[Benefits and risks of mammography screening].
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Adult↗
Biomedical subjects
H Jung
Publications and source records attributed to H Jung.
Membrane topology of helices VII and XI in the lactose permease of Escherichia coli studied by lacY-phoA fusion analysis and site-directed spectroscopy.
The use of lactose permease-alkaline phosphatase fusions (lacY-phoA) demonstrates that the lactose permease of Escherichia coli contains 12 transmembrane domains and that approximately half of a transmembrane domain is required to translocate alkaline phosphatase to the periplasmic surface of the membrane [Calamia, J., & Manoil, C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4937-4941]. We have now used fusion analysis in combination with site-directed spectroscopy to examine more precisely the topology of putative helices VII and XI which contain the interacting residues Asp237 and Lys358, respectively. For this purpose, alkaline phosphatase was fused to alternate amino acid residues in transmembrane domains VII and XI. A sharp increase in alkaline phosphatase activity is observed as the fusion junction proceeds from Try228 to Ile230 in helix VII and from Phe354 to Phe356 in helix XI, suggesting that these residues approximate the middle of the corresponding transmembrane helices. Analysis of fluorescence quenching of the pyrene-labeled single-Cys mutants Asp237 --> Cys or Lys358 --> Cys, as well as measurement of collision frequencies between freely diffusing paramagnetic probes and a nitroxide spin-label at these sites, also indicates that Asp237 and also Asp240, which interacts with Lys319 (helix X), are located in transmembrane domains. However, Asp237 and Asp240 are accessible both from the aqueous phase and from within the membrane. The results provide more direct evidence that the three residues are located within transmembrane helices and suggest that Asp237 and Asp240 are either located near the periplasmic surface of the membrane or exposed within a solvent-filled cleft in the permease.
Alkaline Phosphatase↗
Role of glycine residues in the structure and function of lactose permease, an Escherichia coli membrane transport protein.
By using oligonucleotide-directed, site-specific mutagenesis, the role of 34 Gly residues in the lactose permease of Escherichia coli has been studied systematically. Each of 34 out of a total of 36 Gly residues was replaced with Cys in a functional permease mutant devoid of Cys residues (C-less permease), as previous experiments demonstrate that Gly-402 and Gly-404 can be deleted by truncation of the C-terminus with no loss of activity [Roepe, P. D., et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3992; McKenna, E., et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2969]. Out of the 34 Cys-replacement mutants described, 15 transport lactose with high activity, 16 exhibit decreased but significant ability to catalyze lactose accumulation, and 3 (Gly-64-->Cys, Gly-115-->Cys and Gly-147-->Cys) exhibit no activity whatsoever. The inactive mutants were studied in more detail by replacement of Gly with Ala, Val, or Pro. C-less permease with Gly-115-->Ala or Gly-147-->Ala transports lactose almost as well as the control, while mutants with Val or Pro in place of Gly have little or no capacity to accumulate the disaccharide. In contrast, mutants with Ala, Val, or Pro in place of Gly-64 are inactive. Strikingly, however, when the mutations are placed in the wild-type background, Gly-64-->Ala permease transports lactose, beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside, and methyl 1-thio-beta,D-galactopyranoside 40-60% as well as wild-type permease, while Gly-64-->Val or Gly-64-->Pro permease is inactive toward all of these substrates. The results indicate that although none of the Gly residues in lactose permease is mandatory for activity, the bulk of the side chain at positions 64, 115, and 147, rather than conformational flexibility at these positions, is particularly important.
Amino Acid Sequence↗
NMR and circular dichroism studies of synthetic peptides derived from the third intracellular loop of the beta-adrenoceptor.
The C-terminal part of the third intracellular loop of the beta-adrenoceptor is capable of stimulating adenylate cyclase in the presence of phospholipid vesicles via the stimulatory guanine nucleotide binding protein (Gs) [Palm et al. (1989) FEBS Lett. 254, 89-93]. We have investigated the structure of synthetic peptides corresponding to residues 284-295 of the turkey erythrocyte adrenoceptor in micelles, trifluoroethanol and aqueous solution, by using 2D 1H NMR and CD. In the presence of phospholipid micelles the peptides display a C-terminal alpha-helical region, whereas the N-terminal part was found to be highly flexible.
