High speed on-line DNA sequencing on ultrathin slab gels.
Explore the source record for details and available documents.
Automation↗
Biomedical subjects
J Zimmermann
Publications and source records attributed to J Zimmermann.
Amide isosteres of lexitropsins: synthesis, DNA binding characteristics and sequence selectivity of thioformyldistamycin.
The synthesis and properties of an amide isostere of the antibiotic distamycin, thioformyldistamycin 3 is described. Compound 3 exists predominantly in the E conformation of the thioamide group in freshly prepared DMSO solution but is converted into the Z form, predicted by molecular mechanics to be more stable, on standing for 24 h. The coalescence temperature in DMSO is 110 degrees C by 1H-NMR. The thioformyl moiety of 3 is resistant to both peptidase action and acid treatment. Complementary strand MPE footprinting on a EcoRI/Hind III restriction fragment of pBR322 DNA demonstrated that either E or Z forms of 3 give a single set of footprints very similar to that of the parent antibiotic with strongest protection at TAAG and TATTAT with moderately strong protection at ATTT and AAAA. The strength of binding of 3 and distamycin from delta Tm measurements to either poly.d(AT) or calf thymus DNA is comparable. Molecular modeling predicted a preferred conformation for 3 wherein the C = S bond has a torsional angle of 110 degrees with the pyrrole ring. The energy difference between this conformation and the E form is less than 1 kcal/mole. In contrast the E-form has an energy 17.3 kcal/mole greater than the Z and a value of 26.3 kcal/mole was calculated for the energy barrier between the two isomers.
Base Sequence↗
Monoclonal antibodies to gelonin: production and characterization.
BALB/c mice were immunized with gelonin, a 30 kD glycoprotein (type 1 RIP) from the seeds of Gelonium multiflorum. By polyethylene glycol-induced fusion of isolated spleen cells with the myeloma cell line NS-1, three different hybridomas were obtained. Two of them were found to secrete antibodies of the IgG1 subclass, whereas the third cell line produced antibodies of the IgM type. The IgG1-secreting cell lines were adapted to serum-free medium conditions, and the antibodies were isolated from the culture supernatant. The isolated antibodies recognize independent epitopes on the gelonin molecule. The toxicity of gelonin in reticulocyte lysates was not affected when the protein was incubated with the antibodies. The IgG1s exhibit average affinity constants of about 10(9) M-1 and 10(10) M-1, respectively, as determined by a solid-phase EIA using the avidin-biotin system.
Animals↗
Immunogold labelling of human von Willebrand factor adsorbed to collagen.
von Willebrand factor (vWF) mediates adhesion of platelets to the exposed subendothelium at sites of vascular injury. This function is expressed through binding of vWF to both collagen and receptors on the platelet membrane. We have developed a new method using immunogold staining and electron microscopy, permitting visualization of human vWF adsorbed to collagen fibrils. The electron micrographs revealed strings of gold beads reflecting the polymeric structure of vWF. Our data showed dramatic differences in the binding of vWF to collagens of different sources: high binding density was observed using a collagen preparation isolated from aortic tissue whereas colloidal gold was virtually absent from tendon collagen. Using the immunogold labelling method we demonstrated that high shear rate enhanced vWF binding to aortic collagen.
Adsorption↗
Elevated serum diiodotyrosine (DIT) in severe infections and sepsis: DIT, a possible new marker of leukocyte activity.
Ether link cleavage (ELC) of T4 yielding diiodotyrosine (DIT) has recently been shown in vitro to be the major pathway of T4 metabolism in phagocytosing leukocytes. To evaluate this pathway in vivo and the possible clinical relevance of DIT measurements in diseases with increased leukocyte activity, radioimmunological studies on serum levels of DIT and other thyroid parameters were performed in 125 critically ill patients classified into 3 groups with bacterial infections according to the severity of infection and 1 group without infections. While the pattern of iodothyronine and TSH levels typical for severe nonthyroidal disorders, i.e. decreased total T3 and elevated rT3, normal or decreased total T4 and TSH, and normal free T4, was found in all four groups of intensive care patients studied, elevated serum DIT was observed only in those patients whose clinical course was complicated by severe bacterial infections. Serial measurements revealed a close temporal connection between the infection phase and increased DIT levels. Median values and 16th to 84th percentile ranges (in parentheses) of serum DIT (normal range, 0.02-0.55 nmol/L) were as follows: sepsis, 1.38 (0.32-5.14); severe nonsystemic infections such as peritonitis and abscesses, 3.84 (0.24-17.2); moderate infections such as pneumonia and tracheobronchitis, 0.44 (0.18-1.16); and critical illness without infections, 0.14 (0.08-0.30) nmol/L. These elevations of circulating DIT could neither be correlated with changes in renal function nor attributed to drug effects. The results of the present study do not allow any definitive conclusions to be made about the mechanisms underlying the phenomenon of increased serum DIT levels in infections. Apart from this open question, DIT appears to be a relatively specific serum parameter for the presence and course of severe bacterial inflammations. Its measurement could provide useful clinical information, particularly for monitoring the time course of deep-seated infections.
