Translational outcomes in a full gene deletion of ubiquitin protein ligase E3A rat model of Angelman syndrome.

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by developmental delay, impaired communication, motor deficits and ataxia, intellectual disabilities, microcephaly, and seizures. The genetic cause of AS is the loss of expression of UBE3A (ubiquitin protein ligase E6-AP) in the brain, typically due to a deletion of the maternal 15q11-q13 region. Previous studies have been performed using a mouse model with a deletion of a single exon of Ube3a. Since three splice variants of Ube3a exist, this has led to a lack of consistent reports and the theory that perhaps not all mouse studies were assessing the effects of an absence of all functional UBE3A. Herein, we report the generation and functional characterization of a novel model of Angelman syndrome by deleting the entire Ube3a gene in the rat. We validated that this resulted in the first comprehensive gene deletion rodent model. Ultrasonic vocalizations from newborn Ube3am-/p+ were reduced in the maternal inherited deletion group with no observable change in the Ube3am+/p- paternal transmission cohort. We also discovered Ube3am-/p+ exhibited delayed reflex development, motor deficits in rearing and fine motor skills, aberrant social communication, and impaired touchscreen learning and memory in young adults. These behavioral deficits were large in effect size and easily apparent in the larger rodent species. Low social communication was detected using a playback task that is unique to rats. Structural imaging illustrated decreased brain volume in Ube3am-/p+ and a variety of intriguing neuroanatomical phenotypes while Ube3am+/p- did not exhibit altered neuroanatomy. Our report identifies, for the first time, unique AS relevant functional phenotypes and anatomical markers as preclinical outcomes to test various strategies for gene and molecular therapies in AS.

Enhanced alveolar fluid clearance following 72 h of continuous isoproterenol infusion in rats.

AIM: We wished to determine the effect of continuous ß-receptor stimulation on alveolar fluid clearance and to elucidate the mechanisms behind this effect. METHODS: Alveolar fluid clearance was measured in anaesthetized rats pretreated for 72 h with the ß-agonist isoproterenol (200 µg kg(-1) h(-1) sc) or vehicle. Alveolar fluid clearance in artificially ventilated rats was determined over 1 h by infusion of isotonic Ringer solution containing (125) I-albumin into the lungs. Additionally, alveolar fluid clearance was determined when amiloride or l-cis-diltiazem was added to the solution to block ENaC and cyclic nucleotide-gated (CNG) channels respectively. RESULTS: Isoproterenol treatment induced a 42% increase in alveolar fluid clearance (18.9 ± 1.4%) vs. vehicle (13.3 ± 3.3%). Addition of amiloride resulted in a net decrease of 8% in both groups, while l-cis-diltiazem caused a net decrease of 12% in isoproterenol-treated animals, but only 5% in vehicle-treated animals. Western blotting showed that isoproterenol treatment increased the abundance of the α-ENaC and ß-ENaC subunits (223 ± 51% and 274 ± 55% of vehicle, respectively) but we saw no changes in protein level of the γ-EnaC subunit. CONCLUSION: Continuous ß-adrenoceptor stimulation with isoproterenol enhances alveolar fluid clearance through alternative pathways involving l-cis-diltiazem-sensitive channels.

Proteolytic activation of proteose peptone component 3 by release of a C-terminal peptide with antibacterial properties.

The milk protein proteose peptone component 3 (PP3, also known as lactophorin) is a small phosphoglycoprotein, which is exclusively expressed in the lactating mammary gland. A 23-residue synthetic peptide (lactophoricin, Lpcin S), corresponding to the C-terminal amphipathic α-helix of PP3, has previously been shown to permeabilize membranes and display antibacterial activity. Lactophorin readily undergoes proteolytic cleavage in milk and during dairy processing, and it has been suggested that PP3-derived peptides are part of milk's endogenous defense system against bacteria. Here, we report that a 26-residue C-terminal peptide (Lpcin P) can be generated by trypsin proteolysis of PP3 and that structural and functional studies of Lpcin P indicate that the peptide has antibacterial properties. The Lpcin P showed α-helical structure in both anionic and organic solvents, and the amount of α-helical structure was increased in the presence of lipid vesicles. Oriented circular dichroism showed that Lpcin P oriented parallel to the membrane surface. However, the peptide permeabilized calcein-containing vesicles efficiently. Lpcin P displayed antibacterial activity against Streptococcus thermophilus, but not against Staphylococcus aureus and Escherichia coli. The PP3 full-length protein did not display the same properties, which could indicate that PP3 functions as a precursor protein that upon proteolysis, releases a bioactive antibacterial peptide.

