Showing papers by "Karolinska Institutet published in 1990"

Subjective and Objective Sleepiness in the Active Individual

Abstract: Eight subjects were kept awake and active overnight in a sleep lab isolated from environmental time cues. Ambulatory EEG and EOG were continuously recorded and sleepiness ratings carried out every two hours as was a short EEG test session with eyes open for 5 min and closed for 2 min. The EEG was subjected to spectral analysis and the EOG was visually scored for slow rolling eye movements (SEM). Intrusions of SEM and of alpha and theta power density during waking, open-eyed activity strongly differentiated between high and low subjective sleepiness (the differentiation was poorer for closed eyes) and the mean intraindividual correlations between subjective and objective sleepiness were very high. Still, the covariation was curvilinear; physiological indices of sleepiness did not occur reliably until subjective perceptions fell between "sleepy" and "extremely sleepy-fighting sleep"; i.e. physiological changes due to sleepiness are not likely to occur until extreme sleepiness is encountered. The results support the notion that ambulatory EEG/EOG changes may be used to quantify sleepiness.

Insulin-like growth factors and their binding proteins

Abstract: Article de synthese sur les facteurs de croissance IGF et leurs proteines de liaisons: structure, biosynthese, regulation de la production paracrine et endocrine, recepteurs et transmission du signal intracellulaire, activite biologique in vitro et in vivo chez les mammiferes

Empty MHC class I molecules come out in the cold

Abstract: Major histocompatibility complex (MHC) class I molecules present antigen by transporting peptides from intracellularly degraded proteins to the cell surface for scrutiny by cytotoxic T cells. Recent work suggests that peptide binding may be required for efficient assembly and intracellular transport of MHC class I molecules, but it is not clear whether class I molecules can ever assemble in the absence of peptide. We report here that culture of the murine lymphoma mutant cell line RMA-S at reduced temperature (19-33 degrees C) promotes assembly, and results in a high level of cell surface expression of H-2/beta 2-microglobulin complexes that do not present endogenous antigens, and are labile at 37 degrees C. They can be stabilized at 37 degrees C by exposure to specific peptides known to interact with H-2Kb or Db. Our findings suggest that, in the absence of peptides, class I molecules can assemble but are unstable at body temperature. The induction of such molecules at reduced temperature opens new ways to analyse the nature of MHC class I peptide interactions at the cell surface.

Osteopontin--a possible anchor of osteoclasts to bone.

Abstract: A key event in bone resorption is the binding of osteoclasts to the mineral matrix of bone surfaces. A candidate for mediating this binding is osteopontin, a major cell- and hydroxyapatite-binding protein synthesized by osteoblasts. In support of this hypothesis is the fact that the synthesis of osteopontin is stimulated by calcitriol (1,25-dihydroxy-vitamin(D3), a substance that induces bone resorption. The present study demonstrates that osteopontin is highly enriched at regions of the bone surface where osteoclasts are anchored. Furthermore, the vitronectin receptor, which has known specificity for osteopontin, is shown preferentially localized at the corresponding area of the osteoclast plasma membrane. The results thus support the hypothesis that osteoclasts when resorbing bone are anchored by osteopontin bound both to the mineral of bone matrix and to a vitronectin receptor on the osteoclast plasma membrane.

Sequence differences between glycosylated and non-glycosylated Asn-X-Thr/Ser acceptor sites : implications for protein engineering

Abstract: In N-glycosylated glycoproteins, carbohydrate is attached to Asn in the sequence Asn-X-Ser/Thr, where X denotes any amino acid. However, the presence of this consensus peptide does not always lead to glycosylation. We have compiled an extensive collection of glycosylated and non-glycosylated Asn-X-Thr/Ser sites and present a statistical study based on this data set. Our results indicate that non-glycosylated sites tend to be found more frequently towards the C termini of glycoproteins, and that proline residues in positions X and Y in the consensus Asn-X-Thr/Ser-Y strongly reduce the likelihood of N-linked glycosylation. Beyond this, there are no obvious local sequence features that seem to correlate with the absence or presence of N-linked glycosylation. These findings are discussed in terms of the prediction and engineering of glycosylation sites in secretory proteins.

Molecular cloning and neurotrophic activities of a protein with structural similarities to nerve growth factor: developmental and topographical expression in the brain.

