Effect of temperature on the saccharide composition obtained after alpha-amylolysis of starch

The hydrolysis of starch to low-molecular-weight products (normally characterised by their dextrose equivalent (DE), which is directly related to the number-average molecular mass) was studied at different temperatures. Amylopectin potato starch, lacking amylose, was selected because of its low tendency towards retrogradation at lower temperatures. Bacillus licheniformis alpha-amylase was added to 10% [w/w] gelatinised starch solutions. The hydrolysis experiments were done at 50, 70, and 90 degrees C. Samples were taken at defined DE values and these were analysed with respect to their saccharide composition. At the same DE the oligosaccharide composition depended on the hydrolysis temperature. This implies that at the same net number of bonds hydrolysed by the enzyme, the saccharide composition was different. The hydrolysis temperature also influenced the initial overall molecular-weight distribution. Higher temperatures led to a more homogenous molecular weight distribution. Similar effects were observed for alpha-amylases from other microbial sources such as Bacillus amyloliquefaciens and Bacillus stearothermophilus. Varying the pH (5.1, 6.2, and 7.6) at 70 degrees C did not significantly influence the saccharide composition obtained during B. licheniformis alpha-amylase hydrolysis. The underlying mechanisms for B. licheniformis alpha-amylase were studied using pure linear oligosaccharides, ranging from maltotriose to maltoheptaose as substrates. Activation energies for the hydrolysis of individual oligosaccharides were calculated from Arrhenius plots at 60, 70, 80, and 90 degrees C. Oligosaccharides with a degree of polymerisation exceeding that of the substrate could be detected. The contribution of these oligosaccharides increased as the degree of polymerisation of the substrate decreased and the temperature of hydrolysis increased. The product specificity decreased with increasing temperature of hydrolysis, which led to a more equal distribution between the possible products formed. Calculations with the subsite map as determined for the closely related alpha-amylase from B. amyloliquefaciens reconfirmed this finding of a decreased substrate specificity with increased temperature of hydrolysis. Copyright 1999 John Wiley & Sons, Inc.     

POCUS: mining genomic sequence annotation to predict disease genes

Here we present POCUS (prioritization of candidate genes using statistics), a novel computational approach to prioritize candidate disease genes that is based on over-representation of functional annotation between loci for the same disease. We show that POCUS can provide high (up to 81-fold) enrichment of real disease genes in the candidate-gene shortlists it produces compared with the original large sets of positional candidates. In contrast to existing methods, POCUS can also suggest counterintuitive candidates.     

Bladder injection of "naked" hSlo/pcDNA3 ameliorates detrusor hyperactivity in obstructed rats in vivo

The goal of these studies was to examine the potential utility of bladder instilled K+ channel gene therapy with hSlo cDNA (i.e., the maxi-K channel) to ameliorate bladder overactivity in a rat model of partial urinary outlet obstruction. Twenty-two female Sprague-Dawley rats were subjected to partial urethral (i.e., outlet) obstruction, with 17 sham-operated control rats run in parallel. After 6 wk of obstruction, suprapubic catheters were surgically placed in the dome of the bladder in all rats. Twelve obstructed rats received bladder instillation of 100 microg of hSlo/pcDNA in 1 ml PBS during catheterization, and another 10 obstructed rats received 1 ml PBS (7 rats) or 1 ml PBS containing pcDNA only (3 rats). Two days after surgery cystometry was performed on all animals to examine the characteristics of the micturition reflex in conscious and unrestrained rats. Obstruction was associated with a three- to fourfold increase in bladder weight and alterations in virtually every micturition parameter estimate. PBS-injected obstructed rats routinely displayed spontaneous bladder contractions between micturitions. In contrast, hSlo injection eliminated the obstruction-associated bladder hyperactivity, without detectably affecting any other cystometric parameter. Presumably, expression of hSlo in rat bladder functionally antagonizes the increased contractility normally observed in obstructed animals and thereby ameliorates bladder overactivity. These initial observations indicate a potential utility of gene therapy for urinary incontinence.     

A novel disorder caused by defective biosynthesis of N-linked oligosaccharides due to glucosidase I deficiency

Glucosidase I is an important enzyme in N-linked glycoprotein processing, removing specifically distal alpha-1,2-linked glucose from the Glc3Man9GlcNAc2 precursor after its en bloc transfer from dolichyl diphosphate to a nascent polypeptide chain in the endoplasmic reticulum. We have identified a glucosidase I defect in a neonate with severe generalized hypotonia and dysmorphic features. The clinical course was progressive and was characterized by the occurrence of hepatomegaly, hypoventilation, feeding problems, seizures, and fatal outcome at age 74 d. The accumulation of the tetrasaccharide Glc(alpha1-2)Glc(alpha1-3)Glc(alpha1-3)Man in the patient's urine indicated a glycosylation disorder. Enzymological studies on liver tissue and cultured skin fibroblasts revealed a severe glucosidase I deficiency. The residual activity was <3% of that of controls. Glucosidase I activities in cultured skin fibroblasts from both parents were found to be 50% of those of controls. Tissues from the patient subjected to SDS-PAGE followed by immunoblotting revealed strongly decreased amounts of glucosidase I protein in the homogenate of the liver, and a less-severe decrease in cultured skin fibroblasts. Molecular studies showed that the patient was a compound heterozygote for two missense mutations in the glucosidase I gene: (1) one allele harbored a G-->C transition at nucleotide (nt) 1587, resulting in the substitution of Arg at position 486 by Thr (R486T), and (2) on the other allele a T-->C transition at nt 2085 resulted in the substitution of Phe at position 652 by Leu (F652L). The mother was heterozygous for the G-->C transition, whereas the father was heterozygous for the T-->C transition. These base changes were not seen in 100 control DNA samples. A causal relationship between the alpha-glucosidase I deficiency and the disease is postulated.     

