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J J Janssen

Publications and source records attributed to J J Janssen.

30 records · Page 2Linked to original sources
PubMed1995

Histidine tagging both allows convenient single-step purification of bovine rhodopsin and exerts ionic strength-dependent effects on its photochemistry.

For rapid single-step purification of recombinant rhodopsin, a baculovirus expression vector was constructed containing the bovine opsin coding sequence extended at the 3'-end by a short sequence encoding six histidine residues. Recombinant baculovirus-infected Spodoptera frugiperda cells produce bovine opsin carrying a C-terminal histidine tag (v-opshis6x). The presence of this tag was confirmed by immunoblot analysis. Incubation with 11-cis-retinal produced a photosensitive pigment (v-Rhohis6x) at a level of 15-20 pmol/10(6) cells. The histidine tag was exploited to purify v-Rhohis6x via immobilized metal affinity chromatography. Optimized immobilized metal affinity chromatography yielded a binding capacity of > or = 35 nmol of v-Rhohis6x per ml of resin and purification factors up to 500. Best samples were at least 85% pure, with an average purity of 70% (A280 nm/A500 nm = 2.5 +/- 0.4, n = 7). Remaining contamination was largely removed upon reconstitution into lipids, yielding rhodopsin proteoliposomes with a purity over 95%. Spectral analysis of v-Rhohis6x showed a small but significant red shift (501 +/- 1 nm) compared to wild type rhodopsin (498 +/- 1 nm). The pK alpha of the Meta I<==>Meta II equilibrium in v-Rhohis6x is down-shifted from 7.3 to 6.4 resulting in a significant shift at pH 6.5 toward the Meta I photointermediate. Both effects are reversed upon increasing the ionic strength. FT-IR analysis of the Rho-->Meta II transition shows that the corresponding structural changes are identical in wild type and v-Rhohis6x.

Amino Acid Sequence↗
PubMed1995

Cloning and expression of a cDNA encoding bovine retinal pigment epithelial 11-cis retinol dehydrogenase.

PURPOSE: Identification of a 32-kd protein in the bovine retinal pigment epithelium. METHODS: A bovine retinal pigment epithelium cDNA library was constructed in the bacteriophage lambda ZAP Express. A monoclonal antibody, designated 21-C3/AV, was used to isolate the cDNA encoding the 21-C3/AV antigen. A positive full-length clone, designated 21-C3RDH/CD, was sequenced. Northern blot analysis was used to determine the length of the mRNA and the tissue expression pattern. The entire open reading frame of clone 21-C3RDH/CD was used to isolate a recombinant baculovirus clone and expressed in Spodoptera frugiperda insect cells. Enzymatic activity toward 11-cis retinaldehyde was investigated. RESULTS: The complete nucleotide sequence of 21-C3RDH/CD was obtained. The deduced amino acid sequence reveals homology with short-chain alcohol dehydrogenases. Northern blot analysis detected a 1.2-kb transcript. Although the monoclonal antibody used to isolate 21-C3RDH/CD also reacts with other ocular and nonocular tissues, the authors were unable to demonstrate any reactivity with RNA samples isolated from different (non)ocular tissues. Recombinant baculovirus-infected insect cells synthesized the 21-C3/AV antigen. This protein showed 11-cis retinol dehydrogenase activity. CONCLUSIONS: Homology to the human D-beta-hydroxybutyrate dehydrogenase precursor and other alcohol dehydrogenases shows that 21-C3RDH/CD encodes a short-chain alcohol dehydrogenase. Furthermore, tissue specificity and molecular weight of the antigen suggest that 21-C3RDH/CD encodes the bovine retinal pigment epithelial 11-cis retinol dehydrogenase. Direct proof came from experiments in which we used the baculovirus-based expression system for in vitro synthesis of the protein encoded by 21-C3RDH/CD. Protein extracts obtained from recombinant baculovirus-infected insect cells were found capable of reducing 11-cis retinaldehyde.

Alcohol Dehydrogenase↗
PubMed1994

Numerical calculation of energy deposition by high-energy electron beams: III. Three-dimensional heterogeneous media.

The phase space time evolution model of Huizenga and Storchi and Morawska-Kaczyńska and Huizenga has been modified to accommodate calculations of energy deposition by arbitrary electron beams in three-dimensional heterogeneous media. This is a further development aimed at the use of the phase space evolution model in radiotherapy treatment planning. The model presented uses an improved method to control the evolution of the phase space state. This new method results in a faster algorithm, and requires less computer memory. An extra advantage of this method is that it allows the pre-calculation of information, further reducing calculation times. Typical results obtainable with this model are illustrated with the cases of (i) a 20 MeV pencil beam in a water phantom, (ii) a 20 MeV 5 x 5 cm2 beam in a water phantom containing two air cavities, and (iii) a 20 MeV 5 x 5 cm2 beam in a water phantom containing an aluminium region. The results of the dose distribution calculations are in good agreement with and require significantly less computation time than results obtained with Monte Carlo methods.

