Developmental changes in tibia and humerus of goose: morphometric, densitometric, and mechanical analysis.

The skeletal system of young animals undergoes a series of intensive and rapid changes. In this study, we aimed to verify the hypothesis that geese exhibit a distinct pattern of bone growth compared to gallinaceous species. Specifically, we hypothesised that geese would experience an accelerated growth rate in the humerus bone, which can be attributed to the increased wing mobility facilitated by their rearing in free-range systems. This need for access to both ground and water environments contributes to the unique demands placed on their skeletal development. We focused on evaluating the mechanical properties and geometry of the humerus as the forelimb bone, and the tibia as the hindlimb bone. The 320 geese used in this study were divided into 12 groups according to sex (females and males) and age (0-,1-,3-,6-,8-,12-,14-week-old). To assess bone mechanical properties, a three-point bending test was performed, along with densitometry and morphological measurements. The tibiae of the geese showed the most intensive growth until 6 weeks of age and then stabilised. The wing bones (humerus) showed only slight changes in the first weeks after hatching, and then a rapid growth between the third and sixth week, both in terms of mechanical and morphological properties. This is most likely due to a change in the geese's living environment during this period, i.e., allowing them to leave their enclosures and enter open space, which gives them the opportunity to use their wings, resulting in the rapid growth of the wing bones to support increasing muscle mass in this area. This study increases our understanding of bone growth and development in domesticated birds, specifically waterfowl species, and highlights the importance of rearing methods on the proper bone development and functionality of the entire skeletal system, and thus, on their welfare.

Prenatal acrylamide exposure results in time-dependent changes in liver function and basal hematological, and oxidative parameters in weaned Wistar rats.

Acrylamide (ACR) is a toxic compound commonly found in fried, baked and heat-processed starchy foods. The current study investigated the time-dependent effects of maternal exposure to non-toxic ACR doses on the oxidative stress, liver function, and basal blood morphology of the rat offspring. Pregnant, Wistar rats were randomly divided into the control group or the groups administrated with ACR (3 mg/kg b.w./day): long exposure for 15 days, medium exposure for 10 days and short exposure for 5 days during pregnancy. Body mass, blood morphology and hematology, serum concentrations of growth hormone, IGF-1, TNF-α, IL-1ß, IL-6 and insulin, liver histomorphometry, liver activity of beclin1, LC2B and caspase3, markers of oxidative stress and the activity of antioxidative enzymes in blood serum and the liver were measured in offspring at weaning (postnatal day 21). Even short prenatal exposure to ACR led to oxidative stress and resulted in changes in liver histomorphometry and upregulation of autophagy/apoptosis. However, the most significant changes were observed following the long period of ACR exposure. This study has shown for the first time that ACR is responsible for changes in body mass in a time-dependent manner, which could lead to more serious illnesses like overweight and diabetes later in life.

The Canadian Neuromuscular Disease Registry 2010-2019: A Decade of Facilitating Clinical Research Througha Nationwide, Pan-NeuromuscularDisease Registry.

We report the recruitment activities and outcomes of a multi-disease neuromuscular patient registry in Canada. The Canadian Neuromuscular Disease Registry (CNDR) registers individuals across Canada with a confirmed diagnosis of a neuromuscular disease. Diagnosis and contact information are collected across all diseases and detailed prospective data is collected for 5 specific diseases: Amyotrophic Lateral Sclerosis (ALS), Duchenne Muscular Dystrophy (DMD), Myotonic Dystrophy (DM), Limb Girdle Muscular Dystrophy (LGMD), and Spinal Muscular Atrophy (SMA). Since 2010, the CNDR has registered 4306 patients (1154 pediatric and 3148 adult) with 91 different neuromuscular diagnoses and has facilitated 125 projects (73 academic, 3 not-for-profit, 3 government, and 46 commercial) using registry data. In conclusion, the CNDR is an effective and productive pan-neuromuscular registry that has successfully facilitated a substantial number of studies over the past 10 years.

Histomorphometrical changes in intestine structure and innervation following experimental fumonisins intoxication in male Wistar rats.

