Showing papers by "Karolinska Institutet published in 1991"

A new generation of animal cell expression vectors based on the Semliki Forest virus replicon.

Abstract: We have developed a novel DNA expression system, based on the Semliki Forest virus (SFV) replicon, which combines a wide choice of animal cell hosts, high efficiency and ease of use. DNA of interest is cloned into SFV plasmid vectors that serve as templates for in vitro synthesis of recombinant RNA. The RNA is transfected with virtually 100% efficiency into animal tissue culture cells by means of electroporation. Within the cell, the recombinant RNA drives its own replication and capping and leads to massive production of the heterologous protein while competing out the host protein synthesis. The expression system also includes an in vivo packaging procedure whereby recombinant RNA is packaged into infectious virus particles using cotransfection with packaging-deficient helper RNA molecules. The resulting high titer recombinant virus stock can be used to infect a wide range of animal cells with subsequent high expression of the heterologous gene product, but without expression of any structural proteins of the helper. The infected cells produce protein for up to 75 hours post infection after which the heterologous product can constitute as much as 25% of the total cell protein. The general utility of the system is demonstrated through the expression of human transferrin receptor, mouse dihydrofolate reductase, chick lysozyme and Escherichia coli beta-galactosidase.

GPI-anchored cell-surface molecules complexed to protein tyrosine kinases.

Abstract: Binding of ligand or antibody to certain cell-surface proteins that are anchored to the membrane by glycophosphatidylinositol (GPI) can cause activation of leukocytes. However, it is not known how these molecules, which lack intracellular domains, can transduce signals. The GPI-linked human molecules CD59, CD55, CD48, CD24, and CD14 as well as the mouse molecules Thy-1 and Ly-6 were found to associate with protein tyrosine kinases, key regulators of cell activation and signal transduction. A protein tyrosine kinase associated with the GPI-linked proteins CD59, CD55, and CD48 in human T cells, and with Thy-1 in mouse T cells was identified as p56lck, a protein tyrosine kinase related to Src. This interaction of GPI-linked molecules with protein tyrosine kinases suggests a potential mechanism of signal transduction in cells.

Microdialysis--principles and applications for studies in animals and man.

Abstract: . Microdialysis is a technique for sampling the chemistry of the individual tissues and organs of the body, and is applicable to both animal and human studies. The basic principle is to mimic the function of a capillary blood vessel by perfusing a thin dialysis tube implanted into the tissue with a physiological liquid. The perfusate is analysed chemically and reflects the composition of the extracellular fluid with time due to the diffusion of substances back and forth over the membrane. Microdialysis is thus a technique whereby substances may be both recovered from and supplied to a tissue. The most important features of microdialysis are as follows: it samples the extracellular fluid, which is the origin of all blood chemistry; it samples continuously for hours or days without withdrawing blood; and it purifies the sample and simplifies chemical analysis by excluding large molecules from the perfusate. However, the latter feature renders the technique unsuitable for sampling large molecules such as proteins. The technique has been extensively used in the neurosciences to monitor neurotransmitter release, and is now finding application in monitoring of the chemistry of peripheral tissues in both animal and human studies.

Fatty acid composition of brain phospholipids in aging and in Alzheimer’s disease

Abstract: The two major phospholipid classes, namely, phosphatidylethanolamines (PE) and phosphatidylcholines (PC), were studied in four different regions of human brain,i.e., in frontal gray matter, frontal white matter, hippocampus and in pons. The fatty acid (FA) compositions of these phospholipids were found to be specific for the different regions. PC contains mostly saturated and 18∶1 FA, while PE is rich in polyunsaturated FA. Aging has no influence on the FA compositions, while in Alzheimer’s disease (AD) PE is modified in all four regions, particularly in frontal gray matter and in hippocampus. The abundance of the major monounsaturated FA of PE, 18∶1, is not significantly altered in Alzheimer’s disease, but there is a substantial increase in the relative amounts of the saturated components 14∶0, 16∶0 and 18∶0. This is paralleled by a decrease in the polyunsaturated FA 20∶4, 22∶4 and 22∶6. It is not clear whether the changes observed are specific for AD. Changes in saturated/polyunsaturated FA ratio are likely to influence cellular function, which in turn may cause certain neural deficiencies. The findings do not support the hypothesis that AD reflects an accelerated aging process.

