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Biomedical subjects

E Unger

Publications and source records attributed to E Unger.

At least 55 records · Page 3Linked to original sources
PubMed1997

Sequence-specific interactions of minor groove binders with the 154 base pair HindIII-RsaI restriction fragment of cDNA of the human Tau 40 protein involved in pathology of Alzheimer's disease.

The DNA minor groove binders netropsin, distamycin and four structurally related bisquaternary ammonium heterocycles (BQA), SN 6999, SN 6570, SN 6132 and SN 6131, were investigated for sequence-specific interactions with the 154 base pair fragment of cDNA of the human Tau 40 protein (h Tau 40 protein), involved in pathology of Alzheimer's disease. The base sequences 5' AATCTT 3', 5' AATATT 3' and 5' TTTCAATCTTTTTATTT 3' were identified as ligand specific binding sites and demonstrate the obvious dA.dT binding preference. Footprinting titration experiments were performed to estimate sequence-specific binding constants (KA). The KA-values were in the order of 10(6)M-1 and dependent on DNA base sequence as well as ligands used. The highest values estimated were for netropsin (KA = 5.0 x 10(6)M-1) and the quinoline derivative SN 6999 (KA = 6.2 x 10(6)M-1) binding to the sequence 5' ATAAT 3'. Microscopic binding constants are determined by the base sequence rather than by the length of dA.dT stretches. In the extended dA.dT run, 5' TTTCAATCTTTTTATTT 3', netropsin and distamycin binding tolerates the presence of two dG.dC base pairs, as indicated by nearly unaffected footprints. In contrast, the failure of BQAs to form footprints demonstrates their significantly decreased binding selectivity.

Alzheimer Disease↗
PubMed1997

Phase I study of direct gene transfer of an allogeneic histocompatibility antigen, HLA-B7, in patients with metastatic melanoma.

PURPOSE: To determine the safety, toxicity, and efficacy of direct intratumoral injection of an allogeneic major histocompatibility complex (MHC) class I gene, HLA-B7, in a cationic lipid vector (Allovectin-7; Vical Inc, San Diego, CA) in patients with metastatic melanoma. PATIENTS AND METHODS: Seventeen HLA-B7-negative patients were treated with intralesional injection of Allovectin-7. Twelve patients received a single intralesional injection containing 10 micrograms (four patients), 50 micrograms (five patients), or 250 micrograms (three patients) of plasmid DNA. Five patients received two or three injections of 10 micrograms DNA to a single tumor site at 2-week intervals. Tumor biopsies pretherapy and 2 and 4 weeks after gene injection were obtained to determine expression of the plasmid by polymerase chain reaction (PCR), reverse transcriptase (RT)-PCR, flow cytometry, and immunohistochemistry. RESULTS: Toxicities were related to technical aspects of the injections or biopsies. These included pain, hemorrhage, pneumothorax, and hypotension. Two patients were hospitalized overnight for observation. Seven patients (50%) had tumor responses insofar as the injected nodule decreased > or = 25% by radiologic or physical examination. One patient with a single site of disease achieved a complete remission. Ninety-three percent of the patients' post-gene therapy biopsies contained HLA-B7 plasmid DNA, mRNA, or protein. CONCLUSION: Intratumoral injection of the allogeneic histocompatibility gene, HLA-B7, in a lipid vector can be performed safely at plasmid DNA doses < or = 250 micrograms. The safety profile and biologic activity of this therapy warrants further studies to define the mechanism of action, predictors of response, and antitumor efficacy of this approach.

Adult↗
PubMed1997

Human gene therapy for melanoma: CT-guided interstitial injection.

