Intestinal lactase (beta-galactosidase) and other glycosidase activities in suckling and adult tammar wallabies (Macropus eugenii)

The activities of various glycosidases in homogenates of the small intestinal mucosa of two adult and 18 suckling tammar wallabies (M. eugenii) aged from 6 to 50 weeks were investigated. Lactase (beta-D-galactosidase), beta-N-acetylglucosaminidase, alpha-L-fucosidase and neuraminidase activities were high during the first 34 weeks post partum and then declined to very low levels. Maltase, isomaltase, sucrase and trehalase activities were very low or absent during the first 34 weeks, and then increased. The lactase activity was unusual in being greater in the distal than the middle or proximal thirds of the intestine, and in its low pH optimum (pH 4.6), inhibition by p-chloromercuribenzene sulfonate but not by Tris, and lack of cellobiase activity. These properties are those of a lysosomal acid beta-galactosidase rather than of a brush border neutral lactase. The maltase activity had the characteristics of a lysosomal acid alpha-glucosidase early in lactation and of a brush border neutral maltase in adult animals. The significance of these findings is discussed in relation to changes in dietary carbohydrates during weaning and to the mode of digestion of milk carbohydrates by the pouch young.     

Intestinal lactase and other disaccharidase activities of a suckling crabeater seal (Lobodon carcinophagus)

1. The intestinal disaccharidase activities of a suckling crabeater seal were investigated. 2. Lactase, maltase, isomaltase and cellobiase activities were readily detected but trehalase and sucrase activities were absent. 3. The intestinal homogenates were separated into a soluble (S2) fraction and a particulate brush border (P2) fraction. The lactase activities of the two fractions had different properties corresponding to those of an acid and a neutral beta-galactosidase respectively. Approximately two-thirds of the total lactase activity measured at pH 6.0 was due to the acid beta-galactosidase. 4. The isomaltase and cellobiase activities were found almost exclusively in the particulate fractions but about one third of the maltase activity was in the S2 fraction. This soluble maltase activity appeared to be due to an acid maltase.     

Intake of milk by suckling echidnas (Tachyglossus aculeatus)

Estimation of milk intake in the sucklings of three captive echnidnas, Tachyglossus aculeatus, and in one living in its natural habitat were made by determination of sodium turnover rate and sodium content of the milk. The mean rate of sodium influx of the sucklings was 2.48 +/- 1.31 mmol/kg X day. The daily rates of milk consumption and growth rates were variable with a mean weight increment of 0.41 +/- 0.10 g for each ml of milk consumed. There is a wide disparity in growth rates of echidna young that is related to differences in the body weights of the mothers.     

Cortisone and thyroxine modulate intestinal lactase and sucrase mRNA levels and activities in the suckling rat.

Glucocorticoids and thyroxine modulate postnatal intestinal sucrase and lactase activities. Whether changes in enzyme activity are accompanied by changes in enzyme mRNA levels were determined in day 6 rats given thyroxine, cortisone, or thyroxine plus cortisone and killed 3 days later. Cortisone induced precocious expression of jejunal sucrase activity which was enhanced when cortisone plus thyroxine was administered; sucrase mRNA changed in parallel. Jejunal lactase activity was unaffected by thyroxine and was increased after cortisone, but not after thyroxine plus cortisone. Jejunal lactase mRNA levels increased equally after cortisone or after cortisone plus thyroxine. Thus, cortisone induces coordinated increases in sucrase and lactase activities and in corresponding mRNA levels. Thyroxine only enhances cortisone induced sucrase expression and antagonizes cortisone by depressing lactase activity post-translationally.     

Year-long presence of Eimeria echidnae and absence of Eimeria tachyglossi in captive short-beaked echidnas ( Tachyglossus aculeatus )

The short-beaked echidna ( Tachyglossus aculeatus ) is 1 of 5 extant species of monotreme, found only in Australia and Papua New Guinea. The aim of this study was to identify the species of coccidia present and establish a range of subclinical Eimeria spp. (Coccidia: Apicomplexa) oocyst shedding in echidnas from eastern Australia over 18 mo. The coccidia were detected in 89% (49/55) of fecal samples from 12 long-term monitored and healthy captive echidnas, 75% (3/4) of 4 healthy long-term captive echidnas, 83% (5/6) of 6 short-term captive echidnas, and 60% (6/10) of 10 wild echidnas. Echidnas captive for 4 to 23 yr shed 100-46,000 oocysts g(-1) of E. echidnae and remained clinically healthy during this study. Sub-adult and adult wild, and short-term captive, echidnas shed oocysts of both E. echidnae and E. tachyglossi . The lack of coccidia in juvenile short-beaked echidnas suggests these animals are probably non-immune and should not be placed in environments heavily contaminated with oocysts. In addition, no oocysts were found in captive long-beaked echidnas ( Zaglossus bartoni bartoni , n = 2) housed at Taronga Zoo. This study represents an important step in understanding the host-parasite interaction between coccidia and short-beaked echidnas.     

