cane borer, Diatraea saccharalis Fabricius (Lepidoptera: Pyralidae). The three enzymes have similar Mr (58,000; 61,000; 61,000), pI (7.5, 7.4, and 7.4) and optimum pH (6.7, 6.3, and 7.2) and were resolved by hydrophobic chromatography. The beta-glycosidases prefer beta-glucosides to beta-galactosides, have four subsites for glucose binding and hydrolyse glucose-glucose beta-1,3 linkages better than beta-1, 4- or beta-1,6 linkages. betaGly1 and 2 were completely purified, whereas betaGly3 was isolated with a contaminant peptide that has no activity upon beta-glycosides.By using competing substrates, it was shown that betaGly 1 and 3 have one active site, whereas betaGly2 has two, one hydrolyzing natural and the other synthetic substrates. betaGly2 is the only D. saccharalis beta-glycosidase that can efficiently hydrolyse prunasin, the glycoside remaining after glucose removal from the plant glycoside amygdalin and that liberates the cyanogenic mandelonitrile. As shown elsewhere, betaGly2 activity is reduced when D. saccharalis is reared in amygdalin containing diets. From the results, we propose that the physiological role of betaGly 1 and 3 is the digestion of oligo- and disaccharides derived from hemicelluloses and of betaGly2 is glycolipid hydrolysis.Free energy relationships showed that D. saccharalis betaGly3 and Tenebrio molitor (Coleoptera) betaGly1 have active sites that bind similarly the transition states formed with different substrates. The same is also true for the active sites of D. saccharalis betaGly1 and T. molitor betaGly2. This suggests that active sites of similar enzymes are probably homologous, displaying nearly identical bonds between active site amino acids and substrate moieties.     

Characterization of a beta-glycosidase highly active on disaccharides and of a beta-galactosidase from Tenebrio molitor midgut lumen

The midgut of the yellow mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae) larvae has four beta-glycosidases. The properties of two of these enzymes (betaGly1 and betaGly2) have been described elsewhere. In this paper, the characterization of the other two glycosidases (betaGly3 and betaGly4) is described. BetaGly3 has one active site, hydrolyzes disaccharides, cellodextrins, synthetic substrates and beta-glucosides produced by plants. The enzyme is inhibited by amygdalin, cellotriose, cellotetraose and cellopentaose in high concentrations, probably due to transglycosylation. betaGly3 hydrolyzes beta 1,4-glycosidic linkages with a catalytic rate independent of the substrate polymerization degree (k(int)) of 11.9 s(-1). Its active site is formed by four subsites, where subsites +1 and -1 bind glucose residues with higher affinity than subsite +2. The main role of betaGly3 seems to be disaccharide hydrolysis. BetaGly4 is a beta-galactosidase, since it has highest activity against beta-galactosides. It can also hydrolyze fucosides, but not glucosides, and has Triton X-100 as a non-essential activator (K(a)=15 microM, pH 4.5). betaGly4 has two active sites that can hydrolyze p-nitrophenyl beta-galactoside (NPbetaGal). The one hydrolyzing NPbetaGal with more efficiency is also active against methylumbellipheryl beta-D-galactoside and lactose. The other active site hydrolyzes NPbetaFucoside and binds NPbetaGal weakly. BetaGly4 hydrolyzes hydrophobic substrates with high catalytical efficiency and is able to bind octyl-beta-thiogalactoside in its active site with high affinity. The betaGly4 physiological role is supposed to be the hydrolysis of galactolipids that are found in membranes from vegetal tissues. As the enzyme has a hydrophobic site where Triton X-100 can bind, it might be activated by membrane lipids, thus becoming fully active only at the surface of cell membranes.     

