[Comparison of a sulfate-free polyethylene glycol (PEG) solution with a standard solution for colonoscopy preparation]. / Vergleich einer Sulfat-freien Polyäthylenglykol(PEG)-Lösung mit einer Standardlösung für die Koloskopievorbereitung.

In order to ameliorate the unpleasant salty taste of a standard PEG-electrolyte lavage solution and thus improving acceptance of colonoscopy cleansing, a new sulfate-free, low sodium PEG-solution has been developed as long as 10 years ago. Comparative studies regarding acceptance showed conflicting results. This study was intended to compare the standard solution L (Cololyt) with the sulfate-free solution S (Colo-Sol) on a prospective basis in a collective of average gastroenterology outpatients. The 144 patients who underwent colonoscopy ingested 1 liter of solutions L and S each on the evening before and another liter of the preferred solution (preference) in the morning of the day of examination. Moreover the taste of both solutions was rated (grading scale 0-10). Tolerance and efficacy were registered as well. Preference and thus acceptance presented very well-balanced. Solution L was preferred as frequent (by 50.7% of patients) as solution S (49.3%) which was originally claimed to be better tasting. The respective preferred solutions were rated equal with mean +/- SD values of 6.6 +/- 2.2 for L and 7.0 +/- 1.9 for S. Efficacy and tolerance did not differ from a clinical point of view.

Therapy for vaginismus: in vivo versus in vitro desensitization.

OBJECTIVE: To verify the effectiveness of desensitization exercises with dilators in the treatment of vaginismus, while comparing 2 therapeutic variations with differing instruction procedures ("in vivo" versus "in vitro") during the desensitization exercises. METHOD: A consecutive random sample of 44 female outpatients in sexual therapy were divided into groups for either treatment on a random basis. All patients were treated until the symptoms abated on the basis of a structured therapy program. The stability of the therapeutic success was verified through follow-up interviews. RESULTS: Forty-three (97.2%) of the patients were able to have sexual intercourse after an average of 6.3 therapeutic sessions. One-third reported an increase in their sexual desire. Thirty-four of 39 patients (87.2%) would be prepared to repeat the therapy. There were no significant differences in the success and the required number of consultations for the cure between the 2 groups investigated. CONCLUSIONS: Desensitization through exercises with dilators is an effective method for treating vaginismus. The choice of instruction procedure can be left to the patient.

Temperature-responsive size-exclusion chromatography using poly(N-isopropylacrylamide) grafted silica.

Silica-based packing materials induce non-specific interactions with proteins in aqueous media because of the nature of their surface, mainly silanol groups. Therefore, the silica surface has to be modified in order to be used as stationary phase for the High Performance Size-Exclusion Chromatography (HPSEC) of proteins. For this purpose, porous silica beads were coated with hydrophilic polymer gels (dextrans of different molecular weights) carrying a calculated amount of diethyl-aminoethyl groups (DEAE). Actually, as shown by HPSEC, these dextran modified supports minimize non-specific adsorption for proteins and pullulans in aqueous solution. Then, in order to change the pore size in response to temperature, temperature responsive polymer of poly(N-isopropylacrylamide) (PIPAAm) was introduced into the surface of dextran-DEAE on porous silica beads. The structure of these supports before and after modification was alternately studied by Scanning Electronic Microscopy (SEM) and Scanning Force Microscopy (SFM). An adsorption of radiolabelled albumin was performed to complete our study. Silica modifications by dextran-DEAE and PIPAAm improve the neutrality of the support and minimize the non-specific interactions between the solid support and proteins in solution. At low temperature, the support having PIPAAm exhibits a high resolution domain in HPSEC and finally permits a better resolution of proteins and pullulans. At higher temperature, hydrophobic properties of PIPAAm produce interactions with some proteins and trigger off a slight delay of their elution time.

A mutant of human insulin-like growth factor II (IGF II) with the processing sites of proinsulin. Expression and binding studies of processed IGF II.

A mutant of human insulin-like growth factor II (IGF II) was constructed by site-directed mutagenesis: the nucleotides coding for Ser33 and Ser39 were changed to yield Arg and Lys, respectively, thus creating two pairs of basic residues, Arg-Arg and Lys-Arg, as flanking sequences of the remaining C domain. [Arg33, Lys39]IGF II was expressed in NIH-3T3 cells as a processed two-chain peptide with a deletion of amino acid residues 37-40 and crosslinked by three disulfide bonds. This des(37-40)[Arg33]IGF II showed 3.6-fold and 7.4-fold reduced affinities to the type 1 and type 2 IGF receptor overexpressing cells, respectively, whereas the thymidine incorporation potency was the same as that of wild-type IGF II. We speculate that the discrepancy between the reduced binding to the type 1 IGF receptor and the full thymidine incorporation potency is due to the 6.1-fold reduced affinity of the expressed mutant to the co-expressed IGF binding protein 3 (IGFBP-3). The results suggest that des(37-40)[Arg33]IGF II assumes a conformation very similar to IGF II, and that the entire length of the C domain is not essential for biological activity.