Bacterial Infections↗
[The value of symptoms and clinical findings in cervical Chlamydia trachomatis infection].
The frequency of symptoms in the case histories of 68 female patients with and of 313 without Chlamydia trachomatis infections was investigated. Dysuria, vaginal discharge and/or burning/itching in the genital region were reported by 50% (n = 34) of chlamydia-positive women and 53.7% (n = 168) of chlamydia-negative women. Clinical investigation of the chlamydia-positive patients revealed discharge in 83.8% (n = 57), contact bleeding in 38.3% (n = 26) and ectopia in 27.9% (n = 19). The corresponding investigations in chlamydia-negative women disclosed discharge in 72.8% (n = 228) women, contact bleeding in 19.8% (n = 62) and ectopia in 16.8% (n = 52). The results were significantly different as far as contact bleeding and ectopia were concerned. Only when yellow or whitish yellow discharge was distinguished from clear discharge was the difference between the two groups significant (66.7% vs 43.9%). Significant numbers of polymorphonuclear leucocytes (greater than 4 per high power field with oil immersion) were found in 52.9% of the chlamydia-positive women, as against 23% of the chlamydia-negative women. Overall, 48.7% of the women with a C. trachomatis infection were found to have notes of symptoms in the history and signs of cervicitis revealed by clinical investigation. Symptoms only were found in 1 woman, while in 36.8% of the patients signs only were found. Neither symptoms nor signs were mentioned by 13.3% of the women. These results indicate that microbiological detection of the infectious agent is obligatory for the diagnosis of C. trachomatis cervicitis.
Bacteriological Techniques↗
[The value of symptoms and findings in urethral Chlamydia trachomatis infection].
The history and the results of clinical investigations in 74 patients with and 262 men without urethral Chlamydia (C.) trachomatis infection were evaluated: symptoms such as dysuria, discharge and/or burning/itching in the genital region were reported by 77% (n = 57) of the patients with an infection and 63.4% (n = 166) of the men without an infection. Upon clinical investigation, discharge was found in 55.4% (n = 41) of the chlamydia-positive patients and in 47.7% (n = 125) of the chlamydia-negative men. Only when yellow, whitish yellow and clear discharge were distinguished from one another was the difference between the two groups of patients (56.1% versus 12%) significant. Microscopic signs of urethritis, i.e. significant numbers of polymorphonuclear leukocytes (greater than 4 per high-power field and oil immersion) were detectable in the smears of 59.5% (n = 44) of the chlamydia-positive patients, in contrast to 15.6% (n = 41) in the smears of chlamydia-negative men. Overall, 67.6% (n = 50) of the patients with a C. trachomatis infection reported symptoms in the anamnesis combined with signs of urethritis upon clinical and microscopic investigation. There were 9.5% (n = 7) of the patients who either mentioned symptoms or showed clinical signs, and 13.5% (n = 10) who neither mentioned symptoms nor showed clinical signs. These results indicate that microbiological detection of the infectious agent is obligatory for diagnosis of urethral infection with C. trachomatis.
Bacteriological Techniques↗
Automated fluorescent sequencing of lambda DNA.
Explore the source record for details and available documents.
Bacteriophage lambda↗
One label, one tube, Sanger DNA sequencing in one and two lanes on a gel.
Explore the source record for details and available documents.
Base Sequence↗
Automated fluorescent sequencing of cosmid DNA.
Explore the source record for details and available documents.
Autoanalysis↗
A simplified protocol for fast plasmid DNA sequencing.
Explore the source record for details and available documents.
Base Sequence↗
Automated DNA sequencing of the human HPRT locus.
The complete sequence of 57 kb of the human HPRT locus has been determined using automated fluorescent DNA sequencing. The strategy employed increasingly directed sequencing methods: A randomly generated M13 library was sequenced to generate contiguous overlapping sets of sequences (contigs). M13 clones at the ends of these contigs were further sequenced using M13 (universal and reverse) and custom oligonucleotide primers to order the contigs and to complete the sequencing project. The human HPRT sequence includes 1676 bp 5' and 15,238 bp 3' to exons 1 and 9, respectively. The sequence contains 49 representatives of the Alu repeat, along with several other types of repetitive sequences. The Alu sequences exhibit a biased orientation, with those sequences in the first half of the locus oriented in the minus direction relative to transcription of the gene (3'----5' = 77%, P less than 0.005) and those sequences in the latter half of the locus oriented randomly (5'----3' = 67%, P less than 0.5). The development and performance of the sequencing strategy and the features of the human HPRT gene are presented.