Genes for Epidermal Growth Factor Receptor, Transforming Growth Factor α, and Epidermal Growth Factor and Their Expression in Human Gliomas in Vivo

Abstract: Anomalies of the epidermal growth factor receptor (EGFR) gene, including amplification, rearrangement, and overexpression, have been reported in malignant human gliomas in vivo. In vitro glioma cell lines coexpress EGFR and at least one of its ligands, transforming growth factor alpha, suggesting the existence of an autocrine growth stimulatory loop. We have studied the tumor tissue from 62 human glioma patients and examined the structure and quantity of the EGFR gene and its transcripts, as well as the quantity of the receptor protein. In addition we have examined the genes and transcripts coding for the pre-pro forms of epidermal growth factor and transforming growth factor alpha, the two endogenous EGFR ligands. EGFR gene amplification was detected in 16 of the 32 malignancy grade IV gliomas (glioblastoma) studied (50%), but only in 1 of 30 gliomas of lesser malignancy grade (I-III). All tumors with an amplified gene overexpressed EGFR mRNA. More than one-half (62.5%) of the glioblastomas with amplified EGFR genes also showed coamplification of rearranged EGFR genes and concomitant expression of aberrant mRNA species. Overexpression, without gene amplification, was observed in some of the low grade gliomas, and aberrant EGFR transcripts were also seen in some cases without gene amplification or detected gene rearrangements. mRNA expression for one or both of the pre-pro forms of the ligands was detected in every tumor studied. Thus, several mechanisms for the activation of the EGFR-mediated growth stimulating pathway are possible in human gliomas in vivo: expression of a structurally altered receptor that may have escaped normal control mechanisms; and/or auto-, juxta-, or paracrine stimulating mechanisms involving coexpression of receptor and ligands, with or without overexpression of the receptor.

Stimulation of high-affinity adenosine A2 receptors decreases the affinity of dopamine D2 receptors in rat striatal membranes.

Abstract: Since high-affinity adenosine A2 receptors (A2a) are localized exclusively in dopamine-rich regions in the central nervous system and mediate inhibition of locomotor activity, we have examined the effect of A2a receptor activation on D1 and D2 receptor binding in membrane preparations of the rat striatum. The A2a agonist 2-[p-(2-carboxyethyl)phenethylamino]-5'- N-ethylcarboxamidoadenosine (CGS 21680) increased the Kd of the dopamine D2 agonist L-(-)-N-[3H]propylnorapomorphine without affecting the Bmax. The increase in Kd was maximal (40%) at 30 nM CGS 21680. CGS 21680 (30 nM) decreased the dopamine-induced inhibition of [3H]raclopride (a D2 antagonist) binding due to an increase (about 3-fold) in KH and KL, the dissociation constants of high- and low-affinity binding sites. The effects of CGS 21680 were antagonized by the adenosine antagonist 8-phenyltheophylline (10 microM). (-)-N6-(2-Phenylisopropyl)adenosine produced an effect similar to that of CGS 21680, provided the concentration used was high enough to stimulate A2a receptors (300 nM). GTP (50 microM) also decreased the dopamine-induced inhibition of [3H]raclopride binding but, in contrast to CGS 21680, GTP decreased the proportion of D2 receptors in the high-affinity state. CGS 21680 (30 nM) did not affect the Kd or Bmax of [3H]raclopride and failed to affect ligand binding to D1 receptors. Thus, stimulation of A2a receptors potently reduces the affinity of D2 agonist binding sites within the plasma membrane of striatal neurons. This A2a-D2 interaction may underlie the neuroleptic-like actions of adenosine agonists and the enhancing effects of adenosine antagonists, such as caffeine, on locomotor activity.