Fumonisins are highly toxic metabolites produced by Fusarium proliferatum and Fusarium verticillioides. Little is known about the effects of a chronic low level of fumonisins on intestinal structure and innervation in monogastric animals, even though the intestine is the first organ exposed to fumonisins. The influence of the most prevalent strains of fumonisins, FB1 and FB2, on intestinal and liver morphology, the enteric nervous system and intestinal epithelial cell prolif- eration was investigated in an experimental rat model of fumonisin intoxication. Adolescent (5-weeks-old), male Wistar rats were randomly divided into a control group (C group) not treated with fumonisins or intoxicated with fumonisins (FB group). FB1 together with FB2 were daily administered intragastrically at a dose of 90 mg/kg body weight for 21 days. The damaging effect was assessed by determination of the activity of ALAT and AspAT. Samples from the small intes- tine and liver were taken and blood samples were collected to determine the activity of gamma- -glutamyl transferase (GGT) and amylase. The exposure to FBs resulted in histopathological degenerative alterations in hepatocytes, including mild vacuolar degeneration and ballooning. FB exposure was also toxic in the duodenum and jejunum, where significant changes in morphology, cell proliferation, collagen wall fibres and innervation were observed. Taken together, the results obtained strengthen the hypothesis that chronic exposure to FBs could induce intestinal damage, including damage to the enteric nervous system and may have consequences for general health.

Analysis of mechanical properties of bones and tendons shows that modern hybrid rye can be introduced to corn-wheat based diet in broiler chickens as an alternative energy source irrespective of xylanase supplementation.

This study focused on analyzing the effects of inclusion of modern hybrid rye to corn-wheat diet on mechanical properties of bones and tendons. A total of 224 broiler chickens were fed a diet without rye inclusion or a diet containing 15% of hybrid rye cv. Brasetto. The diets were either unsupplemented or supplemented with xylanase (minimum activity 1000 FXU/g, dose 200 mg/kg of feed). Each dietary group consisted of 56 birds. On day 42, selected chickens (n = 7 from each group) were slaughtered. Tibia were analyzed for mineralization, geometry, and biomechanical characteristics of bone mid-diaphysis. The mechanical properties of digital flexor III tendon were also assessed. Bone mineral density and bone ash percentage did not differ when both diets were given without xylanase. Enzyme supplementation increased bone mineral density (P < 0.01) in both dietary groups, whereas bone ash percentage (P < 0.01) increased only for corn-wheat diet. Rye inclusion had no effect on bone mid-shaft geometrical traits related to tibia weight-bearing capacity (cross-sectional area, cortical index, and mean relative wall thickness). Performed bending test showed no effect of hybrid rye inclusion on bone mechanical endurance. When xylanase was supplemented, bone length (P < 0.01) and weight (P < 0.05) decreased, whereas yield load (P < 0.01), stiffness (P < 0.05), Young modulus (P < 0.05), elastics stress (P < 0.01), and ultimate stress (P < 0.01) increased, irrespective of rye presence. The tendon tensile strain test showed that in corn-wheat diet enzyme supplementation positively influenced rupture force (P < 0.05) and tendon stiffness (P < 0.01). Xylanase supplementation increased the value of energy required to tendon rupture, irrespective of rye inclusion (P < 0.05). Study showed that modern hybrid rye varieties can be introduced to corn-wheat diets of broiler chickens in the aspect of animal welfare related to the development and homeostasis of musculoskeletal system, irrespective of xylanase supplementation. The enzyme addition positively affected biomechanical properties of bones and tendons.

Structural changes in the small intestine of female turkeys receiving a probiotic preparation are dose and region dependent.