OBJECTIVE: Our intent is to describe the role of CT in the intratumoral injection of Allovectin-7 (Vical, San Diego, CA), an allogeneic class I major histocompatibility complex antigen, HLA-B7, formulated with cationic lipid, in the treatment of metastatic malignant melanoma. MATERIALS AND METHODS: Ten patients with metastatic malignant melanoma were treated with gene therapy in which we used CT-guided intratumoral injection of plasmid DNA containing the HLA-B7 gene. This therapy was part of a phase I gene therapy trial in patients with metastatic melanoma. CT guidance was chosen as an accurate way to direct gene delivery in patients with deep, impalpable lesions. Tumor locations included pulmonary, mediastinal, hepatic, adrenal, and paracaval sites. Patients in the CT protocol underwent baseline CT studies. Examinations were repeated 2, 4, and 8 weeks after gene therapy and thereafter at 3-month intervals. Both injected and noninjected tumors were measured. CT-guided injections of 10, 50, or 250 micrograms of plasmid DNA were performed with 22-gauge spinal needles. Injection volumes were between 1.0 and 4.0 ml, depending on tumor size. CT-guided core biopsy specimens were obtained (with 18- or 20-gauge needles) from the selected tumor before therapy and 2, 4, and 8 weeks after therapy to assess HLA-B7 plasmid DNA and gene expression. Peripheral blood was analyzed for cytotoxic T lymphocytes directed against HLA-B7. RESULTS: CT-guided intratumoral injections were successful in delivering genetic material to all patients with impalpable tumors. Significant responses (as defined by a decrease of 25% or more in the product of the length and width of the injected tumor) were observed in six of the 10 patients. One of these six patients who had a solitary lesion remains free of disease 19 months after gene therapy. HLA-B7 protein expression was detected in 89% of biopsy specimens, and plasmid DNA and messenger RNA were detected in 56% and 22% of biopsy specimens, respectively. CONCLUSION: CT provides a safe, accurate, and efficacious way to monitor and assess tumor progression and response, and it provides guidance for biopsies and intratumoral injections during gene therapy. Significant responses in injected tumors of six of the 10 patients in our study suggest that further clinical trials of this gene therapy are warranted.

Adult↗
PubMed1997

Computer aided adjustment of the phrenic pacemaker: automatic functions, documentation, and quality control.

Electrical stimulation of the phrenic nerves of patients with complete ventilatory insufficiency with the Vienna respiratory pacemaker has been in clinical use since 1983. During the adjustment of stimulation parameters with this device, the following problems have occurred: for some measurements like the recruitment curve, series of complete inspiration cycles have to be stimulated, which causes the danger of muscle fatigue for unconditioned patients. The documentation is completed predominantly by hand, taking time and increasing the possibility of error. As a first step to solve these problems, we developed a new stimulation and measurement system. It consists of a PC with data acquisition hardware, the necessary sensors, and amplifier circuitry. The implanted stimulator is controlled via the parallel interface. The new system offers some advantages: computer control shortens the time for measurement and documentation, and the stress on the patient and the risk of error is reduced; synchronized measurement makes it possible to use single stimulation pulses instead of bursts and ramps to reduce diaphragm fatigue; digital signal processing improves measurement results and reproducibility; and help functions and self tests are provided, together with a graphical user interface. We used sensors for air flow, diaphragm EMG, and acceleration, on up to 8 channels simultaneously. Combined sample rates of up to 100 kS/s were possible. The system could be adapted for other uses involving functional electrical stimulation with our implantable nerve stimulators. Using this equipment saves a lot of effort, and the adjustment process can be focused on improved stimulation results and better performance for the patient. Current research is studying implementation of automatic functions like acquisition of stimulation thresholds. This could result in a predominantly automated adjustment of the phrenic pacemaker and even in a closed-loop controlled system in the future.

Computer Simulation↗
PubMed1996

Deletion of a B800-850 light-harvesting complex in Rhodospirillum molischianum DSM119 leads to "revertants" expressing a B800-820 complex: insights into pigment binding.

A B800-850 light-harvesting complex (also called LH2) deficient strain of Rhodospirillum molischianum was constructed by replacing a portion of the LH2 gene cluster by a kanamycin resistance gene cartridge. The LH2 deficient strain was characterized spectroscopically and by Southern blot analysis. Surprisingly, pseudorevertants were obtained which express a B800-820 complex which could not be observed in the wild type. This B800-820 complex was isolated and characterized. It consists of an alpha- and a beta subunit with 56 and 45 amino acid residues, respectively. The amino acid sequences of both subunits are extremely similar to those of the corresponding B800-850 complex. Resonance Raman spectroscopy shows that in the B800-820 complex the two 2-acetylcarbonyl groups of the bacteriochlo-rophyll a (BChl a) molecules absorbing at 820 nm are free from hydrogen bond interactions, whereas one of the two 2-acetylcarbonyl groups of the pair of BChl a molecules absorbing at 850 nm of the B800- 850 complex is involved in hydrogen bonds. These different protein- pigment interactions are due to the replacement of alpha Trp43 in the B800-850 complex by a Phe in the B800- 820 complex. Comparison of the amino acid sequences of the B800-850 and B800-820 complexes of Rs. molischianum and Rhodopseudomonas acidophila reveals a conserved motif comprised of three amino acid residues. Molecular modeling using the known LH2 structure of Rps. acidophila Ac 10050 indicates that this motif might be important for the precise structural arrangement of the native complex and fine tuning of its spectroscopic properties.

