Biomedical subjects
C C Cunningham
Publications and source records attributed to C C Cunningham.
Gelsolin overexpression enhances neurite outgrowth in PC12 cells.
The rational design of therapies for treating nerve injuries requires an understanding of the mechanisms underlying neurite extension. Neurite motility is driven by actin polymerization; however, the mechanisms are not clearly understood. One actin accessory protein, gelsolin, is involved with remodeling the cytoskeleton, although its role in cell motility is unclear. We report a two-fold upregulation of gelsolin upon differentiation with nerve growth factor. Cells that were genetically modified to overexpress gelsolin have longer neurites and a greater neurite motility rate compared to controls. These data suggest that gelsolin plays an important role in neurite outgrowth.
A Phase I trial of H-ras antisense oligonucleotide ISIS 2503 administered as a continuous intravenous infusion in patients with advanced carcinoma.
BACKGROUND: Abnormal expression of Ras proteins frequently is found with oncogenic transformation making ras a promising therapeutic target. ISIS 2503 is a 20-base antisense phosphorothioate oligodeoxyribonucleotide that specifically downregulates H-ras expression and inhibits tumor cell growth in preclinical studies. Here, the authors report an initial clinical study of the safety and tolerability of an intravenous infusion of ISIS 2503 in patients with advanced cancer. METHODS: A continuous intravenous infusion of ISIS 2503 was administered for 14 days every 3 weeks to 23 patients with a variety of solid tumors refractory to standard therapy. The dose of ISIS 2503 was increased in sequential cohorts of patients, as toxicity allowed, until a final dose of 10.0 mg/kg/day of body weight was reached. Toxicity was scored by the National Cancer Institute's Common Toxicity Criteria, and tumor response was monitored after every two treatment cycles. Pharmacokinetic studies were performed in some of the patients up to, and including, the final dose of 10 mg/kg/day/day of body weight. Levels of H-ras mRNA expression also were determined in the circulating lymphocytes of some patients by quantitative reverse transcriptase-polymerase chain reaction. RESULTS: A total of 23 patients received 63 cycles of ISIS 2503 at escalating doses to 10.0 mg/kg/day without dose-limiting toxicity and only minimal side effects. Four patients had stabilization of their disease for 6-10 cycles. No consistent decreases in H-ras mRNA levels were observed in peripheral blood lymphocytes. CONCLUSIONS: ISIS 2503, an antisense oligonucleotide against H-ras, was well tolerated as a single agent at doses up to 10.0 mg/kg/day by 14-day continuous intravenous infusion. Several patients had stabilization of disease, suggesting that ISIS 2503 had some tumor growth inhibitory effects and future trials of ISIS 2503 in combination with chemotherapy should be considered.
Cell permeant polyphosphoinositide-binding peptides that block cell motility and actin assembly.
Polyphosphoinositides (PPIs) affect the localization and activities of many cellular constituents, including actin-modulating proteins. Several classes of polypeptide sequences, including pleckstrin homology domains, FYVE domains, and short linear sequences containing predominantly hydrophobic and cationic residues account for phosphoinositide binding by most such proteins. We report that a ten-residue peptide derived from the phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding region in segment 2 of gelsolin, when coupled to rhodamine B has potent PIP(2) binding activity in vitro; crosses the cell membrane of fibroblasts, platelets, melanoma cells, and neutrophils by a process not involving endocytosis; and blocks cell motility. This peptide derivative transiently disassembles actin filament structures in GFP-actin-expressing NIH3T3 fibroblasts and prevents thrombin- or chemotactic peptide-stimulated actin assembly in platelets and neutrophils, respectively, but does not block the initial [Ca(2+)] increase caused by these agonists. The blockage of actin assembly and motility is transient, and cells recover motility within an hour after their immobilization by 5-20 microm peptide. This class of reagents confirms the critical relation between inositol lipids and cytoskeletal structure and may be useful to probe the location and function of polyphosphoinositides in vivo.