Characterization of human mucin (MUC15) and identification of ovine and caprine orthologs.

The glycoprotein MUC15 (mucin 15) was initially isolated from the bovine milk fat globule membrane. The present work demonstrates the existence of immunologically similar proteins ( approximately 130 kDa) in ovine, caprine, porcine, and buffalo milk samples. Purification and N-terminal amino acid sequencing confirmed the presence of ovine and caprine MUC15 orthologs in milk fat globule membranes. Expression of MUC15 in human milk was demonstrated by immunostaining ( approximately 150 kDa) as well as by mass spectrometry. Screening of a human multiple tissue expression array showed abundant MUC15 gene expression in placenta, salivary gland, thyroid gland, trachea, esophagus, kidney, testis, and the leukemia K-562 cell line. Furthermore, moderate expression was seen in the pancreas, adult and fetal lung, fetal kidney, lymph node, adult and fetal thymus, and parietal lobe. Structural motifs for interactions (epidermal growth factor receptor and Src homology 2 domains) are identified in the intracellular region. Implication of the mucin in signal transduction and the potential physiological function of MUC15 are discussed.

Characterization of carbohydrate structures of bovine MUC15 and distribution of the mucin in bovine milk.

The present work reports the characterization of carbohydrate structures and the distribution of the newly identified mucin MUC15, a highly glycosylated protein associated with the bovine milk fat globule membrane (MFGM). Distribution of MUC15 was investigated in various fractions of bovine milk by densitometric scanning of Western blots. In raw milk, MUC15 was shown to constitute 0.08% (wt) of the protein and approximately 1.5% (wt) of the MFGM-associated proteins. Surprisingly, this study showed that in addition to the fat-containing fractions, such as MFGM and buttermilk, MUC15 was present in nonfat-containing fractions as well, such as skim milk and whey. Compositional and structural studies of the carbohydrates of bovine milk MUC15 showed that the glycans are composed of fucose, galactose, mannose, N-acetylgalactosamine, N-acetylglycosamine, and sialic acid. The carbohydrate was shown to constitute 65% of the total molecular weight, and the molar ratios of the individual sugars to protein of the O-linked glycans were determined. The glycan structures of MUC15 were further studied by enzymatic deglycosylation experiments using different endo- and exoglycosidases as well as a panel of lectins. The N-linked glycans were shown to contain mainly hybrid-type N-glycans. In addition, the N-glycans were shown to be sialylated and contain terminal poly-lactosamine structures. The O-linked glycans were found to constitute some unsubstituted Core-1 structures and a substantial number of sialylated Core-1 O-linked glycans. By comparing the results of peanut agglutinin lectin binding, enzymatic deglycosylation, and monosaccharide composition analysis, we concluded that bovine MUC15 also contains more complex O-glycans containing high amounts N-acetylglucosamine residues. Furthermore, a small subset of the O-linked glycans is decorated with lactosamine on their terminal ends.

Microalbuminuria in type 2 diabetic patients: a prospective follow-up study.

We have studied 46 patients, 30 men and 16 women, with type 2 (non-insulin-dependent) diabetes in a follow-up period of 6-52 months (mean 30 months). The patients were consecutively entered in the study from the out-patient diabetic clinic. None had urinary tract infections nor proteinuria at entry. Investigations were done every 3 months during the first year and after that every 6 months. At entry 16 patients (35%) had microalbuminuria and a further 16 patients developed microalbuminuria and 16 proteinuria. The systolic blood pressure was higher in men with microalbuminuria compared to men without microalbuminuria. The glomerular filtration rate decreased with time for patients with microalbuminuria without change in plasma creatinine. The C-peptide concentration was higher in the hypertensive patients compared to non-hypertensive and the same was found for the triglyceride concentration. During the observation period the various complications increased in frequency (retinopathy, cardiomyopathy, neuropathy, angiopathy and hypertension) without significant relation to the presence of microalbuminuria or proteinuria. During the observation period nine patients died mainly due to cardiovascular events.

