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Biomedical subjects

K Miller

Publications and source records attributed to K Miller.

At least 253 records · Page 14Linked to original sources
PubMed1997

Purification of ADAM 10 from bovine spleen as a TNFalpha convertase.

We have purified a protease with characteristics of TNFalpha convertase from bovine spleen membranes. Peptide sequencing of the purified protein identified it as ADAM 10 (Genbank accession no. Z21961). This metalloprotease cleaves a recombinant proTNFalpha substrate to mature TNFalpha, and can cleave a synthetic peptide substrate to yield the mature TNFalpha amino terminus in vitro. The enzyme is sensitive to a hydroxamate inhibitor of MMPs, but insensitive to phosphoramidon. In addition, cloned ADAM 10 mediates proTNFalpha processing in a processing-incompetent cell line.

ADAM Proteins↗
PubMed1997

High-level expression of the prohormones proenkephalin, pro-neuropeptide Y, proopiomelanocortin, and beta-protachykinin for in vitro prohormone processing.

Prohormone substrates are required for investigation of the proteolytic processing of prohormones and proproteins into active peptide hormones and neurotransmitters. However, the lack of prohormone proteins has been a limiting factor in elucidating proteolytic mechanisms for conversion of prohormones into active peptides. Therefore, in this study, cloned cDNAs encoding the prohormones proenkephalin (PE), pro-neuropeptide Y (pro-NPY), pro-opiomelanocortin (POMC), and beta-protachykinin (beta-PT) were utilized to express recombinant prohormones in Escherichia coli. High-level expression of milligrams of prohormones was achieved with the pET3c expression vector utilizing the T7 promoter for production of PE, pro-NPY, and POMC, as demonstrated by SDS-PAGE gel electrophoresis, Western blots, and 35S-methionine labeling. In addition, beta-PT was expressed at high levels as fusion proteins with the maltose-binding protein and glutathione S-transferase by the pMAL-c and pGEX-2T expression vectors, respectively. Relative rates of processing by the established processing proteases "prohormone thiol protease" (PTP), 70-kDa aspartyl protease, and PC1/ 3 and PC2 (PC, prohormone convertase) were examined with purified PE, pro-NPY, and POMC. Distinct preferences of processing enzymes for different prohormones was demonstrated. PTP preferred PE and pro-NPY substrates, whereas little processing of POMC was detected. In contrast, the 70-kDa aspartyl protease cleaved POMC more readily than pro-NPY or PE. However, PC1/3 and PC2 prefer POMC as substrate. Demonstration of selectivity of processing enzymes for prohormone substrates illustrates the importance of expressing recombinant prohormones for in vitro processing studies.

Animals↗
PubMed1997

Expression, purification, and characterization of an activated cytokine-suppressive anti-inflammatory drug-binding protein 2 (CSBP2) kinase from baculovirus-infected insect cells.

An activated form of the human cytokine-suppressive anti-inflammatory drug-binding protein 2 (CSBP2) kinase was expressed in Spodoptera frugiperda (SF9) cells from a baculovirus vector. To maximize expression and to facilitate purification of the recombinant protein, CSBP2 kinase was expressed as a carboxy-terminal fusion protein to glutathione S-transferase (GST). Under optimal conditions, 2-3 mg of GST-CSBP2 could be obtained per liter of infected cell culture. The fusion protein was easily purified from the soluble fraction of the total cell lysate under nondenaturing conditions by using a glutathione-Sepharose 4B affinity resin. As expected, the purified GST-CSBP2 fusion protein was approximately 68 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and reacted with antibodies directed toward either the GST or the CSBP amino terminus. To obtain activated CSBP2, SF9 cells were coinfected with two recombinant baculovirus vectors: one that directed the synthesis of the GST-CSBP2 fusion protein and a second vector that directed the synthesis of a constitutively active form of the CSBP activating kinase, MKK3. Coexpression of GST-CSBP2 kinase with the MKK3 activator increased GST-CSBP2 activity 8- to 10-fold based on the ability of GST-CSBP2 to phosphorylate the substrate, myelin basic protein (MBP), and the ATF2 transcription factor, in vitro. Moreover, activated GST-CSBP2 was capable of activating a bacterially derived mitogen-activated protein kinase-activating protein kinase 2 in vitro. The activity of insect-derived GST-CSBP2 was also inhibited by the CSBP inhibitor, SB202190. We anticipate that the preparation and purification techniques described in this study will facilitate further biochemical characterization of this kinase.