Abstract: We have used a pool of degenerate oligonucleotides representing all possible codons in regions of homology between brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) to prime rat hippocampal cDNAs in the polymerase chain reaction. The amplified DNA included a product with significant similarity to NGF and BDNF, which was used to isolate a 1020-nucleotide-long cDNA from a rat hippocampal library. From the nucleotide sequence, a 282-amino-acid-long protein with approximately 45% amino acid similarity to both pig BDNF and rat NGF was deduced. In the adult brain, the mRNA for this protein was predominantly expressed in hippocampus, where it was confined to a subset of pyramidal and granular neurons. The developmental expression in brain showed a clear peak shortly after birth, 1 and 2 weeks earlier than maximal expression of BDNF and NGF, respectively. It was also expressed in several peripheral tissues with the highest level in kidney. The protein, transiently expressed in COS cells, was tested on chicken embryonic neurons and readily stimulated fiber outgrowth from explanted Remak's ganglion and, to a lesser extent, the nodose ganglion. A weak, but consistent, fiber outgrowth response was also seen in the ciliary ganglion and in paravertebral sympathetic ganglia. Moreover, the protein displaced binding of NGF to its receptor, suggesting that it can interact with the NGF receptor. Thus, this factor, although structurally and functionally related to NGF and BDNF, has unique biological activities and represents a member of a family of neurotrophic factors that may cooperate to support the development and maintenance of the vertebrate nervous system.

Prognostic subgroups in B-cell chronic lymphocytic leukemia defined by specific chromosomal abnormalities.

Abstract: Background and methods Specific chromosomal abnormalities have been shown to affect the overall survival of patients with acute leukemia, but the possibility that specific chromosomal defects may influence the course of B-cell chronic lymphocytic leukemia (CLL) is controversial. We assessed this possibility as follows: blood mononuclear cells from 433 patients with B-cell CLL in five European centers were cultured with B-cell mitogens, and banded metaphases were studied. Results Three hundred ninety-one patients could be evaluated cytogenetically, and 218 had clonal chromosomal changes. The most common abnormalities were trisomy 12 (n = 67) and structural abnormalities of chromosome 13 (n = 51; most involving the site of the retinoblastoma gene) and of chromosome 14 (n = 41). Patients with a normal karyotype had a median overall survival of more than 15 years, in contrast to 7.7 years for patients with clonal changes. Patients with single abnormalities (n = 113) did better than those with complex karyotypes (P less than 0.001). Patients with abnormalities involving chromosome 14q had poorer survival than those with aberrations of chromosome 13q (P less than 0.05). Among patients with single abnormalities, those with trisomy 12 alone had poorer survival than patients with single aberrations of chromosome 13q (P = 0.01); the latter had the same survival as those with a normal karyotype. A high percentage of cells in metaphase with chromosomal abnormalities, indicating highly proliferative leukemic cells, was associated with poor survival (P less than 0.001). Cox proportional-hazards analysis identified age, sex, the percentage of cells in metaphase with chromosomal abnormalities, and the clinical stage of the disease (Binet classification system) as independent prognostic variables. Conclusions Chromosomal analysis provides prognostic information about overall survival in addition to that supplied by clinical data in patients with B-cell CLL.

Regulation of differentiated properties and proliferation of arterial smooth muscle cells.

Abstract: D uring recent years, it has become increasingly evident that arterial smooth muscle cells occur in at least two distinct states, usually referred to as a synthetic and a contractile phenotype. Synthetic-state cells have a fibroblast-like appearance, and their main functions are to proliferate and to produce extracellular matrix components. They are found in the embryo and the young growing organism, where they take part in the formation of the vessel wall. Contractile-state cells have a musclelike appearance and contract in response to chemical and mechanical stimuli. They predominate in the vessels of adults and are primarily involved in the control of blood pressure and flow. However, these cells are able to return to a synthetic phenotype, and this appears to be an important early event in atherogenesis. This review briefly summarizes the present knowledge concerning the regulation of differentiated properties and proliferation of arterial smooth muscle cells. Most of the discussion will deal with studies on cultured cells, which so far are the most abundant. Particular attention will be paid to the role of extracellular matrix components like fibronectin and laminin and of polypeptide mitogens like platelet-derived growth factor (PDGF). It is believed that future studies in this area will help to widen our understanding of vasculogenesis and the initial stages in the pathogenesis of atherosclerosis.

D2 dopamine receptors in neuroleptic-naive schizophrenic patients. A positron emission tomography study with [11C]raclopride.