Gut microbiota have been shown to play a critical role in the maintenance of host health. Probiotics, which regulate gut microbiota balance, could serve as an effective alternative to antibiotic growth promoters. Since changes in the gastrointestinal tract, caused by a variety of different strains, groups and amounts of microorganisms, may be reflected in its histological structure, the aim of the present study was to examine the effects of rising doses of a mixed probiotic preparation on the structure and development of the small intestine of female turkeys. Eighty, three-day-old, healthy, female turkeys (Big-6 breed) were used in the current (16-week) study. The turkeys were randomly allocated to four weight-matched (59.70 ± 0.83 g) groups (n = 20), according to probiotic treatment dose (0, 107 cfu•g-1, 108 cfu•g-1 or 109 cfu•g-1, in 500 g•1000 kg-1) (cfu - a colony-forming unit). Three, non-genetically modified strains of probiotic cultures obtained from poultry, four bacterial and one yeast culture, were used. Histomorphometric analysis of the structure of the small intestinal wall of the duodenum and jejunum was performed. All probiotic doses used in the current study exerted a beneficial effect on the histological structure of the small intestine; however, the observed effect was dose and region dependent. Significant increases in villi height, crypt depth, villi and crypt width, mucosa thickness, epithelial height, enterocyte number, absorption surface and intestinal ganglia geometric indices were observed, specifically in the duodenum of birds receiving an intermediate dose of probiotic (108 cfu•g-1). The probiotic doses used in the current study differed significantly in their effect on the small intestine (P < 0.01), with the intermediate dose (108 cfu•g-1) significantly improving 58% of the parameters assessed, compared to the control. The duodenum was more susceptible to the favourable effects of the probiotic than the jejunum (56% v. 31% improvement in the parameters assessed) (P < 0.01). The weakest favourable effect was observed in the group that received the highest dose of probiotic.

Intestinal mucosa develops in a sex-dependent manner in Japanese quail (Coturnix japonica) fed Saccharomyces cerevisiae.

1. The aim of study was to investigate whether the impact of the yeast Saccharomyces cerevisiae on the histological structure of the intestine, innervation of the small intestine wall, and basal biochemical serum parameters in Japanese quail was sex dependent. 2. One-day-old healthy male and female Japanese quail were fed either a basal diet containing no yeast (control group) or the basal diet plus 1.5% (15 g/kg of diet) of yeast (S. cerevisiae inactivated by drying). Samples from the duodenum and jejunum were taken from each bird at the age of 42 days. Blood samples were collected at this age and the concentrations of glucose, total protein, creatinine, uric acid, lipid profile (total cholesterol, low density lipoproteins (LDL), high density lipoproteins (HDL) and triacylglycerols (TG)), alanine aminotransferase (ALAT), aspartate aminotransferase (AspAT), lactate dehydrogenase (LDH), amylase (AMY), calcium, phosphorus and iron were determined. 3. Female quail fed diets supplemented with yeast had significantly lower total cholesterol and amylase activity than the control females. The concentration of HDL was higher in the male quail than in the females, irrespective of the treatment. An opposite effect was observed in LDL. The diet treatments influenced the activity of AspAT, which was significantly less in the male quail fed diets with 1.5% yeast. 4. Supplementation with S. cerevisiae increased the myenteron, submucosa and mucosa thickness, villus length and thickness and size of absorptive surface, while the number of villi and enterocytes were decreased in the duodenum in males. Female quail showed an increased absorptive surface in the jejunum. The Meissner (submucosal) plexuses were influenced by the feeding and sex to a greater extent than the Auerbach plexus (in the muscularis propria). 5. The results demonstrated that S. cerevisiae (1.5%) in the diet caused significant positive effects in Japanese quail, exerting an effect on the morphology of the small intestine in a sex-dependent manner.

Effects of maternal treatment with ß-hydroxy-ß-metylbutyrate and 2-oxoglutaric acid on femur development in offspring of minks of the standard dark brown type.

The aim of the study was to evaluate the effect of the diet, mother type and sex of the offspring on the mechanical and geometric parameters of long bones as well as bone tissue density in minks. Primiparous and multiparous dams were supplemented with ß-hydroxy ß-methylbutyrate (a metabolite of leucine, at the daily dosage of 0.02 g/kg of body weight) and/or 2-oxoglutaric acid (a precursor of glutamine, at the daily dosage of 0.4 g/kg of body weight) during gestation. The diet did not influence bone tissue density and the length of the humerus. An increase in the length of the femur was noted in male offspring delivered by multiparous dams. The diet resulted in an increase in the weight of the humerus in males from multiparous dams and a decrease in offspring from primiparous dams. Heavier femora were noted in male offspring delivered by both types of dams. The maximum elastic strength of the humerus was higher in the offspring delivered by multiparous than primiparous dams, irrespective of the offspring sex. The diet resulted in reduction in the ultimate strength of the femur in the male offspring delivered by primiparous dams. Only females born by multiparous dams, irrespective of the diet, showed a significant increase in the cross-sectional area of the humerus, while a significant decline was noted in males delivered by multiparous dams and in all the offspring delivered by primiparous dams. An increase in the cross-sectional area of the femur was noted in the offspring delivered by multiparous dams, while reduction was observed in the offspring delivered by primiparous dams. These results have shown for the first time that the presence of ß-hydroxy-ß-methylbutyrate or 2-oxoglutaric acid in the diet of pregnant primiparous or multiparous dams unambiguously affects the geometry and mechanical properties of offspring's long bones.

