Effect of acutely increased left ventricular afterload on work output from the right ventricle in conscious dogs.

OBJECTIVE: To determine the effect of acute increments in left ventricular afterload on the stroke work output of the right ventricle in vivo. METHODS: After pharmacologic attenuation of autonomic reflexes, left and right ventricular pressure-volume data were obtained in 9 conscious dogs during vena caval occlusions performed before and during aortic constriction. RESULTS: The relationship between right ventricular stroke work and end-diastolic volume during vena caval occlusion was highly linear (r = 0.97 +/- 0.02), but the slope decreased by 20% +/- 13% during aortic constriction sufficient to increase left ventricular mean ejection pressure by 25% +/- 14% (P <.05). The volume-axis intercept remained constant. Similarly, the slope of the linear relationship between right ventricular free wall regional segment work and end-diastolic segment length declined by 22% +/- 10% during aortic constriction (P <.05), without significant change in the length-axis intercept. The reduction in both global and regional right ventricular stroke work at any given preload with increased left ventricular afterload was due entirely to decreased right ventricular stroke volume and free wall shortening, because right ventricular mean ejection pressure was unchanged. Additional experiments were performed in 5 open-chest dogs to produce a greater reduction in left ventricular free wall shortening than observed with aortic constriction by transient constriction of the left circumflex coronary artery. However, this intervention had no effect on right ventricular free wall segment work output. CONCLUSION: Increased left ventricular afterload decreases global and regional right ventricular stroke work at any given preload, a direct, negative systolic ventricular interaction.

[Semen analysis results and air pollution in the group of men from infertile couples in the Lower Silesia in 1977-2000]. / Analiza wplywu zanieczyszczen powietrza na obraz nasienia mezczyzn z nieplodnych par w Regionie Dolnoslaskim w latach 1977-2000.

OBJECTIVES: To evaluate the relation among the concentration of selected air pollution and seminal parameters, examined from 1977 to 2000. DESIGN: Semen analysis and air pollution results were retrospectively evaluated. MATERIALS AND METHODS: We have analysed semiograms from 1363 men from infertile couples inhabiting Lower Silesia. Seminal volume, sperm concentration, percentage of pathologic sperms were measured in all men. Estimation of mean seminal volume, total sperm number, sperm motility, and percentage of pathologic sperms per year was performed. Average values for NO2, CO, SO2 and dust concentrations during the study were taken from official sources. RESULTS AND CONCLUSIONS: We showed the statistically significant increase in the percentage of pathological sperms (R2 = 0.9, p < 0.05), the slight increase in the semen volume(R2 = 0.4, p < 0.05). The total sperm count in the semen samples revealed a slight increase (R2 = 0.25, p < 0.05). The sperm concentrations and the percentage of motile sperms remained relatively stable and relatively constant. Statistically important decrease in NO2 (R2 = 0.85, p < 0.05), SO2 (R2 = 0.96, p < 0.05) and dust concentration (R2 = 0.89, p < 0.05) and no change in CO concentration was revealed. There is no correlation among concentrations of dust, NO2, SO2, CO and the increase in percentage of pathologic sperms.

[Analysis of newborns' health status in twin pregnancies according to the duration of pregnancy and its delivery based on the materials of Reproduction and Obstetrics Clinic in 1995-1999]. / Analiza stanu noworodków w ciazach blizniaczych w zaleznosci od czasu trwania ciazy i sposobu jej rozwiazania w materiale Kliniki Rozrodczosci i Poloznictwa we Wroclawiu w latach 1995-1999.

On the ground of the analysis of 79 twin pregnancies, the valuation of newborns' condition in Apgar's scale, according to the delivery means, has been executed. The lack of differences between the mean values of the scale has been ascertained. There was no indication of any difference between the condition of the twin I and II. Furthermore, there has been executed a detailed comparative analysis of the newborns' condition in the four periods of the pregnancy duration: 23-27, 28-32, 33-37 and 38-42 weeks of gestation. It has been ascertained that the newborns delivered through the abdominal delivery were in better condition than those born in the spontaneous delivery, in the 28-32 weeks of gestation period. The use of rather intraspinal rather than general anesthesia in the 33-37 weeks period gave better results by improving the newborns' condition. Moreover, there has been stated a similar condition of the newborns delivered through either spontaneous or abdominal delivery with intraspinal anesthesia in the periods of 33-37 and 38-42 weeks of gestation.

