Charge engineering of a protein domain to allow efficient ion-exchange recovery.

We have created protein domains with extreme surface charge. These mutated domains allow for ion-exchange chromatography under conditions favourable for selective and efficient capture, using Escherichia coli as a host organism. The staphylococcal protein A-derived domain Z (Zwt) was used as a scaffold when constructing two mutants, Zbasic1 and Zbasic2, with high positive surface charge. Far-ultraviolet circular dichroism measurements showed that they have a secondary structure content comparable to the parental molecule Zwt. Although melting temperatures (Tm) of the engineered domains were lower than that of the wild-type Z domain, both mutants could be produced successfully as intracellular full-length products in E. coli and purified to homogeneity by ion-exchange chromatography. Further studies performed on Zbasic1 and Zbasic2 showed that they were able to bind to a cation exchanger even at pH values in the 9 to 11 range. A gene fusion between Zbasic2 and the acidic human serum albumin binding domain (ABD), derived from streptococcal protein G, was also constructed. The gene product Zbasic2-ABD could be purified using cation-exchange chromatography from a whole cell lysate to more than 90% purity.

Genetic investigation of the porphobilinogen deaminase gene in Swedish acute intermittent porphyria families.

A total of 12 mutations associated with acute intermittent porphyria (AIP) have been detected in the porphobilinogen deaminase gene in Swedish AIP families. Three of them are newly discovered and unique to the Swedish population: a splice mutation in intron 6 (int6+1), a missense mutation in exon 11 (Gly216Asp) and a TG deletion in exon 14.

Characterization and regulation of the nonerythroid porphobilinogen deaminase promoter.

The non-erythroid porphobilinogen deaminase (E.C. 4.3.1.8) promoter was investigated according to sequence changes and transcriptional activity. The minimal promoter sequence required for maximal transcription, was localized by deletion mapping to -243 to -115 relative to the translational start site. A negative transcriptional element was found between -55 and +1, indicating a repression mechanism. A new polymorphism was identified, at position -235 within the minimal promoter, both in acute intermittent porphyria patients and healthy control subjects. The polymorphic variants did not affect expression of cloned promoter fragments under the conditions used.

Four mutations in the porphobilinogen deaminase gene in patients with acute intermittent porphyria.

We have detected four different mutations in the porphobilinogen deaminase (PBGD) gene in acute intermittent porphyria (AIP) families from England, Norway, and Sweden. A splicing mutation in the first position of intron 8 (Int8 + 1) was found in a family from England and a missense mutation in exon 12 (Glu250) was detected in a Norwegian family. Two mutations were identified in Swedish families, one splicing mutation in the first position of intron 3 (Int3 + 1) and one missense mutation in exon 8 (Pro119).

Diagnosis of acute intermittent porphyria in northern Sweden: an evaluation of mutation analysis and biochemical methods.

OBJECTIVE: To validate the use of a recently observed guanine to adenine mutation in exon 10 in the porphobilinogen deaminase (PBGD) gene as a diagnostic marker of acute intermittent porphyria (AIP). To evaluate the efficiency of the traditional biochemical diagnostic methods. DESIGN: Matched and blinded case-control study (1:4). SETTING: A primary health care centre in Arjeplog, the National Porphyria Research Unit and a department of clinical genetics in Stockholm. SUBJECTS: A total of 48/49 (98%) patients over the age of 15 years living in Arjeplog with AIP, diagnosed according to standard clinical and biochemical criteria. For each AIP patient, four controls were matched for age, sex and geographical area and 164/196 (86%) participated. In the validity study, 35 patients were selected as indisputable AIP gene carriers, according to strict biochemical criteria, and 92 matched controls were selected with strict exclusion criteria. MAIN OUTCOME MEASURES: Validity, specificity and sensitivity of DNA diagnosis for this AIP mutation. Specificity and sensitivity of traditional biochemical methods. RESULTS: Validity study: the mutation was found in all 35 individuals classified as carriers of AIP. None of the 92 controls had the mutation. Evaluation study: all 48 AIP gene carriers, diagnosed by traditional methods, had the mutation, as had one of the control persons. In an inconclusive group of five persons with heredity for AIP, two had a positive DNA test. CONCLUSIONS: The PBGD mutation analysis was found to have full specificity and sensitivity and can be used as the sole diagnostic method in the family complex studied, representing the major AIP mutation in Sweden. The traditional diagnostic methods, used in optimal combinations, work in most cases, but they do not show high precision. However, they must be used when the specific mutation in the PBGD gene is not known.

Two new mutations in the porphobilinogen deaminase gene and a screening method using PCR amplification of specific alleles.

Acute intermittent porphyria (AIP) is attributable to defects in the porphobilinogen deaminase (PBGD) gene. Two new mutations have been found in the PBGD gene in Swedish families. The first is a G to A splice mutation in the last position of intron 9. A screening method using allele-specific amplification has been designed for the rapid detection of this mutation. The second mutation is a C to T substitution in exon 10, changing Arg201 to Trp. This mutation can be detected by restriction enzyme cleavage.

