Comparative studies on beta-glucan hydrolases. Isolation and characterization of an exo(1 yields 3)-beta-glucanase from the snail, Helix pomatia.

An exo-β-glucan hydrolase, present in the digestive juice of the snail, Helix pomatia, has been purified to homogeneity by chromatography on Bio-Gel P-60, Sephadex G-200, DEAE-cellulose, and DEAE-Sephadex. The enzyme degrades β-(1 → 3)-linked oligosaccharides and polysaccharides, rapidly and to completion, or near completion, yielding glucose as the major product of enzyme action. Mixed linkage (1→3; 1→4)-β-glucans are also extensively degraded and β-(1→6)- and β-(1→4)-linked glucose polymers are slowly degraded by the enzyme. This enzyme differs from other exo-β-glucanases, reported previously, in the broadness of its substrate specificity. The K_m values for action on laminarin and lichenin are respectively 1.22 and 2.22 mg/ml; the maximum velocity of action on laminarin is approximately twice that on lichenin. The enzyme has a molecular weight of 82,000 as determined by polyacrylamide gel electrophoresis. Maximum activity is exhibited at pH 4.3 and at temperatures of 50–55 °C.     

The utilization of bromodeoxyuridine incorporation into DNA for the analysis of cellular kinetics

Recently developed differential staining techniques based on the incorporation of bromodeoxyuridine (BUdR) into DNA permits the unequivocal identification of metaphase cells which have replicated once, twice, and three or more times. This technique has the potential of being utilized in the examination of kinetics of dividing cell populations. This potential is examined in a phytohemagglutinin-stimulated lymphocyte system. Determinations of the effect of increasing concentrations of BUdR on the distribution of metaphase cells between different generation cycles reveals no inhibition of cellular kinetics below 35 μM. The ability to distinguish third generation metaphase cells from subsequent generations is examined through the determination of “labelled” centromeric regions. The applicability of this system to current cellular kinetics is discussed.     

Different timing of increases in activities of four X-chromosome linked enzymes during the cell cycle of synchronized human lymphoblasts

A permanent human lymphoblast culture was synchronized with repetitive thymidine blocks, and the changes in the levels of activity of four X-chromosome-linked enzymes were followed during the cell cycle. The four enzymes studied were phosphoglycerate kinase (PGK), α-galactosidase (α-Gal), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), and glucose-6-phosphate dehydrogenase (G6PD). The levels of PGK and α-Gal activities increased simultaneously in G1 and in S, while HGPRT and G6PD increased close together in middle and late S. Therefore, different control mechanisms may be involved in the increases of the activities of these two sets of enzymes.     

Scanning molecular sieve chromatography of interacting protein systems. IV. The difference profile method as applied to the Gibbs-Duhem expression in the analysis of the dimer-tetramer equilibria of oxyhemoglobin A

The recently-developed large zone difference profile method in scanning molecular sieve chromatography is applied to the analysis of the Gibbs-Duhem expression in the tetramer-dimer equilibrium of human oxyhemoglobin A. The preferential binding term and solvation parameters of the Hofmeister anion phosphate are examined. Results indicate that as the concentration of phosphate ions increase, a hydrated phosphate is formed which enhances the association by perturbing the solvation layer of the hemoglobin molecules. The standard free energy change at a given Hofmeister anion activity of InA(x) = -3.2476 is 9.4 +/- 0.2 kcal mole . DeltaG degrees at InA(x) = -1.2711 is 10.90 +/- 0.05 kcal mole , suggesting that approximately 11 kcal are required to dissociate one mole of tetramer into dimer.     

Subcellular fractionation of pig platelets

Subcellular components were obtained from pig platelets, disrupted by means of a French press and separated into 4 primary fractions. The granule fraction (10 000 g) was subjected to a sucrose gradient fractionation. Primary fractions and the granule subfractions were studied electron microscopically and biochemically by following the distribution of markers of membranes, lysosomes of α-granules, mitochondria and dense granules. With this technique of platelet homogenization, 80% of the serotonin and 93% of the β-N-acetylglucosaminidase were found to be particulate. In the gradient, mitochondria were sharply banded in a fraction (density 1.16–1.17) having a specific activity 10–100 times higher than the other fractions of the gradient. Serotonin-containing granules were found in a pellet of density greater than 1.27 and contained 60% of the serotonin and adenine nucleotides of the granule fraction. The lysosome markers that were monitored, acid phosphatase and β-N-acetylglucosaminidase, exhibited different distribution patterns. Acid phosphatase showed the highest specific activity in the microsomal fraction with only 2.8% in the granule fraction, and this latter amount also appeared to be associated with membranes upon further fractionation. β-N-Acetylglucosaminidase was present in both the granule fraction and in the microsomal fraction with nearly the same specific activity. However, that present in the granule fraction was clearly associated with granules that distributed over a wide range of densities on a sucrose gradient. The calcium distribution was followed to attempt to determine its subcellular location; 19% was found in the same subfraction as the serotonin-containing granules, but at least 50% of the particulate calcium was associated with granules distinctly separate from the storage granules.     