Effects of replacing soybean meal with chickpea seeds in the diet on mechanical and thermal properties of tendon tissue in broiler chicken.

The efficiency of the musculoskeletal system of broiler chickens, in particular during locomotion and in ensuring its supportive function, depends directly on the adequate function and mechanical endurance of soft tissues, including tendons. However, little is known whether the properties of musculoskeletal soft tissues can be influenced by changes of dietary protein. We substituted soybean meal with raw chickpea seeds as the primary protein source in the diet and studied the effects it had on the mechanical and thermal properties of drumstick tendons in broiler Ross 308 chickens. In the experiment, 160 chicks were divided into 2 groups, receiving in their diet either soybean meal (n = 80) or chickpea seeds (n = 80). The experiment lasted 42 days. The physical condition of the drumstick tendons was analyzed on the basis of a tensile test and the results of thermal denaturation as measured by a differential scanning calorimetry. The mechanical evaluation of tendon tensile strength of the broilers fed with chickpea seeds demonstrated an increase in the ultimate strain (for over 22%, P < 0.04) and total energy absorbed by the tendon until rupture (for over 57%, P < 0.05) as when compared to the group fed with soybean meal. Thermal analysis demonstrated alterations in tendon collagen cross-linking as transition onset temperature decreased (from 63.8 to 61.8°C, P < 0.001), whereas the calorimetric enthalpy increased (from 16.2 to 22.1 J⋅g-1, P < 0.05) in the group fed with chickpea seeds. In summary, this study demonstrated that dietary protein source can impact the physical properties of tendons and showed that thermal analysis can be a useful tool for studying the effect of nutrition on the development and structural changes in tendons of broiler chickens.

Long-bone properties and development are affected by caponisation and breed in Polish fowls.

1. The aim of the study was to investigate the effects of caponisation on bone development of males of two native breeds in Poland. 2. The weight, length and cross-sectional area of tibiae and femora were measured, densitometric measurements and tests of strength were determined and dimensions were calculated. 3. Breed and caponisation did not influence bone weight and length. Higher mechanical strength of the femur was found in entire males, mainly in the Polbar breed. Tibial strength was reduced in capons of the Green Partridge breed. Maximum elastic strength was greater in the Polbar, irrespective of caponisation. Bone cross-sectional area was influenced by breed, while caponisation reduced femoral bone mineral density in both breeds. 4. Caponisation thus increased growth rate but had adverse effects on bone development. 5. Caponisation had fewer negative effects in the Polbar than in the Greenleg Partridge.

The influence of dexamethasone administered prenatally on cartilage of newborn spiny mouse (Acomys cahirinus) offspring.

Considering the negative effects of glucocorticoid treatment, especially during fetal development it is important to investigate effectors decreasing such disadvantages. The aim of this study was to investigate the effect of prenatally administered dexamethasone (Dex), a synthetic glucocorticoid, on the histomorphometry of the femur in the offspring of spiny mice. The study was performed on 24 pregnant spiny mice. The time of the experiment included the prenatal period between the 20th day of gestation until birth (pregnancy lasts on average of 36-38 days). The mice from the experimental group received dexamethasone per os in a dose of 125 mg/kg birth weight daily. At the end, the newborns from the experimental and control group were weighted and euthanized. Maternal Dex treatment resulted in a 17% decrease in birth weight in newborns. Dex administration significantly reduced the thickness of the hypertrophy zone of the growth plate by 34% and total thickness by 8,7%. In addition, Dex decreased the number of cells in the articular cartilage by 27% and significantly decreased their diameter by 5%. Dex also affected the structure and spatial distribution of thick and thin collagen fibers, lowering the proportion of thin fibers compared with the control group. Moreover, Dex treatment considerably lowered the amount of proteoglycans in articular and growth cartilages. Exposure to glucocorticoids in pregnant spiny mice affects cartilage development by accelerating maturity of collagen fibers and growth plate, presumably along with further disruption of longitudinal growth of long bones.