Chronic ethanol consumption and liver glycogen synthesis.
Chronic ethanol consumption results in a dramatic decrease in liver glycogen concentrations, which could be related to either a depressed rate of synthesis or an increased rate of breakdown. Earlier studies suggested that there is not an increase in the rate of glycogenolysis as glycogen phosphorylase activities are not elevated. In the present study it was observed that the incorporation of radiolabeled glucose into glycogen was significantly depressed in hepatocytes from ethanol-fed rats under both anaerobic and aerobic conditions. Chronic ethanol consumption decreased the total glycogen synthase (a + b) activity, which correlated closely with a loss in glycogen synthase protein. However, glycogen synthase messenger RNA levels were not depressed, which indicated posttranscriptional modifications affecting both activity and protein levels. The concentration of glucose transporter 1 was also decreased due to ethanol consumption, but glucose transporter 2 levels were not altered. This latter result suggests that glucose transport in the perivenous region of the liver lobule may be decreased in chronic ethanol consumers. The alterations in glucose transport protein and glycogen synthesis observed in this study may contribute to lowered glycogen synthesis, but do not appear to account for the magnitude of the decreases in glycogen levels and rate of synthesis. Indeed, ethanol effects on glycogen metabolism are likely to be exerted at several levels, including posttranslational modulation of enzyme activities.
The optical stretcher: a novel laser tool to micromanipulate cells.
When a dielectric object is placed between two opposed, nonfocused laser beams, the total force acting on the object is zero but the surface forces are additive, thus leading to a stretching of the object along the axis of the beams. Using this principle, we have constructed a device, called an optical stretcher, that can be used to measure the viscoelastic properties of dielectric materials, including biologic materials such as cells, with the sensitivity necessary to distinguish even between different individual cytoskeletal phenotypes. We have successfully used the optical stretcher to deform human erythrocytes and mouse fibroblasts. In the optical stretcher, no focusing is required, thus radiation damage is minimized and the surface forces are not limited by the light power. The magnitude of the deforming forces in the optical stretcher thus bridges the gap between optical tweezers and atomic force microscopy for the study of biologic materials.
Comparison of the 1969 and 1993 standardizations of the Bayley Mental Scales of Infant Development for infants with Down's syndrome.
The Bayley Scales of Infant Development (BSID) were re-standardized in 1993 (BSID-II). The present study reports a comparison of the two versions with infants with Down's syndrome (DS). The BSID-II was used for 93 assessments of 54 children with DS (age range = 7-43 months). Comparisons were made with the 1969 standardization for 42 of these assessments, and for 45 assessments of 20 typically developing children aged between 6 and 24 months. The 1993 standardization produced significantly lower mean differences of 1.0 months mental age and 8.4 points mental development index for infants with DS, and 1.5 months mental age and 9.2 points mental development index for the typically developing group. Nineteen per cent more infants with DS had scores below two standard deviations and there was a larger decrement for lower functioning children. Both groups of children have to perform at a higher level to achieve the same relative score on BSID-II compared to BSID. This indicates that caution should be used in comparing cohorts of children tested on different versions of the Bayley scales. In addition, concerns are highlighted regarding the rules for establishing basal and ceiling levels for BSID-II for children with developmental delays.
Ethanol and oxidative stress.
This article represents the proceedings of a workshop at the 2000 ISBRA Meeting in Yokohama, Japan. The chair was Albert Y. Sun. The presentations were (1) Ethanol-inducible cytochrome P-4502E1 in alcoholic liver disease, by Magnus Ingelman-Sundberg and Etienne Neve; (2) Regulation of NF-kappaB by ethanol, by H. Matsumoto, Y. Nishitani, Y. Minowa, and Y. Fukui; (3) Chronic ethanol consumption increases concentration of oxidized proteins in rat liver, by Shannon M. Bailey, Vinood B. Patel, and Carol C. Cunningham; (4) Antiphospholipids antibodies and oxidized modified low-density lipoprotein in chronic alcoholic patients, by Tomas Zima, Lenka Fialova, Ludmila Mikulikova, Ptr Popov, Ivan Malbohan, Marta Janebova, and Karel Nespor; and (5) Amelioration of ethanol-induced damage by polyphenols, by Albert Y. Sun and Grace Y. Sun.