Amino Acid Sequence↗
PubMed1996

Localization of G-proteins in macrophages and E. coli during phagocytosis.

Heterotrimeric GTP-binding proteins (G-proteins) have been shown to play an important role in cellular signalling. However, G-protein involvement in the intracellular spreading of bacterial pathogens is still poorly understood. In this study, antibodies, that recognize G-protein alpha-subunits (anti-G alpha), were used to investigate the localization of G-proteins in the macrophage-like cell line P388D1 and E. coli, also in their L-forms, during phagocytosis. In E. coli, anti-G alpha-binding sites were detected preferably in the cell wall and septa of the whole bacterial forms as well as in the cytoplasm of L-forms. Western blotting of bacterial lysates demonstrated protein bands with positive immunoreaction to antibodies against Gs alpha, Gi alpha, and Gcommon alpha with a higher affinity to the antibody against Gs alpha. Immunoreaction with the anti-Gs alpha-antibody was markedly higher in pathogenic strains of E. coli. Because of the conserved structure in all GTP-binding proteins which seem to derive from a single primordial protein involved in signal transduction mechanisms, it is reasonable to assume that some anti-Ga-positive proteins in E. coli might be related to G-proteins of higher organisms. A putative candidate for bacterial G-proteins seems to be a 36 kDa protein. Enhancement in G-protein immunostaining in the cytoplasm of macrophages around the internalized bacteria testifies to the involvement of G-proteins in mediation of endocytosis responses of phagocytes.

Animals↗
PubMed1996

[Pruritometer 1: Portable measuring system for quantifying scratching as an objective measure of cholestatic pruritus].

Objective assessment of subjective symptoms such as pruritus always presents problems, which can often be resolved only indirectly. The objectification of pruritus was necessitated by a study on the efficacy of a serotonin antagonist used as treatment of cholestatic pruritus. In the present paper, a portable measuring system for the indirect objective assessment of pruritus via the quantification of scratching is described. A piezoelectric scratch-vibration sensor for attachment to the middle finger of the patient's dominant hand was developed. A sensor interface detects the scratching signals and generates pulses that are then summed in an adapted sports watch. The entire system-Pruritometer 1- is worn by the patient like a wrist watch, and is characterized by ease of handling. Acceptance by the patient is reported to be good. A statistically good correlation between measured (Pruritometer) and visually counted scratches was demonstrated. Pruritometer 2, which will enable scratch frequency and intensity distribution over time to be determined, is presently being developed.

Cholestasis, Intrahepatic↗
PubMed1996

Determination of microscopic binding constants at individual DNA base sequences for the minor groove binders Hoechst 33258, DAPI and pentamidine.

Footprinting titration experiments have been used to estimate microscopic binding constants (KA) for interactions of the ligands Hoechst 33258, DAPI and pentamidine with the 167-mer of the EcoRl-Rsal restriction fragment of the plasmid pBR322. The symmetric base sequence AATTAA yielded the lowest KA values while binding sites which contain an AAA-segment displayed higher binding constants. The accommodation of an adjacent G-C base pair to the ATTT-sequence does not significantly interfere with the stability of the DNA-drug complex. The footprint pattern of the compound Hoechst 33258 shows a peculiarity. Complex formation at low drug concentrations (up to 0.5 mu M) is accompanied by distinct protection sites in the DNA fragment against DNAasel digestion, while at higher drug concentration (up to 5.0 mu M) at the same sites the cleavage activity of the enzyme is enhanced. This is discussed in terms of a conformational change of DNA induced by two concentration-dependent binding modes (AT-specific minor groove binding at low drug concentration and GC-specific interaction at a higher one).

Base Sequence↗
PubMed1995

Scanning force microscopy of microtubules and polymorphic tubulin assemblies in air and in liquid.