Amino Acid Sequence↗
Factors predicting the risk of relapse after antiepileptic drug discontinuation in children with partial seizures.
The purpose of this study is to identify possible factors which could influence the seizure recurrence after anti-epileptic drug (AED) withdrawal in children with partial epilepsy. AED was discontinued in 82 children who had been free of partial epileptic seizures for 2.0-11.0 years (mean 4.7 years). Twenty-four patients (29.3%) had a relapse from a few days to 6.1 years (mean 1.2 years) after AED discontinuation. Significantly more common in children who relapsed were: younger age at beginning of AED withdrawal, occurrence of complicated febrile convulsions (5/24 vs 1/58, P < 0.01), abnormal neurological examination (8/24 vs 8/58, P < 0.05), delayed psychomotor development (7/24 vs 7/58, P < 0.05), focal slowing (6/24 vs 3/58, P < 0.01) and focal epilepti-form discharges (7/24 vs 6/58, P < 0.05) in the last EEG before AED discontinuation. Between the two groups no statistical significant differences concerning the age at onset of seizures, the duration at AED therapy after the last seizure, the familial occurrence of epilepsy and background EEG abnormalities in the last EEG before AED discontinuation were found. On the basis of EEG, occurrence of febrile convulsions, and neurological and developmental examination it may be possible to predict which children have the best chance to remain free of recurrence after AED discontinuation.
Anticonvulsants↗
[Health risks due to radiation exposure].
Recent data analyses of several ongoing epidemiological studies, in particular the Life Span Study of Hiroshima and Nagasaki, have led to new estimates of risks due to radiation-induced cancer. The risk coefficients published by the International Commission on Radiological Protection (ICRP) in 1991 are 0.5% Sv-1 for leukaemia and 4.5% Sv-1 for solid cancers. For adults the mean latency periods for radiation-induced leukaemia are about 15 years, for solid cancers more than 40 years. Using the organ specific risk coefficients several examples are given for risk estimates for some frequently applied radiological procedures. The results are compared with various generally known risks of everyday life. Some specific problems involved in risk communication are considered.
Adolescent↗
Correlation between thermal radiosensitization and slowly rejoined DNA strand breaks in CHO cells.
The effect on the repair of slowly rejoined strand breaks was studied in CHO cells using the alkaline unwinding technique. Heat (45 degrees C, 20 min) combined with a X-ray dose of 9 Gy was found to result in an increased half-time of repair but also in an increased number of slowly rejoined strand breaks. When a time interval at 37 degrees C was inserted between irradiation and heat, the half-time of repair was not altered, whereas the number of slowly rejoined strand breaks as measured 300 min after irradiation decreased with increasing time interval between the two treatments. The half-time of 18 +/- 2 min suggested that the additionally formed, slowly rejoined strand breaks arise from a certain type of radiation-induced DNA base lesions with repair of which is modified by heat. The effect of X-irradiation combined with heat was also studied for cell survival. When irradiation and heat were separated by an incubation at 37 degrees C, cell survival increased with a half-time of 20 +/- 2 min, which is similar to that measured for the number of additional, slowly rejoined strand breaks. For a great variety of combined treatments, the reduction in cell survival correlates well with the enhanced number of slowly rejoined strand breaks measured 300 min after irradiation. This positive correlation and the similarity in the half-times mentioned above suggests that thermal radiosensitization results from the number of additional, slowly rejoined strand breaks formed when irradiation was combined with heat.
Animals↗
[Work in the refugee camp of Mogunga/Goma-Zaire].
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Democratic Republic of the Congo↗
Cysteine 148 in the lactose permease of Escherichia coli is a component of a substrate binding site. 1. Site-directed mutagenesis studies.