Synthesis and glycosidase inhibitory activities of 2-(aminoalkyl)pyrrolidine-3,4-diol derivatives

Several 2-(aminomethyl)-and 2-(2-aminoethyl)-pyrrolidine-3,4-diol derivatives have been assayed for their inhibitory activities towards glycosidases. Good inhibitors of alpha-mannosidases must have the (2R,3R,4S) configuration and possess 2-(benzylamino)methyl substituents. Stereomers with the (2S,3R,4S) configuration are also competitive inhibitors of alpha-mannosidases, but less potent as they share the configuration of C(1), C(2), C(3) of beta-D-mannosides rather than that of alpha-D-mannosides. Interestingly, (2S,3R,4S)-2-[2-[(4-phenyl)phenylamino]ethyl]pyrrolidine-3,4-diol (12g) inhibits several enzymes, for instance alpha-L-fucosidase from bovine epididymis (K(i)=6.5microM, competitive), alpha-galactosidase from bovine liver (K(i)=5microM, mixed) and alpha-mannosidase from jack bean (K(i)=102microM, mixed). Diamines such as (2R,3S,4R)-2-[2-(phenylamino) or 2-(benzylamino)ethyl]pyrrolidine-3,4-diol (ent-12a, ent-12b) inhibit beta-glucosidase from almonds (K(i)=13-40microM, competitive).     

Absence of beta-galactosidase (lactase) activity from intestinal brush borders of suckling macropods: implications for mechanism of lactose absorption

1. Neutral beta-galactosidase (lactase) activity was absent from crude brush borders of small intestines of three species of suckling macropods (kangaroos and wallabies), even though the intestinal mucosal homogenates had high beta-galactosidase activities. 2. These activities were entirely due to an intracellular acid beta-galactosidase, probably located in lysosomes. 3. The results suggest that the absorptive-digestive mechanism for lactose in macropods is fundamentally different from that in eutherian mammals.     

Sucrose-dependent increase in oligosaccharide production and associated glycosidase activities in the potato aphid Macrosiphum euphorbiae (Thomas)

Osmotic stress due to high sucrose concentration was imposed on the potato aphid, Macrosiphum euphorbiae (Thomas), in an artificial diet situation. Thin layer chromatography resolved melezitose and six additional honeydew oligosaccharides. Significantly higher levels of these oligosaccharides were recovered from the honeydew of this species in response to feeding on increased dietary sucrose concentrations, pointing to an osmoregulatory response. In addition, carbohydrase activities from the potato aphid were described. A degree of specificity toward hydrolysis of the sucrose substrate over other α-glucosyl sugars was demonstrated. Also, sucrose was optimal for the formation of the oligosaccharides. A strong α-galactosidase activity was found and transgalactosylation ability was indicated. These major trends in carbohydrase activity were also found in the foxglove aphid, Acyrthosiphon solani (Kaltenbach), and the oleander aphid, Aphis nerii Fonscolombe.     

Quantification of glycosidase activities in selected yeasts and lactic acid bacteria

Using a model system, the activities of α-L-arabinofuranosidase, β-glucosidase, and α-L-rhamonopyranosidase were determined in 32 strains of yeasts belonging to the genera Aureobasidium, Candida, Cryptococcus, Hanseniaspora, Hansenula, Kloeckera, Metschnikowia, Pichia, Saccharomyces, Torulaspora and Brettanomyces (10 strains); and seven strains of the bacterium Leuconostoc oenos. Only one Saccharomyces strain exhibited β-glucosidase activity, but several non-Saccharomyces yeast species showed activity of this enzyme. Aureobasidium pullulans hydrolyzed α-L-arabinofuranoside, β-glucoside, and α-L-rhamnopyranoside. Eight Brettanomyces strains had β-glucosidase activity. Location of enzyme activity was determined for those species with enzymatic activity. The majority of β-glucosidase activity was located in the whole cell fraction, with smaller amounts found in permeabilized cells and released into the growth medium. Aureobasidium pullulans hydrolyzed glycosides found in grapes. Received 02 February 1999/ Accepted in revised form 26 June 1999     

Field energetics of free-living, lactating and non-lactating echidnas (Tachyglossus aculeatus)

We measured daily energy expenditure (DEE) and water turnover rates in lactating and non-lactating short beaked echidnas (Tachyglossus aculeatus) using the doubly labelled water technique during the lactation period in spring. Reproductively inactive echidnas were on average significantly heavier (median: 3354 g; range: 2929-3780 g; N=4) than lactating females (median: 2695 g; range: 2690-2715 g; N=3) during the equivalent time period. The median water flux rate of lactating echidnas (152 ml day(-1); range: 120-198 ml day(-1)) did not differ significantly from that of non-lactating females (170 ml day(-1); range: 128-227 ml day(-1)). The median DEE of echidnas that were lactating was 645 kJ day(-1) (range: 581-850 kJ day(-1)), which was not different from the median DEE of non-reproductive control females (763 kJ day(-1); range: 720-766 kJ day(-1)). Lactating females somehow compensate for the energy costs of milk production, resulting in a daily energy budget that is not different from that of non-reproductive females. At least part of their energy minimising strategy could involve the use of moderate heterothermy, allowing a greater proportion of daily energy expenditure to diverted to milk production.     

