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Biomedical subjects

H Jung

Publications and source records attributed to H Jung.

At least 55 records · Page 3Linked to original sources
PubMed1999

B-myb proto-oncogene products interact in vivo with each other via the carboxy-terminal conserved region.

Using the yeast two-hybrid assay and in vivo binding assay, we investigated whether B-myb oncogene products (B-myb) can associate with each other. Specificity tests of the yeast two-hybrid system showed a self-association of B-myb proteins in yeast. Cotransfection experiments demonstrated that B-myb proteins form a complex in vivo. Deletion analysis revealed that this binding was sufficiently mediated by the carboxy-terminal conserved region of B-myb. In addition, the B-myb self-association is directly dependent on the amount of expressed B-myb in cells and slightly increased by the dephosphorylation state. These results suggested that B-myb could form a complex and influence its transcriptional activity.

Cell Line↗
PubMed1999

Role of conserved Arg40 and Arg117 in the Na+/proline transporter of Escherichia coli.

The Na+/proline transporter of Escherichia coli (PutP) is a member of a large family of Na+/solute symporters. To investigate the role of Arg residues which are conserved within this family, Arg40 at the cytoplasmic end of transmembrane domain (TM) II and Arg117 in cytoplasmic loop 4 of PutP are subjected to amino acid substitution analysis. Removal of the positive charge at position 40 (PutP-R40C, Q, E) leads to a dramatic decrease of the V(max) of Na(+)-coupled proline uptake (1-10% of PutP-wild-type). The reduced transport rates are accompanied by decreased apparent affinities of the transporter for Na+ and Li+ while the apparent affinity for proline is only slightly altered. Furthermore, single Cys PutP-R40C reacts with N-ethylmaleimide (NEM), and this reaction is partially inhibited by proline and more efficiently by Na+ ions. Remarkably, NEM modification of Cys40 inhibits Na(+)-driven proline uptake almost completely while facilitated influx of proline into deenergized cells is stimulated by this reaction, suggesting an at least partially uncoupled phenotype under these conditions. These results suggest that Arg40 is located close to the site of ion binding and is important for the coupling of ion and proline transport. The observations confirm the functional importance of TM II described in earlier studies [M. Quick and H. Jung (1997) Biochemistry 36, 4631-4636]. In contrast to Arg40, Arg117 is apparently not important for function of the mature protein. The low transport rates observed upon substitution of Arg117 (PutP-R117C, K, Q) can at least partially be attributed to reduced amounts of PutP in the membrane. However, once inserted into the membrane, PutP containing Arg117 replacements shows a stability comparable to the wild-type as indicated by pulse-chase experiments. These observations suggest that Arg117 plays a crucial role at a stage prior to complete functional insertion of PutP into the membrane, i. e., by stabilizing a folding intermediate.

Amino Acid Sequence↗
PubMed1999

Age-related changes in ultrastructural features of cathepsin B- and D-containing neurons in rat cerebral cortex.

The present study examines age-related changes in the subcellular localization of cathepsin B (cath B) and cathepsin D (cath D), as well as morphological features of the cathepsin-immunoreactive (ir) neurons in rat cerebral cortex. Sprague-Dawley rats were studied at 3 and 26 months. By immunoelectron microscopy cath B- or cath D-immunoreactivities were found in many, but not all, pyramidal neurons. In young rat cerebral cortical neurons, cath B was observed not only in lysosomal systems such as multivesicular bodies, dense bodies, and lipofuscin granules, but also in extralysosomal sites. By contrast, cath D was confined mainly to lysosomal systems in young rats. In aged rats, cath B showed a similar pattern in its subcellular localization compared to the young control, but some of the dense bodies containing cath B was closely apposed to the outer nuclear envelope. These cells exhibited a relatively normal appearance. Regardless of subcellular localization, approximately 10% of cath B-ir neurons displayed ultrastructural disturbances presumed to indicate an early stage of degeneration. The nucleus was indented, nuclear boundary was indistinct, nuclear pore structures appeared separately with high frequency, and the endoplasmic reticulum appeared to be affected. In addition to its presence in lysosomal structures, cath D-immunoreactivity in aged cerebral cortex was noted prominently in the cytosol as diffuse granules. About 37% of cath D-ir cells showed this age-related change. Among the neurons with the diffusely scattered form of cath D, approximately 70% of cells exhibited the degenerating features. These cells were characterized by large amounts of diffuse cath D, reduced cellular size, loss of the nuclear boundary, scattered nuclear pore structures, an often fragmentation of the nucleus, disturbances of endoplasmic reticular system, and in advanced stages, condensed nucleus and poor preservation of almost cytoplasmic organelles. Though some of these features were also found in cath B-ir neurons, findings of overt degeneration, such as fragmented and condensed nuclei and impaired almost cytoplasmic organelles, were generally not observed in cath B-ir neurons. In addition, lipofuscin aggregates containing cath D were observed frequently in the extracellular space close to sites of ruptured plasma membrane, whereas in the sections stained with anti-cath B antibodies, large-sized lipofuscin aggregates showed only very weak or no cath B-immunoreactivity at all. Taken together, the present results suggest that cath D and cath B may be regulated differently and play their specific roles in the aging of the brain, especially, the change in location of cath D from the lysosomal system to the cytosol in the aged brain may play an important role in age-related cell death.