We have investigated microtubules (MTs) and polymorphic assemblies, formed in vitro from isolated microtubule protein, by scanning force microscopy (SFM) in air and in liquid. Immobilization of MTs was achieved by placing a drop of the assembly solution on a polylysine-coated coverslip. After washing with taxol and air drying, the characteristic microtubular fibrous morphology appeared in the SFM. The MTs formed a network similar to that obtained by transmission electron microscopy (TEM). A height of approximately 9.5 nm for dried MTs was computed from the surface topography. Glutaraldehyde fixation of the MTs yielded higher structures (approximately 14 nm), which swelled to approximately 20 nm after rehydration, a value close to the MT diameter of approximately 25 nm determined from TEM images of ultrathin sections. The protofilament pattern of the MTs and surface attached MT-associated proteins were not apparent from SFM, although the height along the long axis of the MTs appeared slightly modulated. In addition to MTs, various polymorphic tubulin assemblies including ribbons, hoops and double-walled MTs were visualized by SFM.

Air↗
PubMed1995

MR imaging of the retroperitoneum.

The multiplanar and soft-tissue capabilities of MR imaging make it a valuable diagnostic tool for the retroperitoneal space and its organs. In addition, by virtue of its location, the retroperitoneum is well suited for study by this modality. Appropriate techniques, including the use of contrast, are discussed as are the normal and pathologic appearances of the organs and areas of the retroperitoneum.

Contrast Media↗
PubMed1994

In vitro production of proteoglycans in the articular-epiphyseal cartilage of growing pigs.

The failure of cartilage mineralization in osteochondrotic cartilage may be due to an impaired proteoglycan production. The in vitro production of proteoglycans was therefore studied in the joint cartilage of growing pigs, aged 9-18 weeks, after incubation of cartilage samples with 35S-sulfate. Cartilage was obtained from different areas of the femoral condyles and samples from these areas were further divided into three layers, where the superficial layer contains articular cartilage and the basal layers consist of growth cartilage. There was no significant difference in the overall amount of 35S-proteoglycans synthesized in different areas of the condyles. However, the total production of 35S-proteoglycans per mg tissue was highest in the basal layer in all areas. This was not due to a larger number of cells; the superficial layer contained more DNA per mg tissue than the basal layer. Gel chromatography on Sepharose CL-2B of the cartilage extracts, which resulted in the separation of large proteoglycans (Kav approximately 0.4) from proteoglycans of small hydrodynamic size (Kav approximately 0.8), showed that the relative amount of large proteoglycans increased with the distance from the articular surface. Again, no difference in the relative amounts of large and small proteoglycans were found when cartilage from different areas were compared. Osteochondrotic cartilage was detected in the pigs aged 12-18 weeks. In areas where osteochondrotic cartilage were present, the total production of 35S-proteoglycans was lowered and the relative amount of large proteoglycans was less than that found in the adjoining areas devoid of osteochondrotic lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1994

The usefulness of a contrast agent and gradient-recalled acquisition in a steady-state imaging sequence for magnetic resonance imaging-guided noninvasive ultrasound surgery.

RATIONALE AND OBJECTIVES: The ability of magnetic resonance imaging to detect small temperature elevations from focused ultrasound surgery beams was studied. In addition, the value of a contrast agent in delineating the necrosed tissue volume was investigated. MATERIALS AND METHODS: Gradient-recalled acquisition in a steady state (GRASS) T1-weighted images were used to follow the temperature elevation and tissue changes during 2-minute sonications in the thigh muscles of 10 rabbits. The effects of the treatment on the vascular network was investigated by injecting a contrast agent bolus before or after the sonication. RESULTS: The signal intensity decreased during the sonication, and the reduction was directly proportional to the applied power and increase in temperature. The signal intensity returned gradually back to baseline after the ultrasound was turned off. Injection of the contrast agent increased the signal intensity in muscle, but not in the necrosed tissue. The dimensions of the delineated tissue volume were the same as measured from the T2-weighted fast-spin-echo images and postmortem tissue examination. CONCLUSIONS: These results indicate that magnetic resonance imaging can be used to detect temperature elevations that do not cause tissue damage and that contrast agent can be used to delineate the necrosed tissue volume.

Animals↗