Microalbuminuria in type 2 diabetic patients: a cross-sectional study of frequency, sex distribution and relation to hypertension.

We studied 112 type 2 diabetic patients. Fourteen patients had frank proteinuria, and 37 of the remaining 98 had microalbuminuria which was more frequent in men than in women (P < 0.02). Hypertension was found in 47 of the patients, equally distributed between sexes. Male diabetics with microalbuminuria had higher systolic blood pressure than diabetics without microalbuminuria (P < 0.02). Body mass index was higher in both sexes with hypertension compared to patients without hypertension. In the hypertensive men plasma C-peptide values were higher compared to patients without hypertension (P < 0.01) irrespective of the presence of microalbuminuria. A positive correlation between blood pressure and C-peptide was found (P < 0.01) in the men. We suggest that gender should be taken into account in the analysis and interpretation of microalbuminuria in type 2 diabetes.

Collagen distribution in developing experimentally induced granulation tissue. A morphometric study.

Viscose cellulose sponges were implanted subcutaneously on the back of full-grown Sprague-Dawley rats. Seven, 14, 21, 28, 42, 60 and 90 days after implantation, groups of 12 animals decapitated and the sponges were removed and processed for light microscopy. Five microns sections were stained with Picro-Sirius Red. Morphometry was performed on the zone of ingrowth and the collagen. The intersectional variation in the morphometrically determined collagen density within the sponges was below 20%. The hydroxyproline content was determined biochemically in 5 microns sections of sponges implanted for 14, 42, 60 and 90 days. A positive correlation (rho = 0.79, p less than 0.0001) was observed between the biochemically and morphometrically determined collagen contents. The morphometric determinations showed a steady increase in the granulation tissue ingrowth. At day 60 the ingrowth was complete. There was an increasing collagen density from days 7 and 14 through days 21 and 28, followed by a nearly steady state up to day 90 and a significantly higher collagen density peripherally than centrally in the day 42 sponges. The study has shown that morphometric collagen determination at light microscopical level using Sirius Red-stained sections may add quantitative data describing the dynamic changes in collagen content and distribution within developing granulation tissue.

Gallbladder stones classified by chemical analysis of cholesterol content. Frederiksberg, 1987-1988.

Gallstones from 80 cholecystectomies and 81 autopsies were chemically analysed and showed a trend of decreasing cholesterol content with increasing age (p = 0.00009). The frequency of cholesterol stones (cholesterol content greater than 70%) was higher in operated women (81%) than in operated men (33%) (p = 0.0006) and in the total autopsy material (42%). The study supports the theory that the predominance of gallstone disease in women is an effect of the preponderance of cholesterol stones. The accuracy of estimates of cholesterol content of gallstones from the appearance of the cut surface was low. When the chemical analysis was used as a key, only one in three was correct. There was a tendency to underestimate cholesterol content.

Lipid metabolism and coagulation of two contraceptives: correlation to serum concentrations of levonorgestrel and gestodene.

The effects of two triphasic oral contraceptives containing the same amount of ethinylestradiol (EE) in combination with levonorgestrel (LNG) or gestodene (GES), respectively, on lipid metabolism and coagulation were studied. Serum concentrations of GES and LNG were determined at the same time. Thirty-three healthy women were randomized into two groups receiving either of the preparations. Before treatment and in the 3rd and the 6th cycle, blood samples were drawn in the morning while subjects were still in bed to obtain basal conditions. HDL2-cholesterol decreased in the LNG group but was unchanged in the GES group, whereas apolipoprotein A1 increased in the GES but not in the LNG group. Antithrombin III decreased in the GES group but was unchanged in the LNG-treated women. Factor VII increased in both groups, but more in the GES group. It is concluded that GES has a positive influence on lipid metabolism, and has a slightly negative influence on coagulation but the latter is more likely to be without clinical relevance. The positive influence of GES compared to LNG on lipids is probably due to its lower androgenicity and not to differences in bioavailability.