Amino Acid Sequence↗
Molecular recognition and binding of a GC site-avoiding thiazole-lexitropsin to the decadeoxyribonucleotide d-[CGCAATTGCG]2: 1H-NMR evidence for thiazole intercalation.
The structural and dynamic aspects of the interaction of the thiazole containing lexitropsin (1) with an oligodeoxyribonucleotide were studied by high field 1H-NMR spectroscopy. Complete assignment of the 1H-NMR resonances of lexitropsin 1 was accomplished by 2D-NMR techniques. The complexation-induced chemical shifts and NOE cross peaks in the NOESY map of the 1:1 complex of lexitropsin (1) and d-[CGCAATTGCG]2 reveal that the thiazole ring of the lexitropsin (1) intercalates between dA4.A5 bases and the rest of the ligand resides in the minor groove of the AT rich core of decamer, thus occupying the 5'-AATT sequence on the DNA. Intercalation of the thiazole moiety of the drug has been detected by the presence of intermolecular NOEs both in the major and the minor groove of the decamer helix. The absence of intranucleotide NOEs between base protons and H1'/H2' protons suggested local unwinding of the binding site on the DNA. From COSY and NOESY methods of 2D-NMR, it was established that the N-formyl (amino) terminus of the thiazole lexitropsin (1) is projecting into the major groove towards A5H8 while the amidinium terminus lies in the minor groove towards the T7G8 base pairs of the opposite strand. The expected intranucleotide NOEs confirmed that the decadeoxyribonucleotide in the 1:1 complex exists in a right handed B-conformation. The presence of exchange signals along the binding site 5'-AATT indicated an exchange of the bound drug process wherein the rate of exchange between the two equivalent sites was estimated to be congruent to 130 s-1 at 30 degrees C and with delta G degrees of 62.4 kJ mol-1. Force field and Pi calculations permitted a rationalization of the experimentally observed binding mode in terms of preferred conformation of the ligand and repeat length in lexitropsins compared with the DNA receptor.
Base Composition↗
Sequence specific molecular recognition and binding by a GC recognizing Hoechst 33258 analogue to the decadeoxyribonucleotide d-[CATGGCCATG]2: structural and dynamic aspects deduced from high field 1H-NMR studies.
The non-exchangeable and imino proton NMR resonances have been assigned of the 1:1 complex of an analogue 2 of Hoechst 33258 1 bound to the decadeoxyribonuycleotide d-[CATGGCCATG]2 by a combination of NOE difference, COSY and NOESYPH techniques. In contrast to Hoechst 33258 which recognizes 5'-AATT sequences exclusively, analogue 2 possesses structural features designed to permit the recognition of GC sites. The NOESY and 1D-NOE experiments place the drug in the minor groove and it is located on the 5'-CCAT sequence. The orientation of the drug in the groove is such as to place the N-methylpiperazine terminus at a GC site. Cross-correlation peaks in the NOESY experiment show that the DNA duplex retains its right-handed B form, similar to that in the free decamer. Specific NOEs locate the benzoxazole moiety on the 5'-CCAT and are consistent with the pyridine nitrogen forming a new hydrogen bond to G(4)-2NH2 at 5'-CCAT. The drug appears to undergo rotation around the C9-C10 bond, at a rate comparable with NMR time scale, even after binding. Variable temperature 1H-NMR studies established that the DNA is thermally stabilized as a result of the drug binding. The drug binding is a dynamic process involving exchange between the equivalent 5'-CCAT sites at approximately 60s-1 with delta G degree of 65 kJ mol-1 at 308K. The experimental evidence is in accord with a slide-swing mechanism for this process.
Base Sequence↗
Extracardial effects of oral ibopamine versus furosemide in patients with mild or moderate heart failure. A double-blind, randomized trial.