[Analysis of twin pregnancies on the ground of women hospitalized in the Department of Reproduction and Obstetrics Medical University in Wroclaw in 1995-1999]. / Analiza ciaz blizniaczych w oparciu o material Kliniki Rozrodczosci i Poloznictwa AM we Wroclawiu w latach 1995-1999.

The aim of this study was the general valuation of the course and the delivery means of the twin pregnancies. The research material composed of 83 from among 5540 pregnant women hospitalized in the Department of Reproduction and Obstetrics Medical University of Wroclaw in the years 1995-1999. The mean body mass values and the condition of the newborns have been analyzed on the ground of Apgar's scale, according to the date of delivery. In the period between 23 and 42 week of pregnancy a very high correlation between fetus' body mass and a high correlation between Apgar's scale and the pregnancy's duration has been ascertained. These values have also been estimated in particular periods: 23-27, 28-32, 33-37 and 38-42 weeks of gestation. Statistical analysis didn't indicate any difference between the mean values of Apgar's scale of the newborns from the periods of 33-37 and 38-42 weeks of gestation. There was no evidence of differences either in Apgar's scale values or in the twins' I and II body masses, as well in the whole examined group as in particular periods.

Biosynthesis of ganglioside mimics in Campylobacter jejuni OH4384. Identification of the glycosyltransferase genes, enzymatic synthesis of model compounds, and characterization of nanomole amounts by 600-mhz (1)h and (13)c NMR analysis.

We have applied two strategies for the cloning of four genes responsible for the biosynthesis of the GT1a ganglioside mimic in the lipooligosaccharide (LOS) of a bacterial pathogen, Campylobacter jejuni OH4384, which has been associated with Guillain-Barré syndrome. We first cloned a gene encoding an alpha-2, 3-sialyltransferase (cst-I) using an activity screening strategy. We then used nucleotide sequence information from the recently completed sequence from C. jejuni NCTC 11168 to amplify a region involved in LOS biosynthesis from C. jejuni OH4384. The LOS biosynthesis locus from C. jejuni OH4384 is 11.47 kilobase pairs and encodes 13 partial or complete open reading frames, while the corresponding locus in C. jejuni NCTC 11168 spans 13.49 kilobase pairs and contains 15 open reading frames, indicating a different organization between these two strains. Potential glycosyltransferase genes were cloned individually, expressed in Escherichia coli, and assayed using synthetic fluorescent oligosaccharides as acceptors. We identified genes encoding a beta-1, 4-N-acetylgalactosaminyl-transferase (cgtA), a beta-1, 3-galactosyltransferase (cgtB), and a bifunctional sialyltransferase (cst-II), which transfers sialic acid to O-3 of galactose and to O-8 of a sialic acid that is linked alpha-2,3- to a galactose. The linkage specificity of each identified glycosyltransferase was confirmed by NMR analysis at 600 MHz on nanomole amounts of model compounds synthesized in vitro. Using a gradient inverse broadband nano-NMR probe, sequence information could be obtained by detection of (3)J(C,H) correlations across the glycosidic bond. The role of cgtA and cst-II in the synthesis of the GT1a mimic in C. jejuni OH4384 were confirmed by comparing their sequence and activity with corresponding homologues in two related C. jejuni strains that express shorter ganglioside mimics in their LOS.

[Changes in morphological picture of the semen in the group of men from infertile couples in the Lower Silesia in 1977-1993]. / Zmiany w morfologicznym obrazie nasienia mezczyzn z bezdzietnych par malzenskich, mieszkanców regionu dolnoslaskiego, w latach 1977-1993.

OBJECTIVES: To reveal the trends of the particular semen parameters changes during the 17 years period. DESIGN: Semen analysis results were retrospectively evaluated. MATERIALS AND METHODS: We have analysed semiograms from 618 men from infertile couples in 1977-1993. Regression analysis of the results from years of observation was performed. RESULTS AND CONCLUSIONS: We showed the statistically important increase in the percentage of pathological sperms (R2 = 0.89, p < 0.0001), the slight increase in the semen volume (R2 = 0.30, p < 0.02). The total sperm count in the semen samples revealed a slight increase (R2 = 0.33, p < 0.02). The sperm concentrations and the percentage of the motile sperms remained relatively stable and relatively constant.