Animals↗
PubMed1997

Expression of CD44V2 in transitional cell carcinoma of the urinary bladder and in urine.

CD44 is the principal cell surface receptor for hyaluronate. Variant forms of the receptor, produced by alternative splicing, have been found to be associated with tumor progression in a variety of cancers. Based on investigations at the RNA level, it has recently been proposed that expression of CD44 variant V2 was present in urothelial cancer but not in normal urothelium. Since a distinctive marker for urothelial cancer would be extremely useful, frozen sections of normal urothelium and urothelial cancer were examined for expression of standard CD44 and CD44V2. Frozen sections of specimens of 35 patients with transitional cell carcinoma of the bladder, 16 specimens of normal bladder and 5 ureters were examined. Immunohistochemical staining was performed using a polyclonal antibody to CD44V2 (PAB CD44V2), a monoclonal antibody to CD44V2 (MAB CD44V2) and a monoclonal antibody to CD44S (MAB CD44S). CD44V2 and CD44S were also measured in lysates of urine sediments from 21 patients by enzyme-linked immunoabsorbent assay (ELISA). All investigated transitional cell carcinomas expressed CD44V2. There was no differentiation between invasive and noninvasive carcinoma. CD44V2 was also expressed in normal urothelium. Standard CD44 was expressed by the transitional cell carcinoma, normal urothelium, musculature and interstitial tissue. The amount of CD44V2 and CD44S in lysates of urine sediments is not correlated to diagnosis. In contrast to investigations at the RNA level, CD44V2 on the protein level seems not to be a distinctive marker for urothelial cancer. Therefore, CD44V2 will not be a useful diagnostic marker for detection of transitional cell carcinoma.

Antibody Specificity↗
PubMed1997

Ileocutaneous fistula formation following laparoscopic polypropylene mesh hernia repair.

A rare case of enterocutaneous fistula caused by chronic erosion of polypropylene mesh after laparoscopic repair of a recurrent inguinal hernia is described. Successful treatment was achieved by fistulectomy, total resection of the implanted mesh, and small-bowel segmental resection. The patient recovered well postoperatively, and at follow-up 18 months later, the herniorrhaphy has remained intact. This complication needs to be added to the differential diagnosis in patients who present inflammation, abscess formation, or cutaneous fistula following laparoscopic hernia repair.

Aged↗
PubMed1997

Delayed diagnosis of malignant tumors missed at laparoscopic cholecystectomy.

BACKGROUND: The aim of this study was to compare the significance of routine examinations prior to laparoscopic cholecystectomy (LC) with intraoperative abdominal investigation. Preoperative evaluation becomes increasingly important when laparoscopic procedures are performed for the removal of gallstones because other intraabdominal diseases may coexist in these patients, mimicking biliary tract disease. METHODS: Over the last 6 years, we treated 816 patients with symptomatic cholecystolithiasis using LC. Prior to surgery, routine tests such as upper abdominal ultrasonography, chest radiography, and standard laboratory blood tests were carried out. RESULTS: Despite these routine tests, coexisting colonic cancers escaped detection in four out of 816 cases. This indicates a risk of more "missed pathologies" during the course of laparoscopic operations compared to standard laparotomy. CONCLUSION: The risk of missing coexisting diseases during laparoscopic operations has to be minimized by placing additional emphasis on careful evaluation of anamnesis. Physical examination and additional laboratory tests--such as analysis of tumor markers and blood in the stool--combined with complete abdominal ultrasonography, gastroscopy, and/or complete colonoscopy should be performed prior to LC.

Adenocarcinoma↗
PubMed1997

Lipid specificity and location of the sterol carrier protein-2 fatty acid-binding site: a fluorescence displacement and energy transfer study.