Abstract: • Several groups have reported increased densities of D2dopamine receptors in the basal ganglia of schizophrenic brains postmortem. The significance of this finding has been questioned, since an upregulation of receptor number may be a neuronal response to neuroleptic drug treatment. We have used positron emission tomography and [11C]raclopride to examine central D2dopamine receptor binding in 20 healthy subjects and 18 newly admitted, young, neuroleptic-naive patients with schizophrenia. An in vivo saturation procedure was applied for quantitative determination of D2dopamine receptor density (Bmax) and affinity (Kd). When the two groups were compared, no significant difference in Bmaxor Kdvalues was found in the putamen or the caudate nucleus. The hypothesis of generally elevated central D2dopamine receptor densities in schizophrenia was thus not supported by the present findings. In the patients but not in the healthy controls, significantly higher densities were found in the left than in the right putamen but not in the caudate nucleus.

Changes in cortical extracellular levels of energy-related metabolites and amino acids following concussive brain injury in rats.

Abstract: The aim of this study was to measure extracellular chemical changes in the cerebral cortex in response to compression contusion trauma in rats. Energy-related metabolites (i.e., lactate, pyruvate, adenosine, inosine, and hypoxanthine) and amino acids were harvested from the extracellular fluid (ECF) using microdialysis and analyzed by high-performance liquid chromatography. The measurements were performed in cortical tissue, where neuronal injury occurs in this model. The severity of the trauma was varied by using different depths of impact: mild trauma, 1.5 mm; severe trauma, 2.5 mm. The trauma induced a dramatic increase in the ECF levels of energy-related metabolites that was conditioned by the severity of the insult. The ECF level of taurine, glutamate, aspartate, and γ-aminobutyric acid (GABA) also rose markedly, while other amino acids did not change significantly. The results suggest that the trauma induced a transient, profound focal disturbance of energy metabolism in the cortical tissue, probabl...

Biochemical mechanisms for oxygen free radical formation during exercise.

Abstract: The biochemical mechanisms behind skeletal muscle soreness and damage with muscular overuse have remained unclear. Recently, however, a growing amount of evidence indicates that free radicals play an important role as mediators of skeletal muscle damage and inflammation. During exercise, two of the potentially harmful free radical generating sources are semiquinone in the mitochondria and xanthine oxidase in the capillary endothelial cells. During high intensity exercise the flow of oxygen through the skeletal muscle cells is greatly increased at the same time as the rate of ATP utilisation exceeds the rate of ATP generation. The metabolic stress in the cells causes several biochemical changes to occur, resulting in a markedly enhanced rate of production of oxygen free radicals from semiquinone and xanthine oxidase. During normal conditions free radicals are generated at a low rate and subsequently taken care of by the well developed scavenger and antioxidant systems. However, a greatly increased rate of free radical production may exceed the capacity of the cellular defence system. Consequently, a substantial attack of free radicals on the cell membranes may lead to a loss of cell viability and to cell necrosis and could initiate the skeletal muscle damage and inflammation caused by exhaustive exercise.

Cloning and functional analysis of multiply spliced mRNA species of human immunodeficiency virus type 1.

Abstract: We have used the polymerase chain reaction technique to clone the small multiply spliced mRNA species produced after infection of human cells by a molecular clone of human immunodeficiency virus type 1 (HIV-1). We identified six Rev-expressing mRNAs, which were generated by the use of two splice acceptors located immediately upstream of the rev AUG. The class of small mRNAs included 12 mRNAs expressing Tat, Rev, and Nef. In addition, HIV-1 produced other multiply spliced mRNAs that used alternative splice sites identified by cloning and sequencing. All of these mRNAs were found in the cytoplasm and should be able to produce additional proteins. The coding capacity of the tat, rev, and nef mRNAs was analyzed by transfection of the cloned cDNAs into human cells. The tat mRNAs produced high levels of Tat, but very low levels of Rev and Nef. All the rev mRNAs expressed high levels of both Rev and Nef and were essential for the production of sufficient amounts of Rev. Therefore, HIV-1 uses both alternatively spliced and bicistronic mRNAs for the production of Tat, Rev, and Nef proteins.

Muscle activity and coordination in the normal shoulder : an electromyographic study

Abstract: Muscle activity and coordination in ten shoulders were studied in five healthy subjects using electromyography (EMG) recorded during standardized loaded movements, i.e., flexion, extension, abduction, external rotation, and internal rotation at 0 degrees, 45 degrees, and 90 degrees of abduction. Bipolar surface and intramuscular fine-wire electrodes were used, and the EMG signal was low-pass filtered, full-wave rectified, and time-averaged. Activity from the subscapularis, supraspinatus, infraspinatus, pectoralis major (sternoclavicular part), the anterior, middle, and posterior parts of the deltoid, and the latissimus dorsi was recorded in parallel. In order to allow a comparison of the activity in a subject's different muscles and the activity in specific muscles between different individuals, the EMG was normalized. Muscle activity occurred simultaneously in muscles producing the movement and in antagonistic muscles. Coordination due to muscle contractions plays a significant role in stabilizing the shoulder joint. The infraspinatus, subscapularis, and latissimus dorsi acted as stabilizers during flexion; the subscapularis acted as a stabilizer during external rotation and with the supraspinatus during extension.