Monte Carlo techniques in medical radiation physics

Abstract: The author's main purpose is to review the techniques and applications of the Monte Carlo method in medical radiation physics since Raeside's review article in 1976. Emphasis is given to applications where proton and/or electron transport in matter is simulated. Some practical aspects of Monte Carlo practice, mainly related to random numbers and other computational details, are discussed in connection with common computing facilities available in hospital environments. Basic aspects of electron and photon transport are reviewed, followed by the presentation of the Monte Carlo codes widely available in the public domain. Applications in different areas of medical radiation physics, such as nuclear medicine, diagnostic X-rays, radiotherapy physics (including dosimetry), and radiation protection, and also microdosimetry and electron microscopy, are presented. Actual and future trends in the field, like Inverse Monte Carlo methods, vectorization of codes and parallel processors calculations are also discussed.

Afferent regulation of locus coeruleus neurons: anatomy, physiology and pharmacology

Abstract: Tract-tracing and electrophysiology studies have revealed that major inputs to the nucleus locus coeruleus (LC) are found in two structures, the nucleus paragigantocellularis (PGi) and the perifascicular area of the nucleus prepositus hypoglossi (PrH), both located in the rostral medulla. Minor afferents to LC were found in the dorsal cap of the paraventricular hypothalamus and spinal lamina X. Recent studies have also revealed limited inputs from two areas nearby the LC, the caudal midbrain periaqueductal gray (PAG) and the ventromedial pericoerulear region. The pericoeruleus may provide a local circuit interface to LC neurons. Recent electron microscopic analyses have revealed that LC dendrites extend preferentially into the rostromedial and caudal juxtaependymal pericoerulear regions. These extracoerulear LC dendrites may receive afferents in addition to those projecting to LC proper. However, single-pulse stimulation of inputs to such dendritic regions reveals little or no effect on LC neurons. Double-labeling studies have revealed that a variety of neurotransmitters impinging on LC neurons originate in its two major afferents, PGi and PrH. The LC is innervated by PGi neurons that stain for markers of adrenalin, enkephalin or corticotropin-releasing factor. Within PrH, large proportions of LC-projecting neurons stained for GABA or met-enkephalin. Finally, in contrast to previous conclusions, the dorsal raphe does not provide the robust 5-HT innervation found in the LC. We conclude that 5-HT inputs may derive from local 5-HT neurons in the pericoerulear area. Neuropharmacology experiments revealed that the PGi provides a potent excitatory amino acid (EAA) input to the LC, acting primarily at non-NMDA receptors in the LC. Other studies indicated that this pathway mediates certain sensory responses of LC neurons. NMDA-mediated sensory responses were also revealed during local infusion of magnesium-free solutions. Finally, adrenergic inhibition of LC from PGi could also be detected in nearly every LC neuron tested when the EAA-mediated excitation is first eliminated. In contrast to PGi, the PrH potently and consistently inhibited LC neurons via a GABAergic projection acting at GABAA receptors within LC. Such PrH stimulation also potently attenuated LC sensory responses. Finally, afferents to PGi areas that also contain LC-projecting neurons were identified. Major inputs were primarily autonomic in nature, and included the caudal medullary reticular formation, the parabrachial and Kolliker-Fuse nuclei, the PAG, NTS and certain hypothalamic areas.(ABSTRACT TRUNCATED AT 400 WORDS)

Hla class ii-associated genetic susceptibility in multiple sclerosis : a critical evaluation

Abstract: Multiple sclerosis (MS) has, since the 1970s, been known to be associated with the HLA-Dw2 and -DR2 specificities in Caucasian Europeans and North Americans. By the use of genomic typing techniques, the association has been specified to be with the DRw15,DQw6,Dw2, i.e. the DRB1*1501-DQA1*0102-DQB1*0602 haplotype. A significant DPw4 association in Scandinavian MS patients has been described in one report. However, this association has not been confirmed in several subsequent studies with patients from the same and other ethnic groups. During the last few years several reports, based on serological, RFLP and PCR-SSO data, have suggested that the HLA class II-associated MS susceptibility gene(s) may be more closely associated with the DQ than with the DR subregion. The observations that the HLA-DQB1 genes of MS patients share long stretches of sequence motifs and also carry DQA1 alleles encoding glutamine at position 34 of the DQ alpha chain have received considerable attention. It has been suggested that the susceptibility to develop MS might be determined by the corresponding DQ alpha-beta heterodimers either encoded in cis or in trans. We have investigated these issues in a large group of Swedish MS patients (n = 179). We found that the associations with the suggested DQB1 sequences and position 34 of the DQ alpha chain were due to linkage disequilibrium and secondary to the association with the DRw15,DQw6,Dw2 haplotype (p less than 10(-9) and p less than 10(-8), respectively). No overrepresentation of the implicated DQ alpha-beta heterodimers was observed in DRw15,DQw6,Dw2-negative patients.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the small 6,000-molecular-weight membrane protein modulates virus release