Ibopamine is a novel oral dopamine analogue with vasodilatory, positive inotropic and diuretic effects. In a double-blind, randomized study, the drug was investigated in 12 patients (mean age 49 +/- 10 years, 8 male, 4 female) with mild or moderate heart failure (NYHA classes II:8 patients, III:4 patients). Effects of single oral doses of 200 mg ibopamine, of 40 mg furosemide and of 200 mg ibopamine + 40 mg furosemide were compared in each patient at 3-day intervals. 1 h after administration, systolic and diastolic blood pressure increased from 120 +/- 11 to 124 +/- 9 and from 76 +/- 5 to 81 +/- 6 mm Hg in the ibopamine group. During 4 h after drug ingestion, urinary flow was significantly raised from 124 +/- 81 to 228 +/- 166 ml/2 h in the ibopamine group (p less than 0.05), while the administration of furosemide (with or without ibopamine) resulted in several folds increases of urinary flow. After ibopamine, the 2-h creatinine clearance rose from 123 +/- 73 to 131 +/- 85 ml/min (not significant). Sodium and potassium excretion remained essentially unchanged by ibopamine, while effects of furosemide were several folds of those of ibopamine. Plasma renin activity was lowered to 65% by ibopamine (p less than 0.01). No additive effects of ibopamine in the presence of furosemide were observed for all parameters tested. These results indicate that ibopamine has smaller renal effects than furosemide with regard to water diuresis and kaliuresis. These effects of ibopamine could reflect direct changes of renal function or secondary effects of neurohumoral origin. Ibopamine does not produce undesirable renal side effects, but affects the neurohumoral status favourably. This drug, thus, could be useful as an adjuvant therapy in mild heart failure.
Administration, Oral↗
Automated sequencing of fluorescently labelled DNA by chemical degradation.
A new general method for sequencing fluorescently labelled DNA by chemical degradation has been developed. It is based on the observation that fluorescein attached via a mercaptopropyl or aminopropyl linker arm to the 5'-phosphate of an oligonucleotide is stable during the reactions commonly used in chemical cleavage procedures. DNA to be degraded is first enzymatically synthesized in vitro by annealing and extending a fluorescently labelled primer thereby introducing the fluorescent label at the 5'-end of the fragment. The newly synthesized fluorescently labelled DNA is then chemically degraded using: (a) a set of four different cleavage reactions; or (b) only one reaction comprising methylation of G-residues followed by a partial cleavage with piperidine in the presence of sodium chloride. The fluorescent degradation products are loaded on either four lanes or one lane of the gel, respectively, and the emitted fluorescence detected online during electrophoresis. In the 'four reactions/four lanes' method 200-350 bp (base pairs) can be read from the labelled end. The 'one reaction/one lane' method, in which the nucleotide sequence is determined by measuring different signal intensities following the rule G greater than A greater than C greater than T, currently yields around 100-200 bp of sequence per sample.
Automation↗
[Renal effects of ibopamine in comparison with furosemide in patients with mild heart failure].
Ibopamine is a novel oral dopamine analogue with positive inotropy and diuretic effects. In a double-blind, randomized study, the drug was investigated in 10 patients (mean age 49 +/- 10 years, six male, four female) with mild heart failure (NYHA classes II: six patients, III: four patients). Effects of single oral doses of 200 mg ibopamine, of 40 mg furosemide, and of 200 mg ibopamine plus 40 mg furosemide were compared in each patient at 3-day-intervals. One h after application, systolic and diastolic blood pressure increased from 119 +/- 11 to 124 +/- 8, and from 75 +/- 4 to 80 +/- 6 mm Hg (p less than 0.01) in the ibopamine group, while changes in both other groups and changes of the heart rate were insignificant. During 2 h after drug ingestion urinary flow was raised from 124 +/- 81 to 227 +/- 166 ml/2 h in the ibopamine group (p less than 0.05), while the application of furosemide (with or without ibopamine) resulted in several fold increases of urinary flow. After ibopamine, the 2-h-creatinine-clearance rose from 123 +/- 73 to 130 +/- 85 ml/min (not significant). Sodium excretion remained unchanged by ibopamine, potassium excretion was increased from 2.9 +/- 1.7 to 4.0 +/- 3.3 mmol/h (p less than 0.05), while effects of furosemide were several fold of those of ibopamine. Atrial natriuretic factor concentrations in plasma increased significantly after ibopamine and after ibopamine plus furosemide (p less than 0.01), but remained constant after furosemide alone.(ABSTRACT TRUNCATED AT 250 WORDS)
Atrial Natriuretic Factor↗
[Tumoral calcinosis].
Tumoral calcinosis is a distinct entity, which is rarely seen in Europe and North America but much more common in black Africans. Typical symptoms are calcified nodules, which grow while remaining asymptomatic and are found in the tissues adjacent to the large joints of the body. Histologically there is collagen necrobiosis initially, which results in aggregates of densely calcified material. The aetiology is unknown, but the condition is probably a form of dystrophic calcification caused by mechanical injury. A patient with tumoral calcinosis is presented, and the clinical and histological findings are described.
Aged↗