Translational repression of E2F1 mRNA in carcinoma in situ and normal testis correlates with expression of the miR-17-92 cluster.
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Animals↗
Biomedical subjects
J E Nielsen
Publications and source records attributed to J E Nielsen.
MEP recruitment curves in multiple sclerosis and hereditary spastic paraplegia.
OBJECTIVE: Axons remodel at multiple levels after a single inflammatory lesion in the spinal cord, which can contribute to recovery. The primary aim of this study was to investigate whether the MEP response as function of the excitatory strength, here called recruitment curves, may be used in discriminating demyelination from compensated axonal loss. Multiple sclerosis (MS) represents both demyelination and axonal degeneration. Hereditary Spastic Paraplegia (HSP) was included as a model of pure axonal loss. METHODS: To investigate both spinal and cortical recruitment, the methods used for gradual recruitment were two different test paradigms of voluntary pre-activation and stimulus intensity. The MEP-recruitment curves were obtained by means of transcranial magnetic stimulation (TMS) in 29 MS patients, 9 patients with HSP and in 30 healthy controls. RESULTS: Saturated recruitment curves were obtained in all subject groups, muscles and paradigms and were generally found to be identical. The two groups of patients had clinical signs, CMCT changes and reduced MEP amplitude reflecting relevant cortico-spinal disorder. CONCLUSIONS: We conclude that both demyelination and axonal degeneration in the CNS leads to diminished MEP amplitudes and CMCT changes. The recruitment curves of MS and HSP was identical to controls and may not be used for diagnostic or monitoring purposes.
Adult↗
Genome-wide gene expression profiling of testicular carcinoma in situ progression into overt tumours.
The carcinoma in situ (CIS) cell is the common precursor of nearly all testicular germ cell tumours (TGCT). In a previous study, we examined the gene expression profile of CIS cells and found many features common to embryonic stem cells indicating that initiation of neoplastic transformation into CIS occurs early during foetal life. Progression into an overt tumour, however, typically first happens after puberty, where CIS cells transform into either a seminoma (SEM) or a nonseminoma (N-SEM). Here, we have compared the genome-wide gene expression of CIS cells to that of testicular SEM and a sample containing a mixture of N-SEM components, and analyse the data together with the previously published data on CIS. Genes showing expression in the SEM or N-SEM were selected, in order to identify gene expression markers associated with the progression of CIS cells. The identified markers were verified by reverse transcriptase-polymerase chain reaction and in situ hybridisation in a range of different TGCT samples. Verification showed some interpatient variation, but combined analysis of a range of the identified markers may discriminate TGCT samples as SEMs or N-SEMs. Of particular interest, we found that both DNMT3B (DNA (cytosine-5-)-methyltransferase 3 beta) and DNMT3L (DNA (cytosine-5-)-methyltransferase 3 like) were overexpressed in the N-SEMs, indicating the epigenetic differences between N-SEMs and classical SEM.
Carcinoma in Situ↗
Stem cell pluripotency factor NANOG is expressed in human fetal gonocytes, testicular carcinoma in situ and germ cell tumours.
AIMS: NANOG is a key regulator of embryonic stem cell (ESC) self-renewal and pluripotency. Our recent genome-wide gene expression profiling study of the precursor of testicular germ cell tumours, carcinoma in situ testis (CIS), showed close similarity between ESC and CIS, including high NANOG expression. In the present study we analysed the protein expression of NANOG during normal development of human testis and in a large series of neoplastic/dysgenetic specimens. METHODS AND RESULTS: We detected abundant expression of NANOG in CIS and in CIS-derived testicular tumours with marked differences; seminoma and embryonal carcinoma were strongly positive, differentiated somatic elements of teratoma were negative. We provide evidence for the fetal origin of testicular cancer as we detected strong expression of NANOG in fetal gonocytes up to gestational week 20, with subsequent down-regulation occurring earlier than for OCT-4. We detected no expression at the protein level in normal testis. CONCLUSIONS: NANOG is a new marker for testicular CIS and germ cell tumours and the high level of NANOG along with OCT-4 are determinants of the stem cell-like pluripotency of the preinvasive CIS cell. Timing of NANOG down-regulation in fetal gonocytes suggests that NANOG may act as a regulatory factor up-stream to OCT-4.
Adolescent↗
Reciprocal inhibition and corticospinal transmission in the arm and leg in patients with autosomal dominant pure spastic paraparesis (ADPSP).