Aging↗
PubMed1999

Purification and reconstitution of an osmosensor: transporter ProP of Escherichia coli senses and responds to osmotic shifts.

The ProP protein of Escherichia coli is an osmoregulatory H+-compatible solute cotransporter. ProP is activated by an osmotic upshift in both whole cells and membrane vesicles. We are using biochemical and biophysical techniques to explore the osmosensory and catalytic mechanisms of ProP. We now report the purification and reconstitution of the active transporter. Protein purification was facilitated by the addition of six histidine (His) codons to the 3' end of proP. The recombinant gene was overexpressed from the E. coli galP promoter, and ProP-(His)6 was shown to be functionally equivalent to wild-type ProP by enzymatic assay of whole cells. ProP-(His)6, purified by Ni2+ (NTA) affinity chromatography, cross-reacted with antibodies raised against the ProP protein. ProP-(His)6 was reconstituted into Triton X-100 destabilized liposomes prepared with E. coli phospholipid. The reconstituted transporter mediated proline accumulation only if (1) a membrane potential was generated by valinomycin-mediated K+ efflux and (2) the proteoliposomes were subjected to an osmotic upshift (0.6 M sucrose). Activity was also stimulated by DeltapH. Pure ProP acts, in the proteoliposome environment, as sensor, transducer, and respondent to a hyperosmotic shift. It is the first such osmosensor to be isolated.

Bacterial Proteins↗
PubMed1999

Late complications after a combined pre and postoperative (sandwich) radiotherapy for rectal cancer.

BACKGROUND AND PURPOSE: The purpose of this study was to analyse the treatment related side effects, the outcome and the prognostic significance of clinical parameters in two groups of patients with rectal cancer receiving either preoperative or pre and postoperative radiotherapy after radical resection. The authors of this study were not involved in the radiation treatments. PATIENTS AND METHODS: From 1986 to 1990, 63 patients received a combined pre and postoperative (sandwich) radiotherapy. Preoperative irradiation was given in four fractions of 5 Gy each applied within 2 or 3 days. Postoperative irradiation consisted mostly of 15 x 2 Gy (31 patients) but the range was 20-40 Gy. The results were compared with those on 73 patients who only received preoperative radiotherapy in the same time period. The distribution of prognostic factors was not very different between treatment groups. Out of 63 patients in the sandwich group, 22 received concurrent chemotherapy and 18 also received radiotherapy to the liver. Radical surgery usually followed on the day after the last preoperative radiotherapy session. Median follow-up of survivors was 6 years. RESULTS: Local tumour control was 88% after 5 years and 84% after 8 years in the sandwich group, and 90 and 85%, respectively, in the preoperative radiotherapy group. Thus, tumour control was similar for the two radiotherapy regimens applied. However, the percentage of patients suffering from one or more complications after 5 years was 84% in the sandwich and 17% in the preoperative radiotherapy group. The incidence of severe late complications (grade > or = 3) was recorded as a function of time after start of treatment. In the sandwich group the actuarial rates of late complications at 5 years (and the median time to diagnosis) were 53% (27 months) for anorectum, 43% (37 months) for bladder, 28% (51 months) for bone, 19% (36 months) for dermis, 47% (48 months) for ileum, 41% (32 months) for lymphatic and soft tissue, and 44% (53 months) for ureters. CONCLUSIONS: Severe late reactions did not occur within a certain period of time, but continued to appear for at least 10 years after radiotherapy. Sandwich therapy, as given in this series, did not appear to give a greater tumour control than preoperative radiotherapy alone, whereas the rate of complications was drastically enhanced. Thus, the rationale of a sandwich therapy with a long time interval between surgery and postoperative irradiation appears questionable.