Methods designed for the identification and characterization of<Emphasis Type="Italic">in vitro</Emphasis> and<Emphasis Type="Italic">in vivo</Emphasis> chromatin assembly mutants in<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Assembly of DNA into chromatin allows for the formation of a barrier that protects naked DNA from protein and chemical agents geared to degrade or metabolize DNA. Chromatin assembly occurs whenever a length of DNA becomes exposed to the cellular elements, whether during DNA synthesis or repair. This report describes tools to study chromatin assembly in the model systemSaccharomyces cerevisiae. Modifications to anin vitro chromatin assembly assay are described that allowed a brute force screen of temperature sensitive (ts) yeast strains in order to identify chromatin assembly defective extracts. This screen yielded mutations in genes encoding two ubiquitin protein ligases (E3s):RSP5, and a subunit of the Anaphase Promoting Complex (APC),APC5. Additional modifications are described that allow for a rapid analysis and anin vivo characterization of yeast chromatin assembly mutants, as well as any other mutant of interest. Our analysis suggests that thein vitro andin vivo chromatin assembly assays are responsive to different cellular signals, including cell cycle cues that involve different molecular networks. Published: July 3, 2003     

Cyclic hydrostatic compression stimulates chondroinduction of C3H/10T1/2 cells

While the potential for intermittent hydrostatic pressure to promote cartilaginous matrix synthesis is well established, its potential to influence chondroinduction remains poorly understood. This study examined the effects of relatively short- and long-duration cyclic hydrostatic compression on the chondroinduction of C3H/10T1/2 murine embryonic fibroblasts by recombinant human bone morphogenetic protein-2 (rhBMP-2). Cells were seeded at high density into round bottom wells of a 96-well plate and supplemented with 25 ng/ml rhBMP-2. Experimental cultures were subjected to either 1,800 cycles/day or 7,200 cycles/day of 1 Hz sinusoidal hydrostatic compression to 5 MPa (applied 10 min on/10 min off) for 3 days. Non-pressurized control and experimental cultures were maintained in static culture for an additional 5 days. Cultures were then analyzed for alcian blue staining intensity, DNA and sulfated glycosaminoglycan (sGAG) content, and for the rate of collagen synthesis. Whereas cultures subjected to 1,800 pressure cycles exhibited no significant differences (statistical or qualitative) compared to controls, those subjected to 7,200 cycles stained more intensely with alcian blue, contained nearly twice as much sGAG, and displayed twice the rate of collagen synthesis as non-pressurized controls. This study demonstrates the potential for cyclic hydrostatic compression to stimulate chondrogenic differentiation of the C3H/10T1/2 cell line in a duration-dependent manner.     

The peritrophic membrane of Spodoptera frugiperda: secretion of peritrophins and role in immobilization and recycling digestive enzymes

A peritrophin from the Spodoptera frugiperda peritrophic membrane (PM) and microvillar proteins from S. frugiperda anterior midgut cells were isolated and used to raise antibodies in a rabbit. These antibodies, as well as a Tenebrio molitor amylase antibody that cross-reacts with S. frugiperda amylases, and wheat-germ aglutinin were used in immunolocalization experiments performed with the aid of confocal fluorescence and immunogold techniques. The results showed that the peritrophin was secreted by anterior midgut columnar cells in vesicles pinched-off the microvilli (microapocrine secretion). The resulting double membrane vesicles become single membrane vesicles by membrane fusion, releasing peritrophin and part of the amylase and trypsin. The remaining membranes still containing microvillar proteins and membrane-bound amylase and trypsin are incorporated into a jelly-like material associated with PM. Calcofluor-treated larvae lacking a PM were shown to lose the decreasing gradient of trypsin and chymotrypsin observed along the midgut of control larvae. This gradient is thought to be formed by a countercurrent flux of fluid (in the space between PM and midgut cells) that powers enzyme recycling.     

