Human histone H1 variants impact splicing outcome by controlling RNA polymerase II elongation.

Histones shape chromatin structure and the epigenetic landscape. H1, the most diverse histone in the human genome, has 11 variants. Due to the high structural similarity between the H1s, their unique functions in transferring information from the chromatin to mRNA-processing machineries have remained elusive. Here, we generated human cell lines lacking up to five H1 subtypes, allowing us to characterize the genomic binding profiles of six H1 variants. Most H1s bind to specific sites, and binding depends on multiple factors, including GC content. The highly expressed H1.2 has a high affinity for exons, whereas H1.3 binds intronic sequences. H1s are major splicing regulators, especially of exon skipping and intron retention events, through their effects on the elongation of RNA polymerase II (RNAPII). Thus, H1 variants determine splicing fate by modulating RNAPII elongation.

Programmable fusion of liposomes mediated by lipidated PNA.

We recently reported a DNA-programmed fusion cascade enabling the use of liposomes as nanoreactors for compartmentalized chemical reactions. This communication reports an alternative and robust strategy based on lipidated peptide nucleic acids (LiPs). LiPs enabled fusion of liposomes with remarkable 31% efficiency at 50 °C with low leakage (5%).

Efficient liposome fusion mediated by lipid-nucleic acid conjugates.

The fusion of biomembranes with release of encapsulated content in a controlled way is crucial for cell signaling, endo- and exocytosis and intracellular trafficking. Programmable fusion of liposomes and an efficient mixing of their contents have the potential to enable the study of chemical and enzymatic processes in a confined environment and under crowded conditions outside biological systems. We report on DNA-controlled fusion of lipid bilayer membranes using lipid-nucleic acid conjugates (LiNAs) to mediate lipid and content mixing of liposomes. Screening of different membrane anchor and linker structures as well as incubation temperatures led to significantly improved fusion and content mixing compared to reported systems. LiNA designs were optimized by changing lipophilic moieties as membrane anchors, PEG-spacer patterns and by introducing locked nucleic acid (LNA) modifications. Liposome fusion induced by complementary LiNAs results in remarkable efficient content mixing at 37 °C and 50 °C (up to 70%) with low leakage (≤5%).

Form of dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A nonphosphorylated at tyrosine 145 and 147 is enriched in the nuclei of astroglial cells, adult hippocampal progenitors, and some cholinergic axon terminals.

Compelling lines of evidence indicate that overexpression of dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) in subjects with trisomy 21 (Down syndrome[DS]) contributes to the abnormal structure and function of the DS brain. In the present study, we used a novel, phospho-dependent antibody recognizing DYRK1A only with nonphosphorylated tyrosine 145 and 147 (DYRK1A Tyr-145/147P(-)), to investigate the expression pattern of this DYRK1A species in trisomic and disomic human and mouse brains. Immunoblotting and dephosphorylation experiments demonstrated higher levels of DYRK1A Tyr-145/147P(-) in postnatal trisomic brains in comparison with controls (by ∼40%) than those of the DYRK1A visualized by three other N- and C-terminally directed antibodies to DYRK1A. By immunofluorescence, the immunoreactivity to DYRK1A Tyr-145/147P(-) was the strongest in the nuclei of astroglial cells, which contrasted with the predominantly neuronal localization of DYRK1A visualized by the three other antibodies to DYRK1A we used. In addition, DYRK1A Tyr-145/147P(-) was enriched in the nuclei of neuronal progenitors and newly born neurons in the adult hippocampal proliferative zone and also occurred in some cholinergic axonal terminals. Our data show a distinctive expression pattern of DYRK1A forms nonphosphorylated at Tyr-145 and Tyr-147 in the brain tissue and suggest that DS subjects may exhibit not only upregulation of total DYRK1A, but also more subtle differences in phosphorylation levels of this kinase in comparison with control individuals.

Use of iron sucrose in dialysis patients sensitive to iron dextran.

This study aimed to assess the safety and efficacy of iron sucrose in hemodialysis (HD) patients with documented hypersensitivity reactions to iron dextran. Of 205 HD patients who received low molecular weight iron dextran, 15 (7.3%) patients developed documented hypersensitivity reactions. The patients were treated with iron sucrose (100 mg administered as an intravenous push over 5-10 minutes once a week) for 8 weeks. Complete blood count, serum iron, serum ferritin, and parathyroid hormone were measured at the beginning and at the end of the study (except parathyroid hormone). All patients received subcutaneous erythropoietin at a constant dose of 5000 IU twice weekly unless a change was required. All the patients completed the study period and none of them developed hypersensitivity reactions to iron sucrose. The mean hematocrit increased from 23.8% to 32.27% (p < 0.0001), the mean serum ferritin from 185 ng/mL to 599 ng/mL (p < 0.0001), and the mean serum iron from 29.3 ng/dL to 76.8 ng/dL (p = 0.01). We conclude that iron sucrose is safe and effective in HD patients with documented hypersensitivity reactions to low molecular weight iron dextran.