Actuarial Analysis↗
PubMed1999

Comparison of subjective classification of stool consistency and stool water content.

OBJECTIVE: The purpose of this study was to compare the stool consistency categorizations made by 39 adults with fecal incontinence and the percentage of water in their stools determined by lyophilization. METHODS: Subjects collected all stools daily for 8 days during a baseline period and at the end of a fiber treatment period. Stool consistency was recorded as hard and formed, soft but formed, loose and unformed, or liquid. Aliquots of the stools were lyophilized to constant weight. MAIN OUTCOME MEASURES: The main outcome measures were the percentage of stool water among stools in each consistency category and the correlation between subjects' stool consistency categorizations and the percentage of stool water. RESULTS: The subjects were 8 men and 21 women, ranging in age from 30 to 89 years, who were participating in a study of the effectiveness of dietary fiber for treating fecal incontinence. A total of 1023 stool samples were analyzed. Significant differences in the mean percentage of water were found among the 4 stool consistency categories (hard and formed = 68% +/- 0.9%, soft but formed = 74% +/- 0.3%, loose and unformed = 80% +/- 0.4%, and liquid = 85% +/- 0.3%; P < .001). Ninety-six percent of the stools had a percentage of water within 2 SDs of the mean percentage of water of other stools in their consistency category. CONCLUSION: This classification system of stool consistency is a valid and practical measure for clinical studies. It may be useful for clinicians and patients to evaluate outcomes of treatments directed at improving stool consistency.

Adult↗
PubMed1999

Expression and characterization of anti-NCA-95 scFv (CEA 79 scFv) in a prokaryotic expression vector modified to contain a Sfi I and Not I site.

The CEA 79 antibody has been used in bone marrow scintigraphy for the differential diagnosis of skeletal tumors and the evaluation of the bone marrow status of patients with various hematological disorders. The specific localization of radio-labeled CEA 79 antibody in bone marrow depends on its reactivity with NCA-95 (nonspecific cross-reacting antigen-95) present on the surface and in the cytosol of human granulocytes and myelopoietic cells. To make a CEA 79 scFv molecule that would be less immunogenic and more penetrating than the intact mouse immunoglobulin, we constructed a pRSET Sfi I/Not I expression vector. The scFv gene was then excised from a pCANTAB 5 E phage display vector by digestion with Sfi I and Not I and inserted into the pRSET Sfi I/Not I expression vector. Upon transformation of a BL21(DE3)pLysS strain of E. coli, CEA 79 scFv became expressed in inclusion bodies requiring a renaturation process for solubilization. The final yield of CEA 79 scFv was 5 mg per a liter of culture. The refolded CEA 79 scFv exhibited an affinity (Kd = 2.1 x 10(-9) M) equivalent to that of the original CEA 79 antibody (K(d) = 3.3 x 10(-9) M) and the same immunoreactivity to CEA and NCA-95 in Western blots and in immunohistochemical staining experiments.

Amino Acid Sequence↗
PubMed1999

Some hypothalamic hamartomas contain transforming growth factor alpha, a puberty-inducing growth factor, but not luteinizing hormone-releasing hormone neurons.