Selection of antibody single-chain variable fragments with improved carbohydrate binding by phage display.

A single-chain variable fragment (Fv) version of a murine monoclonal antibody, Se155-4, specific for Salmonella serogroup B O-polysaccharide, was used as a model system for testing monovalent phage display as a route for enhancing the relatively low affinities that typify anti-carbohydrate antibodies. Random single-chain Fv mutant libraries generated by chemical and error-prone polymerase chain reaction methods were panned against the serogroup B lipopolysaccharide. Panning of a randomly mutated heavy chain variable domain library indicated selection for improved serogroup B binders and yielded six mutants, five of which showed wild type activity by enzyme immunoassay. Two of these were apparently selected on the basis of better functional single-chain Fv yield in Escherichia coli. A heavy chain mutation (Ile77-->Thr) in one mutant, 3B1, appeared to have a particularly dramatic effect, resulting in yields of approximately 120 mg/liter of functional periplasmic product. The sixth mutant, 4B2, had complementarity determining region 1 (CDR1) and CDR2 mutations and demonstrated 10-fold improved binding, by enzyme immunoassay, relative to the wild type. Extensive analysis of antigen-antibody interactions indicated that the improved binding properties of 4B2 were attributable to a higher association rate constant and interaction with an epitope that is larger than the trisaccharide recognized by the wild type. None of the mutations involved known trisaccharide contact residues; this was consistent with analysis of wild type and mutant single-chain Fvs by titration microcalorimetry. Examination of the structure indicated that two mutations in the heavy chain CDR2 provided improved surface complementarity between the protein and the extended epitope encompassing 2 additional hexose residues. However, introduction of only the CDR2 mutations into the wild type structure failed to confer the improved binding properties of 4B2, indicating an indirect effect by the more distant mutations. Panning of randomly mutated light chain variable domain and full-length single-chain Fv mutant libraries did not yield mutants with improved assembly or binding properties.

Probing the combining site of an anti-carbohydrate antibody by saturation-mutagenesis: role of the heavy-chain CDR3 residues.

The carbohydrate-binding site in Fab fragments of an antibody specific for Salmonella serogroup B O-polysaccharide has been probed by site-directed mutagenesis using an Escherichia coli expression system. Of the six hypervariable loops, the CDR3 of the heavy chain was selected for exhaustive study because of its significant contribution to binding-site topography. A total of 90 mutants were produced and screened by an affinity electrophoresis/Western blotting method. Those of particular interest were further characterized by enzyme immunoassay, and on this basis seven of the mutant Fabs were selected for thermodynamic characterization by titration microcalorimetry. With regard to residues that hydrogen bond to ligand through backbone interactions, Gly102H could not be substituted, while several side chains could be introduced at Gly100H and Tyr103H with relatively little effect on antigen binding. There was, however, a preference for nonpolar side chains at position 103H. Substitution of His101H with carboxylate and amide side chains gave mutants with binding affinities approaching that of the wild type; complete side-chain removal by mutation to Gly was tolerated with a 10-fold reduction in binding constant. Analysis of binding by titration microcalorimetry revealed some dramatic thermodynamic changes hidden by the similarity of the binding constants. Similar effects were observed with residue changes in an Arg-Asp salt-bridge at the base of the loop. These results indicate that alterations to higher affinity anti-carbohydrate antibodies are characterized by an enthalpy-entropy compensation factor which allows for fundamental changes in the nature of the binding interactions but impedes engineering for increases in affinity.

Right ventricular preload recruitable stroke work, end-systolic pressure-volume, and dP/dtmax-end-diastolic volume relations compared as indexes of right ventricular contractile performance in conscious dogs.