Motor learning in man: a positron emission tomographic study

Abstract: We measured regional cerebral blood flow (rCBF) with positron emission tomography to study changes in anatomical structures during the course of learning a complicated finger sequence of voluntary movements. Motor learning was accompanied by rCBF increases in the cerebellum, decreases in all limbic and paralimbic structures, and striatal decreases which changed to striatal increases as the motor skill was learned. Simultaneously, activations of initially contributing non-motor parts of the cerebral cortex vanished. Both cerebellar circuits and striatal circuits appear important for the storage of motor skills in the brain.

Autoreactive T lymphocytes in multiple sclerosis determined by antigen-induced secretion of interferon-gamma.

Abstract: Multiple sclerosis (MS) is a disease with unknown cause characterized by inflammation and demyelination in the central nervous system. Although an autoimmune pathogenesis has been suggested, there are no conclusive data on the number of T cells autoreactive with myelin antigens in MS compared to controls. We showed that T lymphocytes secreting interferon-gamma in response to possible target autoantigens are severalfold more common among PBL mononuclear cells in patients with MS than in patients with aseptic meningitis and tension headache. On average T cells reactive with myelin basic protein (MBP), two different MBP peptides, or with proteolipid protein amounted to 2.7-5.2/10(5) PBL from MS patients. MBP-reactive T cells were still more frequent among mononuclear cells isolated from the cerebrospinal fluid (CSF; 185/10(5) CSF cells). We concluded that T cells reactive with myelin autoantigens are strongly increased in MS. This approach to detect them could allow definition of immunodominant T cell epitopes in individual MS patients, and thereby enable further development towards specific immunotherapy.

Oxidized low density lipoprotein induces differentiation and adhesion of human monocytes and the monocytic cell line U937.

Abstract: Hypercholesterolemia is a major risk factor for development of atherosclerosis. In experimental animals fed a high-cholesterol diet, monocytes adhere to the arterial endothelium and penetrate into the intima where they differentiate into macrophages and ingest lipids thus giving rise to fatty streaks, the earliest type of atherosclerotic plaque. Macrophages express few receptors for normal low density lipoprotein (LDL) but can take up oxidized LDL by way of a scavenger receptor. The present study was designed to investigate the possible role of oxidized LDL in recruitment of resident intimal macrophages. We found that oxidized LDL induced enhanced expression of major histocompatibility complex class II molecules on human monocytes and U937 cells, a well-established system for studies of monocytic differentiation. Oxidized LDL also induced enhanced expression of the surface antigen LeuM3 but caused decreased expression of CD4 antigen, a pattern compatible with expression of a more-differentiated macrophage-like phenotype. Oxidized LDL also initiated aggregation of monocytes and U937 cells and stimulated adhesion of U937 cells to cultured endothelial cells. The results indicate that oxidized LDL may contribute to development of atherosclerosis by inducing adhesion of monocytes to the arterial intima and by stimulating intimal monocytes to differentiate into resident macrophages.

Cleavage-site motifs in mitochondrial targeting peptides.

Abstract: Although mitochondrial targeting peptides lack a common consensus sequence, a certain bias in the positional distribution of amino acids has recently been found These patterns seem to be associated with cleavage of the precursor proteins by matrix processing proteases We have extended the previous studies and found new sequence motifs that are conserved within subgroups of mitochondrial targeting peptides These motifs have certain common themes, indicating that they are associated with cleavage by one single protease Two of the conserved patterns have a high predictive value, but even for sequences that do not possess these patterns, a fairly accurate prediction of the cleavage site is shown to be possible We also suggest that a well-conserved RXY decreases (S/A) pattern may be used to engineer efficiently recognized cleavage sites into uncleaved or artificial mitochondrial targeting peptides

Psychological and psychophysiological effects of shift work

Abstract: The psychophysiology of shift work is mainly related to circadian rhythmicity and sleep-wake phenomena. Individuals on a rotating three-shift or similar system work the night shift at the low phase of circadian rhythm. On retiring to bed in the morning they fall asleep rapidly but are prematurely awakened by their circadian rhythm and exhibit severe sleepiness and reduced performance capacity. In connection with the morning shift the circadian psychophysiology makes it difficult to fall asleep as early as needed during the preceding night. Around 0400 to 0500, when the individuals should rise, they have difficulties awakening because of the sleep loss and the circadian rhythm, which at that point is at its lowest. Subsequently, day work is characterized by sleepiness and reduced performance. It should be emphasized that it does not seem possible to improve one's ability to adjust over time, even with permanent night work. Older age and "morningness" personality are related to higher than average problems in adjusting.