Enhanced consumption of an aversively conditioned taste following the presentation of a “medicine” taste

Rats given presentations of a citric acid solution while recovering from LiCl-induced illness (i.e., a “medicine effect” treatment) subsequently drank more of an aversively conditioned NaCl solution at test, when the NaCl presentation was immediately preceded by citric acid. That is, citric acid passed a summation test of conditioned inhibition. Such an effect was not observed in a group given explicitly unpaired presentations of LiCl and citric acid. It is proposed that enhanced consumption of an aversive taste due to the previous presentation of a “medicine” taste can provide an animal model of human maladaptive behavior in regards to food consumption.     

Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification,Distraction Osteogenesis,or Healing of Critical Sized Defects

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role in healing of bone injuries. In the worst-case scenario mechanical instability can lead to delayed or non-union in humans. However, motion can also stimulate the healing process. In fractures that have motion cartilage forms to stabilize the fracture bone ends, and this cartilage is gradually replaced by bone through recapitulation of the developmental process of endochondral ossification. In contrast, if a bone fracture is rigidly stabilized bone forms directly via intramembranous ossification. Clinically, both endochondral and intramembranous ossification occur simultaneously. To effectively replicate this process investigators insert a pin into the medullary canal of the fractured bone as described by Bonnarens⁴. This experimental method provides excellent lateral stability while allowing rotational instability to persist. However, our understanding of the mechanisms that regulate these two distinct processes can also be enhanced by experimentally isolating each of these processes. We have developed a stabilization protocol that provides rotational and lateral stabilization. In this model, intramembranous ossification is the only mode of healing that is observed, and healing parameters can be compared among different strains of genetically modified mice ^5-7, after application of bioactive molecules ^8,9, after altering physiological parameters of healing ¹⁰, after modifying the amount or time of stabilization ¹¹, after distraction osteogenesis ¹², after creation of a non-union ¹³, or after creation of a critical sized defect. Here, we illustrate how to apply the modified Ilizarov fixators for studying tibial fracture healing and distraction osteogenesis in mice.     

Butanedione treatment reduces receptor binding of a lysosomal enzyme to cells and membranes

Treatment of the lysosomal enzyme, α-L-iduronidase, with 2,3 butanedione, an arginine modifying reagent, under conditions where enzyme activity was unaffected, reduced by 50% the internalization of the enzyme into cultured human fibroblasts. The lowered rate of internalization was a result of a reduced binding of the enzyme to cell surface receptors. The butanedione treatment of α-L-iduronidase caused a 90% reduction of binding when isolated fibroblast membranes were used as the source of receptor. This marked reduction of binding was also seen when membranes from a rat chondrosarcoma were examined. Although there is ample evidence that the receptor recognizes mannose 6-phosphate residues on the enzyme, the results suggest that other structural features, such as arginine moieties, may also be important in iduronidase binding.     

A New Single Chamber Implantable Defibrillator with Atrial Sensing: A Practical Demonstration of Sensing and Ease of Implantation

Implantable cardioverter-defibrillators (ICDs) terminate ventricular tachycardia (VT) and ventricular fibrillation (VF) with high efficacy and can protect patients from sudden cardiac death (SCD). However, inappropriate shocks may occur if tachycardias are misdiagnosed. Inappropriate shocks are harmful and impair patient quality of life. The risk of inappropriate therapy increases with lower detection rates programmed in the ICD. Single-chamber detection poses greater risks for misdiagnosis when compared with dual-chamber devices that have the benefit of additional atrial information. However, using a dual-chamber device merely for the sake of detection is generally not accepted, since the risks associated with the second electrode may outweigh the benefits of detection. Therefore, BIOTRONIK developed a ventricular lead called the Linox^SMART S DX, which allows for the detection of atrial signals from two electrodes positioned at the atrial part of the ventricular electrode. This device contains two ring electrodes; one that contacts the atrial wall at the junction of the superior vena cava (SVC) and one positioned at the free floating part of the electrode in the atrium. The excellent signal quality can only be achieved by a special filter setting in the ICD (Lumax 540 and 740 VR-T DX, BIOTRONIK). Here, the ease of implantation of the system will be demonstrated.     

Human kinesiology

This paper discusses kinesiological aspects of human locomotion in terms of the incomplete morphological adaptation of the body to current lifestyles. Modern instrumentation used for biomechanical analyses is outlined for the most part in the context of human running. Investigations of spatiotemporal and segmental measures by electronic walkways, photography, optoelectronics and angle-angle diagrams are referred to, as is electromyography and direct force measurement. Three commonly identifiable areas of applied kinesiological research, namely sport, industry and medicine, are considered with reference to the prevention, treatment and after-care of injuries whether received on the sporting field, in the work place, or in an automobile accident.