Abnormal expression of synaptic proteins and neurotrophin-3 in the Down syndrome mouse model Ts65Dn.

Down syndrome (DS) results from triplication of the whole or distal part of human chromosome 21. Persons with DS suffer from deficits in learning and memory and cognitive functions in general, and, starting from early development, their brains show dendritic and spine structural alterations and cell loss. These defects concern many cortical brain regions as well as the hippocampus, which is known to play a critical role in memory and cognition. Most of these abnormalities are reproduced in the mouse model Ts65Dn, which is partially trisomic for the mouse chromosome 16 that is homologous to a portion of human chromosome 21. Thus, Ts65Dn is widely utilized as an animal model of DS. To better understand the molecular defects underlying the cognitive and particularly the memory impairments of DS, we investigated whether the expression of several molecules known to play critical roles in long-term synaptic plasticity and long-term memory in a variety of species is dysregulated in either the neonatal brain or adult hippocampus of Ts65Dn mice. We found abnormal expression of the synaptic proteins synaptophysin, microtubule-associated protein 2 (MAP2) and cyclin-dependent kinase 5 (CDK5) and of the neurotrophin-3 (NT-3). Both the neonatal brain and adult hippocampus revealed significant abnormalities. These results suggest that a dysregulation in the expression of neurotrophins as well as proteins involved in synaptic development and plasticity may play a potential role in the neural pathology of DS in humans.

Immunogold study of effects of prenatal exposure to lipopolysaccharide and/or valproic acid on the rat blood-brain barrier vessels.

The involvement of blood microvessels, representing the anatomic site of the blood-brain barrier (BBB), in brain damage induced by prenatal exposure to lipopolysaccharide (LPS) and/or valproic acid (VPA) was studied in four-week-old rats. The immunogold procedure was applied for localization at the ultrastructural level of endogenous albumin and glucose transporter (GLUT-1) in three brain regions: cerebral cortex, cerebellum and hippocampus. Four groups of rats were used: (1) untreated control, (2) prenatally VPA-treated, (3) prenatally LPS-treated, and (4) prenatally LPS- and VPA-treated. The functional state of the BBB was evaluated as follows: (a) by its tightness, i.e., permeability to blood-borne albumin, and (b) by the expression of GLUT-1 in the endothelial cells (ECs). Using morphometry, the labelling density for GLUT-1 was recorded over luminal and abluminal plasma membranes of the ECs, also providing information on their functional polarity. No extensive increase of vascular permeability and/or any considerable dysfunction of the BBB in experimental groups nos. 2 and 3 were observed, although in solitary vascular profiles, increased endocytosis or even transcytosis of albumin by ECs was noted. In experimental group no. 4, some vascular profiles showed scanty leakage (microleakage), manifested by the presence of immunosignals for albumin in the perivascular area. Although some fluctuations in the expression of GLUT-1 occurred in all experimental groups, especially in group no. 3, a most pronounced and significant diminution of the labelling density, in all three regions of the brain, was observed in group no. 4. This finding suggests the synergistic action of prenatally applied LPS and VPA that affects specific transport functions of glucose in the microvascular endothelium. The diminished or disturbed supply of glucose to selected brain regions can be one of the factors leading to previously observed behavioral disturbances in similarly treated rats.

Effectiveness and tolerability of a homoeopathic remedy compared with conventional therapy for mild viral infections.

Treatments for mild viral infections are usually directed at providing symptomatic relief. The effectiveness of the homoeopathic remedy Gripp-Heel was compared with that of conventional treatments in a prospective, observational cohort study in 485 patients with mild viral infections and symptoms such as fever, headache, muscle pain, cough or sore throat. Practitioners specialised in homoeopathy or conventional treatment, or practised both to similar extents. As evaluated by the practitioners, the homoeopathic therapy was effective to similar or greater degree than the conventional therapies: 67.9% of patients were considered asymptomatic at the end of Gripp-Heel therapy vs. 47.9% of patients in the control group. Practitioners judged homoeopathic treatments as 'successful' in 78.1% of cases vs. 52.2% for conventional therapies. Tolerability and compliance were good in both treatment groups, with the verdict 'very good' given for 88.9% of patients in the homoeopathic group vs. 38.8% in the conventional treatment group.