Acrylamide-induced prenatal programming of intestine structure in guinea pig.

Potential effects of prenatal administration of acrylamide (ACR) on postnatal development of the small intestine were not examined experimentally yet. The aim of this study was to establish changes of morphological parameters of the small intestine damaged by prenatal action of ACR in guinea pigs. The 3 mg/kg body weight of ACR was given in drinking water every day during the last 35 days of the pregnancy in guinea pigs. The histomorphometry of the duodenum and jejunum was determined. Immunohistochemical staining with anti cadherin antibody was performed. Maternal treatment with ACR led to the decrease of the expression of cadherin in the epithelium. Maternal ACR treatment increased the number of total, divided and inactive crypt, and the number of damaged villi in the duodenum and jejunum of newborn guinea pigs. The thickness of myenteron and submucosa, mucosa fractal dimension and the depth of crypts in the duodenum were increased by ACR. Additionally, in offspring born by mothers administered with ACR the decrease of villi epithelium thickness and active crypt number was observed. Moreover, ACR decreased goblet cells and inact villi number in the duodenum, mucosa thickness and crypts width in the jejunum. Intestine absorptive surface was affected by ACR in the jejunum as well. Results of measurements showed that maternal ACR treatment had negative influence on small intestine histomorphometry. ACR acting prenatally influenced small intestine nervous plexuses that became enlarged by 2.5 times compared with the control group. In conclusion, our results showed the negative impact of maternal ACR treatment on histological structure, integrity and innervation of small intestine wall as well as on absorptive function of small intestine mucosa.

Morphological changes of the cartilage and bone in newborn piglets evoked by experimentally induced glucocorticoid excess during pregnancy.

The study examined articular and growth plate cartilages as well as bone tissues in the offspring of sows treated with glucocorticoid during the last 45 days of pregnancy (dexamethasone at the dose of 0.03 mg/kg body weight intramuscularly, every second day). The offspring were tested at the birth and basal morphology for both articular and growth plate cartilages, and the histomorphometry of trabeculae of the epiphysis and metaphysis of femur and tibia were established. The concentration of selected cytokines and the activity of bone alkaline phosphatase were determined in blood serum. Maternal dexamethasone (DEX) administration reduced the thickness of proliferative, resting and hypertrophic zones of growth plate of femur and tibia of male piglets when compared with the control. DEX significantly reduced the thickness of the resting zone in both bones. It also elongated proliferative and hypertrophic zones of the growth plate in the femur as well as the hypertrophic zone in the tibia of female piglets when compared with the control group. Moreover, DEX decreased the articular cartilage thickness of the tibia in female piglets and enhanced the articular cartilage thickness of the femur in male piglets. Articular cartilage was highly cellular, and chondrocytes were separated by thin septa of matrix. An analysis of the trabecular bone architecture in male piglets showed a loss of the trabecular bone by thinning and DEX-related increase in trabecular porosity. Moreover, the cortical bone looked similar to the trabeculae because of trabecularization of the cortex. There was a DEX that reduced serum osteocalcin and BAP concentrations in both female and male newborn piglets, whereas the serum IL-1 and Il-6 was reduced only in male piglets. The obtained results demonstrated that DEX administration to sows during the last 45 days of pregnancy might cause the growth to slow and eventually to stop, especially in male piglets. It might lead to an alteration within the cartilage during its normal function, and with the time, arthritic changes can follow.

Postnatal administration of 2-oxoglutaric acid improves articular and growth plate cartilages and bone tissue morphology in pigs prenatally treated with dexamethasone.