Abstract: We report on the construction of a full-length cDNA clone of Semliki Forest virus (SFV). By placing the cDNA under the SP6 promoter, infectious RNA can be produced in vitro and used to transfect cells to initiate virus infection. To achieve efficient transfections, a new protocol for electroporation of RNA was developed. This method gave up to 500-fold improvement over the traditional DEAE-dextran transfection procedure. Since virtually 100% of the cells can be transfected by electroporation, this method is a useful tool for detailed biochemical studies of null mutations of SFV that abolish production of infections virus particles. We used the cDNA clone of SFV to study what effects a deletion of the 6,000-molecular-weight membrane protein (6K membrane protein) had on virus replication. The small 6K protein is part of the structural precursor molecule (C-p62-6K-E1) of the virus. Our results conclusively show that the 6K protein is not needed for the heterodimerization of the p62 and E1 spike membrane proteins in the endoplasmic reticulum, nor is it needed for their transport out to the cell surface. The absence of the 6K protein did, however, result in a dramatic reduction in virus release, suggesting that the protein exerts its function late in the assembly pathway, possibly during virus budding.

Characteristics of short-chain alcohol dehydrogenases and related enzymes.

Abstract: Different short-chain dehydrogenases are distantly related, constituting a protein family now known from at least 20 separate enzymes characterized, but with extensive differences, especially in the C-terminal third of their sequences. Many of the first known members were prokaryotic, but recent additions include mammalian enzymes from placenta, liver and other tissues, including 15-hydroxyprostaglandin, 17 beta-hydroxysteroid and 11 beta-hydroxysteroid dehydrogenases. In addition, species variants, isozyme-like multiplicities and mutants have been reported for several of the structures. Alignments of the different enzymes reveal large homologous parts, with clustered similarities indicating regions of special functional/structural importance. Several of these derive from relationships within a common type of coenzyme-binding domain, but central-chain patterns of similarity go beyond this domain. Total residue identities between enzyme pairs are typically around 25%, but single forms deviate more or less (14-58%). Only six of the 250-odd residues are strictly conserved and seven more are conserved in all but single cases. Over one third of the conserved residues are glycine, showing the importance of conformational and spatial restrictions. Secondary structure predictions, residue distributions and hydrophilicity profiles outline a common, N-terminal coenzyme-binding domain similar to that of other dehydrogenases, and a C-terminal domain with unique segments and presumably individual functions in each case. Strictly conserved residues of possible functional interest are limited, essentially only three polar residues. Asp64, Tyr152 and Lys156 (in the numbering of Drosophila alcohol dehydrogenase), but no histidine or cysteine residue like in the completely different, classical medium-chain alcohol dehydrogenase family. Asp64 is in the suggested coenzyme-binding domain, whereas Tyr152 and Lys156 are close to the center of the protein chain, at a putative inter-domain, active-site segment. Consequently, the overall comparisons suggest the possibility of related mechanisms and domain properties for different members of the short-chain family.