The pathophysiological mechanisms underlying the development of spasticity are not clear, but the excitability of the disynaptic reciprocal inhibitory pathway is affected in many patients with spasticity of different origin. Patients with genetically identified autosomal dominant pure spastic paraparesis (ADPSP) develop spasticity and paresis in the legs, but usually have no symptoms in the arms. Comparison of the spinal and supraspinal control of the legs and arms in these patients may therefore provide valuable information about the pathophysiology of spasticity. In the present study, we tested the hypothesis that one of the pathophysiological mechanisms of spasticity in these patients is abnormal corticospinal transmission and that this may lead to decreased reciprocal inhibition. Ten patients and 15 healthy age-matched control subjects were investigated. The patients were all spastic in the legs (with hyperactive tendon reflexes, increased muscle tone and Babinski sign), but had no neurological symptoms in the arms (except for one patient). Disynaptic reciprocal Ia inhibition of flexor carpi radialis (FCR) and soleus (SOL) motoneurons was measured (as the depression of the background FCR and SOL EMG activity and as the short latency inhibition of the FCR and SOL H-reflex evoked by radial and peroneal nerve stimulation). In addition, the latency of motor evoked potentials (MEPs) in the FCR muscle and the tibialis anterior (TA) muscle was measured. In the patients, the mean reciprocal inhibition was normal in the arms, while it was significantly decreased in the leg compared with the healthy subjects. In the patients, the average latency of MEPs in the FCR muscle was normal, while the latency to the MEP in TA muscle was significantly longer than that found in healthy subjects. Four patients, however, differed from the other patients by having significant reciprocal inhibition in the leg and a significantly shorter latency of TA MEPs than found in the other patients. The six patients without reciprocal inhibition in the leg instead had significant short latency facilitation of the SOL H-reflex and a longer TA MEP latency than seen in the healthy subjects and in the four patients with retained reciprocal inhibition. These findings support the hypothesis that disynaptic reciprocal inhibition and short latency facilitation are involved in the development of spasticity and, furthermore, they suggest a positive correlation between impairment of corticospinal transmission and decrease of reciprocal inhibition/appearance of reciprocal facilitation.
Adult↗
Identification of genes differentially expressed in testes containing carcinoma in situ.
Virtually all testicular germ cell tumours originate from a common precursor, the carcinoma in situ (CIS) cell. The precise nature of the molecular mechanisms leading to CIS remains largely unknown. We performed the first systematic analysis of gene expression in testis with CIS compared to normal testis by the differential display (DDRT-PCR) method, with subsequent analysis by RT-PCR and in situ hybridization (ISH). In tissue containing CIS we identified overexpression of 28 mRNA, some previously reported in CIS and a number of genes not previously described in germ cell neoplasia, including the novel expressed sequence tag (EST) OIC1 (Overexpressed In CIS). The genes could be grouped functionally into genes involved in cell growth, proliferation, differentiation, immunological response, and genes with unknown biological function. Examples of overexpressed genes are SFRP1 that is involved in Wnt signalling and IGFBP6, which is of importance for fetal growth and inhibits cell growth through insulin-like growth factor-II. ISH analysis showed that both mRNA were localized to CIS cells. The results of our search for differentially expressed genes in CIS demonstrated a number of genes linked to testicular development (e.g. DCN, IGFBP6, SFRP1, SALL1), supporting our hypothesis that the origin of CIS is probably associated with disturbances of the fetal development of the testis.
Adolescent↗
Hereditary spastic paraplegia with cerebellar ataxia: a complex phenotype associated with a new SPG4 gene mutation.
Complex forms of hereditary spastic paraplegia (HSP) are rare and usually transmitted in an autosomal recessive pattern. A family of four generations with autosomal dominant hereditary spastic paraplegia (AD-HSP) and a complex phenotype with variably expressed co-existing ataxia, dysarthria, unipolar depression, epilepsy, migraine, and cognitive impairment was investigated. Genetic linkage analysis and sequencing of the SPG4 gene was performed and electrophysiologic investigations were carried out in six individuals and positron emission tomography (PET) in one patient. The disease was linked to the SPG4 locus on chromosome 2p as previously reported for pure HSP. Sequence analysis of the SPG4 (spastin) gene identified a novel 1593 C > T (GLN490Stop) mutation leading to premature termination of exon 12 with ensuing truncation of the encoded protein. However, the mutation was only identified in those individuals who were clinically affected by a complex phenotype consisting of HSP and cerebellar ataxia. Other features noted in this kindred including epilepsy, cognitive impairment, depression, and migraine did not segregate with the HSP phenotype or mutation, and therefore the significance of these features to SPG4 is unclear. Electrophysiologic investigation showed increased central conduction time at somatosensory evoked potentials measured from the lower limbs as the only abnormal finding in two affected individuals with the SPG4 mutation. Moreover, PET of one patient showed significantly relatively decreased regional cerebral blood flow in most of the cerebellum. We conclude that this kindred demonstrates a considerable overlap between cerebellar ataxia and spastic paraplegia, emphasizing the marked clinical heterogeneity of HSP associated with spastin mutations.
