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Biomedical subjects

W Haase

Publications and source records attributed to W Haase.

At least 55 records · Page 3Linked to original sources
PubMed1997

Isolation of proximal and distal tubule cells from human kidney by immunomagnetic separation. Technical note.

After collagenase digestion and Percoll density gradient centrifugation of human renal tissue, tubular epithelial cells of the proximal and the distal segments were isolated with an immunomagnetic method using MACS microbeads. To enrich proximal tubular (PT) cells we used a monoclonal antibody (mAb) against aminopeptidase M (APM, CD 13), specific of the proximal tubule. Distal tubular (DT) cells were isolated through a mAb recognizing Tamm-Horsfall glycoprotein (THG), a specific antigen for the thick ascending limb and the early distal convoluted tubule. Cells of the proximal primary isolate were histochemically strongly positive for aminopeptidase M (98.6%), however, cells of the distal portion were negative (98.7%). Ultrastructural analysis of PTC primary isolates revealed highly preserved brush border microvilli, well-developed endocytosis apparati and numerous mitochondria, whereas DTC primary isolates showed smaller cells with basolateral invaginations and less apical microvilli. Characterization by immunofluorescence indicated the coexpression of cytokeratin and vimentin, whereas staining for desmin, smooth muscle actin, a fibroblast-specific marker and von Willebrand factor was negative. Cultured PT and DT cells displayed different adenylate cyclase responsiveness to hormonal stimulation. PTH (10(-6) M) increased cAMP production in distal cells up to 32.8-fold of the basal level and in proximal only up to 3.5-fold (10(-8) M, DT 14.4x and PT 2.25x). Calcitonin stimulated adenylate cyclase in DT in a dose dependent fashion (10(-6) M, 4.3x; 10(-8) M, 2.25x), whereas only a low calcitonin response was found in PT cells (10(-6) M, 1.6x; 10(-8) M, 1.4x). AVP (10(-6) M) activated the distal cAMP-production only up to 1.9x of the basal level, but the proximal cAMP-production was negligible (only 1.3x the basal level). The data of this study indicate the proximal and distal tubule origin of the cultured cells that were isolated according to their segment-specific antigens.

Animals↗
PubMed1997

[Ophthalmoscopic evaluation of ocular fixation in children up to 2 years old].

BACKGROUND: The examination of fixation on the fundus oculi is one of the main points in the diagnosis of amblyopia. Therefore it is necessary to describe the fixation ability and to clarify at what age children can fixate foveally and at what age this examination can be carried out, considering not only physiological aspects, but also co-operation reasons. PATIENT AND METHODS: Fixation on the fundus oculi was examined in 111 children aged between 1 and 25 months with the ophthalmoscop-star. This examination was carried out in 64 cases by a second and in 15 cases by a third examiner on the same day. RESULTS: During the first 3 months of age children cannot fixate foveally, one can observe instead, a kind of scatter at the posterior pole in a diameter of about 5 degrees. In the course of the 4, and 5, month of age foveal fixation develops. Repeated examinations of the same child on the same day by a 2, and 3, examiner reveal a very high rate of accordance (78%) of their results. CONCLUSIONS: The examination of fixation on the fundus can be carried out at 4 to 5 months of age. At that age one can differentiate quite reliable between a foveal and eccentric fixation as well as scatter at the posterior pole. The rate of accordance 78% with this fixation examination by several examiners shows that same reliability of this examination carried out in school children and adults. Comparing this result with fixation examination carried out with the Haidinger brushes then one must calculate with a fault rate of about 20% when examining with the ophthalmoscope, considering that the examination with Haidinger brushes as a reference examination.

Adult↗
PubMed1997

[Determination of far vision with DIN 58220 (criterion 6/10): computer vision test with high resolution monitor in comparison with vision charts].