Cys 148 in the lactose permease of Escherichia coli has been replaced with hydrophobic (Ala, Val, Ile, Phe), hydrophilic (Ser, Thr), or charged (Asp, Lys) residues, and the properties of the replacement mutants have been analyzed. Although Cys 148 is not essential for transport, the size and polarity of the side chain at this position modifies transport activity and substrate specificity. Thus, small hydrophobic side chains (Ala, Val) generally increase the apparent affinity of the permease for substrate, while hydrophilic side chains (Ser, Thr, Asp) decrease apparent affinity and bulky or positively charged side chains (Phe, Lys) virtually abolish activity. In addition, hydrophilic substitutions (Ser, Thr, Asp) alter the specificity of the permease toward monosaccharides relative to disaccharides. On the basis of these and other observations, it is concluded that Cys 148 is located in a sugar binding site of lac permease and probably interacts hydrophobically with the galactosyl moiety. The postulate receives more direct support from site-directed fluorescence labeling studies presented in the following paper in this issue [Wu, J., & Kaback, H. R. (1994) Biochemistry (following paper in this issue)].
Amino Acid Sequence↗
Dynamics of lactose permease of Escherichia coli determined by site-directed fluorescence labeling.
Recently we described the use of site-directed pyrene labeling of engineered lactose permease containing paired Cys residues to obtain proximity relationships between helices in the C-terminal half of the molecule [Jung, K., Jung, H., Wu, J., Privé, G. G., & Kaback, H.R. (1993) Biochemistry 32, 12273]. Pyrene excimer fluorescence was detected for the double Cys mutants His322-->Cys/Glu325-->Cys, Arg302-->Cys/Glu325-->Cys, and Glu269-->Cys/His322-->Cys, indicating that helix X (His322-->Cys/Glu325-->Cys) is in an alpha-helical conformation and that helices VIII (Glu269-->Cys) and IX (Arg302-->Cys) are close to helix X (His322-->Cys and Glu325-->Cys). In this report, these interactions are used to study dynamic aspects of the permease. Excimer fluorescence between helices VIII and X or helices IX and X is markedly diminished by sodium dodecyl sulfate, while the excimer observed within helix X is unaffected, suggesting that tertiary interactions are disrupted by the denaturant with little effect on secondary structure. Furthermore, excimer fluorescence observed between helices VIII (Glu269-->Cys) and helix X (His322-->Cys) is quenched by Tl+, and the effect is markedly and specifically attenuated by ligands of the permease, suggesting that the pyrene becomes less accessible to the aqueous phase. The reactivity of single Cys residues at positions 269 or 322 was also examined by studying the rate of increase in fluorescence with N-(l-pyrenyl)maleimide. With both mutants, ligands of the permease cause a dramatic increase in reactivity which is consistent with the notion that these positions are transferred into a more hydrophobic environment.(ABSTRACT TRUNCATED AT 250 WORDS)
Cysteine↗
High-resolution one- and two-dimensional 1H MRS of human brain tumor and normal glial cells.
Astrocytoma (WHO grade II, III), glioblastoma, malignant melanoma, and normal glial cell cultures, established from biopsies, were investigated by 1H MRS. At a 1H resonance frequency of 500 MHz (11.75 T) a high spectral resolution was achieved in 1D 1H spectra; in conjunction with 2D shift-correlated (COSY) MRS, resonances of alanine, aspartate, choline, creatine, glutamate, glutamine, hypotaurine, myo-inositol, phosphocreatine, phosphoryl-ethanolamine, phosphoryl-choline, lactate, lysine, N-acetylaspartate, taurine, threonine and valine could be identified. T1 relaxation times for the most prominent compounds are presented. T1 values of lactate ranged between 450 ms and 850 ms. The intensity of the lactate signal revealed differences between individual spectra, but exhibited no correlation between different tumor specimens or degree of malignancy. It was shown that the lactate signal at 1.3 ppm is covered by peaks arising from threonine and fatty acids. The choline signal level varied among spectra of different tumors, among tumors with similar degree of malignancy, and within the same tumor. Further preliminary differences due to aspartate, inositol and glutamine/glutamate were found in 1D and 2D COSY spectra between normal glial cells as well as different tumors. These results indicate that some differences observed in in vivo spectra may be attributable to secondary macroscopic structural changes (hypoxia, necrosis) and not to tumor inherent characteristics. Further correlation between in vivo and in vitro spectroscopy is therefore required.