Activation of LH-releasing hormone (LHRH) secretion, essential for the initiation of puberty, is brought about by the interaction of neurotransmitters and astroglia-derived substances. One of these substances, transforming growth factor alpha (TGFalpha), has been implicated as a facilitatory component of the glia-to-neuron signaling process controlling the onset of female puberty in rodents and nonhuman primates. Hypothalamic hamartomas (HH) are tumors frequently associated with precocious puberty in humans. The detection of LHRH-containing neurons in some hamartomas has led to the concept that hamartomas advance puberty because they contain an ectopic LHRH pulse generator. Examination of two HH associated with female sexual precocity revealed that neither tumor had LHRH neurons, but both contained astroglial cells expressing TGFalpha and its receptor. Thus, some HH may induce precocious puberty, not by secreting LHRH, but via the production of trophic factors--such as TGFalpha--able to activate the normal LHRH neuronal network in the patient's hypothalamus.

Astrocytes↗
PubMed1998

Topology of the Na+/proline transporter of Escherichia coli.

Hydropathy profile analysis of the amino acid sequence of the Na+/proline transporter of Escherichia coli (PutP) suggests that the protein consists of 12 transmembrane domains (TMs) which are connected by hydrophilic loops (Nakao, T., Yamato, I., and Anraku, Y. (1987) Mol. Gen. Genet. 208, 70-75). We have tested this prediction by applying a gene fusion approach in combination with a Cys accessibility analysis and site-specific proteolysis. Characterization of a series of PutP-alkaline phosphatase (PhoA) and PutP-beta-galactosidase (LacZ) hybrid proteins yields a reciprocal activity pattern of the reporter proteins that is in agreement with the topology of TMs III to XII of the 12-helix model. Placement of the PutP-PhoA and PutP-LacZ junction sites closer to the N terminus does not yield conclusive results. As a prerequisite for further topology studies, a functional PutP molecule devoid of all five native Cys residues (Cys-free PutP) is generated. Subsequently, amino acids in Cys-free PutP are replaced individually with Cys, and the accessibility of the sulfhydryl groups is analyzed. Surprisingly, Cys residues placed close to the N terminus of PutP (Ile-3 --> Cys, Thr-5 --> Cys) or into putative TM II (Ser-71 --> Cys, Glu-75 --> Cys) are highly accessible to membrane permeant and impermeant thiol reagents in intact cells. In contrast, Cys at the C terminus (Ser-502 --> Cys) reacts only with the membrane permeant but not with the impermeant reagent in intact cells. These results contradict the 12-helix motif and indicate a periplasmic location of the N terminus whereas the C terminus faces the cytoplasm. In addition, a transporter with Cys in place of Leu-37 (putative periplasmic loop (pL2) shows the same accessibility pattern as the Cys at the C terminus. Furthermore, PutP which has been purified and reconstituted into proteoliposomes in an inside-out orientation, is readily cleaved by the endoproteinase AspN before Asp-33 (pL2), Asp-112 (putative cytoplasmic loop (cL3), Asp-262 (cL7), and Asp-356 (cL9). These results suggest a cytosolic location of Asp-33 and Leu-37, thereby implying the formation of an additional TM formed by amino acids of pL2. Based on these observations, a new secondary structure model is proposed according to which the protein consists of 13 TMs with the N terminus on the outside and the C terminus facing the cytoplasm. The 13-helix structure is discussed as a common topological motif for all members of the Na+/solute cotransporter family.

Amino Acid Sequence↗
PubMed1998

A conserved aspartate residue, Asp187, is important for Na+-dependent proline binding and transport by the Na+/proline transporter of Escherichia coli.

Asp187 in the Na+/proline transporter of Escherichia coli (PutP) is conserved within the Na+/solute cotransporter family. Information on the role of this residue has been gained by amino acid substitution analysis. PutP with Glu, Asn, or Cys in place of Asp187 catalyzed Na+-coupled proline uptake at 75%, 25%, and 1.5%, respectively, of the Vmax of PutP-wild-type while the apparent Km for proline was only slightly altered. Importantly, acetylation or amidoacetylation of an engineered transporter containing a single Cys at position 187 stimulated proline uptake. Strikingly, PutP-D187C exhibited high-affinity proline binding even at very low Na+ concentrations (2 microM) while proline binding to PutP-wild-type, -D187E, and -D187N was strictly dependent on the Na+ concentration. The apparent independence of proline binding from the Na+ concentration can at least partially be attributed to an enhanced Na+ affinity of PutP-D187C. In addition, reaction of PutP containing a single Cys at position 187 with N-ethylmaleimide was inhibited by Na+ but not by Li+ or proline. The results indicate that electrostatic interactions of the amino acid side chain at position 187 in PutP with other parts of the transporter and/or the coupling ion are crucial for active proline transport. It is suggested that Asp187 is located close to the pathway of the coupling ion through the membrane and may be involved in the release of Na+ on the cytoplasmic side of the membrane.