Multicenter evaluation of the phosphorylcholine-coated biodivYsio stent in short de novo coronary lesions: The SOPHOS study

AIMS: The BiodivYsio trade mark stent (Biocompatibles Ltd, Farnham, UK) is coated with a phosphorylcholine (PC)-containing copolymer to confer biocompatibility. The SOPHOS (Study Of PHosphorylcholine coating On Stents) study was designed to assess the safety and efficacy of this novel coronary stent and by indirect comparison to indicate equivalence with other formal stent studies. METHODS AND RESULTS: Patients with angina and a single short (#x2A7F;12 mm) de novo lesion in a native coronary artery of >/=2.75 mm diameter were included. A total of 425 patients were allocated in 24 centers. Clinical data were collected at one-, six- and nine-month follow-up. Angiography was performed before and after the stent implantation. In addition, in the first 200 patients (SOPHOS A) angiography was routinely performed at six months. The following 225 patients (SOPHOS B) were merely followed up clinically. The primary end-point of the study, the six-month MACE-rate (MACE = Major Adverse Cardiac Events) was 13.4% (two cardiac death; five Q-wave/nine non-Q-wave myocardial infarctions (MI); nine CABG and 32 target lesion revascularization (TLR), which is similar to the calculated 15% MACE-rate in comparable reference studies. Secondary end-points included among others restenosis at six months in the SOPHOS A population. The target vessel diameter was 2.98 +/- 0.48 mm. Minimal lumen diameter pre/post procedure and at follow-up was 1.00 +/- 0.32, 2.69 +/- 0.37, 1.91 +/- 0.71 mm, respectively. The binary restenosis rate (>/=50% diameter stenosis at follow-up) was 17.7%. CONCLUSION: The coronary BiodivYsio stent is safe and effective as a primary device for the treatment of native coronary artery lesions in patients with stable or unstable angina pectoris. Clinical and angiographic results are in the statistical range of equivalence with comparable studies with other current stents.     

Endoscopic foreheadplasty: a histologic comparison of periosteal refixation after endoscopic versus bicoronal lift

Endoscopic brow lift techniques using temporary fixation rely on rapid readherence of the periosteum to calvarial bone. Little is known about the histologic events that occur during the early postoperative period after these procedures. An animal study was designed to compare and contrast periosteal fixation to bone and unelevated periosteum, with endoscopic and bicoronal brow lift techniques. One method of temporary fixation is the use of absorbable (polylactic/polyglycolic acid copolymer) LactoSorb screws; a histologic analysis of implanted LactoSorb screws was also performed. Sixteen rabbits underwent brow lifts; eight underwent endoscopic brow lift and fixation with LactoSorb screws without skin excision, and another eight underwent traditional bicoronal brow lift with skin excision and closure under tension. Animals were killed 1, 2, 6, and 12 weeks after the procedures were performed to evaluate the interaction of periosteum and bone and the normal, unelevated periosteum/calvarium interface at a site distant from the operative area. Histologic specimens were examined for the degree of apposition of periosteum to bone and for any fibrous or bony reaction at this interface. Histologic analysis showed various degrees of periosteal fibrosis and fixation to calvarial bone. After an initial phase of minimal periosteal adherence and moderate inflammation, the periosteum became progressively more adherent to bone in both groups, with no significant differences between treatment groups in rates of fixation. Fixation required at least 6 weeks. LactoSorb screws were surrounded by an area of mild inflammation and were progressively hydrolyzed and digested. Periosteal fixation increases over time for bicoronal and endoscopic brow lifts with minimal differences between the two techniques. With this animal model, periosteal adherence to calvarium requires at least 6 weeks with complete adherence by 12 weeks. In addition, the use of absorbable fixation screws seems to be both effective and well tolerated. The histologic changes associated with periosteal healing observed in this study suggest that permanent or semipermanent fixation may improve the accuracy and early postoperative maintenance of forehead advancement.     

A laser-based method for measuring thermal nociception of cattle

We describe a method for measuring nociception in cattle using a CO(2) laser aimed at the caudal aspect of the metatarsi. In Experiment 1, infrared thermography showed that calves responded by lifting their legs when skin temperatures reached 45-55 degrees C. In Experiment 2a, the validity of the method was tested by comparing the response latencies of 14 calves to two power settings (2.25 W vs. 4.5 W) with each setting being applied six times. We found that both leg-lift latencies and tail-flick latencies were lower at the higher power setting, and the calves were more likely to respond by kicking than by simply moving the leg. The standard deviations between and within calves were smaller at the higher power setting, and the large within-calf variation means that at least three tests were required to obtain reliable measures that could differentiate between calves. In Experiment 2b, application of the laser at a range of power settings (2.0, 3.0, 4.0, 4.5, 5.0 and 5.5 W) on 16 calves showed that response latencies decreased as power increased up to 4.5 W, after which no further change occurred. In Experiment 3, the repeatability of the method was evaluated on nine measures with the high power setting (4.5 W). The coefficient of variation associated with repetition of the measures was 36%. In general, we found little change in response latencies with repeated use of the laser, except that responses on the second test tended to be shorter. Experiment 4 showed that ambient temperatures between 16 degrees C and 27 degrees C did not affect response latencies, but these were longer at temperatures of 7 degrees C. We suggest that the method is a useful way of measuring cattle's sensitivity to nociception as the animals need not be restrained and the distance to the animal need not be closely controlled. However, to obtain accurate, valid and reliable measures it is necessary to use a high power setting (4.5 W) and take at least three consecutive measures of the response latency.