Genetic carrier detection in Norwegian families with acute intermittent porphyria.

Early detection of carriers of acute intermittent porphyria (AIP) is of great value as an assistance for correct diagnosis and prevention of attacks. In order to complement traditional biochemical methods, restriction fragment length polymorphism (RFLP) studies as well as analysis for a previously identified point mutation were included in a study of three Norwegian AIP families. Several asymptomatic carriers could be identified, and the study thus demonstrates the usefulness of the combination of biochemical and genetic analysis.

Genetic heterogeneity of the porphobilinogen deaminase gene in Swedish families with acute intermittent porphyria.

Acute intermittent porphyria (AIP) is an autosomal dominant metabolic disorder affecting the enzyme porphobilinogen (PBG) deaminase in the heme biosynthetic pathway. The highest prevalence of the disorder has been observed in Scandinavia, especially in northern Sweden (Lappland) where it occurs with a prevalence of 1 in 1500. Biochemical assays of the activity and concentration of PBG deaminase in red blood cells, haplotyping with 4 intragenic restriction fragment length polymorphisms (RFLPs) (MspI, PstI, BstNI, ApaLI) using the polymerase chain reaction (PCR) and screening for known base substitutions by oligonucleotide probes was performed in 28 Swedish AIP families. There was no close relationship between haplotype, biochemical findings (PBG deaminase activity, enzyme-linked immunosorbent assay [ELISA], and excess urinary excretion of delta-aminolevulinic acid or PBG), and a specific mutation. Three different haplotypes were identified. The haplotype 2/1/1/2 (MspI/PstI/BstNI/ApaLI; +/-/-/+) was found to be the most frequent among gene carriers (P less than 0.001). The disease segregated with the haplotype 2/1/1/2 in the 10 families originating from northern Sweden. All 28 families were screened for three known point mutations. Only one was found to carry one of these mutations. Thus, the genetic background of AIP is heterogeneous in Sweden.

Serum levels of the low molecular weight form of insulin-like growth factor binding protein in healthy subjects and patients with growth hormone deficiency, acromegaly and anorexia nervosa.

The low molecular weight form of insulin-like growth factor binding protein (35 kD IGFBP), determined in serum by radioimmunoassay during non-fasting conditions, was high at birth and declined with increasing age during childhood and adolescence (N = 149). Inverse correlation was found between chronological age and 35 kD IGFBP values (r = -0.61, P less than 0.001) during childhood and adolescence, but no age dependency was found in adult subjects aged 20-66 years (N = 73). The mean and 95% confidence limits of immunoreactive 35 kD IGFBP were 34 micrograms/l and 15-79 micrograms/l, respectively, in healthy adults (N = 73) in whom the blood samples were drawn after a one-night fast. The mean level of the 35 kD IGFBP in patients with acromegaly (19 micrograms/l, N = 23) was decreased by 50% in comparison with healthy adults, whereas a 2-fold elevation of the mean levels was found in both anorexia nervosa patients (70 micrograms/l, N = 13) and adult patients with GH deficiency (69 micrograms/l, N = 22). In patients with anorexia nervosa, the 35 kD IGFBP levels were inversely related to the body mass index (r = -0.65, P less than 0.02).

Serum levels of somatomedins and somatomedin-binding protein in pregnant women with type I or gestational diabetes and their infants.

The serum levels of the low mol wt form of somatomedin-binding protein (SMBP) were 5-fold higher in both diabetic (n = 44) and nondiabetic pregnant women (n = 14) than in nonpregnant women. No difference was found between women with type 1 diabetes and those with gestational diabetes. There was a negative correlation between maternal levels of SMBP during the last trimester and the birth weight percentile of the infants (r = -0.51). There was a 2- to 3-fold elevation of maternal insulin-like growth factor (IGF-I) levels during pregnancy in both diabetic and nondiabetic women. A positive correlation (r = 0.49) was found between maternal IGF-I levels and the birth weight percentiles of their infants. The correlation between the ratio of IGF-I to SMBP, which may reflect the IGF-I available to the placenta, to birth weight percentile was higher (r = 0.57), and the SE of estimate of weight percentile was 23%. The ratio between IGF-I and SMBP in cord blood was correlated with birth weight, although cord blood IGF-I and SMBP values were not. The IGF-II levels in cord serum were 50% higher in the infants of diabetic than in those of nondiabetic mothers. These findings raise the questions of whether maternal SMBP levels influence the amount of IGF-I available for the fetal-placental unit and whether IGF-II participates in glucose homeostasis in the fetus.

Somatomedin levels in pregnancy: longitudinal study in healthy subjects and patients with growth hormone deficiency.