Adenosine Triphosphatases↗
Germ cell differentiation-dependent and stage-specific expression of LANCL1 in rodent testis.
LANCL1 (LanC-like protein 1) is related to the bacterial LanC (lanthionine synthetase C) family, which is involved in the biosynthesis of antimicrobial peptides. Highest expression levels of LANCL1 are found in testes and brain, two organs that exist behind blood-tissue barriers. In the mouse, the establishment of an impermeable blood-testis barrier occurs between day 10-16 post natal (pn). Differential display analysis showed that the expression level of LANCL1 mRNA in mouse testes was very low until day 16 pn, but increased gradually from day 16 pn to reach a maximum on days 22-24 pn followed by a slight reduction from day 26 pn to adult animals. Thus, the expression of LANCL1 mRNA is initiated following the establishment of the blood-testis barrier. In situ hybridisation revealed that LANCL1 mRNA was induced in diplotene spermatocytes, which appear for the first time in mouse testes between days 18 and 20 pn, verifying the expression profile determined by differential display. LANCL1 mRNA level remained high in the meiotic division phase and in early round spermatids, but was down regulated in elongating spermatids and it was undetectable in step 9 elongating spermatids in stage IX (as defined by Russel et al., 1990). The steady decrease in expression level from round spermatids in stage I to elongating spermatids in stage IX suggested that LANCL1 mRNA was not transcribed in spermatids. LANCL1 expression in rat testes was initiated already in pachytene spermatocytes in stage IX, but otherwise similar to mouse.
Animals↗
Possible autocrine loop of the epidermal growth factor system in patients with benign prostatic hyperplasia treated with finasteride: a placebo-controlled randomized study.
OBJECTIVE: To analyse the expression of the epidermal growth factor (EGF) system in prostate tissue and secretions obtained from patients with benign prostatic hyperplasia (BPH) treated with or without finasteride (which primarily targets the androgen-sensitive secretory epithelial cells in the prostate, with little effect on basal epithelial and stromal cells). PATIENTS AND METHODS: The expression of the EGF system was evaluated by enzyme-linked immunosorbent assay and immunohistochemistry in samples of prostate tissue and secretions from patients with BPH randomized for treatment with finasteride or placebo for 3 months before surgery. RESULTS: Prostate tissue expressed the EGF receptor (HER1) and HER2, and the ligands EGF, transforming growth factor alpha (TGFalpha), heparin-binding (HB) EGF, betacellulin and amphiregulin. Treatment with finasteride produced greater concentrations of amphiregulin (P < 0.05) than did placebo, did not change the level of TGFalpha, HER1 and HER2, and tended to decrease the concentration of EGF, betacellulin and HB-EGF in prostate tissue. Using immunohistochemistry, HER1 and TGFalpha were both localized to the basal epithelial cells, and there was a strong positive correlation among the tissue concentrations of HER1, HER2 and TGFalpha. Amphiregulin localized to the luminal secretory epithelium. Prostate secretions contained only EGF, which was at levels approximately 150 times higher than in prostate tissue; treatment with finasteride did not affect the concentration of EGF in prostate secretion. CONCLUSIONS: There were only minor changes in the expression of TGFalpha, HER1 and HER2 after finasteride treatment. This may represent an important system for the continuous growth and homeostasis of the androgen-independent basal epithelial cells in the prostate.
Amphiregulin↗
Effects of finasteride on vascular endothelial growth factor.