Effect of low- and high-frequency TENS on Met-enkephalin-Arg-Phe and dynorphin A immunoreactivity in human lumbar CSF

Abstract: Transcutaneous nerve stimulation (TENS) treatment was given for 30 min to 37 patients divided into 3 groups of 10 patients and 1 group of 7 patients Two groups received low-frequency (2 Hz) and the other 2 groups high-frequency (100 Hz) stimulation A diagnostic lumbar cerebrospinal fluid (CSF) sample was obtained immediately before and after stimulation The CSF samples were subjected to analysis of immunoreactive (ir) opioid peptides, Met-enkephalin-Arg-Phe (MEAP) from preproenkephalin and dynorphin A (Dyn A) from preprodynorphin, respectively Low frequency TENS applied on the hand and the leg resulted in a marked increase (367%, P less than 005) of ir-MEAP but not ir-Dyn A, whereas high-frequency (100 Hz) TENS produced a 49% increase in ir-Dyn A (P less than 001) but not ir-MEAP This is the first report in humans that 2 Hz and 100 Hz peripheral stimulation induces differential release of peptides from preproenkephalin and preprodynorphin, respectively

Neuronal network generating locomotor behavior in lamprey: circuitry, transmitters, membrane properties, and simulation.

Abstract: All patterns of behavior are produced by interacting nerve cells. Although progress has been rapid on the level of the single nerve cell and its different types of ion channels, little or no knowledge is available on how the neural networks underlying different aspects of the vertebrate behavioral repertoire may function on a cellular level. The reason for this condition is that a detailed knowledge about the circuitry is required, for instance how different, relevant nerve cells interact, their properties, the types of synaptic interaction between interneurons, and so forth. Such detailed knowledge has been beyond reach with current techniques for these com­ plex mammalian nervous systems, which have been studied in some detail, like those of rat and cat. Nevertheless, much valuable information has been gathered about these nervous systems concerning which parts of the

Visual size cues in the programming of manipulative forces during precision grip

Abstract: A size-weight illusion was used to examine the role of visual cues in the programming of manipulative forces during the lifting of test objects utilizing the precision grip. Three different boxes of equal weight and unequal size were lifted. These were equipped with an instrumented grip handle to measure the employed grip force, load force (vertical lifting force), force rates and vertical movement. All fifteen subjects participating in the study reported that the smallest box was the heaviest, which is consistent with size-weight illusion predictions. However, the rate of increase of the isometric grip and load forces initially during the lift, the peaks of the grip and load force and the vertical acceleration were all found to increase with the box size. Thus, despite the conscious perception indicating a heavier weight for the small object, the motor program was scaled for a lighter weight. Yet, no differences were found in grip force during the static phase of the lift, where weight related information was apparently available via sensory feedback. Previous studies have reported that the programming of the precision grip is based on somatosensory information gained during previous lifts (Johansson and Westling 1984, 1988a, b). The present study suggests that visual cues are integrated in the programming of manipulative forces during precision grip.

A computerized brain atlas: construction, anatomical content, and some applications.

Abstract: An adjustable computerized atlas of the human brain has been developed, which can be adapted to fit individual anatomy. It is primarily intended for positron emission tomography (PET) but may also be used for single photon emission CT, transmission CT, magnetic resonance imaging, and neuroimaging-based procedures, such as stereotactic surgery and radiotherapy. The atlas is based on anatomical information obtained from brains fixed in situ soon after death. All structures have been drawn in on digitized photos of slices from one cryosectioned brain. The definition and classification of the anatomical structures and divisions are in agreement with the standard textbooks of anatomy, and the nomenclature is that of the Nomina Anatomica of 1965. The boundaries of the cortical cytoarchitectonic areas (Brodmann areas) have been determined using information from several sources, since three-dimensional literature data on their distribution are incomplete, scarce, and partly contradictory. However, no analysis of the cytoarchitectonics of the atlas brain itself has been undertaken. At present the data base contains three-dimensional representations of the brain surface, the ventricular system, the cortical gyri and sulci, as well as the Brodmann cytoarchitectonic areas. The major basal ganglia, the brain stem nuclei, the lobuli of the vermis, and the cerebellar hemispheres are also included. The computerized atlas can be used to improve the quantification and evaluation of PET data in several ways. For instance, it can serve as a guide in selecting regions of interest. It may also facilitate comparisons of data from different individuals or groups of individuals, by applying the inverse atlas transformation to PET data volume, thus relating the PET information to the anatomy of the reference atlas rather than to the patient's anatomy. Reformatted PET data from individuals can thus be averaged, and averages from different categories or different functional states of patients can be compared.

