Improved binding of a bivalent single-chain immunotoxin results in increased efficacy for in vivo T-cell depletion.

Anti-CD3 immunotoxins exhibit considerable promise for the induction of transplantation tolerance in pre-clinical large animal models. Recently an anti-human anti-CD3epsilon single-chain immunotoxin based on truncated diphtheria toxin has been described that can be expressed in CHO cells that have been mutated to diphtheria toxin resistance. After the two toxin glycosylation sites were removed, the bioactivity of the expressed immunotoxin was nearly equal to that of the chemically conjugated immunotoxin. This immunotoxin, A-dmDT390-sFv, contains diphtheria toxin to residue 390 at the N-terminus followed by VL and VH domains of antibody UCHT1 linked by a (G(4)S)(3) spacer (sFv). Surprisingly, we now report that this immunotoxin is severely compromised in its binding affinity toward CD3(+) cells as compared with the intact parental UCHT1 antibody, the UCHT1 Fab fragment or the engineered UCHT1 sFv domain alone. Binding was increased 7-fold by adding an additional identical sFv domain to the immunotoxin generating a divalent construct, A-dmDT390-bisFv (G(4)S). In vitro potency increased 10-fold over the chemically conjugated immunotoxin, UCHT1-CRM9 and the monovalent A-dmDT390-sFv. The in vivo potency of the genetically engineered immunotoxins was assayed in the transgenic heterozygote mouse, tgepsilon 600, in which the T-cells express human CD3epsilon as well as murine CD3epsilon. T-cell depletion in the spleen and lymph node observed with the divalent construct was increased 9- and 34-fold, respectively, compared with the monovalent construct. The additional sFv domain appears partially to compensate for steric hindrance of immunotoxin binding due to the large N-terminal toxin domain.

Expression of an anti-CD3 single-chain immunotoxin with a truncated diphtheria toxin in a mutant CHO cell line.

ADP-ribosylating immunotoxins are generally expressed in Escherichia coli and then refolded in vitro. Because the efficiency of the in vitro refolding process decreases with the number of protein domains and internal disulfide bonds, these immunotoxins have been generally limited to single-chain monovalent structures. We now show that using the hamster cell line CHO K1 RE1.22c (J. M. Moehring and T. J. Moehring, 1979, Somat. Cell Genet. 5, 453-468) that has been mutated to ADP-ribosylation insensitivity, a level of 4 microg/ml of a truncated anti-T cell immunotoxin, DT390-scFvUCHT1, can be secreted into the medium. This immunotoxin is glycosylated at the two potential N-linked glycosylation sites in the toxin moiety: positions 16-18 in the A chain and residues 235-237 in the B chain. The glycosylated immunotoxin is relatively nontoxic (IC(50) 4.8 x 10(-10) M). Removal of the N-linked oligosaccharides by N-glycosidase F treatment or mutations at the two N-linked glycosylation sites results in a highly active immunotoxin with an IC(50) of 4 x 10(-12) M toward CD3(+) Jurkat cells. This is a 12-fold increase in toxicity over the same immunotoxin harvested from E. coli periplasm without refolding. A single Asn(235) Ala mutation that removed the B chain glycosylation was nearly as toxic as the double mutant. This suggests that B chain glycosylation is the major cause for the loss of toxicity.

A specific 3' exonuclease activity of UvrABC.

Specific cutting of undamaged DNA by UvrABC nuclease is observed. It occurs seven nucleotides (nt) from the 3' terminus of oligonucleotides annealed to single-stranded M13 DNA circles. Although the location of the UvrABC cut on undamaged DNA is similar to that of the cut on the 5' side of a damaged DNA site during the dual incision reaction, the cut of undamaged DNA is not an intermediate in the dual incision step. On DNA duplexes with a single AAF adduct, the anticipated cut at the eighth phosphodiester bond 5' of the lesion is present, but extra cuts at 7-nt increments are observed at the 15th and 22nd phosphodiester bonds. We suggest that these additional cuts are made by the UvrABC activity observed on undamaged DNA; such activity is referred to as ABC 3' exonuclease and may play a significant role by providing a suitable gap for RecA-mediated recombinational exchanges during repair of interstrand crosslinks and closely opposed lesions. This ABC 3' exonuclease activity depends on higher concentrations of Uvr proteins as compared with dual incision and may be relevant to reactions that occur when UvrA and UvrB are increased during SOS induction.

UvrAB activity at a damaged DNA site: is unpaired DNA present?