BACKGROUND: The results of nearly automated computerized determination of visual acuity by PC are compared to those of card measurements. The introduction of a new measuring technique should not change the dimension--visual acuity--itself, therefore there should be no difference between the results of the conventional technique and those of the PC. METHODS: Both methods were tested in a group of subjects (N = 100) with vision between 0.1-2.5. The nearly automated technique was developed and established by our department. Like the conventional method it keeps strictly to the DIN rates. For DIN-criterion we chose for the benefit of high reproducibility 6/10. RESULTS: In spite of some deficiencies in the presentation of the Landoltrings on the monitor, our analysis presents a surprisingly good correspondence between the results of the two methods. In the analysis, we are not comparing arithmetic mean values. Instead, we examine the individual difference between the two methods in acuity steps and its these distribution within the group of subjects. No statistically provable difference can be detected. This is valid for both single visual acuity and for visual line acuity, which can also be tested with the computer aided method. CONCLUSION: We conclude that the PC proves to be an equivalent method to the measurement with cards. Thus, it is suitable for the use in daily routine of vision examination with the advantage, that the investigator has less influence on the outcome of the test.

Adult↗
PubMed1996

Glycosylation, palmitoylation, and localization of the human D2S receptor in baculovirus-infected insect cells.

In order to evaluate the baculovirus expression system as a means for high-yield production of homogeneous D2S receptor, we have expressed various D2S receptor constructs in two Spodoptera frugiperda cell lines, a Trichoplusia ni and a Mammestra brassicae cell line. To improve expression yield, the environment of the polyhedrin gene translational initiation site was retained by fusing the first 12 codons of the polyhedrin gene to the 5'-end of the D2S receptor coding sequence. The pharmacological profile of the expressed D2S receptor was similar to that reported for neuronal D2 receptors. Sf9 and Tn cells were best suited for overexpression, yielding about 2 x 10(6) and 4 x 10(6) receptors/cell, respectively, corresponding to 6 pmol/mg of cell protein in Sf9 cells and 10 pmol/mg of cell protein in Tn cells. We have developed a D2 receptor-specific anti-peptide antibody to study glycosylation, palmitoylation, and localization of the heterologously produced receptor. Immunoprecipitation of digitonin/cholate-solubilized receptor from control and tunicamycin-treated Sf9, Tn, and Mb cells revealed an apparent molecular mass of 47-48 kDa for the glycosylated receptor and of 39-40 kDa for the unglycosylated receptor. Although pulse-chase studies showed that glycosylation occurred rapidly and efficiently, the glycosylated receptor only constituted a small fraction of the overall produced receptor protein, which was mainly located intracellularly. The glycosylation of the receptor was of the high-mannose-type in contrast to the complex-type glycosylation found in native tissue. The glycosylated D2S receptor was palmitoylated. Glycosylation, however, was not a prerequisite for palmitoylation which was insensitive to tunicamycin, brefeldin A, and monensin. NH2-terminal addition of the signal sequence of prepromelittin to the D2S receptor increased expression levels 2-3-fold and significantly enhanced membrane insertion and processing, resulting in increased targeting of the synthesized receptor to the plasma membrane.

Animals↗
PubMed1996

[Medicamentous therapy of alcoholic polyneuropathy. Randomized double-blind study comparing 2 vitamin B preparations and a nucleotide preparation].

In a randomized double-blind study involving 303 patients with alcoholic polyneuropathy the efficacy and tolerability of the combinations thiamine/pyridoxine, benfotiamine/pyridoxine, and the nucleotides of cytidine and uridine administered orally for 21 days were compared. Pain and paraesthesia, measured on a visual 10-cm-long analogue scale, as also pallaesthesia and the strength of the dorsiflexion and plantar flexors of the foot, clearly improved in all treatment groups. Clinically relevant differences in efficacy were not observed. All the drugs tested were well tolerated.

Adult↗
PubMed1996

Up-regulation of pressure-activated Ca(2+)-permeable cation channel in intact vascular endothelium of hypertensive rats.