Serum aminoterminal type III procollagen peptide. Relation to biosynthesis of collagen type III in experimentally induced granulation tissue in rats.

Serum aminoterminal type III procollagen peptide was measured in rats during the development of granulation tissue induced by subcutaneous implantation of viscose cellulose sponges. Active collagen type III synthesis in granulation tissue during the first three weeks was accompanied by an increase in serum propeptide level. A positive correlation was observed between the increase in serum propeptide level on the one hand and the increase in granulation tissue collagen type III content and the in vitro formation of tissue 3H-hydroxyproline on the other hand. In some animals the serum propeptide level remained low, despite biochemical signs of collagen synthesis, indicating variations in the release into serum and/or the metabolism of circulating propeptide. The increase in propeptide antigen concentration was mainly due to an elevated content of material with molecular weight equal to or twice that of the propeptide. A minor fraction of the propeptide remained attached to the interstitial collagen fibres in the granulation tissue. The correlation between the serum propeptide level and the biosynthesis of collagen at the site of the focal fibroproliferative process suggests that the serum propeptide level may be a valuable indicator of fibrogenesis and thereby of disease activity in fibrotic conditions.

Collagen type IV and procollagen type III during granulation tissue formation: a serological, biochemical, immunohistochemical and morphometrical study on the viscose cellulose sponge rat model.

The serum concentrations of collagen type IV,7S, collagen type IV,nc1, and aminoterminal type III procollagen peptide immunoreactive components were measured by means of specific radioimmunoassays during development of granulation tissue in rats. The results were compared with tissue deposition of basement membranes and interstitial collagens in the granulation as measured morphometrically. A parallel sequential pattern in tissue deposition of collagen types III and IV, and serum increase of collagen types III- and IV-related fragments, was observed. Serum collagen type IV was less sensitive as a marker for development of granulation tissue than the serum procollagen type III N-peptide. This was in accordance with a low collagen type IV/interstitial collagen ratio in the granulation tissue. However, a cross-sectional study showed that serum collagens types IV,7S and IV,nc1 may be useful as early quantitative indicators of granulation tissue formation. Simultaneously, measurement of collagen type IV- and procollagen type III N-peptide-related antigens in serum provides a differentiated reflection of the dynamic matrix processes in developing granulation tissue.

Serum lipids in hyper- and hypothyroidism before and after treatment.

Total cholesterol (TC), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), triglycerides (TG) and apoprotein A (Apo A) were determined in serum from 21 hyperthyroid patients and 11 hypothyroid patients before and after treatment to euthyroid state. The above-mentioned components were also determined in a reference population. In the hyperthyroid group the concentration of total cholesterol and low density lipoprotein-cholesterol increased significantly upon treatment. High density lipoprotein also increased except for 9 patients also receiving propranolol. The ratio between high density lipoprotein-cholesterol and total cholesterol decreased in the patients receiving propranolol. In the hypothyroid group the values after treatment decreased significantly for cholesterol, high density lipoprotein-cholesterol and low density lipoprotein-cholesterol. The ratio between high density lipoprotein-cholesterol and total cholesterol was unchanged.

Receptor-mediated endocytosis of a ricin-colloidal gold conjugate in vero cells. Intracellular routing to vacuolar and tubulo-vesicular portions of the endosomal system.