Although it was recently recognized that sterol carrier protein-2 (SCP-2) interacts with fatty acids, little is known regarding the specificity of SCP-2 for long-chain fatty acids or branched-chain fatty-acid-like molecules. Likewise the location of the fatty-acid binding site within SCP-2 is unresolved. A fluorescent cis-parinaric acid displacement assay was used to show that SCP-2 optimally interacted with 14-22 carbon chain lipidic molecules: polyunsaturated fatty acids > monounsaturated, saturated > branched-chain isoprenoids > branched-chain phytol-derived fatty acids. In contrast, the other major fatty-acid binding protein in liver, fatty-acid binding protein (L-FABP), displayed a much narrower carbon chain preference in general: polyunsaturated fatty acids > branched-chain phytol-derived fatty acids > 14- and 16-carbon saturated > branched-chain isoprenoids. However, both SCP-2 and L-FABP displayed a very similar unsaturated fatty-acid specificity profile. The presence and location of the SCP-2 lipid binding site were investigated by fluorescence energy transfer. The distance between the SCP-2 Trp50 and bound cis-parinaric acid was determined to be 40 A. Thus, the SCP-2 fatty-acid binding site appeared to be located on the opposite side of the SCP-2 Trp50. These findings not only contribute to our understanding of the SCP-2 ligand binding site but also provide evidence suggesting a potential role for SCP-2 and/or L-FABP in metabolism of branched-chain fatty acids and isoprenoids.

Animals↗
PubMed1997

Circulating soluble adhesion molecules E-cadherin, E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in patients with gastric cancer.

The concentrations of the soluble adhesion molecules E-cadherin, E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were investigated in 45 patients with gastric cancer before treatment and their correlation with clinical, histological and routine laboratory parameters was examined. Data were collected on tumour stage at presentation, presence and sites of metastatic disease, tumour pathology, survival and results of routine laboratory tests. Serum concentrations of ICAM-1 and VCAM-1 were significantly elevated in the patients with gastric cancer in comparison with the group of healthy subjects (P < 0.00001 and P < 0.0001 respectively). Increased serum concentrations of VCAM-1 were associated with locally advanced and metastatic disease whereas ICAM-1 was significantly elevated both in local and in advanced/metastatic disease. Soluble E-cadherin and E-selectin concentrations did not show any significant elevation in gastric cancer patients. Concentrations of soluble adhesion molecules showed significant correlation with each other (except E-selectin and VCAM-1) and with alkaline phosphatase. Soluble ICAM-1 and VCAM-1 were significantly associated with an elevated total white cell count. Patients with elevated VCAM-1 had significantly poorer survival in comparison with patients with normal serum levels (P = 0.0361).

Adult↗
PubMed1997

Neural tube defects in chromosomally normal and abnormal human embryos.

Neural tube defects (NTDs) were recognized in eight out of 91 intact embryos from spontaneous abortions and in one case of an induced abortion following prenatal diagnosis of a chromosomal disorder. Five of the nine cases showed chromosomal abnormalities. Trisomy 18 and triploidy were associated with spina bifida in three cases, trisomy 7 with parieto-occipital encephalocele and monosomy X with spina bifida and iniencephaly in one case. A sixth anencephalic embryo in which chromosomal analysis was not performed showed a malformation pattern highly suggestive for trisomy 18. Discussion focuses on the high rate and the type of chromosomal abnormalities among spontaneously aborted NTD embryos, on the contrasting phenotype of 45,X conceptions and on the morphogenesis of the different neural tube defects in early development. In view of future early endovaginal ultrasound diagnosis, the changing morphological pattern is exemplified, and ranges from apparently hyperplastic to degenerative alterations of the exposed neural tissue.

Chorionic Villi Sampling↗
PubMed1997

A combined inhibitor of matrix metalloproteinase activity and tumour necrosis factor-alpha processing attenuates experimental autoimmune neuritis.