In endothelial cells, stretch-activated cation channels have been proposed to act as mechanosensors for changes in hemodynamic forces. We have identified a novel mechanosensitive pressure-activated channel in intact endothelium from rat aorta and mesenteric artery. The 18-pS cation channel responded with a multifold increase in channel activity when positive pressure was applied to the luminal cell surface with the patch pipette and inactivated at negative pipette pressure. Channel permeability ratio for K+, Na+, and Ca2+ ions was 1:0.98:0.23. Ca2+ influx through the channel was sufficient to activate a neighboring Ca2(+)-dependent K+ channel. Hemodynamic forces are chronically disturbed in arterial hypertension. Endothelial cell dysfunction has been implicated in the pathogenesis of arterial hypertension. In two comparative studies, density of the pressure-activated channel was found to be significantly higher in spontaneously hypertensive rats and renovascular hypertensive rats compared with their respective normotensive controls. Channel activity presumably leads to mechanosensitive Ca2+ influx and induces cell hyperpolarization by K+ channel activity. Both Ca2+ influx and hyperpolarization are known to induce a vasodilatory endothelial response by stimulating endothelial nitric oxide (NO) production. Up-regulation of channel density in hypertension could, therefore, represent a counterregulatory mechanism of vascular endothelium.

Animals↗
PubMed1996

Expression of type II cGMP-dependent protein kinase in rat kidney is regulated by dehydration and correlated with renin gene expression.

cGMP-based regulatory systems are vital for counteracting the renin-angiotensin system (RAS) which promotes volume expansion and high blood pressure. Natriuretic peptides and nitric oxide acting through their second messenger cGMP normally increase natriuresis and diuresis, and regulate renin release; however, the severe pathological state of cardiac heart failure is characterized by elevated levels of atrial natriuretic peptide that are no longer able to effectively oppose exaggerated RAS effects. There is presently limited information on the intracellular effectors of cGMP actions in the kidney. Recently we reported the cloning of the cDNA for type II cGMP-dependent protein kinase (cGK II), which is highly enriched in intestinal mucosa but was also detected for the first time in kidney. In the present study, cGK II was localized to juxtaglomerular (JG) cells, the ascending thin limb (ATL), and to a lesser extent the brush border of proximal tubules. An activator of renin gene expression, the angiotensin II type I receptor inhibitor, losartan, increased cGK II mRNA and protein three to fourfold in JG cells. In other experiments, water deprivation increased cGK II mRNA and protein three to fourfold in the inner medulla where both cGK II, and a kidney specific CI- channel shown by others to be regulated by dehydration, are localized in the ATL. Whereas additional data suggest that cGK I may primarily mediate cGMP-related changes in renal hemodynamics, cGK II may regulate renin release and ATL ion transport.

Animals↗
PubMed1996

A new set of versatile vectors for the heterologous expression of foreign genes using the baculovirus system.

A new set of versatile advanced baculovirus (BV) vectors for the production of fused proteins in insect cells, under the control of the strong polyhedrin promoter of the Autographa california nuclear polyhedrosis virus (AcMNPV), has been constructed. The vectors contain peptide tags which allow immunological detection, as well as purification of recombinant protein produced via the BV expression system.

Amino Acid Sequence↗
PubMed1996

Functional expression of the Aequorea victoria green fluorescent protein in insect cells using the baculovirus expression system.

A DNA fragment encoding the green fluorescent protein (GFP) was isolated via PCR from a jellyfish Aequorea victoria cDNA, cloned and sequenced. Subsequently, a recombinant baculovirus bearing the coding region of the GFP under the transcriptional control of the Autographa californica nuclear polyhedrosis virus (AcMNPV) polyhedrin gene promoter was constructed and isolated. High-level expression of GFP could be easily monitored in Spodoptera frugiperda (Sf9) insect cells after infection with recombinant baculovirus, due to the intrinsic fluorescence (lambda(max) = 508 nm) of the recombinant protein after excitation with blue light (lambda(max) = 400 nm). The functional recombinant GFP displayed an apparent molecular mass of approximately 43 kDa and the fluorescence emission spectrum of the recombinant protein was virtually identical to that of the native green fluorescent protein.

Animals↗
PubMed1996

Identification of NF2 germ-line mutations and comparison with neurofibromatosis 2 phenotypes.