The potential effects of prenatal administration of dexamethasone (DEX) and postnatal treatment with 2-oxoglutaric acid (2-Ox) on postnatal development of connective tissue of farm animals were not examined experimentally. The aim of this study was to establish changes in morphological parameters of bone and articular and growth plate cartilages damaged by the prenatal action of DEX in piglets supplemented with 2-Ox. The 3 mg of DEX was administered by intramuscular route every second day from day 70 of pregnancy to parturition and then piglets were supplemented with 2-Ox during 35 days of postnatal life (0.4 g/kg body weight). The mechanical properties, BMD and BMC of bones, and histomorphometry of articular and growth plate cartilages were determined. Maternal treatment with DEX decreased the weight by 48%, BMD by 50% and BMC by 61% of the tibia in male piglets while such action of DEX in female piglets was not observed. DEX led to thinning of articular and growth plate cartilages and trabeculae thickness and reduced the serum GH concentration in male piglets. The administration of 2-Ox prevented the reduction of trabeculae thickness, the width of articular and growth plate cartilages in male piglets connected with higher growth hormone concentration compared with non-supplemented male piglets. The result showed that the presence of 2-Ox in the diet had a positive effect on the development of connective tissue in pigs during suckling and induced a complete recovery from bone and cartilage damage caused by prenatal DEX action.

Bone development of suckling piglets after prenatal, neonatal or perinatal treatment with dexamethasone.

In mammals, the release from growth-inhibiting conditions results in catch-up growth. To investigate animal evidence for whether prenatal dexamethasone (DEX) treatment leads to the development of growth restriction especially reduced mineralization of skeleton, and release from it leads to the phenomenon of catch-up, piglets were prenatally exposed to DEX (3.0 mg/sow per day(-2)) during the last 24 days of prenatal life and tested further in two different ways: discontinued at birth and continued administration of DEX (0.5 mg/kg day(-2)) to piglets through 30 days of neonatal life. Using dual energy X-ray absorptiometry methods, bone mineral density (BMD) and bone mineral content (BMC) were measured. The three-point bending test was applied to determine the mechanical properties of the bones. Furthermore, geometric properties of the bones were assessed. Serum concentration of osteocalcin (OC) was determined. Histomorphological analysis of the ribs was also performed. The consequences of neonate DEX treatment and in utero DEX exposure were reflected in a dramatic decrease of BMD, BMC and blood serum OC concentration and geometric parameters of piglets' bones. Prenatal action of DEX during the last 24 days of pregnancy resulted in continued neonatal modification of bone tissues, thus diminishing bone quality, and negatively influenced structural development and mechanical properties, finally increasing the risk of fractures of ribs and limb bones. Prenatal DEX treatment limited to the last 24 days of foetal life did not reduce the term birth weight and the growth of suckling piglets followed up to 30 days of neonatal life, and catch-up in bone mineralization did not occur.

Alpha-ketoglutarate protects the liver of piglets exposed during prenatal life to chronic excess of dexamethasone from metabolic and structural changes.

Glucocorticoids play a role in the origin of the features of the metabolic diseases. Alpha-ketoglutarate (AKG) is defined as glutamine homologue and derivative, conditionally an essential amino acid. In the liver, glutamine serves as a precursor for ureagenesis, gluconeogenesis and acute phase protein synthesis The aim of the study was to determine the effect of AKG administered to piglets prenatally exposed to dexamethasone, on the structure of the liver and its metabolic function. Sows were administered with dexamethasone (3 mg/sow/48 h) from day 70 of pregnancy to the parturition, and then after the birth, the piglets were divided into the group administered with AKG (0.4 g/kg body weight) or physiological saline. Biochemical markers, lysozyme and ceruloplasmin serum activities, concentrations of selected free amino acids, macro- and microelements and histomorphometry of the liver tissue were determined. The total cholesterol concentrations in the sows and their newborns from the Dex groups were higher by 72% and 64%, respectively, compared with the control groups. Triacylglycerol concentration was higher by 50% in sows from the Dex group and 55% in the new-born piglets. Alpha-ketoglutarate administered to the piglets after prenatal influence of dexamethasone lowered the total cholesterol concentration by 40%, and enhanced aspartate by 41%, serine by 76%, glutamate by 105%, glutamine by 36%, glycine by 53% and arginine by 105%, as well as methionine and cystathionine, but increased the sulphur concentration compared with the control (p < 0.01). Intracellular space D decreased after AKG administration in comparison with the piglets from Dex/Control group not treated with AKG. Postnatal administration of AKG had a protective effect on liver structure, and lowered the total cholesterol concentration in piglets prenatally exposed to dexamethasone, and also influenced selected macro- and microelement serum concentrations and amino acids plasma concentration.