Matrix metalloproteinases (MMPs) and the cytokine tumour necrosis factor (TNF)-alpha are implicated in the pathology of inflammatory demyelinating diseases of the CNS, and may also be involved in peripheral demyelinating diseases such as acute inflammatory demyelinating polyradiculoneuropathy. We have tested an inhibitor of MMP activity and TNF-alpha processing, BB-1101, in experimental autoimmune neuritis (EAN), an animal model of Guillain-Barré syndrome. Treatment with BB-1101 from the time of immunization prevented the development of EAN, and when given from the onset of symptoms, it significantly reduced disease severity. These results indicate that MMPs and/or TNF-alpha are involved in the pathogenesis of EAN, and that drugs of this type may have potential as novel therapeutic agents in the therapy of peripheral nervous system demyelinating diseases.

Animals↗
PubMed1997

Expression of functional mitochondrial creatine kinase in liver of transgenic mice.

The mitochondrial isoform of creatine kinase (MiCK) is localized to the mitochondrial intermembrane space, and its precise role in vivo is still actively being investigated. Here, we report a transgenic mouse model in which MiCK is expressed in liver, a tissue that does not normally express significant levels of CK. Expression of the genomic clone for human, ubiquitous MiCK was controlled by the promoter/enhancer region of the transthyretin gene. Three of seven founder mice were chosen to establish lines and had MiCK activity values ranging from 13 to 269 micromol x min(-1) x g wet wt(-1). Differential centrifugation and histochemical staining demonstrated that >90% of the CK activity is localized to the mitochondrial intermembrane space. An unusual mitochondrial morphology characterized by an angular nature to the membranes was detected using electron microscopy in the transgenic line expressing the highest levels of MiCK. Increasing hepatic total creatine levels led to a return to normal mitochondrial morphology. 31P-nuclear magnetic resonance spectroscopy demonstrated that the expressed MiCK is capable of producing and utilizing phosphocreatine. These mice will be useful for investigating gain of function effects of MiCK in cellular energetics.

Animals↗
PubMed1997

Leydig cell tumor--comparison of results of radical and testis-sparing surgery in a single center.

Between January 1981 and December 1995, 15 patients were treated for Leydig cell tumor at our institution -- 12 by radical orchiectomy, 3 by tumor enucleation. All patients were contacted to assess the long-term outcome depending on the treatment initially chosen. Follow-up ranged from 8 to 161 months (mean 56). In no case was progressive disease documented, in 1 case local recurrence was witnessed 4 months after tumor enucleation despite negative resection margins. We conclude that a small Leydig cell tumor can safely be managed by local enucleation alone.

Adult↗
PubMed1997

Side effects of chemotherapy for advanced urothelial carcinoma with etoposide and ifosfamide.

OBJECTIVES: To date, chemotherapy for advanced urothelial carcinoma has been associated with only moderate therapeutic success and minimal extension of survival. This, coupled with the occurrence of serious side effects and the resulting reduced quality of life, underscores the need for new chemotherapeutic agents. A first study investigating combination therapy with etoposide and ifosfamide reported not only comparable therapeutic effectiveness but, due primarily to the elimination of the cisplatin component, a reduction in nephrotoxic side effects. In the present study, therefore, patients with advanced urothelial carcinoma and minor compromised renal function received combination chemotherapy with etoposide and ifosfamide. METHODS: Fourteen patients with advanced urothelial carcinoma underwent chemotherapy with a combination of etoposide and ifosfamide. On days 1-5, patients received 1,500 mg/m2 ifosfamide and 120 mg/m2 etoposide. The next corresponding cycle was started on day 22. RESULTS: A number of serious side effects were observed. These consisted predominantly of high-grade myelosuppression requiring therapy, as well as disturbances in the central nervous system and impairment of renal function. Due to severe side effects, chemotherapy had to be prematurely terminated in 8 of 14 patients (57%). The efficacy of therapy, however, was observed in patients completing the treatment regimen. CONCLUSIONS: The new combined chemotherapy with etoposide and ifosfamide shows efficacy in the treatment of advanced urothelial carcinoma. This, however, is overshadowed by the high rate of serious side effects leading to premature interruption of therapy.

Aged↗