Three indexes developed originally to assess left ventricular contractile performance were applied instead to the right ventricle (RV) in 11 conscious dogs: the relation between stroke work and end-diastolic volume (EDV), termed the preload recruitable stroke work (PRSW) relation; the end-systolic pressure-volume (ESPV) relation; and the maximum dP/dt (dP/dtmax)-EDV relation. The reproducibility, inotropic sensitivity, chronotropic sensitivity, and afterload sensitivity of these RV relations were compared. RV volume was determined with an ellipsoidal shell subtraction model from orthogonal dimensions measured by sonomicrometry. RV transmural pressure was measured with micromanometers. After autonomic blockade, preload was varied by repeated, transient vena caval occlusions before and during partial occlusion of the main pulmonary artery, after release of the pulmonary arterial occlusion, after calcium infusion, and over a range of heart rates induced by atrial pacing. The slope and volume-axis intercept of the PRSW relation were more reproducible (SD/mean, 7.8 +/- 3.3% and 6.2 +/- 4.1%, respectively) than the slope and volume-axis intercept of the ESPV relation (10.1 +/- 6.7% and 23.0 +/- 31.3%, both p less than 0.05) or the slope and volume-axis intercept of the dP/dtmax-EDV relation (43.4 +/- 70.4% and 153.8 +/- 184.6%, both p less than 0.05). The slope of the PRSW relation increased 32 +/- 17% (p less than 0.05) after calcium infusion, but the volume-axis intercept did not change significantly. In contrast, the slopes of the ESPV and dP/dtmax-EDV relations did not change significantly after calcium infusion, but the volume-axis intercepts decreased significantly (both p less than 0.05). Despite a 71 +/- 26% increase in mean RV ejection pressure during partial occlusion of the main pulmonary artery, the slopes and volume-axis intercepts of both the PRSW and dP/dtmax-EDV relations did not change significantly, but the slope of the ESPV relation increased 45 +/- 22% (p less than 0.05) without significant change in the volume-axis intercept. None of the relations demonstrated significant chronotropic sensitivity. The PRSW relation is the preferred index of RV contractile performance because 1) it is the most reproducible, 2) its slope alone sensitively detects changes in contractile state, and 3) unlike the ESPV relation, it is relatively insensitive to afterload.

Scanning of key residues in antibody binding sites by two saturation-mutagenesis approaches.

The complementarity-determining region 3 of the heavy chain (CDRH3) generally contributes the most to antibody-antigen binding. His101H in CDRH3 of the antibody Se155-4, which is specific for a trisaccharide epitope of Salmonella serotype B O-antigen, was mutated systematically into all nineteen other amino acids by a double mutation approach. Enzyme immunoassay (EIA) and affinity chromatography showed that the Asn, Gln, Gly and Ser mutants exhibited moderate to strong activity. Some mutants, such as Thr and Pro, had weak binding activity, while the acidic and hydrophobic amino acid substitutions resulted in complete loss of activity. A second mutation approach which randomly changed a selected residue into all other nineteen amino acids, while precluding wild-type transformants, is also described.

Crystal structure of T4-lysozyme generated from synthetic coding DNA expressed in Escherichia coli.

The polypeptide produced by expressing a chemically synthesized gene coding for the amino-acid sequence of T4-lysozyme has been crystallized and subjected to X-ray diffraction. The crystal structure has been refined to a standard R-factor of 0.191 for data between 8 and 2 A resolution. The refined model is essentially the same as the well-known structure of wild-type T4-lysozyme determined previously by Matthews et al. (1987). Some small changes in the C-terminal region, which is important in maintaining the folded structure, have been noted. In addition to confirming that the synthetic gene product is very close to the wild type, this structure provides a benchmark for protein engineering experiments on the folding and the catalytic activity of this molecule by the method of gene synthesis.

Hierarchical strategy for protein folding and design: synthesis and expression of T4 lysozyme gene and two putative folding mutants.

A T4 lysozyme-coding DNA sequence of 495 bp was chemically synthesized and cloned by ligation of 26 deoxyribooligonucleotide fragments in two steps with a linearized plasmid followed by transformation. On selection by colony hybridization and DNA sequence analysis, clone pTLY.10 was identified to contain a complete T4 lysozyme synthetic DNA. On expression under lac-promoter, unfused T4 lysozyme was obtained in approximately 4-6% yield. The design and synthesis of two putative folding mutants, flexible (Gly-Gly-Gly) and rigid (Asn-Asp-Gly) at position 73-74-75, were based on hierarchical principles. Both mutants lost enzymatic activity of the wildtype. These results are readily understandable if the hierarchical organization of the structure is taken into account. A possible explanation is that the catalytic sites are blocked in both mutants.

Observation of a possible pause mutant in the total synthesis of T4 lysozyme.