[Glomerulonephritis with transient C3 hypoclompimentemia and endotheliomesangial glomerulonephritis in childhood. A long-term experience]. / Glomerulonephritis mit transitorischer C3-Hypokomplementämie und endotheliomesangiale Glomerulonephritis im Kindesalter. Eine Langzeiterfahrung.

62 children (20 girls and 42 boys, ranging in age between 3 and 15 years), presenting with acute hypocomplementemic glomerulonephritis or morphologically confirmed endotheliomesangial glomerulonephritis, were admitted to the University Children's Hospital, Berne from 1970 to 1991. The annual incidence of cases of acute hypocomplementemic glomerulonephritis was stable during the study period. The site of the antecedent infection was the throat in 26 patients, upper respiratory tract in 15, the skin in 9, and unknown in 10. The latent period ranged from 0.5 to 3.5 weeks. 41 patients developed hypertension and 17 renal failure. Hypertensive complications were observed in 6 patients and remitted completely in 5 cases. A nephrotic syndrome (edema, proteinuria of 40 mg/[m2.h], albuminemia < 25 g/l) was observed in 11 patients. Microscopic hematuria persisted in many patients for one year or more. Proteinuria remitted in all but one patient, who was found to have Alport syndrome. This study shows the stable frequency of hypocomplementemic glomerulonephritis since 1970, its good prognosis, and the importance of the measurement of C3-complementemia in children presenting with acute glomerulonephritis.

Mutants of human insulin-like growth factor II (IGF II). Expression and characterization of truncated IGF II and of two naturally occurring variants.

Insulin-like growth factor II (IGF II) and four structural analogs, constructed by site-directed mutagenesis, were expressed as protein A fusion proteins in Escherichia coli BL21pLysS cells, cleaved with cyanogen bromide and purified by affinity chromatography and HPLC. Two mutants (Ser29 substituted by Arg-Leu-Pro-Gly, and Ser33 substituted by Cys-Gly-Asp) represent two naturally occurring variants of IGF II. The other two mutants, (7-67)IGF II and (9-67)IGF II, are truncated at the amino-terminus in analogy to the naturally occurring des(1-3)IGF I ('truncated IGF I'). These mutants were tested for their binding affinities to type-1 and type-2 IGF receptors, to IGF binding protein-3 (IGFBP-3) and for their stimulation of thymidine incorporation into DNA. The affinities of the Ser29 and Ser33 mutants to the type-1 IGF receptor were 85% and 39%, respectively, compared to wild-type IGF II, those of (7-67)IGF II and (9-67)IGF II 96% and 15%, respectively. The potencies of the Ser33 and the (9-67) mutant to stimulate thymidine incorporation into DNA correlated closely with the affinities to the type-1 IGF receptor, whereas the bioavailability of the Ser29 mutant was lower and that of the (7-67) mutant higher than the type-1 receptor binding, possibly due to interferences with endogenously secreted IGFBPs. The affinities of the Ser29 and Ser33 mutants to the type-2 IGF receptor were 110% and 71%, respectively, those of the two truncated mutants 25% and 23%, respectively. The affinity of the Ser29 mutant to IGFBP-3 was increased to 171%, whereas those of the Ser33 mutant and the two truncated mutants were reduced (34%, 10% and 19%, respectively).

Mutants of human insulin-like growth factor II: expression and characterization of analogs with a substitution of TYR27 and/or a deletion of residues 62-67.

Five structural analogs of human insulin-like growth factor II (IGF II), [Leu27]IGF II, [Glu27]IGF II, des(62-67)IGF II, des(62-67)[Leu27]IGF II and des(62-67)[Glu27]IGF II were constructed by site-directed mutagenesis and expressed as protein A fusion proteins in E. coli BL21 pLysS cells, cleaved with CNBr and purified by affinity chromatography and HPLC. These mutants were tested for their binding affinities to type 1 and type 2 IGF receptors, to IGF binding protein-3 (IGFBP-3) and for their stimulation of thymidine incorporation into DNA. [Leu27]IGF II exhibits an affinity to the type 2 IGF receptor close to that of wild-type IGF II, but has lost completely the affinity to the type 1 IGF receptor. The results further suggest that the D domain, which is close to Tyr27, forms part of the binding region for the type 1 IGF receptor.

Mutants of human insulin-like growth factor II with altered affinities for the type 1 and type 2 insulin-like growth factor receptor.

With the aim to produce insulin-like growth factors (IGF) with enhanced specificity for the type 1 or type 2 IGF receptors, three mutants of IGF II have been prepared and expressed in NIH-3T3 cells. IGF II mutated at Tyr27 to Leu and Glu showed a 25- and 54-fold decrease in affinity for the type 1 IGF receptor and a 3.4- and 9.2-fold decrease in affinity for the type 2 IGF receptor. IGF II mutated at Phe48 to Glu showed a 18-fold decrease in affinity for the type 2 IGF receptor and a 2.8-fold decrease in affinity for the type 1 IGF receptor. These affinities were measured in radioreceptor assays using type 1 or 2 IGF receptor overexpressing cells. Data obtained on receptor cross-linking and thymidine incorporation assays confirmed the results of the radioreceptor assays. It is concluded that mutations of Tyr27 preferentially decrease binding to the type 1 IGF receptor and of Phe48 to the type 2 IGF receptor, either by the loss of a residue involved in receptor binding or by preferentially destabilizing the region involved in receptor binding.