Combination of enzyme analysis, allele-specific PCR and sequencing to detect mutations in the GALT gene.

Newborn screening can identify patients with classical galactosaemia, and their diagnosis needs to be confirmed with assay of the activity of galactose-1-phosphate uridyltransferase (GALT). Unfortunately, in many cases the results can be ambiguous and further testing is required. Here we report a combination of biochemical analysis of GALT enzyme activity and mutation analysis of the most common mutations in the corresponding gene. Samples (n = 243) submitted for confirmatory testing for classical galactosaemia were analysed simultaneously for GALT enzyme activity and allele-specific PCR/fragment analysis for seven mutations and two polymorphisms in the GALT gene (mutations IVS2-2A>G, p.S135L, p.T138M, p.L195P, p.K285N, p.Q188R, p.Y209C; polymorphisms p.N314D, p.L218L). Mutation detection accorded with biochemical analysis in 93% of samples. Subsequently, a total of 34 samples with either discordant results between the above methods or low enzyme activity were fully sequenced, identifying previously reported pathogenic mutations and seven novel variations (p.P185H, p.R201C, p.E220K, p.R223S, p.I278N, p.L289F and p.L218X) in the GALT gene. This approach further increased concordance between genetic and biochemical analysis to 99% of all alleles tested. Our results indicate that DNA testing can help to verify biochemical enzymatic data and improve distinction of borderline enzyme activities where a patient may still benefit from treatment.

Controlled secretion into the culture medium of a hybrid beta-glucanase by Acetobacter methanolicus mediated by the kil gene of Escherichia coli located on a Tn5-derived transposon.

A Tn5-based transposon bearing the kil gene (killing protein), mediating controlled export of periplasmic proteins into the culture medium, was constructed (Tn5-KIL3). This transposon contained the kil gene of the ColEl plasmid under the growth-phase-dependent promoter of the fic gene (filamentation induced by cAMP) of Escherichia coli, an interposon located upstream of kil, a kanamycin/neomycin-resistance gene, a multiple cloning site and the mob site. The transposition of Tn5-KIL3 to Acetobacter methanolicus showed a moderate transposition frequency (10(-5) -10(-6). By insertion of a Bacillus hybrid beta-glucanase (bgl) as a model protein into the transposon (Tn5-LF3) it was shown that the secretion function as well as the gene of the target protein had been transferred to and stably integrated into the chromosome of A. methanolicus, and that the transposition of Tn5-LF3 was non-specific. beta-Glucanase was highly overexpressed and secreted into the medium during stationary phase. Total and extra-cellular production of beta-glucanase varied depending on the integration site of the transposon. The viability of the bacterial cells was not affected, and cell lysis did not occur.

The kil gene of the ColE1 plasmid of Escherichia coli controlled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase.

Heterologous gene products produced by Escherichia coli cells can be exported into the culture medium by the action of the kil gene of the ColE1 plasmid, which encodes a bacterial release protein. The kil gene was fused with the stationary-phase promoter of the fic gene of E. coli, and a secretion cassette (Kil-Km cassette) containing the regulated kil gene, the Km-resistance gene, and multiple cloning sites for the integration of target genes was constructed. Using the gene for beta-glucanase (bgl) as a target gene, it was shown that the protein produced was only secreted into the medium during the stationary phase. Quasi-lysis and lethality were not observed. The primary effect of the induction of the kil gene was the overproduction of beta-glucanase. The total amount produced per milliliter of bacterial culture was almost threefold higher than that of the corresponding Kil- control. The protein pattern of periplasm and culture medium was analyzed before and after induction of the kil gene expression, indicating that the release of periplasmic proteins is semiselective. This secretion system is the first to use a growth-phase-regulated promoter for the expression of the kil gene.