A DNA of 495 bp coding for T4-lysozyme was chemically synthesized and cloned in Escherichia coli. On DNA sequence analysis, clones pTLY.10 and pTLY.9 were identified to contain identical and complete T4-lysozyme coding sequences except that pTLY.9 had an additional 23 bp inverted repeat DNA at the 3'-end of the coding sequence. On expression and purification under similar conditions, T4-lysozymes from these two clones showed different degrees of retention time on HPLC as well as in the rate of enzymatic reaction. We speculate that this difference could be due to the generation of a pause mutant of T4-lysozyme in pTLY.9 under the influence of 3'-inverted repeat DNA that alters the rate of protein synthesis.

Retention of enzymatic activity by N-terminal domain (1-78) T4-lysozyme: expression of synthetic DNA in Escherichia coli.

DNA of 235 b.p. coding for N-terminal domain (1-78) T4-lysozyme was synthesized and cloned by ligating twelve synthetic fragments with a linearized plasmid pUCE8 followed by transformation. On expression in E. coli strain JM103 cells, colonies containing the synthetic DNA were found to be lytic. On purification, clone ptly. 23-5 was found to contain polypeptide (M.W. 10,500), corresponding to N-terminal domain, its dimeric and aggregate form. It was identified by amino acid sequence analysis of the dimeric form.

Transposition studies of mini-Mu plasmids constructed from the chemically synthesized ends of bacteriophage Mu.

We describe below the chemical synthesis of the right and left ends of bacteriophage Mu and characterize the activity of these synthetic ends in mini-Mu transposition. Mini-Mu plasmids were constructed which carry the synthetic Mu ends together with the Mu A and B genes under control of the bacteriophage lambda pL promoter. Derepression of pL leads to a high frequency of mini-Mu transposition (5.6 X 10(-2) which is dependent on the presence of the Mu ends and the Mu A and B proteins. Five deletion mutants in the Mu ends were tested in the mini-Mu transposition system and their effects on transposition are described.

"In vitro" method of assembling a synthetic gene.

Without prior in vitro enzymatic ligation a DNA duplex was assembled successfully by directly transforming competent cells with a mixture containing six synthetic complimentary oligodeoxyribonucleotides and a linearized plasmid. One out of 100 transformants was positive in colony hybridization with one of the synthetic fragment probe. The sequence of the DNA duplex inserted into the plasmid was confirmed by dideoxy sequencing method.

A novel deletion found during cloning of a synthetic palindromic DNA.

A 212-bp palindromic DNA comprising two copies of the left end of bacteriophage Mu was assembled from chemically synthesized oligonucleotides and inserted into plasmid pUC9. When cloned and propagated in Escherichia coli, the palindrome was found to be unstable and was generally lost. However, in a few cases, a precise, asymmetric deletion of one half of the insert was observed. This pattern of deletion suggests that the symmetry axis region of the palindrome was involved as recognition site in the deletion process.

The human preproinsulin gene: synthesis, cloning, gene modification, and expression studies.

A 355 base pair (bp) DNA sequence coding for human preproinsulin has been assembled by joining a synthetic DNA leader sequence coding for 24 preregion amino acids to the previously synthesized DNA duplex of 277 bp constituting the sequence of BCA chain. It was next cloned in M13 mp8 single-stranded bacteriophage and subjected to site-specific mutagenesis and phase shifting to allow its inducible expression under lac operator control. An affinity leader sequence of 25 bp has been added in an attempt to facilitate purification of the preproinsulin.

Synthesis of a human insulin gene. VII. Synthesis of preproinsulin-like human DNA, its cloning and expression in M13 bacteriophage.

A 74-bp DNA sequence coding for the pre sequence of human preproinsulin and containing EcoRI termini was synthesized by the chemical enzymatic method, joined with previously synthesized proinsulin DNA, and cloned in the M 13mp8 vector. A clone pNB82 -121 was identified by DNA sequence which confirmed the correct orientation of the pre sequence to the proinsulin DNA. The EcoRI site at the junction of pre- and proinsulin DNA was eliminated by removing a triplet ATT using a synthetic 19-mer primer. To simplify preproinsulin isolation and to study its expression in the M 13 system, a 25-bp affinity leader sequence coding for (glu)7 was inserted at the remaining EcoRI site; this put the preproinsulin DNA in a correct reading frame with the AUG initiation codon of beta-galactosidase. Preproinsulin was expressed under lac promoter control as analyzed by a radioimmunoassay (RIA) against C-peptide.