Incidence in Sweden and clinical features of familial hemophagocytic lymphohistiocytosis.

Abstract: We retrospectively studied the incidence of familial hemophagocytic lymphohistiocytosis (FHL) in children during the 16-year period 1971-86. First, all departments of pediatrics, pathology, and infectious diseases were enquired for children with FHL or disorders resembling FHL. Secondly, the causes of death of all children who died during the study period in Sweden (n = 19,542) were also investigated. Files and histological specimens were further studied in selected children. By using a set of inclusion/exclusion criteria, we found 32 children with FHL. The incidence was 1.2/1,000,000 children per year. One child per 50,000 live borns developed FHL during this period. The sex ratio was close to 1:1. Prominent early clinical signs were fever (91%), splenomegaly (84%), hepatomegaly (90%), rash (43%), and lymph node enlargement (42%). Neurological symptoms, which developed in 47%, could totally dominate the clinical picture and develop prior to other symptoms and signs. Common laboratory findings were pancytopenia, hypertriglyceridemia, hypofibrinogenemia, elevated serum transaminases, hyperbilirubinemia, hyponatremia, hypoalbuminemia, and a moderate spinal fluid pleocytosis. Chest X-ray often revealed mostly discrete pulmonary infiltrates. FHL is an underdiagnosed disease and in only 11/32 children was diagnosis made during their lifetime. It is important to be aware of the disorder as potential therapy now exists.

Influence of eccentric actions on skeletal muscle adaptations to resistance training.

Abstract: Three different training regimens were performed to study the influence of eccentric muscle actions on skeletal muscle adaptive responses to heavy resistance exercise. Middle-aged males performed the leg press and leg extension exercises two days each week. The resistance was selected to induce failure within six to twelve repetitions of each set. Group CON/ECC (n = 8) performed coupled concentric and eccentric actions while group CON (n = 8) used concentric actions only. They did four or five sets of each exercise. Group CON/CON (n = 10) performed twice as many sets with only concentric actions. Eight subjects did not train and served as controls. Tissue samples were obtained from m. vastus lateralis using the biopsy technique before and after 19 weeks of training, and after four weeks of detraining. Histochemical analyses were performed to assess fibre type composition, fibre area and capillarization. Training increased (P less than 0.05) Type IIA and decreased (P less than 0.05) Type IIB fibre percentage. Only group CON/ECC increased Type I area (14%, P less than 0.05). Type II area increased (P less than 0.05) 32 and 27%, respectively, in groups CON/ECC and CON/CON, but not in group CON. Mean fibre area increased (P less than 0.05) 25 and 20% in groups CON/ECC and CON/CON, respectively. Capillaries per fibre increased (P less than 0.05) equally for Type I and Type II fibres. Capillaries per fibre area for both fibre types, however, increased (P less than 0.05) only in groups CON and CON/CON. The changes in fibre type composition and capillary frequency were manifest after detraining.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscle activation during maximal voluntary eccentric and concentric knee extension

Abstract: The aim of this investigation was to study the relationships among movement velocity, torque output and electromyographic (EMG) activity of the knee extensor muscles under eccentric and concentric loading. Fourteen male subjects performed maximal voluntary eccentric and concentric constant-velocity knee extensions at 45, 90, 180 and 360 degrees.s-1. Myoelectric signals were recorded, using surface electrodes, from the vastus medialis, vastus lateralis and rectus femoris muscles. For comparison, torque and full-wave rectified EMG signals were amplitude-averaged through the central half (30 degrees-70 degrees) of the range of motion. For each test velocity, eccentric torque was greater than concentric torque (range of mean differences: 20%-146%, P less than 0.05). In contrast, EMG activity for all muscles was lower under eccentric loading than velocity-matched concentric loading (7%-31%, P less than 0.05). Neither torque output nor EMG activity for the three muscles changed across eccentric test velocities (P greater than 0.05). While concentric torque increased with decreasing velocity, EMG activity for all muscles decreased with decreasing velocity (P less than 0.05). These data suggest that under certain high-tension loading conditions (especially during eccentric muscle actions), the neural drive to the agonist muscles was reduced, despite maximal voluntary effort. This may protect the musculoskeletal system from an injury that could result if the muscle was to become fully activated under these conditions.