Effect of a single embryonic exposure to alcohol on glucose transporter (GLUT-1) distribution in brain vessels of aged mouse.

Distribution of glucose transporter (GLUT-1) in brain microvascular endothelium, representing the anatomic site of the blood-brain barrier (BBB), was studied with electron microscopy in 24-month-old mice, which had been exposed prenatally (on 9th day of gestation) to a single teratogenic dose of ethanol. Offspring of mice that had received an equivalent volume of isocaloric dextrose served as controls. Sections of brain samples embedded at low temperature in hydrophilic resin Lowicryl K4M were exposed to anti-GLUT-1 antiserum followed by gold-labelled secondary antibodies. By using morphometry, the labelling density was recorded over luminal and abluminal plasma membranes of the endothelial cells of blood microvessels supplying four brain regions: cortex, hippocampus, cerebellum and olfactory bulb. We found that the density of immunosignals for GLUT-1, represented by colloidal gold particles, was unchanged in the olfactory bulb and slightly lowered in the abluminal plasmalemma of the vascular endothelium in the cerebral cortex of the ethanol-treated mice. In contrast, statistical analysis using Mann-Whitney U-test revealed that in the hippocampus and cerebellum, the density of immunolabelling of both plasma membranes of microvascular endothelial cells was significantly lowered in the ethanol-treated mice. These findings suggest that prenatally applied ethanol had a different influence on the vasculature supplying different brain regions. In effect, the inefficient supply of glucose to selected brain regions can be one of the factors leading to the previously observed deficit in long-term memory in a similar alcohol-treated group of mice.

Fmr1 knockout mouse has a distinctive strain-specific learning impairment.

The Fmr1 gene knockout mouse is a model for the human Fragile X mental retardation syndrome. Fmr1 knockout mice with a C57BL/6-129/OlaHsd hybrid background have been reported to have only a very mild deficiency in learning the Morris water maze task. We compared the effect of this knockout mutation on learning in mice with either an FVB/N-129/OlaHsd hybrid background or a C57BL/6 background. When FVB-129 mice were tested in a cross-shaped water maze task, the knockout mice showed a pronounced deficiency in their ability to learn the position of a hidden escape platform in comparison to normal littermates. In contrast, knockout mice with a C57BL/6 background learned the maze just as well as their normal littermates. Fear conditioning did not reveal differences between knockout and normal mice in either background. These results show that silencing the Fmr1 gene clearly interfered with learning a specific visuospatial task in FVB/N-129 hybrid mice but not in C57BL/6 mice. The strain dependence may model the influence of genetic background in the human Fragile X syndrome.

Micromolar concentrations of steroids and of aldosterone antagonists inhibit the outwardly rectifying chloride channel with different kinetics.

We used the patch-clamp technique to analyse the open/close kinetics of single, outwardly rectifying, intermediate-conductance (ORIC) Cl- channels from cultured epithelial cells under control conditions and in presence of different inhibitors. As observed previously in excised inside/out patches under control conditions, the switching kinetics were characterized by one open-state time constant (tau0 is approximately 30 ms) and three closed-state time constants (tau(cl)is approximately = to 0.2 ms, tau(c2) is approximately = 2 ms and tau(c3) is approximately = 60 ms). Aldosterone, six further steroids and two aldosterone antagonists inhibited channel open probability (NPo) concentration dependently with the potency at 10 micromol/l increasing in the sequence: hydrocortisone, corticosterone, P-oestradiol, cortisone, aldosterone, testosterone, progesterone, canrenone, spironolactone. Although all substances decreased tau(o), neither the steroids nor the aldosterone antagonists affected tau(cl), tau(c2) or tau(c3) or induced additional transitions with additional time constants. Instead, the steroids increased the prevalence of tau(c2) in the dwell-time histograms and the aldosterone antagonists increased the prevalence of tau(c3), both in a concentration-dependent manner. These observations may be explained by a model in which one open state leads to one of three closed states with rate constants alpha, beta and gamma, and in which beta or gamma increase under the influence of steroids or aldosterone antagonists, respectively. Cytosol, which contains a Cl- channel inhibitor of unknown molecular structure, (Krick et al., Pflügers Arch 418:491, 1991) was also tested, but the results did not conform to the blocker mechanisms described above. This shows that there are even further modes of channel inhibition and argues against the cytosolic Cl- channel inhibitor being a steroid.

Effects of valproate derivatives II. Antiepileptic efficacy in relation to chemical structures of valproate sugar esters.