Astrocytoma↗
A conformational change in the lactose permease of Escherichia coli is induced by ligand binding or membrane potential.
Lactose transport in membrane vesicles containing lactose permease with a single Cys residue in place of Val 315 is inactivated by N-ethylmaleimide in a manner that is stimulated by substrate or by a H+ electrochemical gradient (delta microH+; Sahin-Tóth M, Kaback HR, 1993, Protein Sci 2:1024-1033). The findings are confirmed and extended in this communication. Purified, reconstituted Val 315-->Cys permease reacts with N-ethylmaleimide or hydrophobic fluorescent maleimides but not with a membrane impermeant thiol reagent, and beta-galactosides specifically stimulate the rate of labeling. Furthermore, the reactivity of purified Val 315-->Cys permease is enhanced by imposition of a membrane potential (delta psi, interior negative). The results indicate that either ligand binding or delta psi induces a conformational change in the permease that brings the N-terminus of helix X into an environment that is more accessible from the lipid phase.
Cysteine↗
Light-evoked arachidonic acid release in the retina: illuminance/duration dependence and the effects of quinacrine, mellitin and lithium. Light-evoked arachidonic acid release.
Arachidonic acid (AA) is the precursor molecule of a variety of cellular lipid mediators that interact with retinal physiology. In this study, we investigated the time- and illuminance-dependence of the release of AA in the rat retina in vitro in control and lithium-pretreated rats. We also studied the effects of the specific phospholipase A2 (PLA2) inhibitor quinacrine and the specific PLA2 stimulator mellitin on the release of AA. Isolated rat retinas were labelled with 3H-AA for 90 min in vitro in darkness and the incorporation of AA into retinal phospholipids was monitored by thin-layer chromatography. The release of 3H-AA in the incubation medium was determined under different illuminance and timing conditions, with the addition of quinacrine and mellitin, and after pretreatment of the animals with lithium. Light exposure of the prelabelled isolated retinas evoked up to a two-fold increase in AA release compared with retinas incubated for the same time in darkness. The AA release was dependent on illuminance time (10,000 1x white fluorescent light for 0.25, 2, 5 and 10 min) and illuminance level (0, 100, 1000, 5000, and 10,000 1x for 10 min). Complete rhodopsin bleaching occurred after 2 min at 10,000 1x. Quinacrine significantly suppressed the light-elicited AA release whereas mellitin increased the release of AA in dark-adapted and light-exposed retinas. Lithium pretreatment, which is known to potentiate light-evoked rod outer segment disruptions, significantly augmented the light-evoked AA release. Our results confirm a light-stimulated release of AA in the retina.(ABSTRACT TRUNCATED AT 250 WORDS)
Administration, Oral↗
Crotonobetaine reductase from Escherichia coli--a new inducible enzyme of anaerobic metabolization of L(-)-carnitine.
Crotonobetaine reductase from Escherichia coli 044 K74 is an inducible enzyme detectable only in cells grown anaerobically in the presence of L(-)-carnitine or crotonobetaine as inducers. Enzyme activity was not detected in cells cultivated in the presence of inducer plus glucose, nitrate, gamma-butyrobetaine or oxygen, respectively. Fumarate caused an additional stimulation of growth and an increased expression of crotonobetaine reductase. The reaction product, gamma-butyrobetaine, was identified by autoradiography. Crotonobetaine reductase is localized in the cytoplasm, and has been characterized with respect to pH (pH 7.8) and temperature optimum (40-45 degrees C). The Km value for crotonobetaine was determined to be 1.1 x 10(-2M). gamma-Butyrobetaine, D(+)-carnitine and choline are inhibitors of crotonobetaine reduction. For gamma-butyrobetaine (Ki = 3 x 10(-5M)) a competitive inhibition type was determined. Various properties suggest that crotonobetaine reductase is different from other reductases of anaerobic respiration.