Structural and functional relations among thioredoxins of different species.

Abstract: Three-dimensional models have been constructed of homologous thioredoxins and protein disulfide isomerases based on the high resolution x-ray crystallographic structure of the oxidized form of Escherichia coli thioredoxin. The thioredoxins, from archebacteria to humans, have 27-69% sequence identity to E. coli thioredoxin. The models indicate that all the proteins have similar three-dimensional structures despite the large variation in amino acid sequences. As expected, residues in the active site region of thioredoxins are highly conserved. These include Asp-26, Ala-29, Trp-31, Cys-32, Gly-33, Pro-34, Cys-35, Asp-61, Pro-76, and Gly-92. Similar residues occur in most protein disulfide isomerase sequences. Most of these residues form the surface around the active site that appears to facilitate interactions with other enzymes. Other structurally important residues are also conserved. A proline at position 40 causes a kink in the alpha-2 helix and thus provides the proper position of the active site residues at the amino end of this helix. Pro-76 is important in maintaining the native structure of the molecule. In addition, residues forming the internal contact surfaces between the secondary structural elements are generally unchanged such as Phe-12, Val-25, and Phe-27.

Development of human precision grip. I: Basic coordination of force.

Abstract: The coordination of manipulative forces was examined while children and adults repeatedly lifted a small object between the thumb and index finger. Grip force, load force (vertical lifting force), grip force rate and the vertical position of the test object were continuously measured. In adults, the force generation was highly automatized and was nearly invariant between trials. After a preload phase in which the grip was established, the grip and load forces increased in parallel under isometric conditions until the load force overcame the force of gravity and the object started to move. During this loading phase, the force rate profiles were essentially bell shaped and single peaked, suggesting that the force increases were programmed as one coordinated event. Children below the age of two exhibited a prolonged preload phase and a loading phase during which the grip and load forces did not increase in parallel. A major increase in grip force preceded the increase in load force, and at the start of the loading phase, the grip force was usually several Newtons (N). The force rate profiles were multi peaked with stepwise force increases most likely allowing peripheral feedback to play an important role in the control of the forces. After the age of two, the grip force increased less during the preload phase. The loading phase was more regularly characterized by a parallel increase of the grip force and load force and the duration of the various phases decreased. The older children programmed the forces in one force rate pulse indicating the emergence of an anticipatory strategy. Yet, the mature coordination of forces was not fully developed until several years later.(ABSTRACT TRUNCATED AT 250 WORDS)

The antioxidant butylated hydroxytoluene protects against atherosclerosis.

Abstract: Rabbits fed a 1% cholesterol diet with or without the antioxidant butylated hydroxytoluene (BHT) developed typical atherosclerotic lesions. The addition of BHT gave higher levels of total cholesterol (+40%), triglycerides (+250%), low density lipoprotein (LDL), and very low density lipoprotein (VLDL) in plasma. Despite the lower plasma lipid levels, the degree of atherosclerosis of the aortic surface was considerably higher in rabbits fed cholesterol than in the group treated with cholesterol and BHT. The mean atherosclerotic involvement was 18.6 +/- 4.4% in the former group and 5.9 +/- 1.7% in the latter group (p = 0.02). In all animals, there was a high correlation between the area of the arterial lesion and cholesterol content (r = 0.96). Serum levels of cholesterol autooxidation products (7-ketocholesterol and cholesterol 5 alpha,6 alpha-epoxide) were lower in the group of rabbits treated with BHT (p less than 0.005). Serum levels of vitamin E were slightly higher in the BHT group. There was no significant difference in the clearance of beta-VLDL between the two treatment groups after using either beta-VLDL from cholesterol-fed animals or beta-VLDL from BHT-fed animals. The results are in accord with the contention that oxidative modification of lipoproteins is important for the development of atherosclerosis and that antioxidants may have a protective effect. At present, however, other explanations cannot be completely excluded, for example, effects of antioxidants on immunologic factors or monocyte adhesion.