Algorithms↗
PubMed1994

Characterization of monoclonal antibodies recognizing retinal pigment epithelial antigens.

PURPOSE: To purify and characterize retinal pigment epithelial proteins exhibiting uveitogenic characteristics after immunization of Lewis rats, a broad panel of monoclonal antibodies directed against retinal pigment epithelial (RPE) protein antigens, was isolated. METHODS: Bovine RPE detergent extracts were used to isolate monoclonal antibodies against RPE antigens. Species and tissue specificity within the eye was tested through immunocytochemical analysis. Western blot analysis was used to determine the molecular weight of the RPE antigens. RESULTS: Several RPE-reactive antibodies were obtained. At least four monoclonal antibodies were isolated that reacted with different RPE antigen types. Against most of the antigens more than one hybridoma cell line was isolated. Two hybridoma lines were isolated producing antibodies, which on immunocytochemical analysis showed strong reactivity with the RPE and eye muscle tissue. The latter monoclonal antibody recognizes a 32 kD protein in RPE cells. Five monoclonal antibodies recognize a protein with an apparent molecular weight of 65 kD. One cell line was isolated that produced antibodies showing an irregular reaction pattern with both iris and ciliary body. CONCLUSIONS: RPE cells, striated eye muscle and smooth muscle cells share a 32 kD antigenic protein. This antigen is present in almost all ocular epithelial cells. Based on reaction patterns on Western blot and immunocytochemical analysis, there are at least three different 65 kD RPE antigens, two of which are RPE-specific and one of which is also present in the kidney epithelium.

Animals↗
PubMed1990

Development and degeneration of retina in rds mutant mice: immunoassay of the rod visual pigment rhodopsin.

Development and loss of photoreceptor cells in mice, afflicted by the rds (retinal degeneration slow) gene, was analyzed by measuring the ocular visual pigment content as rhodopsin (spectroscopy) and opsin (immunoassay). With regard to the postnatal age, where opsin was just detectable, and to the initial rate of opsin synthesis, the mutants did not strongly deviate from the normal animals. The final maximal visual pigment level was, however, about half of normal for the heterozygous mutants and about 3% of normal for the homozygous mutants, both in the pigmented and in the albino strain. In the pigmented normal or heterozygous mutant the (rhod)opsin levels remain stable up to at least 1 year of age. For the corresponding albino animals this was only observed up to 9 months of age. Thereafter the level declines. In the homozygous mutants, maximal opsin levels were observed at about 3 weeks postnatal. Subsequently, this level gradually declined to about 40% in the pigmented and about 15% in the albino mutant. The results indicate that the rds gene does not directly affect the biosynthetic pathways of opsin. The physiological effect of the rds gene is aggravated by photodamage for which the albino animal is particularly susceptible.

Aging↗
PubMed1990

Asp83, Glu113 and Glu134 are not specifically involved in Schiff base protonation or wavelength regulation in bovine rhodopsin.

Site-specific mutagenesis was employed to investigate the proposed contribution of proton-donating residues (Glu, Asp) in the membrane domains of bovine rhodopsin to protonation of the Schiff base-linking protein and chromophore or to wavelength modulation of this visual pigment. Three point-mutations were introduced to replace the highly conserved residues Asp83 by Asn (D83N), Glu113 by Gln (E113 Q) or Glu134 by Asp (E134D), respectively. All 3 substitutions had only marginal effects on the spectral properties of the final pigment (less than or equal to 3 nm blue-shift relative to native rhodopsin). Hence, none of these residues by itself is specifically involved in Schiff base protonation or wavelength modulation of bovine rhodopsin.

Animals↗
PubMed1990

Defecography in patients with anorectal disorders. Which findings are clinically relevant?