Ethanol and protein metabolism.
This article represents the proceedings of a workshop at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Carol C. Cunningham and Victor R. Preedy. The presentations were (1) Ribosomal content, ribosomal localization and the levels of ribosomal protein mRNA and rRNA in rat skeletal muscle exposed to ethanol, by Alistair G. Paice, John E. Hesketh, Timothy J. Peters, and Victor R. Preedy; (2) Altered hepatic mitochondrial ribosome structure after chronic ethanol administration, by Vinood B. Patel and Carol C. Cunningham; (3) Clinical aspects of hepatic protein metabolism and alcohol, by Elena Volpi; and (4) Effects of oral intake of alanine plus glutamine on ethanol metabolism and ethanol-related depression in motor activity, by Kazunori Mawatari, H. Masaki, M. Mori, and Kunio Torii.
Chronic ethanol consumption alters the glutathione/glutathione peroxidase-1 system and protein oxidation status in rat liver.
BACKGROUND: Alcohol-induced liver damage is associated with oxidative stress, which might be linked to disturbances in liver antioxidant defense mechanisms. The effect of chronic ethanol consumption on the mitochondrial and cytosolic glutathione/glutathione peroxidase-1 (GSHPx-1) system and oxidative modification of proteins was therefore studied in the rat. METHODS: Male Sprague-Dawley rats were fed liquid diets that provided 36% total calories as ethanol for at least 31 days. Pair-fed controls received isocaloric diets with ethanol calories substituted with maltose-dextrins. Mitochondrial and cytosolic fractions were prepared from livers and assayed for GSHPx-1 and glutathione reductase activities and total and oxidized concentrations of glutathione. Catalase activity was measured in the postmitochondrial supernatant. Levels of GSHPx-1, lactate dehydrogenase, and the beta subunit of the F1 portion of the ATP synthase protein were determined by western blot analysis. Concentrations of mitochondrial and cytosolic protein carbonyls were measured to assess ethanol-induced oxidation of proteins. RESULTS: Chronic ethanol consumption significantly decreased cytosolic and mitochondrial GSHPx-1 activities by 40% and 30%, respectively. Levels of GSHPx-1 protein in cytosol were unaffected by ethanol feeding, whereas there was a small decrease in GSHPx-1 protein levels in mitochondria isolated from ethanol-fed rats. Glutathione reductase activities were increased in both intracellular compartments and catalase activity was increased as a consequence of ethanol exposure. Cytosolic total glutathione was mildly decreased, whereas ethanol feeding increased mitochondrial levels of total glutathione. Chronic ethanol feeding significantly increased both cytosolic and mitochondrial concentrations of protein carbonyls by 30% and 60%, respectively. CONCLUSIONS: This study demonstrates that chronic ethanol-induced alterations in the glutathione/GSHPx-1 antioxidant system might promote oxidative modification of liver proteins, namely those of the mitochondrion, which could contribute to the adverse effects of ethanol on the liver.
Physiochemical properties of rat liver mitochondrial ribosomes.