To study the activity of the Escherichia coli UvrA and UvrB nucleotide excision repair proteins during the formation of the pre-incision complex at a damaged DNA site, we used substrates with modifications around a single 2-(acetylamino)fluorene (AAF) lesion. Based on the release of AAF-containing oligonucleotides from a single-stranded DNA circle, we conclude that during interaction with our substrates UvrAB introduces changes in DNA which are localized at the lesion and are limited to 1-3 bp. Since these changes might include a denaturation of DNA at the lesion site and, consequently, a bubble structure might be present in a pre-incision complex, we studied incision activity of UvrABC excinuclease on substrates with 1-4 unpaired bases next to an AAF adduct. Opening more than one base on either or both sides of the lesion caused a significant decrease in the incision activity of UvrABC, but did not change the position of the incision sites. We conclude that the UvrAB action leading to a pre-incision complex does not include the formation of a bubble intermediate generated by extensive denaturation of base pairs.

The limited strand-separating activity of the UvrAB protein complex and its role in the recognition of DNA damage.

The recognition by Escherichia coli Uvr nucleotide excision repair proteins of a variety of lesions with diverse chemical structures and the presence of helicase activity in the UvrAB complex which can displace short oligonucleotides annealed to single-stranded DNA led to a model in which this activity moves UvrAB along undamaged DNA to damaged sites where the lesion blocks further translocation and the protein-DNA pre-incision complex is formed. To evaluate this mechanism for damage recognition, we constructed substrates with oligonucleotides of different lengths annealed to single-stranded DNA circles and placed a single 2-(acetylamino)fluorene (AAF) lesion either on the oligonucleotide or on the circle. For the substrates with no lesion, the UvrAB complex effectively displaced a 22-mer but not a 27-mer or longer fragments. The presence of AAF on the oligonucleotide significantly increased the release of the 27-mer but oligomers of 30 or longer were not separated. Placing the lesion on the circular strand did not block the release of the fragments. Instead, the releasing activity of UvrAB was stimulated and also depended on the length of the annealed oligonucleotide. These observations do not agree with the predictions of a damage recognition mechanism that depends on helicase-driven translocation. Most likely, the strand-separating activity of UvrAB is a consequence of local changes occurring during the formation of a DNA-protein pre-incision complex at the damaged site and is not due to translocation of the protein along undamaged DNA to locate a lesion.

Recombinational analysis of a natural noncytopathic human immunodeficiency virus type 1 (HIV-1) isolate: role of the vif gene in HIV-1 infection kinetics and cytopathicity.

Two molecularly cloned coisolates of human immunodeficiency virus type 1 (HIV-1) have been found to exhibit different phenotypes of viral expression, either rapid and cytopathic (N1T-A virus) or delayed and noncytopathic (N1T-E virus [X. Ma, K. Sakai, F. Sinangil, E. Golub, and D. J. Volsky, Virology 176:184-194, 1990]). To identify the viral genetic elements responsible for these phenotypes, we prepared reciprocal recombinants in different regions of N1T-A and N1T-E viral genomes. Infectivity experiments with the recombinant viruses revealed that the rapid/cytopathic (N1T-A-like) phenotype assorted cleanly with the V1f-coding region and Vif expression. The smallest HIV-1 DNA region that conferred the complete phenotypic switch was a 284-bp NdeI-StuI fragment within the vif open reading frame. Nucleotide sequence analysis revealed a 35-bp deletion starting at nucleotide 218 in the N1T-E vif gene. A 23-kDa Vif protein was detected by immunoblotting using Vif-specific antiserum in extracts of cells infected with N1T-A but not N1T-E virus. No detectable vif protein was found in association with sedimented particles of either virus. Cotransfection of a eucaryotic vif expression plasmid with N1T-E DNA complemented the N1T-E defect; rapid/cytopathic infection similar to that in N1T-A-transfected cells was observed. We conclude that Vif controls the rate, and consequently the cytopathic outcome, of HIV-1 infection.

[The concentration of glucose in the amniotic fluid in a high risk pregnancy group]. / Kontsentratsiia gliukozy v amnioticheskoi zhidkosti u beremennykh gruppy vysokogo riska.

Amniotic fluid glucose levels were measured in 125 pregnant women included in high-risk group in respect of perinatal pathology. In the reference group the measurements have demonstrated an agreement between glucose concentrations in the amniotic fluid and gestation term and an inverse relationship between pregnancy progress and glucose level, whereas in diabetics this relationship was direct. Fetal developmental defects were associated with elevated glucose content in the amniotic fluid, concomitant placental abnormalities were conducive to reduction of these levels, and no correlations between this parameter and pregnancy terms were observed.

[The prenatal diagnosis of obstructive uropathy and its course in the postnatal period]. / Prenatal'naia diagnostika obstruktivnoi uropatii i ee techenie v postnatal'nom periode.

The results of ultrasound investigation of 24 pregnant women at pregnancy term of 22-39 weeks, who had a developmental anomaly of the urinary system of a fetus been revealed, and as well the findings of complex postnatal examination of 24 children at the age of from 10 days to 12 months are presented. In 62.5% of fetuses, obstructive uropathy was revealed, in 37.5%--cystic anomaly of the kidneys. At the postnatal period, obstructive uropathy was characterized by progressive development of retentive changes in the upper urinary tract requiring the operative correction, or by their stable dilation subject to conservative treatment.