Apocrine secretion of amylase and exocytosis of trypsin along the midgut of Tenebrio molitor larvae

Amylase and trypsin were purified from Tenebrio molitor midgut larvae and used to raise antibodies in a rabbit. A Western blot of T. molitor midgut homogenates, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using amylase and trypsin antisera, showed only bands co-migrating with the purified enzymes. The antisera were used to localize the enzymes by immunofluorescence and immunogold labeling. Amylase occurs in a few regularly disposed anterior midgut cells. Non-amylase-secreting anterior midgut cells are proposed to be water-absorbing cells based on morphology and dye movements. Amylase is found inside vesicles originating from Golgi areas that seem to fuse together before their release along with the now disorganized apical cytoplasm (apocrine secretion). Trypsin precursors are observed inside small vesicles near the apical plasma membrane of posterior midgut cells, suggesting an exocytic mechanism of secretion, followed by putative trypsin activation. Apocrine secretion is thought to be an adaptation to enhance the dispersion of secretory vesicle contents released from a water-absorbing epithelium, whereas exocytosis is an efficient secretory mechanism in a water-secreting epithelium.     

Alpha-galactosidases from the larval midgut of Tenebrio molitor (Coleoptera) and Spodoptera frugiperda (Lepidoptera)

There are three midgut alpha-galactosidases (TG1, TG2, TG3) from Tenebrio molitor larvae that are partially resolved by ion-exchange chromatography. The enzymes have approximately the same pH optimum (5.0), pl value (4.6) and Mr value (46000-49000) as determined by gel filtration or native electrophoresis run in polyacrylamide gels with different concentrations. Substrate specificities and functions were proposed for the major T. molitor midgut alpha-galactosidases (TG2 and TG3) based on chromatographic, carbodiimide inactivation, Tris inhibition, and on substrate competition data. Thus, TG2 would hydrolyse alpha-1,6-galactosaccharides, exemplified by raffinose, whereas TG3 would act on melibiose and apparently also on digalactosyldiglyceride, the most important compound in the thylacoid membranes of chloroplasts. Most galactoside digestion should occur in the lumen of the first two thirds of T. molitor larval midguts, since alpha-galactosidase activity predominates there. Spodoptera frugiperda larvae have three midgut alpha-galactosidases (SG1, SG2, SG3) partially resolved by ion-exchange chromatography. The enzymes have similar pH optimum (5.8), pl value (7.2) and Mr value (46000-52000), and at least the major alpha-galactosidase must have an active carboxyl group in the active site. Based on data similar to those described for T. molitor, SG1 and SG3 should hydrolyse melibiose and SG3 should digest raffinose and, perhaps, also digalactosyldiglyceride. The midgut distribution of alpha-galactosidase activity supports the proposal that alpha-galactosidase digestion occurs at the surface of anterior midgut cells in Spodoptera frugiperda larvae.     

Purification, molecular cloning, and properties of a beta-glycosidase isolated from midgut lumen of Tenebrio molitor (Coleoptera) larvae

Two beta-glycosidases (M(r) 59k) were purified from midgut contents of larvae of the yellow mealworm, Tenebrio molitor (Coleoptera: Tenebrionidae). The two enzymes (betaGly1 and betaGly2) have identical kinetic properties, but differ in hydrophobicity. The two glycosidases were cloned and their sequences differ by only four amino acids. The T. molitor glycosidases are family 1 glycoside hydrolases and have the E379 (nucleophile) and E169 (proton donor) as catalytic amino acids based on sequence alignments. The enzymes share high homology and similarity with other insect, mammalian and plant beta-glycosidases. The two enzymes may hydrolyze several substrates, such as disaccharides, arylglucosides, natural occurring plant glucosides, alkylglucosides, oligocellodextrins and the polymer laminarin. The enzymes have only one catalytic site, as inferred from experiments of competition between substrates and sequence alignments. The observed inhibition by high concentrations of the plant glucoside amygdalin, used as substrate, is an artifact generated by transglucosylation. The active site of each purified beta-glycosidase has four subsites, of which subsites +1 and +2 bind glucose with more affinity. Subsite +2 has more affinity for hydrophobic groups, binding with increasing affinities: glucose, mandelonitrile and nitrophenyl moieties. Subsite +3 has more affinity for glucose than butylene moieties. The intrinsic catalytic constant calculated for hydrolysis of the glucose beta-1,4-glucosidic bond is 21.2 s(-1) x M(-1). The putative physiological role of these enzymes is the digestion of di- and oligosaccharides derived from hemicelluloses.