In the present study, the physiochemical properties of rat liver mitochondrial ribosomes were examined and compared with Escherichia coli ribosomes. The sedimentation and translational diffusion coefficients as well as the molecular weight and buoyant density of rat mitochondrial ribosomes were determined. Sedimentation coefficients were established using the time-derivative algorithm (Philo, J. S. (2000) Anal. Biochem. 279, 151-163). The sedimentation coefficients of the intact monosome, large subunit, and small subunit were 55, 39, and 28 S, respectively. Mitochondrial ribosomes had a particle composition of 75% protein and 25% RNA. The partial specific volume was 0.688 ml/g, as determined from the protein and RNA composition. The buoyant density of formaldehyde-fixed ribosomes in cesium chloride was 1.41 g/cm(3). The molecular masses of mitochondrial and E. coli ribosomes determined by static light-scattering experiments were 3.57 +/- 0.14 MDa and 2.49 +/- 0.06 MDa, respectively. The diffusion coefficient obtained from dynamic light-scattering measurements was 1.10 +/- 0.01 x 10(-7) cm(2) s(-1) for mitochondrial ribosomes and 1.72 +/- 0.03 x 10(-7) cm(2) s(-1) for the 70 S E. coli monosome. The hydration factor determined from these hydrodynamic parameters were 4.6 g of water/g of ribosome and 1.3 g/g for mitochondrial and E. coli ribosomes, respectively. A calculated hydration factor of 3.3 g/g for mitochondrial ribosomes was also obtained utilizing a calculated molecular mass and the Svedberg equation. These measurements of solvation suggest that ribosomes are highly hydrated structures. They are also in agreement with current models depicting ribosomes as porous structures containing numerous gaps and tunnels.
Optical deformability of soft biological dielectrics.
Two counterpropagating laser beams were used to significantly stretch soft dielectrics such as cells. The deforming forces act on the surface between the object and the surrounding medium and are considerably higher than the trapping forces on the object. Radiation damage is avoided since a double-beam trap does not require focusing for stable trapping. Ray optics was used to describe the stress profile on the surface of the trapped object. Measuring the total forces and deformations of well-defined elastic objects validated this approach.
Effects of chronic ethanol feeding on the protein composition of mitochondrial ribosomes.
Chronic ethanol feeding has been shown to decrease the number of functionally active mitochondrial ribosomes by 55%. In this work, 55S mitochondrial ribosomes were isolated from rat liver and their constitutive proteins characterized by two-dimensional polyacrylamide gel electrophoresis and quantified by densitometry. A total of 86 proteins were found to be associated with the mitochondrial ribosome. This compares with 70 isolated from cytoplasmic ribosomes. In addition, mitochondrial ribosomal proteins were found to be significantly less basic than their cytoplasmic counterparts. Chronic ethanol feeding was found to significantly decrease the levels of a number of constitutive proteins of the mitochondrial ribosome when compared to those isolated from pair-fed controls. Sucrose density gradient analyses revealed a significant decrease in the number of intact 55S ribosomes. It is suggested that ethanol-elicited alterations in specific constitutive proteins of the mitochondrial ribosome may lead to impaired assembly of the monosome and that this may result in lower levels of those displaying functional activity.
The information needs and understanding of 5-10-year old children with epilepsy, asthma or diabetes.
This exploratory study compared the information needs and understanding of 25 5-10-year olds with epilepsy with those of 10 children with asthma and 10 with diabetes (of the same age range). The children were interviewed whilst attending specialist clinics by the first author and were unaware of her professional status. All the children had access to specialist nurses and their families had received literature about their condition. The interview covered five main areas: knowledge of their condition, psychological effects, medication, restrictions on lifestyle, where they obtained their information and if they had unanswered questions. The children with epilepsy had far more unanswered questions and felt excluded from discussions with doctors. They also appeared reluctant to tell their friends their diagnosis and, at such a young age, felt stigmatized by their condition. The results highlight a contrast in the understanding of children with epilepsy when compared with those with asthma or diabetes. It is proposed that if a simple biological model were used to explain epilepsy this could aid children's understanding and reduce their reluctance to disclose their diagnosis.
Parents' reports of young people with Down syndrome talking out loud to themselves.
Despite parental concerns about young people with Down syndrome talking out loud to themselves (using private speech), there is virtually no research literature on this behavior. In that which exists, investigators have largely interpreted the behavior within a pathological framework. An alternative perspective is that self-talk is developmentally appropriate for these young people. Parents of 78 young people with Down syndrome, age 17 to 24 years, were asked whether their offspring had ever used private speech. Results confirm the universality of private speech and its developmental pattern. No association was found between private speech and behavior problems, communication difficulties, or social isolation. Talking out loud to self by young people with Down syndrome should be seen as adaptive, and not an indication of pathology.