Somatomedin (Sm) levels throughout pregnancy were determined in a longitudinal study of four normal women and three patients with GH deficiency by use of the RIA for Sm-A, a newly developed RIA for insulin-like growth factor 2 (IGF-2), and the placenta RRA for Sm-A. In both normal women and those with GH deficiency, there was a continuous rise of immunoreactive Sm-A throughout pregnancy. During the third trimester the levels were 2-fold elevated above the level in nonpregnant age-matched normal subjects. No change of immunoreactive IGF-2 levels was found in the normal pregnant women, whereas an increase from low to normal levels was found in GH-deficient patients during pregnancy. The placenta RRA-Sm-A did not detect the increase of Sm-A immunoactivity in the normal pregnant women, whereas the levels were normalized in GH-deficient patients. After delivery a rapid fall of Sm levels occurred in patients with GH deficiency. The calculated half-lives for immunoreactive Sm-A and IGF-2 were 27 and 52 h, respectively. The birth weights of the seven children were significantly (P less than 0.05) correlated to both the individual peak and the mean maternal value of immunoreactive Sm-A during the last trimester. The present findings indicate that the production of both IGF-1 and IGF-2 related peptides during pregnancy is independent of maternal pituitary GH production.

Cell-surface immunoglobulin and insulin receptor expression in an EBV-negative lymphoma cell line and its EBV-converted sublines.

Membrane Ig and insulin receptors were assayed in the EBV-negative Ramos lymphoma line and its EBV-converted sublines by surface fluorescence. 125I-protein A-binding assay, immunoprecipitation, and insulin receptor assay. The original Ramos line expressed surface IgM but not IgD and had a low concentration of insulin receptors. Ten of 10 converted lines expressed IgD and a variable but usually high insulin-binding capacity. Molecular weight analysis of immunoprecipitated Ig showed the presence of 2 mu-chains and 2 delta-chains. One of each heavy chain type could be characterized as membrane Ig, whereas the other was cytoplasmic. Antisera against the lambda-chain precipitated mu and delta heavy chains as well, indicating that the delta-chain expressed on the membrane of the EBV-converted Ramos lines is bound to the lambda light chain. The results show that the differentiation of a Burkitt lymphoma line is not completely "frozen" and can be induced to change in a more "activated" direction by EBV conversion.

Comparative clinical evaluation of lofepramine and imipramine. Psychiatric aspects.

Lofepramine, an imipramine analogue, was compared with imipramine in a multicentre, double-blind clinical trial. The 62 patients (31 in each of two treatment groups) had a depressive syndrome that normally would have been treated with a tricyclic antidepressant. These patients had not received any adequate treatment for this present depressive episode. After a wash-out period and once a week during treatment (up to 5 weeks), routine laboratory tests and electrocardiograms was done. The dosage was 50 mg t.i.d. for imipramine and 70 mg t.i.d. for lofepramine. Depression ratings with the Cronholm-Ottosson depression rating scale were performed before treatment and once weekly for 3 weeks and then in the 5th week. The last four ratings were combined with rating of side-effects. In the 5th week of treatment 15 out of 31 in the lofepramine group and 18 out of 31 in the imipramine group had recovered. This difference was not significant, nor did the median values of individual symptoms differ between the groups. The side-effects were moderate and the two groups only differed significantly in the items "dry mouth" and accommodation disturbances in favour of lofepramine. The drug compliance was checked by plasma levels of desmethylimipramine in the imipramine group, parent compound, and "apparent" desmethylimipramine in the lofepramine group. The relationship between plasma drug levels, the effect on noradrenaline uptake in vitro and amelioration discussed in Siwers et al. (1977) in this issue. The clinical outcome in the two groups did not differ significantly; interpretation of this result is discussed in relation to the reliability and selection of patients.

Comparative clinical evaluation of lofepramine and imipramine. Pharmacological aspects.

A multicentre comparative clinical evaluation of lofepramine, an imipramine analogue, and imipramine has been made with double-blind technique and fixed dosage (lofepramine 70 mg t.i.d., imipramine 50 mg t.i.d.). Plasma was drawn after 3 weeks for determination of noradrenaline-uptake inhibitory capacity of the parent compound and/or its active metabolites. Plasma concentrations of lofepramine and desmethylimipramine (DMI) were determined in the same samples. The concentrations of lofepramine in the whole material were low (5-27 ng/ml) except for one patient who had a level of 53 ng/ml. In both groups of patients there was an almost 40-fold range in the plasma levels of DMI or apparent DMI. The patients were rated for severity of depression before treatment, then once weekly for 3 weeks and finally during the fifth week. For further information concerning the psychiatric aspects, see d'Elia et al. in this issue (1977). A significant correlation was found between the concentrations of DMI and the noradrenaline-uptake inhibitory capacity in the plasma samples. No correlations were found between uptake inhibitory capacity of plasma samples and the amelioration scores.