The structure effect relationships of derivatives of the antiepileptically active ester of valproate (VPA) 3,4:5,6-Di-O-isopropylidene-1-O-(2-propylpentanoyl)-D-mannitol (1) have been studied using intracellular recording to record the membrane potential of single neurons (buccal ganglia, Helix pomatia). Epileptiform activity was induced by the epileptogenic drug pentylenetetrazol. The effects of several derivatives on epileptiform activity were compared with those of the relay compound 1. Most of the synthesized agents decreased the duration of paroxysmal depolarization shifts (PDS) and increased their repetition rate. It was considered that a decreased the duration of PDS is antiepileptic and an increased repetition rate is pro-epileptic. Compared with the effects of compound 1, the following relationships were found: (1) Derivatives containing glucitol or galactitol were of similar antiepileptic potency. (2) Introduction of pyranoses or furanoses rendered the substances inactive or even pro-epileptic. (3) VPA in position 1 and 6 at the sugar acted as an antiepileptic whereas in position 3 and 4 it proved to be ineffective. (4) Replacement of VPA by ethylhexanoyl reduced the antiepileptic potency slightly and pivaloyl strongly. (5) Replacement of isopropylidene bridges by penta-O-acetyl or cyclohexylidene residues led to largely inactive substances. (6) Compounds having isopropylidene bridges in position 2,4;3,5 proved to be antiepileptic whereas bridges especially in positions 2,3:4,5 slightly enhanced epileptic activities.

Haemophilus influenzae stimulates ICAM-1 expression on respiratory epithelial cells.

Epithelial cells interact directly with bacteria in the environment and play a critical role in airway defense against microbial pathogens. In this study, we examined the response of respiratory epithelial cells to infection with nontypable Haemophilus influenzae. Using an in vitro cell culture model, we found that epithelial cell monolayers released significant quantities of IL-8 and expressed increased levels of ICAM-1 mRNA and surface protein in response to H. influenzae. In contrast, levels of IL-1beta, TNF-alpha, and MHC class I were not significantly affected, suggesting preferential activation of a specific subset of epithelial genes directed toward defense against bacteria. Induction of ICAM-1 required direct bacterial interaction with the epithelial cell surface and was not reproduced by purified H. influenzae lipooligosaccharide. Consistent with a functional role for this response, induction of ICAM-1 by H. influenzae mediated increased neutrophil adherence to the epithelial cell surface. Furthermore, in an in vivo murine model of airway infection with H. influenzae, increased epithelial cell ICAM-1 expression coincided with increased chemokine levels and neutrophil recruitment in the airway. These results indicate that ICAM-1 expression on human respiratory epithelial cells is induced by epithelial cell interaction with H. influenzae and suggest that an ICAM-1-dependent mechanism can mediate neutrophil adherence to these cells independent of inflammatory mediator release by other cell types. Direct induction of specific epithelial cell genes (such as ICAM-1 and IL-8) by bacterial infection may allow for rapid and efficient innate defense in the airway.

Infantile handling eliminates reversal learning deficit in rats prenatally exposed to alcohol.

Prenatal exposure to ethanol results in learning deficits and alters physiological response to stress. Neonatal handling and stimulation. on the other hand, produce long-lasting physiological and behavioral changes in response to stress. To determine whether early handling, consisting of daily separation and tactile stimulation for the first 3 weeks, can modify fetal alcohol effects on learning ability of young adult rats, offspring of rats chronically exposed to ethanol throughout pregnancy and control animals were trained in a T-maze to learn a position response and then to reverse the learned response. The nonhandled, ethanol-treated rats were deficient on reversal, but the ethanol-treated rats that were handled during the first 3 weeks of postnatal development showed no deficit in learning to reverse their previously learned responses. Postnatal handling had no effect on acquisition in alcohol-treated rats. Neither reversal nor acquisition was affected by infantile handling in pair-fed or normal control animals. Early handling may have eliminated the reversal deficit in the ethanol-treated offspring by altering their physiological and behavioral reactivity to stress.

[Long-term outcome of incontinence and prolapse surgery with reference to multiple endogenous and exogenous risk factors for the female pelvic floor]. / Langzeitergebnisse der Inkontinenz- und Deszensuschirurgie unter Berücksichtigung multipler endogener und exogener Belastungsfaktoren des weiblichen Beckenbodens.