Neurofibromatosis 2 (NF2) is an autosomal inherited disorder that predisposes carriers to nervous system tumors. To examine genotype-phenotype correlations in NF2, we performed mutation analyses and gadolinium-enhanced magnetic resonance imaging of the head and full spine in 59 unrelated NF2 patients. In patients with vestibular schwannomas (VSs) or identified NF2 mutations, the mild phenotype was defined as < 2 other intracranial tumors and < or = 4 spinal tumors, and the severe phenotype as either > or = 2 other intracranial tumors of > 4 spinal tumors. Nineteen mutations were found in 20 (34%) of the patients and were distributed in 12 of the 17 exons of the NF2 gene, including intron-exon boundaries. Seven mutations were frameshift, six were nonsense, four were splice site, two were missense, and one was a 3-bp in frame deletion. The nonsense mutations included one codon 57 and two codon 262 C-->T transition in CpG dinucleotides. The frameshift and nonsense NF2 mutations occurred primarily in patients with severe phenotypes. The two missense mutations occurred in patients with mild phenotypes, and three of the four splice site mutations occurred in families with both mild and severe phenotypes. Truncating NF2 mutations are usually associated with severe phenotypes, but the association of some mutations with mild and severe phenotypes indicates that NF2 expression is influenced by stochastic, epigenetic, or environmental factors.

DNA Mutational Analysis↗
PubMed1996

[Ophthalmologic spectrum of neurofibromatosis type 2 in childhood].

BACKGROUND: Neurofibromatosis type 2 (NF2) is a disorder with autosomal dominant inheritance which leads to tumor growth in the central and peripheral nervous system. In affected adult patients there is a typical association with ocular abnormalities like juvenile cataract. METHODS: Ophthalmologic investigation was carried out in ten children aged one to fourteen years with suspected NF2. The diagnosis was confirmed by further clinical examination and-in one patient-by segregation analysis. RESULTS: Nine of these ten children showed ocular abnormalities such as juvenile subcapsular cataracts, retinal hamartomas, optic nerve sheath tumors, fibrotic maculopathies as well as one case of a perineural calcification of the optic nerve and one case of a cerebral hamartoma on the ground of the third ventricle. DISCUSSION: In six children ophthalmological symptoms were the presenting symptom of the disease. The knowledge of these symptoms allows for the diagnosis of NF2 in children who present with isolated ocular deficits or with other typical criteria of the disease. The early diagnosis of the disease may lead to an improved prognosis with regard to preservation of hearing by surgery of bilateral vestibular schwannoma which occur in more than 90% of the NF2-patients.

Adolescent↗
PubMed1996

The neuroimaging and clinical spectrum of neurofibromatosis 2.

Neurofibromatosis 2 (NF2) is an autosomal dominant disease predisposing to multiple tumors of the central and peripheral nervous system. Bilateral vestibular schwannomas are the hallmark of the disease. To define the clinical spectrum of the disease, we performed gadolinium-enhanced magnetic resonance imaging of the brain and spine as well as neurological, dermatological, and ocular examinations in 48 patients with NF2 diagnosed with the National Institutes of Health diagnostic criteria. Patients were ascertained from patient workshops and publications and from referral as a result of vestibular schwannoma surgery. Vestibular schwannomas were found in 46 patients (96%, 43 bilateral and 3 unilateral), spinal tumors were found in 43 (90%), posterior subcapsular cataracts were found in 30 (63%), meningiomas were found in 28 (58%), and trigeminal schwannomas were found in 14 (29%). The presenting symptoms included hearing loss or tinnitus in 15 patients (31%), multiple or nonspecific symptoms in 15 (31%), skin tumors in 12 (25%), and ocular symptoms in 6 (13%). When the complete spine was imaged, spinal tumors were more common in patients with NF2 than has previously been reported. This is a noteworthy finding, because spinal tumors are a major cause of NF2 morbidity and mortality.