We have prepared a conjugate (Ri-Au) of the toxic plant protein ricin and colloidal gold (particle size 5 nm) and used it for internalization studies in monolayer cultures of Vero cells. The Ri-Au conjugate was very stable, with only little release of ricin ([125I]Ri) from the gold particles within a pH range of 4.5-8.0. Within 2 h at 37 degrees C, only very little intracellular degradation of the ricin preparation ([125I]Ri-Au) occurred. The cells bound the same proportion of native ricin ([125I]Ri) and Ri-Au from the medium, and the kinetics of toxicity (decrease in cellular incorporation of [3H]leucine) of [125I]Ri and [125I]Ri-Au were also comparable. At 4 degrees C, the cell-surface binding of Ri-Au was continuous and distinct, as revealed by electron microscopy. This binding was specific, since almost no Ri-Au surface binding occurred at 4 degrees C in the presence of 0.1 M lactose or 1 mg/ml native (unlabelled) ricin. Within the first 30 min of warming prelabelled cells to 37 degrees C, the amount of surface-associated Ri-Au decreased considerably (from 150 to 60 gold particles per micron cell surface in 40 nm sections). Coated pits and vesicles were involved in the internalization of Ri-Au, and within 5-30 min at 37 degrees C Ri-Au had been delivered to vacuolar and tubulo-vesicular portions of the endosomal system, and later also to lysosomes. Analysis of very thin (ca 20 nm) serial sections revealed that most of the tubulo-vesicular elements were separate structures not connected to the membrane of the vacuolar portion. Data here presented indicate that our ricin conjugate, like many "physiological' ligands and viruses, is internalized by receptor-mediated endocytosis via the coated pit-endosomal pathway.

Cholesterol saturation of bile after jejunoileal bypass increases more with a 1:3 than with a 3:1 jejunoileal ratio.

Previous suggested, surprisingly, that after jejunoileal bypass surgery the lithogenicity of the bile, assessed by its cholesterol saturation and rate of gallstone formation, increases more with a 1:3 than with a 3:1 jejunoileal ratio of the functioning segment. The present study re-evaluates this by examining fasting bile samples drawn from duodenum after cholecystokinin stimulation in 34 obese patients without gallstones, who either were waiting for or had had bypass surgery, with a 1:3 or 3:1 jejunoileal ratio, 3,9, or 15 months earlier. In all groups, the cholesterol content exceeded the solubilizing capacity of the bile as determined on the basis of total lipid concentration and content of phospholipid relative to bile acids. The cholesterol supersaturation increased with bypass surgery as such, increased more with a 1:3 than with a 3:1 jejunoileal ratio, decreased with time after surgery, and reached the preoperative level at 15 months. Assuming a total lipid concentration of 10 g/dl in the bile did not change this pattern. Our results indicate that during the period of weight loss after bypass surgery the lithogenicity of gallbladder bile increases more with a 1:3 than with a 3:1 jejunoileal ratio.

Determination of total 3 alpha-hydroxy bile acids in serum.

The usual techniques for determination to total 3 alpha-hydroxy bile acids in serum involving liquid-solid extraction of the bile acids with the adsorbent XAD-2 and fluorimetric measurement of NADH generated from the reaction with a NAD-linked 3 alpha-hydroxysteroid dehydrogenase are evaluated and improved. The influence of different types of enzyme preparations on the results is examined. The results with the improved technique are compared to the results obtained with another method, avoiding extraction of the bile acids before the enzymatic reaction which is followed by fluorimetric measurement of resorufin, produced by transfer of the hydrogen of the generated NADH by diaphorase to resazurin. No significant difference between the results with the two types of methods was found. The concentration of total 3 alpha-hydroxy bile acids in serum of 46 fasting 'healthy' individuals aged 17 to 82 years is estimated. 30 were females, of whom 10 were taking estrogen-containing oral contraceptives, and 16 were males. Mean +/- standard deviation in all the females was 3.0 +/- 1.1 micromol/l, and in the males 4.0 +/- 1.9 micromol/l. There was no significant difference between any of the groups.

Criteria for selection of patients for medical treatment (chenodeoxycholic acid therapy) of gallstones.

With the introduction of medical treatment (chenodeoxycholic acid therapy) of cholesterol gallstones, the prediction of the gallstone type, cholesterol--non-cholesterol stones, (i.e. cholesterol predominating or not), has become important. In 24 consecutive patients admitted for surgery because of gallstones, the value of various criteria for differentiation between the two types of stones was assessed. It is concluded that the combined requirements of radiolucency of the stones and a cholesterol saturation index in duodenal bile above 1.00 constitutes a fairly reliable method for selection of patients for dissolution therapy with chenodeoxycholic acid.