OBJECTIVE: Finasteride has been shown to reduce prostate bleeding in patients with benign prostatic hyperplasia (BPH). The mechanisms behind this are not known, but it has been suggested that finasteride reduces bleeding by inhibiting angiogenesis in the prostate. Studies in animals have shown that castration rapidly induces involution of the prostate vasculature, and androgen-stimulated prostate growth may be angiogenesis dependent. The objective of this study was to explore the response to finasteride on the vasculature and the expression of vascular endothelial growth factor (VEGF), a potent regulatory factor of angiogenesis in human prostate tissue. MATERIAL AND METHODS: Patients with BPH were randomly assigned to 3 months of treatment either with finasteride (5 mg/day) or placebo before undergoing transurethral resection of the prostate (TURP). Prostate tissue VEGF expression was quantified by Western blot and the vascular density determined in Factor VIII immunostained tissue sections. Serum concentrations of VEGF were measured with ELISA technique. RESULTS: Patients treated with finasteride (n = 15) showed a decrease in prostate tissue VEGF(165) expression compared with placebo (n = 13) treated patients (p < 0.05), but the vascular density and the serum VEGF levels were unaffected. CONCLUSIONS: This study shows that finasteride treatment decreases VEGF expression in the human prostate.
5-alpha Reductase Inhibitors↗
Dissecting the electrostatic interactions and pH-dependent activity of a family 11 glycosidase.
Previous studies of the low molecular mass family 11 xylanase from Bacillus circulans show that the ionization state of the nucleophile (Glu78, pK(a) 4.6) and the acid/base catalyst (Glu172, pK(a) 6.7) gives rise to its pH-dependent activity profile. Inspection of the crystal structure of BCX reveals that Glu78 and Glu172 are in very similar environments and are surrounded by several chemically equivalent and highly conserved active site residues. Hence, there are no obvious reasons why their apparent pK(a) values are different. To address this question, a mutagenic approach was implemented to determine what features establish the pK(a) values (measured directly by (13)C NMR and indirectly by pH-dependent activity profiles) of these two catalytic carboxylic acids. Analysis of several BCX variants indicates that the ionized form of Glu78 is preferentially stabilized over that of Glu172 in part by stronger hydrogen bonds contributed by two well-ordered residues, namely, Tyr69 and Gln127. In addition, theoretical pK(a) calculations show that Glu78 has a lower pK(a) value than Glu172 due to a smaller desolvation energy and more favorable background interactions with permanent partial charges and ionizable groups within the protein. The pK(a) value of Glu172 is in turn elevated due to electrostatic repulsion from the negatively charged glutamate at position 78. The results also indicate that all of the conserved active site residues act concertedly in establishing the pK(a) values of Glu78 and Glu172, with no particular residue being singly more important than any of the others. In general, residues that contribute positive charges and hydrogen bonds serve to lower the pK(a) values of Glu78 and Glu172. The degree to which a hydrogen bond lowers a pK(a) value is largely dependent on the length of the hydrogen bond (shorter bonds lower pK(a) values more) and the chemical nature of the donor (COOH > OH > CONH(2)). In contrast, neighboring carboxyl groups can either lower or raise the pK(a) values of the catalytic glutamic acids depending upon the electrostatic linkage of the ionization constants of the residues involved in the interaction. While the pH optimum of BCX can be shifted from -1.1 to +0.6 pH units by mutating neighboring residues within the active site, activity is usually compromised due to the loss of important ground and/or transition state interactions. These results suggest that the pH optima of an enzyme might be best engineered by making strategic amino acid substitutions, at positions outside of the "core" active site, that electrostatically influence catalytic residues without perturbing their immediate structural environment.
Amino Acid Sequence↗
Optimizing the hydrogen-bond network in Poisson-Boltzmann equation-based pK(a) calculations.
pK(a) calculation methods that are based on finite difference solutions to the Poisson-Boltzmann equation (FDPB) require that energy calculations be performed for a large number of different protonation states of the protein. Normally, the differences between these protonation states are modeled by changing the charges on a few atoms, sometimes the differences are modeled by adding or removing hydrogens, and in a few cases the positions of these hydrogens are optimized locally. We present an FDPB-based pK(a) calculation method in which the hydrogen-bond network is globally optimized for every single protonation state used. This global optimization gives a significant improvement in the accuracy of calculated pK(a) values, especially for buried residues. It is also shown that large errors in calculated pK(a) values are often due to structural artifacts induced by crystal packing. Optimization of the force fields and parameters used in pK(a) calculations should therefore be performed with X-ray structures that are corrected for crystal artifacts.
Amino Acid Sequence↗
Increased intracortical facilitation in patients with autosomal dominant pure spastic paraplegia linked to chromosome 2p.