Adolescent↗
PubMed1996

Use of nanogold- and fluorescent-labeled antibody Fv fragments in immunocytochemistry.

Recombinant antibody fragments are emerging as a versatile tool in both basic research and medical therapy. We describe the procedures for direct labeling of engineered antibody fragments (Fv) with fluorescein or nanogold and their use in fluorescence and immunoelectron microscopy, respectively. The Fv fragments were produced in Escherichia coli, purified by one-step Strep tag affinity chromatography, chemically labeled with the marker, and employed in microscopy to localize epitopes on the membrane protein bacteriorhodopsin in purple membranes of Halobacterium halobium and the cytochrome c oxidase of Paracoccus denitrificans. In both cases, methods involving directly labeled antibody fragments show results identical to those in which antibodies or Fv fragments are detected by a secondarily labeled conjugate. The multifunctional design of the recombinant Fv fragments, however, offers more all-around applications in immunocytochemistry. The directly labeled Fv fragments, half the size of an Fab fragment, are at the molecular level the smallest antibody fragments yet described for visualization of biomolecules in microscopy.

Escherichia coli↗
PubMed1996

Pharmacological and biochemical characterization of the mouse 5HT5A serotonin receptor heterologously produced in the yeast Saccharomyces cerevisiae.

The cDNA for the mouse 5HT5A receptor has been functionally expressed in the unicellular yeast Saccharomyces cerevisiae. The NH2-terminal end of the receptor gene was fused to the Bacillus macerans (1-3, 1-4)-beta-glucanase signal sequence to ensure proper membrane insertion and to the c-myc epitope to permit immunological detection of the heterologously expressed protein. In the resulting episomal yeast expression plasmid pCNNmm5HT5A the modified 5HT5A gene is under the transcriptional control of the endopeptidase B gene promoter (PRB1). After transformation of the vector into the protease deficient S. cerevisiae strain cI3-ABYS-86, recombinant clones were examined for the presence of functional receptor by radioligand binding using [3H]LSD. Whole cells as well as crude membrane preparations of recombinant clones showed saturable binding of the receptor with a KD of approximately 2.2 nM. The pharmacological properties for the heterologous expressed receptor, estimated by ligand-displacement studies using certain serotonin agonists and antagonists, were comparable to those reported for the receptor expressed in mammalian systems. Western blot analysis of membranes prepared from a recombinant clone using the monoclonal antibody 9E10, directed against the c-myc epitope of the modified receptor, revealed an apparent molecular mass of about 43 kDa for the receptor expressed in S. cerevisiae. Glycosylation of the receptor was analysed by EndoH digestion. A heat shock of recombinant yeast significantly increased the number of specific binding sites per cell and also improved the affinity of the receptor. Immunogold electron microscopy was used to study the localization of the heterologously expressed protein within the yeast cells.

Amino Acid Sequence↗
PubMed1995

Expression of functional mouse 5-HT5A serotonin receptor in the methylotrophic yeast Pichia pastoris: pharmacological characterization and localization.

The methylotrophic yeast Pichia pastoris was tested for heterologous expression of the mouse 5-HT5A receptor. Three different expression plasmids were constructed where the cDNA of the receptor was cloned under the transcriptional control of the highly inducible promoter of the P. pastoris alcohol oxidase 1 (AOX1) gen. The expression plasmids differed with respect to the signal sequences used for N-terminal fusion. In two cases the coding region was additionally fused to the c-myc tag to permit immunological detection of the receptor. Expression of functional receptor after transformation of strain GS115 was detected by radioligand binding using [3H]LSD. The construct with the best expression levels in strain GS115 was used for transformation of the protease deficient strain SMD1163. Here, the expression level was 2-8 times higher. Whole cells as well as crude membrane preparations of recombinant clones showed saturable binding of [3H]LSD with a Kd of approximately 1.9 nM. Receptor concentrations of approximately 22 pmol/mg membrane protein revealed the potential of the P. pastoris expression system for high level expression of membrane proteins. The pharmacological properties were comparable to those reported for the receptor expressed in mammalian systems.

Animals↗