Amino Acid Sequence↗
PubMed1998

High-performance liquid chromatographic assay for a new fasciolicide agent, alphaBIOF10, in biological fluids.

This paper describes a high-performance liquid chromatographic method with ultraviolet absorbance detection at 304 nm for the determination of 6-chloro-5-(1-naphthyloxy)-2-methylthio benzimidazole (alphaBIOF10) -- a new fasciolicide agent -- and its sulphoxide (SOalphaBIOF10), in plasma and urine. It requires 2 ml of biological fluid, an extraction using Sep-Pak cartridges, and methanol for drug elution. Analysis is performed on a microBondapak C18 (10 microm) column, using methanol-acetonitrile-water (40:30:30, v/v) as the mobile phase. Results showed that the assay is sensitive: 7.2 ng/ml for alphaBIOF10 and SOalphaBIOF10 in plasma and 3.6 ng/ml for both compounds in urine. The response was linear between 0.195 and 12.5 microg/ml. Maximum intra-day coefficient of variation was 5.3%. Recovery obtained was 97.8% for both alphaBIOF10 and SOalphaBIOF10. In urine, recovery was 99.6% and 93.1% for alphaBIOF10 and SOalphaBIOF10 respectively. The method was used to perform a preliminary pharmacokinetic study in two sheep and was found to be satisfactory.

Animals↗
PubMed1998

Unidirectional reconstitution and characterization of purified Na+/proline transporter of Escherichia coli.

A simple approach for large-scale purification and unidirectional reconstitution of the Na+/proline transporter of Escherichia coli (PutP) is described. The procedure is based on the insertion of a highly polar peptide composed of 17 amino acids including a 6His tag at the C-terminus of the transporter. Purification of the hybrid protein is achieved by Ni+-NTA affinity (purity >95%) and ion exchange chromatography (purity >99%). The purified transporter is reconstituted into preformed, detergent-destabilized liposomes. Detergent is removed slowly by adsorption to polystyrene beads. The highest activities [Vmax = 1.1 x 10(3) nmol min-1 (mg of protein)-1] are measured when Triton X100 is used for liposomes destabilization at a concentration corresponding to the onset of lipid solubilization. Site-directed labeling of PutP and site-specific proteolytic cleavage indicate that the transporter is inserted into proteoliposomes in an inside-out orientation. Reconstituted PutP is able to accumulate proline against a concentration gradient in the presence of an inwardly directed electrochemical Na+ or Li+ gradient, while a pH gradient does not affect transport. The apparent proline affinity of PutP in proteoliposomes is similar to the value determined with intact cells. Interestingly however, the apparent Na+ affinity of reconstituted PutP is reduced by a factor of about 25 compared to cells, suggesting a lower cation affinity on the cytosolic side of PutP relative to the outside.

Amino Acid Transport Systems, Neutral↗
PubMed1998

Topology and function of the Na+/proline transporter of Escherichia coli, a member of the Na+/solute cotransporter family.

The Na+/proline transporter of Escherichia coli (PutP) is a member of the Na+/solute contransporter family (SCF) and catalyzes the uptake of proline by a Na+ dependent transport mechanism. Hydropathy profile analysis suggests that the protein consists of 12 transmembrane domains (TMs) that traverse the membrane in zigzag fashion connected by hydrophilic loops. However, analysis of a series of putP-phoA (PutP-alkaline phosphatase) and putP-lacZ (PutP-beta-galactosidase) fusions and site-directed labeling of the transporter indicate a 13-helix motif with the N-terminus on the outside and the C-terminus facing the cytoplasm. The findings are discussed with respect to a common topological motif for all members of the SCF. Furthermore, amino acid substitution analysis indicates that the N-terminal part of PutP is important for ion binding. Thus, Asp55 (putative TM II) is essential for transport and proposed to interact directly with Na+. The functional importance of TM II is further confirmed by the observation that replacement of Arg40, Ser50, Ala53, or Ser57 alters transport kinetics dramatically.