Amino acid sequence of PR-39. Isolation from pig intestine of a new member of the family of proline-arginine-rich antibacterial peptides.

Abstract: We recently isolated from pig intestine and characterized a 31-residue antibacterial peptide named cecropin P1 with activity against Escherichia coli and several other Gram-negative bacteria. The isolation involved a number of batch-wise steps followed by several chromatography steps. The continued investigation of these antibacterial peptides has now yielded another antibacterial peptide with high activity against both E. coli and Bacillus megaterium. Amino acid analysis showed a very high content of proline (49 mol%) and arginine (26 mol%), an intermediate level of phenylalanine and low levels of leucine, tyrosine, isoleucine, and glycine. The primary structure was determined by a combination of Edman degradation, plasma desorption mass spectrometry and C-terminal sequence analysis by carboxypeptidase Y degradation using capillary zone electrophoresis for detection of liberated residues. The calculated molecular mass was 4719.7 Da, which is in excellent agreement with 4719 Da obtained by plasma desorption mass spectrometry. The peptide was named PR-39 (proline-arginine-rich with a size of 39 residues). The lethal concentration of the peptide was determined against six Gram-negative and four Gram-positive strains of bacteria.

Comparison of acid ethanol extraction and acid gel filtration prior to IGF-I and IGF-II radioimmunoassays: improvement of determinations in acid ethanol extracts by the use of truncated IGF-I as radioligand.

Abstract: Insulin-like growth factor binding proteins interfere in the IGF-I and -II radioimmunoassays. In an attempt to overcome this problem, we have compared the use of truncated IGF-I, with reduced IGFBP affinity, and IGF-I as radioligands for IGF-I RIA measurements in serum separated by acid gel filtration or acid ethanol extraction followed by cryo-precipitation. With truncated IGF-I as radioligand the IGF-I measurements in acid gel filtrates and acid ethanol extracts were significantly correlated in healthy subjects (N = 42, r = 0.91, p less than 0.001) and in patients with acromegaly (N = 10, r = 0.85, p less than 0.01), GH deficiency (N = 10, r = 0.88, p less than 0.001) or Type I diabetes mellitus (N = 10, r = 0.90, p less than 0.001). In contrast, the IGF-I concentrations in acid ethanol extracts determined with IGF-I as radioligand did not correlate with those in acid gel filtrates using truncated IGF-I radioligand in patients with acromegaly (r = 0.61, NS) or GH deficiency (r = 0.46, NS). In the latter group the mean IGF-I concentrations measured in acid ethanol extracts were erroneously elevated by 112%. Low-affinity antibodies used for IGF-II RIA determinations failed to give reliable results in acid ethanol extracts from patients with Type I diabetes mellitus or GH deficiency. In conclusion, erroneously high IGF-I concentrations owing to binding of the radioligand to IGFBPs not completely removed by acid ethanol extraction can be avoided by the use of truncated IGF-I as radioligand.

Meat, cooking methods and colorectal cancer: a case-referent study in Stockholm.

Abstract: The associations between methods of cooking meats and colorectal cancer were examined in a population-based case- referent study performed in Stockholm in 1986-1988. The study included 559 cases and 505 referents. Total meat intake, frequent consumption of brown gravy, and a preference for heavily browned meat surface each independently increased the risk for colorectal cancer. The relative risks (RR) were higher for rectal than for colon cancer, and for boiled meat (RR colon = 1.7, RR rectum = 2.7) than for meat fried with medium or lightly browned surface (RR colon = 0.8, RR rectum = 1.1), but the highest risks were for meat fried with heavily browned surface (RR colon = 2.8, RR rectum = 6.0). The analyses were adjusted for year of birth, gender and fat intake. Further adjustments for total energy, dietary fiber intake, body mass and physical activity had little or no influence on the results. The findings suggest that, in addition to frequent meat intake, a heavily browned meat surface formed when frying meat at high temperatures is important in the etiology colorectal cancer.