The binding sites of insulin-like growth factor I (IGF I) to type I IGF receptor and to a monoclonal antibody. Mapping by chemical modification of tyrosine residues.

The surface topography of IGF I(insulin-like growth factor I) was investigated by chemical modification of amino acid residues in free IGF I and bound to type I IGF receptor or to monoclonal antibody MAB43. Tyrosine residues were modified either by chloramine-T or lactoperoxidase catalyzed iodination. In the free IGF I molecule, all 3 tyrosine residues, A19 (Tyr-60), B25 (Tyr-24), and C2 (Tyr-31), were iodinated. Monoclonal antibody MAB43 protected IGF I against modification at tyrosine residue A19, and in the type I IGF receptor-IGF I complex, all 3 tyrosine residues were shielded against iodine incorporation. These results allow the prediction of the binding domains in the IGF I molecule. The minimal receptor binding site in IGF I would include amino acid residues B25 to C2 and, possibly, the C-terminal part of the A-domain with tyrosine residue A19.

Characterization of affinity-purified type I insulin-like growth factor receptor from human placenta.

The binding affinities of type I IGF receptor, purified to near homogeneity from human placental membranes, were characterized. For this receptor preparation, free of type II IGF receptor and essentially free of insulin receptor, dissociation constants of Kd = 0.05 nM for IGF I and of Kd = 0.2 nM for IGF II (linear Scatchard plots) were determined. Competitive binding studies indicated a cross-reactivity of approximately 40% for IGF II to the type I IGF receptor.

Purification of the type I insulin-like growth factor receptor from human placenta.

The type I IGF receptor from human placental membranes was purified to near homogeneity by affinity chromatography on IGF I-Sepharose. SDS-polyacrylamide gel electrophoresis of the affinity purified type I IGF receptor demonstrated a high molecular weight protein with Mr greater than or equal to 300,000 under non-reducing conditions. After reduction with 2-mercaptoethanol two protein bands were found of Mr = 125,000 and 95,000, representing the alpha- and beta-subunits of the receptor molecule, respectively. A co-purification of the insulin receptor through the IGF I-affinity column could be avoided by a preincubation step with insulin.

Amino acid sequence of a variant pro-form of insulin-like growth factor II.

Human serum contains, in addition to the "classical" 7.5-kDa insulin-like growth factors (IGFs) I and II, small amounts of larger IGF-II. A 10-kDa IGF-II was isolated by gel filtration, immunoaffinity chromatography, and reversed-phase HPLC. Upon amino acid sequence determination, a substitution of Cys-Gly-Asp for Ser-33 was found as well as a COOH-terminal extension of 21 residues (E peptide). These sequence differences suggest that 10-kDa IGF-II is a precursor of a variant IGF-II. Since the substitution is not located at a known intron/exon hinge region, the finding of this variant IGF-II is evidence for the presence of more than one gene for IGF-II.

Reconstitution of human erythrocyte membrane acetylcholinesterase in phospholipid vesicles. Analysis of the molecular forms by cross-linking studies.

Unilamellar lipid vesicles were formed upon removal of Triton X-100 with Amberlite XAD-2 from a mixture of egg phosphatidylcholine and Triton-solubilized pure human erythrocyte membrane acetylcholinesterase. A majority of large (230 nm diameter) vesicles together with a minor population of smaller (30 nm diameter) strictures were observed in freeze-fracture electron micrographs. Reconstitution experiments performed with [phenyl-3H(n)]-Triton X-100 showed that only one detergent molecule per 600 lipid molecules was present in the vesicles. Density gradient centrifugation showed co-sedimentation of acetylcholinesterase with the lipid vesicles. About 60% of the incorporated enzyme was directed towards the vesicle exterior and could be partially degraded by papain. Mainly dimeric acetylcholinesterase was found when the reconstituted or, alternatively, the lipid-free but Triton-solubilized enzyme were cross-linked with glutaraldehyde. Aggregates were observed when the detergent-depleted oligomeric forms of the enzyme were cross-linked. The results thus indicate that mainly the dimeric enzyme form is present in a phospholipid environment.

Molecular forms of purified human erythrocyte membrane acetylcholinesterase investigated by crosslinking with diimidates.

Several molecular forms of human erythrocyte membrane acetylcholinesterase have been studied after crosslinking with bifunctional diimidates. The crosslinked products were analysed by centrifugation on linear sucrose density gradients containing Triton X-100. Molecular weights of covalently linked oligomers were estimated by sodium dodecylsulfate gel electrophoresis. It was shown that acetylcholinesterase crosslinked in absence of Triton X-100 consists of molecular forms built up by dimeric protomers. These dimers were identical with the enzymatically active species sedimenting with 6.5S in linear sucrose density gradients.