[Invasive methods of prenatal diagnosis of congenital and hereditary pathology]. / Invazivnye metody v prenatal'noi diagnostike vrozhdennoi i nasledstvennoi patologii.

The paper presents an experience with invasive prenatal diagnostic tests in 409 pregnant women at a high risk for perinatal diseases. Techniques of amniocentesis, chorionic villus sampling, placenta biopsy, chordocentesis, cardiac fetal puncture are described and their efficacy and safety compared. Indications and contraindications for the invasive procedures are reviewed. Concomitant use of noninvasive and invasive techniques is suggested for improving the efficacy of prenatal diagnosis.

[Skin cholesterol level and its correlation with the lipid indicators of the blood serum in healthy persons and in patients with ischemic heart disease]. / Soderzhanie kholesterina v kozhe i ego korreliatsiia s lipidnymi pokazateliami syvorotki krovi u zdorovykh liudei i bol'nykh ishemicheskoi bolezn'iu serdtsa.

Skin free and esterified cholesterol in the forearm and serum lipid levels were examined in 42 male coronary patients and 46 normal male subjects. Irrespective of the type of dislipoproteinemia or normolipidemia, total cholesterol and its free fraction appear to be significantly increased in coronary patients, as compared to normal subjects. There were no significant correlations between the examined skin and serum parameters, either in coronary patients, or in the controls. Possible use of skin cholesterol measurement in the diagnosis of lipid disturbances leading to atherosclerosis is discussed.

[Effect of 17 alpha-ethinyl estradiol on the blood serum lipid indices of rats]. / Vliianie 17 al'fa-étiniléstradiola na lipidnye pokazateli syvorotki krovi krys.

The administration of 17-ethinyl estradiol (0.25 mg/kg) to male Wistar rats caused on the 3rd day a decrease of the levels of free and esterified cholesterol, blood serum triglycerides and also a reduction of the content of esterified cholesterol with respect to total cholesterol while the lecithin-cholesterol-acyltransferase activity appeared to be unchanged. The effect of ethinyl estradiol on blood lipid parameters against the background of lipemia induced by triton WR 1339 was reduced.

[Changes in the activity of enzymes of cholesterol esters synthesis and hydrolysis in rat adrenals after treatment with 17 alpha-ethinyl estradiol]. / Izmenenie aktivnosti fermentov sinteza i gidroliza éfirov kholesterina v nadpochechnikakh krysy pri vozdeistvii 17 alpha-étiniléstradiola.

A study was made of the effect of hypocholesterolemia induced by 3-day administration of 17 alpha-ethinyl estradiol on the activity of lysosomal and cytoplasmatic cholesterol esterases, acyl-CoA: cholesterol acyltransferase and on the free and esterified cholesterol concentrations in the rat adrenals. A decrease in the content of esterified cholesterol in the adrenal tissue was accompanied by a decrease in the activity of acyl-CoA: cholesterol acyltransferase and by an increase in the activity of lysosomal cholesterol esterase. The activity of cytoplasmatic cholesterol esterase was not changed significantly. The data obtained were discussed with relation to the synthesis of steroid hormones in the rat adrenals.

[Effect of 17 alpha-ethinylestradiol on the activity of enzymes synthesizing and hydrolyzing cholesterol esters in the rat liver]. / Vliianie 17 al'fa-étiniléstradiola na aktivnost' fermentov sinteza igidroliza éfirov kholesterina v pecheni krysy.

Intraperitoneal injection of 25 micrograms/100 g body weight of 17 alpha-ethinyl estradiol to rats was shown to decrease serum cholesterol and to increase hepatic cholesterol. The rise in the level of non-labeled and C14-labeled free and esterified cholesterol in hepatic homogenate, as well as in lysosomal and cytosol fractions was accompanied by reduced activity of acyl-CoA-cholesterol acyltransferase and increased activity of lysosomal cholesterol esterase, as compared with the controls. The activity of cytoplasmic cholesterol esterase remained practically unchanged. Fistula bile of treated rats collected during 30 min was analyzed for the concentration of free cholesterol and bile acids. It has been shown that treatment of rats with 17 alpha-ethinyl estradiol caused an increase in hepatic cholesterol elimination via bile pathways.

Can Ca2+-dependent competence be repeatedly induced in the same Escherichia coli cells?

With the help of devised multicycle consecutive transformation (MCT) it is shown that Ca2+-dependent competence can be repeatedly induced in the same population of Escherichia coli cells. The same fraction of cells is induced to competence and transformed during MCT. In contrast to the results on classical transformation with mixed DNA preparations, no double transformants are observed in MCT. The competent cells and transformants are found to be more fragile than nontransformed cells. The latter are represented presumably by the cells that have not absorbed exogenous plasmid DNA. The results suggest that there is strong interference between plasmid DNAs during MCT, and that the presence of exogenous DNA makes the cells more sensitive to the apparently harmful procedure of repeated competence induction.