A phase I trial of c-Raf kinase antisense oligonucleotide ISIS 5132 administered as a continuous intravenous infusion in patients with advanced cancer.
Raf proteins play a central role in the mitogen-activated protein kinase signaling pathway and hence are involved in oncogenic transformation and tumor cell proliferation. ISIS 5132 is a 20-base antisense phosphorothioate oligodeoxyribonucleotide that specifically down-regulates c-raf expression. We report here an initial study of the safety and tolerability of an i.v. infusion of ISIS 5132 in patients with advanced cancer. A continuous i.v. infusion of ISIS 5132 was administered for 21 days every 4 weeks to 34 patients with a variety of solid tumors refractory to standard therapy. The dose of ISIS 5132 was increased in sequential cohorts of patients, as toxicity allowed, until a final dose of 5.0 mg/kg body weight was reached. Toxicity was scored by common toxicity criteria, and tumor response was monitored. Pharmacokinetic studies were performed for 30 patients treated at doses of < or =4.0 mg/kg/day. The initial dose of ISIS 5132 was 0.5 mg/kg body weight and was successfully increased incrementally to 5.0 mg/kg body weight. Toxicities through the 4.0 mg/kg dose level were not dose limiting. Side effects were minimal and could not be specifically related to ISIS 5132. Two patients had prolonged stabilization of their disease, and one patient with ovarian carcinoma had a significant response with a 97% reduction in CA-125 levels. ISIS 5132, an antisense oligonucleotide against c-raf, was well tolerated at doses up to and including 4.0 mg/kg/day by 21-day continuous i.v. infusion and demonstrated antitumor activity at the doses tested.
Chronic ethanol, oxygen tension and hepatocyte energy metabolism.
Hepatocytes from ethanol-fed animals, isolated from either whole liver or the periportal or perivenous regions of the lobule, exhibited an ethanol-related decrease in energy state only when they were oxygen deficient. This was accompanied by an ethanol-related decrease in hepatocyte viability. Both periportal and perivenous hepatocytes from ethanol-fed rats demonstrated increased respiration. The observations reported here are consistent with an ethanol-induced increase in oxygen utilization which could render the perivenous region of the lobule relatively oxygen deficient in the intact liver. This oxygen deficit may cause the decreases in energy state and cell viability associated with chronic ethanol consumption. Ethanol-associated loss in hepatocyte viability appeared to correlate better with a decrease in energy state than with an increase in the products of oxidative stress. An investigation of the association between viability and cellular malondialdehyde levels revealed no effects of chronic ethanol consumption on MDA levels in hepatocytes that demonstrated ethanol-related decreases in cell viability.
Contributions of dietary carbohydrate and ethanol to alterations in liver glycogen levels and glycolytic activity.
Hepatic glycogen levels are decreased in rats as a consequence of chronic ethanol consumption. In earlier studies ethanol (36% of total calories consumed) replaced carbohydrate in the ethanol-containing diet, thus leading to the possibility that the decreases in liver glycogen were a result of limited dietary carbohydrate. In the present study, rats were administered ethanol in low-carbohydrate (LC) or high-carbohydrate (HC) diets to determine if lowered dietary carbohydrate contributes to the decrease in glycogen levels associated with ethanol consumption. The glycogen content of isolated hepatocytes was not different between rats fed LC or HC in control or ethanol-containing diets. Lactate and pyruvate were measured to determine the effects of dietary carbohydrate and ethanol on glycolytic activity, and were not significantly altered by changes in the levels of dietary carbohydrate. However, ethanol-containing diets resulted in decreased concentrations of hepatic glycogen, lactate, and pyruvate as compared with controls in both LC and HC diets. These observations demonstrate that decreases in glycogen content and lactate + pyruvate concentrations are due to chronic ethanol consumption rather than a carbohydrate deficiency, when carbohydrate is maintained above 10% of total calories.