Mutations in the translated region of the lactase gene (LCT) underlie congenital lactase deficiency

Congenital lactase deficiency (CLD) is a severe gastrointestinal disorder characterized by watery diarrhea in infants fed with breast milk or other lactose-containing formulas. We initially assigned the CLD locus by linkage and linkage disequilibrium on 2q21 in 19 Finnish families. Here we report the molecular background of CLD via characterization of five distinct mutations in the coding region of the lactase (LCT) gene. Twenty-seven patients out of 32 (84%) were homozygous for a nonsense mutation, c.4170T-->A (Y1390X), designated Fin(major). Four rare mutations--two that result in a predicted frameshift and early truncation at S1666fsX1722 and S218fsX224 and two point mutations that result in substitutions Q268H and G1363S of the 1,927-aa polypeptide--confirmed the lactase mutations as causative for CLD. These findings facilitate genetic testing in clinical practice and enable genetic counseling for this severe disease. Further, our data demonstrate that, in contrast to common adult-type hypolactasia (lactose intolerance) caused by a variant of the regulatory element, the severe infancy form represents the outcome of mutations affecting the structure of the protein inactivating the enzyme.     

Heat tolerance of short-beaked echidnas (Tachyglossus aculeatus) in the field

(1) Echidnas occur throughout the hot arid zone of Australia yet laboratory studies have concluded that they are ill equipped physiologically to manage T_a higher than 35°C. (2) Consequently, it is generally assumed that echidnas must rely on behavioural thermoregulation, being nocturnal in hot weather and seeking less extreme microclimates during the day. (3) By monitoring T_b of echidnas in the field and relating these to T_a within their day time shelters in Western Queensland during summer, this study showed that echidnas are able to tolerate T_a of 35–40°C in hollow logs for up to 10 h. (4) Further, as T_b remains < T_a in these situations, echidnas may have physiological mechanisms for dealing with the heat after all.     

Enzymes of galactose metabolism in livers of suckling and adult tammar wallabies (Macropus eugenii) and other marsupials

The activities of galactokinase, hexose-1-phosphate uridylyl transferase and UDPglucose 4-epimerase in homogenates of livers of two adult and 20 suckling tammar wallabies aged from 6 to 50 weeks were investigated. The activities of all three enzymes were high until 24-30 weeks post partum, after which they declined to low levels. The activities of the three liver enzymes were high in pouch young of six other species of marsupial. Comparison of the activities of the three liver enzymes in suckling tammar wallabies with those in suckling rats showed no difference between the two species in regard to galactokinase and uridylyl transferase, but the UDPglucose 4-epimerase activity in tammar wallabies was approximately double than found in rats. This may be related to the high galactose content of tammar wallaby milk compared with rat milk. In suckling tammar wallabies, the liver had higher enzyme activities than other tissues studied. It is concluded that, contrary to the suggestion of Stephens et al. (1975), pouch young marsupials are not deficient in their ability to metabolize galactose.     

Screening for glycosidase activities of lactic acid bacteria as a biotechnological tool in oenology

The aim of this study was to evaluate the ability from a number of lactic acid bacteria isolated from different sources to produce glycosidase enzymes. Representative isolates (225) from clusters obtained after genotyping, using randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis, of 1,464 isolates, were screened for β-D-glucosidase activity. Thirty-five of them were selected for subsequent analysis. These strains were able to hydrolyze α-D-glucopyranoside, β-D-xylopyranoside and α-L-arabinofuranoside although β-D-glucosidase activity was the predominant activity for 22 of the selected strains. Only some of them did so with α-L-rhamnopyranoside. All of these were from wine samples and were identified as belonging to the Oenococcus oeni species using Amplification and Restriction Analysis of 16S-rRNA gene (16S-ARDRA). When the influence of pH, temperature and ethanol or sugars content on β-D-glucosidase activity was assayed, a strain-dependent response was observed. The β-D-glucosidase activity occurred in both whole and sonicated cells but not in the supernatants from cultures or obtained after cell sonication. Strains 10, 17, 21, and 23 retained the most β-D-glucosidase activity when they were assayed at the conditions of temperature, pH, ethanol and sugar content used in winemaking. These results suggest that these strains could be used as a source of glycosidase enzymes for use in winemaking.