There are at least seven clinically indistinguishable but genetically different types of autosomal dominant pure spastic paraplegia (ADPSP). In this study we investigated electrophysiological characteristics in patients with ADPSP linked to chromosome 2p (SPG4). Twelve patients from six different families with ADPSP linked to chromosome 2p and 15 control persons were included. Electromyography (EMG), motor and sensory nerve conduction, and motor evoked potentials using single and paired transcranial magnetic stimulation (PTMS) was performed. From the peripheral nervous system we found signs of motor and sensory axonal neuropathy. Motor evoked potentials disclosed greatly reduced corticospinal tract conduction velocity and amplitude of evoked potentials to the lower extremities indicating that the very marked spasticity predominantly seems to rely on dysfunction of the fast conducting axons of the pyramidal tract. PTMS showed an increased intracortical facilitation (ICF), which may reflect an impaired function of gamma-aminobutyric acid (GABA)-controlled interneuronal circuits in the motor cortex, alternatively an increased glutamatergic transmission or a compensatory recruitment of a larger number of neurones with corticospinal projections.
Adult↗
The determinants of alpha-amylase pH-activity profiles.
The glycosyl hydrolases present a large family of enzymes that are of great significance for industry. Consequently, there is considerable interest in engineering the enzymes in this family for optimal performance under a range of very diverse conditions. Until recently, tailoring glycosyl hydrolases for specific industrial processes mainly involved stability engineering, but lately there has also been considerable interest in engineering their pH-activity profiles. We mutated four neutral residues (N190, F290, N326 and Q360) in the chimeric Bacillus Ba2 alpha-amylase to both charged and neutral amino acids. The results show that the pH-activity profile of the Ba2 alpha-amylase can be changed by inserting charged residues close to the active site. The changes in the pH-activity profile for these neutral --> charged mutations do not, however, correlate with the predictions from calculations of the p K(a) values of the active site residues. More surprisingly, the neutral --> neutral mutations change the pH-activity profile as much as the neutral --> charged mutations. From these results, it is concluded that factors other than electrostatics, presumably the dynamic aspects of the active site, are important for the shape of the pH-activity profiles of the alpha-amylases.
Amino Acids↗
Motor evoked potentials from the external anal sphincter in patients with autosomal dominant pure spastic paraplegia linked to chromosome 2p.
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Adult↗
Protein engineering of bacterial alpha-amylases.
alpha-Amylases constitute a very diverse family of glycosyl hydrolases that cleave alpha1-->4 linkages in amylose and related polymers. Recent structural and mutagenic studies of archeael, mammalian and bacterial alpha-amylases have resulted in a wealth of information on the catalytic mechanism and on the structural features of this enzyme class. Because of their high thermo-stability, the Bacillus alpha-amylases have found widespread use in industrial processes, and much attention has been devoted to optimising these enzymes for the very harsh conditions encountered there. Stability has been a major area of focus in this respect, and several remarkably stable bacterial alpha-amylases have been produced by bioengineering techniques. Protein engineering studies of pH-activity profiles and of substrate specificities have also been initiated, although without much success. In the coming years it is likely, however, that the focus of alpha-amylase engineering will shift from engineering stability to these new areas.
Bacillus↗
[Prednisolone treatment of respiratory syncytial virus infection. A randomized, controlled trial of 147 children].
OBJECTS: Our objective was to evaluate the effect of systemic prednisolone as an adjunct to conventional treatment with beta 2-agonist, fluid replacement and respiratory support in hospitalized infants younger than 24 months with respiratory syncytial virus (RSV) infection. METHODS: The study was randomized, double-blind and placebo-controlled. During the winter of 1995-96, 147 infants less than two years of age hospitalized with RSV infection were allocated to treatment with either systemic prednisolone mixture 2 mg/kg daily or placebo for 5 days. RESULTS: Our main outcome measures were: 1. Acute effect variables: duration of stay in hospital, use of medicine and supportive measures while in hospital. 2. At follow-up one month after discharge: duration of illness, start in day care center, morbidity and use of medicine. 3. At follow-up one year after discharge: morbidity, use of medicine and skin prick tests with allergens. CONCLUSION: Prednisolone treatment had no effect on any of the outcome measures. We find our results in agreement with the largest studies reported earlier; therefore, corticosteroid, whether by systemic route or by inhalation, should not be prescribed to infants with RSV infection.
Anti-Inflammatory Agents↗
Synchronization of lower limb motor units in spastic patients.
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Electromyography↗