Anaerobiosis↗
[Congenital choanal atresia and surgical correction].
43 cases with congenital choanal atresia were observed in over 20 years. The indications for surgery of congenital choanal atresia and the different surgical approaches are discussed in detail. The transnasal approach should be performed in emergency situations or as first-aid treatment; the transmaxillary approach cannot be recommended any longer. The transpalatinal approach with the head in inclined position under microsurgical conditions has proved to be the surgical procedure of choice in most situations. 40 patients were operated on during infancy or childhood and followed up subsequently. 5 children showed bilateral choanal atresia; the other patients had unilateral complete atresia. In 9 patients a transnasal approach was performed, in 7 cases there was recurrence of complete stenosis; after transpalatinal re-operations, relapse occurred in one case only. One patient was operated on via the transmaxillary approach. In 30 cases the transpalatinal approach was the method of operation; relapse was seen in 6 cases only and could be revised by transpalatinal approach, excepted for one case. Our own experience and the present results show that the transpalatinal approach is suitable for adults as well as newborn and infants. On account of the better view of the operating area and optimal conditions for reconstruction, the transpalatinal approach is safer and long-term results are good.
Adolescent↗
Sequential method of estimating the LD50 using a modified up-and-down rule.
In this paper, the original up-and-down method, modified up-and-down method, the Robbins-Monro method, and a fixed-sample Spearman-Kärber method are compared for the point estimator as well as the confidence interval of LD50. In particular, three different designs of the modified up-and-down approach based on the combination of experiments on one test space and reduced test space are investigated. The standard normal distribution and chi-square distribution are used as tolerance distributions. Simulation results indicate that the modified up-and-down method tends to be somewhat better than the original up-and-down method in terms of the mean squared error under normal tolerance distribution. In case of chi-square distribution, the modified method is shown to be substantially better when the test space is wide and the initial dose is further away from the LD50.
Lethal Dose 50↗
The lactose permease meets Frankenstein.
The lactose permease (lac) of Escherichia coli is a paradigm for membrane transport proteins. Encoded by the lacY gene, the permease has been solubilized, purified to homogeneity, reconstituted into phospholipid vesicles and shown to catalyse the coupled translocation of beta-galactosides and H+ with a stoichiometry of unity. Circular dichroism and other spectroscopic approaches demonstrate that the purified permease is about 80% helical. Based on hydropathy analysis of the primary amino-acid sequence, a secondary structure has been proposed in which the protein has 12 hydrophobic domains in alpha-helical conformation that traverse the membrane in zigzag fashion connected by hydrophilic loops. A variety of other approaches are consistent with the model and demonstrate that both the N and C termini are on the inner surface of the membrane, and studies on an extensive series of lac permease/alkaline phosphatase fusion proteins provide exclusive support for the topological predictions of the 12-helix motif. This presentation concentrates on the use of site-directed fluorescence spectroscopy to study structure-function relationships in the permease.
Amino Acid Sequence↗
[Pain assessment and therapy in bronchial carcinoma].
In the period from 1983-1991 133 patients (102 men, 31 women) with lung cancer were treated in our pain clinic for 8083 days. Pain was associated with tumour infiltration in 86% of patients and related to therapy in 15%. Even in 6 of 8 patients who were admitted with a diagnosis of "postthoracotomy syndrome" and in all 4 patients with "postradiation syndrome" local recurrence was diagnosed during follow-up. All 17 cases of brachial plexus lesions were caused by local tumour spread. Symptomatic treatment according to WHO guidelines resulted in good pain relief in 92% of patients and on 82% of days. The incidence of dyspnea decreased from 51% of the patients to 16%. Strong opioids were used on 56% of treatment days. Parenteral or spinal administration of opioids was necessary on 3% of days only.
Adult↗