To evaluate the results and clinical impact of defecography in patients with anorectal disorders, 100 results of defecographic examinations from 92 patients were reviewed. The defecographic results were screened for the anorectal angle, defined both at rest and during straining, perineal descent, and abnormalities of the rectal configuration during straining. Anal manometry, saline infusion test, rectal capacity measurement, and anal electromyography (EMG) were also performed. There was a significant difference (P less than 0.001) both at rest (22 degrees) and during straining (12 degrees) between the two anorectal angle measurements. Incontinent patients had a larger anorectal angle, both at rest and during straining, than continent patients (P less than 0.04), but with a large overlap. The anorectal angle was not influenced by gender or age. An abnormal rectal configuration was found in 62 defecographic examinations. From the 8 patients with rectopexy performed for a large rectocele or intussusception, incontinent patients with an intussusception had the best results. In four patients, anal EMG showed an increased activity of the external sphincter during straining. Two of these four patients had abnormal defecograhic results. No correlations were found between anorectal angle and the other function tests. In conclusion, the anorectal angle lacks clinical relevance. In patients with defecation problems, defecography may be indicated whenever other investigations (physical examination, anal manometry, anal EMG) have excluded local pathology or a spastic pelvic floor syndrome. In these situations, defecography could detect an intussusception, which could easily be treated with rectopexy.

Aged↗
PubMed1989

Soiling: anorectal function and results of treatment.

Forty-five patients with soiling but without faecal incontinence were evaluated by means of anorectal function investigations (anal manometry, rectal capacity and saline infusion test). The causes of soiling and the effect of treatment on both soiling and anorectal function were studied. The results were compared with a control group of 161 patients without soiling or incontinence. The diagnoses were haemorrhoids (10), mucosal prolapse (7), rectal prolapse (6), fistulae (5), proctitis (3), faecal impaction (2), rectocele with intussusception (2), scars after fistulectomy (2) and others (8). Simple inspection and proctoscopy were generally sufficient to establish a diagnosis. For two patients the diagnosis rectocele was made after defaecography. Anorectal test results did not differ between the soiling and control group, did not contribute to establish a diagnosis and did not change after treatment. Only patients with a rectal prolapse had abnormal results in anorectal function tests: a low basal sphincter pressure and a limited continence reserve. Appropriate therapy resulted in complete recovery (44%) or improvement of symptoms (29%).

Anal Canal↗
PubMed1989

The external anal sphincter. Relationship between anal manometry and anal electromyography and its clinical relevance.

Anal manometry and anal electromyography (EMG) were performed in 45 patients to evaluate the external anal sphincter. Their symptoms were soiling (N = 6), incontinence (N = 10), and obstipation (N = 19). Clinical diagnoses were previous anal surgery (N = 16), rectal prolapse--partial, total, intussusception (N = 16), puborectalis syndrome (N = 4), neurologic disorders (N = 3), and others (N = 6). The relationship between the maximum squeeze pressure (MSP) measured with anal manometry and the maximum (voluntary) contraction pattern (MCP) and signs of denervation (DEN) measured with anal EMG were examined. The correlation coefficient between MSP and MCP was 0.55 (P less than .001) and between MSP and DEN 0.13 (NS). A normal MSP always showed a normal MCP, a normal MCP showed an abnormal MSP in 43 percent only. In conclusion, the clinical value of anal EMG seems limited. Assessment of an additional anal EMG seems indicated in incontinent patients with previous anal surgery with a low MSP to estimate muscle function, whenever anal surgery is considered. Anal EMG during straining can easily confirm the clinical diagnosis of puborectalis syndrome.

Adolescent↗
PubMed1988

Synthesis of functional bovine opsin in insect cells under control of the baculovirus polyhedrin promoter.

In vitro expression of cDNA encoding bovine opsin is accomplished using the baculovirus expression vector system. Full-length opsin was synthesized which was recognized by poly- and monoclonal antisera raised against bovine rhodopsin. Upon infection with a recombinant virus, 1 x 10(6) insect cells produced up to 3 micrograms opsin. Incubation of the in vitro synthesized opsin with 11-cis retinal produced a hydroxylamine-stable, photosensitive pigment.

Animals↗
PubMed1985

Immunoassay of rod visual pigment (opsin) in the eyes of rds mutant mice lacking receptor outer segments.

In 020/A mice, homozygous for the retinal degeneration slow (rds) gene, the photoreceptor cells fail to develop outer segments, and in the absorption spectra of retinal extracts the rhodopsin peak is lacking. Application of an enzyme-linked immunoassay using antisera against bovine opsin shows, however, that opsin is present in the homozygous mutant retina (0.010 nmol/eye) at 3% of the level of the normal retina (0.38 nmol/eye) of Balb/c mice. In the retina of heterozygous mice the opsin level (0.19 nmol/eye) is about half of the normal. Detection of opsin in the rds mutant retina demonstrates the functional basis for the reported electroretinographic response and light-mediated reduction in cyclic nucleotide levels in this mutant.

Animals↗