Amino Acid Sequence↗
PubMed1998

Several GABAA receptor subunits are expressed in LHRH neurons of juvenile female rats.

Gamma aminobutyric acid (GABA), the dominant inhibitory neurotransmitter in brain, is involved in the developmental regulation of LHRH secretion. Morphological studies in rodents have demonstrated that LHRH neurons are innervated by GABA-containing processes, suggesting that LHRH secretion is under direct transsynaptic GABAergic control. While GABA acts through two different receptors, GABAA and GABAB, to exert its effects, it appears that GABAA receptors are able to mediate both inhibitory and stimulatory effects of GABA on LHRH neurons. GABAA receptors are heterooligomeric ligand-gated anion channels that exhibit a diverse array of functional and pharmacological properties. This diversity is determined by the structural heterogeneity of the receptors, which are assembled from the combination of different classes of subunits with multiple isoforms. Although several studies have described the effect of GABAA receptor stimulation on LHRH and/or gonadotropin release in prepubertal animals, nothing is known about the receptor subunits that may be expressed in LHRH neurons at this phase in development. Double immunohistofluorescence followed by confocal laser microscopy revealed that subsets of prepubertal LHRH neurons are endowed with alpha 1, alpha 2, beta 2/3, and gamma 2 GABAA receptor subunits. Combined immunohistochemistry for LHRH neurons and in situ hybridization for GABAA subunit mRNAs confirmed that the genes encoding the alpha 1, alpha 2, beta 3 and gamma 2 subunits, but not the gamma 1 subunit, are expressed in LHRH neurons. Notwithstanding the relative insensitivity of these methods, both the immunohistochemical and hybridization histochemical approaches employed indicate that only a fraction of LHRH neurons are endowed with GABAA receptors. This arrangement suggests that those LHRH neurons bearing the appropriate GABAA receptors are responsible for either the entire secretory response to direct GABAergic inputs or for its initiation.

Animals↗
PubMed1998

Rhinophyma in literature and the arts.

The nose is the prominent part of our face. Its look often determines a good part of its owner's image. Rhinophyma is a malformation of the nose of unknown etiology that is often mistakenly associated with drinking alcohol. This rare but definitely eye-catching nasal deformation has found interest not only among doctors but among poets and artists throughout the ages. We attempt to follow this disease from the old Maya culture up to the present century, using known pieces of art, as well as literary quotations.

Europe↗
PubMed1998

Rhinophyma: plastic surgery, rehabilitation, and long-term results.

Rhinophymas are characterized by slowly progressive enlargement of the nasal skin that will not resolve spontaneously. The usual indication for treatment has plastic cosmetic and functional reasons, above all in advanced cases with an obstruction of the nasal respiration or reduction of the visual field. Treatment of rhinophyma consists of surgical removal of the hyperplastic alterations. It should always be carried out by an experienced rhinosurgeon, because of possible complications and injury to the more deeply situated nasal structures. Different surgical procedures have been described, such as excision with primary suture or extirpation with plastic covering of the defect by free transplants, subcutaneous rhinophyma resection, as well as decortication with peeling off the proliferations, dermal abrasion, or dermal shaving. In addition, there are various abrasion procedures with abrasive cylinders, burrs, or wire brushes. The methods of exfoliation and abrasive polishing can be effectively combined. Care should be taken to preserve follicular epidermal islets from the more deeply situated layers of the skin. The follicular epithelium left behind is the point of departure for re-epithelization of the wound surface. If decortication is too deep, injuries to the perichondrium or the nasal cartilage may arise, leading to cosmetically unattractive scar formations and necessitate plastic surgery. The author's own method, which involves a combined procedure with peeling or dermal abrasion, remodeling with abrasive cylinders, as well as preoperative injection into the nasal tumor masses and a subsequent covering of the wound area with fibrin glue, is shown with reference to several examples of more than 60 cases. The cosmetic and long-term results are excellent.

Aged↗