OBJECTIVE: The long-term results following different types of operations for urinary stress incontinence (minimum follow-up: 18 months) as well as multiple risk factors for the pelvic floor with regard to the results of surgery are reviewed in a retrospective study. STUDY DESIGN: Between 1980 and 1992 1283 patients underwent surgery because of urinary stress incontinence with or at the University Women's' Hospital in Heidelberg. The data of 478 patients, 430 of those following primary surgery and 48 following recurrent surgery, were evaluated from questionnaires with regard to their risk profile and long-term results. RESULTS: 57% of patients following primary surgical therapy and 37% following recurrent surgery were cured for longer than 5 years or since the operation. A cure or improvement of the incontinence could be observed in 80% following primary and in 73% following recurrent surgery. Long-term results were significantly unfavorable, if the patient was exposed to one or several of the following risk factors: 1. Strain at work by carrying weights more than 5 kg 2. At least medium-hard housework, psychological tensions in the private sphere or idle women, who take a car for shopping 3. Double strain with at least medium-high burden with house-work and physical strain at work CONCLUSION: Long-term results following primary urinary stress incontinence surgery are influenced by certain risk-factors.

[Long-term outcome of incontinence and prolapse surgery at the Heidelberg University Gynecologic Clinic 1980-1992]. / Langzeitergebnisse der Inkontinenz- und Deszensuschirurgie an der Universitäts-Frauenklinik Heidelberg von 1980-1992.

OBJECTIVE: Long-term results after different types of operations for urinary stress incontinence (minimum follow-up: 18 months) as well as multiple risk factors for the pelvic floor were analysed in a retrospective study. STUDY DESIGN: Between 1980 and 1992 1283 patients underwent surgery because of urinary stress incontinence at the University Women's Hospital in Heidelberg. The data of 478 patients, 430 of these after primary and 48 after recurrent surgery, were evaluated by questionnaires with regard to the long-term-results. RESULTS: 57% of patients after primary surgical therapy and 37% after recurrent surgery were cured for longer than 5 years or since the operation. A cure or improvement of the incontinence could be observed in 80% after primary and in 73% after recurrent surgery. Among the vaginal approaches for primary surgery the hysterectomy combined with colporrhaphy was most successful (60% cured or more than 5 years continent, 80.5% at least improved). The Burch colposuspension revealed even better results among the abdominal approaches (64% cured or longer than 5 years continent, 86% at least improved) compared to the Marshall-Marchetti-Krantz procedure with a cure rate of 33%. For therapy of the recurrent urinary incontinence the abdominal Burch colposuspension showed the best results with cure rates of 50% and cure or improvement in 75%. Therefore the abdominal approach seems to be superior to vaginal techniques such as sling operations (33% cure rate, 67% at least improved) or only re-colporrhaphy (27% cure rate, 78% at least improved). CONCLUSION: For primary incontinence the hysterectomy with vaginal repair or the Burch colposuspension have proved to be most successful. For recurrent urinary incontinence the abdominal colposuspension (Burch procedure) seems to be superior to other approaches.

Protective effects of fetal neocortical transplants on cognitive function and neuron size in rats with congenital micrencephaly.

The rat with micrencephaly, produced by prenatal exposure to methylazoxymethanol, provides a useful model to study neurobehavioral abnormalities associated with congenital brain defects. The micrencephalic animals have a life-long learning impairment. As they age, their already impaired learning competence deteriorates further. To determine whether the aging-associated functional deterioration could be ameliorated by a neural transplant, micrencephalic rats bearing solid transplants of normal fetal neocortical tissue since infancy were evaluated on a visual pattern discrimination learning at 15 months and a spatial navigation test at 24 months of age. The transplant-bearing rats learned both tasks significantly better than the micrencephalic rats without transplants. Morphometric analyses revealed that cortical pyramidal neurons were larger in the transplant-bearing rats than in micrencephalic rats without transplants. The life-long presence of a transplant appeared to have protected the micrencephalic brain against aging-associated deterioration. This is the first demonstration that a neural transplant, placed in a congenitally defective infant brain, can ameliorate aging-associated cognitive deficits.

Cl- channel inhibition by glibenclamide is not specific for the CFTR-type Cl- channel.

As long as the question of which channels are responsible for cAMP-mediated epithelial Cl- secretion remains unsolved, it is still important to search for specific inhibitors that might help to relate macroscopic to microscopic events. Following the report by Sheppard and Welsh (J Gen Physiol 100: 573, 1992) that glibenclamide inhibits whole-cell Cl- currents in genetically manipulated fibroblasts expressing the cystic fibrosis transmembrane conductance regulator (CFTR), we have studied the effect of glibenclamide on different types of Cl- channels of HT29 and T84 cells at the single-channel level. Our results confirm that micromolar concentrations of glibenclamide inhibit the linear, low-conductance Cl-channel, which appears to represent CFTR and show that the inhibition results from a typical flicker block. However, the same concentrations of glibenclamide inhibit also the outwardly rectifying intermediate conductance Cl- channel which, potentially, may contribute to transepithelial Cl- secretion.