The role of the delta-lysin gene (hld) in the regulation of virulence genes by the accessory gene regulator (agr) in Staphylococcus aureus.

Abstract: The synthesis of at least 14 extracellular toxins and enzymes in Staphylococcus aureus is regulated by a set of trans-acting elements from the agr (accessory gene regulator) locus. We have shown that the delta-lysin gene (hld) that is transcribed from a promoter immediately upstream of the agr locus, and which is positively controlled by agr, is part of this regulatory system. Deletion replacement mutagenesis of the chromosomal hld gene had the same pleiotropic effect on the synthesis of several virulence factors as agrA mutations. Characteristically, these mutants had an almost complete block in the synthesis of alpha-toxin, serin- and metalloprotease, whereas synthesis of protein A was greater than 10-fold higher than in the parental strain. Corresponding changes in the levels of alpha-toxin and protein A mRNAs were demonstrated by northern blotting experiments. The effects of the hld deletion mutation could be fully complemented by the hld gene on a plasmid. A plasmid insertion mutation in the 3' non-coding region of hld had a similar effect on exoprotein synthesis, indicating a role of the hld transcript in the regulation of exoprotein synthesis. This was confirmed by the finding that the effects of alpha-toxin and protein A synthesis by the hld deletion replacement mutation could be fully complemented by a hld allele in which we had introduced an early stop codon in the delta-lysin structural gene. However, the mutant hld allele could not complement the defect in production of extracellular proteases, indicating that delta-lysin may act in conjunction with its mRNA to regulate the expression of some exoprotein genes.

Force and EMG signal patterns during repeated bouts of concentric or eccentric muscle actions

Abstract: Healthy males (n = 14) performed three bouts of 32 unilateral, maximal voluntary concentric (CON) or eccentric (ECC) quadriceps muscle actions on separate days. Surface electromyography (EMG) of the m. vastus lateralis (VL) and m. rectus femoris (RF) and torque were measured. Integrated EMG (IEMG), mean (MPF) and median power frequencies and torque were averaged for seven separate blocks of four consecutive muscle actions. Torque was greater (P less than 0.05) for ECC than for CON muscle actions at the start of exercise. It did not decline throughout ECC exercise, but decreased (P less than 0.05) markedly for each bout and over bouts of CON exercise. Thus, torque overall was substantially greater (P less than 0.05) for ECC than for CON exercise. At the start of exercise IEMG of VL or RF was greater (P less than 0.05) for CON than for ECC muscle actions. This was also true for overall IEMG activity during exercise. The IEMG increased (P less than 0.05) modestly for both muscles during each bout of CON or ECC muscle actions, but did not change for the VL over bouts. The IEMG of RF decreased (P less than 0.05) modestly over CON but not ECC exercise bouts. At the beginning of the first bout of exercise the IEMG/torque ratio was twofold greater (P less than 0.05) for CON than ECC muscle actions. The ratio of IEMG/torque increased (P less than 0.05) markedly during CON but did not change during ECC exercise. Thus, by the end of the third bout there was a fivefold difference (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Contrasting developmental and tissue-specific expression of alpha and beta thyroid hormone receptor genes.

Abstract: Thyroid hormones and their receptors (TRs) have critical functions in development. Here we show that a chicken TR beta cDNA clone encodes a receptor with a novel, short N-terminal domain. In vitro-expressed TR beta protein bound thyroid hormone with similar affinity as the chicken TR alpha. Comparison of expression of TR alpha and TR beta mRNAs throughout chicken development until 3 weeks post-hatching revealed ubiquitous expression of TR alpha mRNAs (in 14 different tissues) with some variations in levels, from early embryonic stages. In contast, expression of TR beta mRNA was restricted, occurring notably in brain, eye, lung, yolk sac and kidney, and was subject to striking developmental control, especially in brain where levels increased 30-fold upon hatching. Levels also sharply increased in late embryonic lung, but were relatively high earlier in embryonic eye and yolk sac. RNase protection analyses detected no obvious mRNAs for alpha and beta TRs with variant C-termini as demonstrated previously for the rat TR alpha gene. The data suggest a general role for TR alpha and specific developmental functions for TR beta, and that thyroid-dependent development involves temporal and tissue-specific expression of the TR beta gene.