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Biomedical subjects

R M Robinson

Publications and source records attributed to R M Robinson.

At least 19 recordsLinked to original sources
PubMed2001

IGF-I stimulation of extracellular acidification is not linked to cell proliferation for autocrine cells.

Insulin-like growth factor-I (IGF-I) increases extracellular acidification rate (ECAR), a measure correlated with proliferation for nonautocrine cells. To evaluate the effect of autocrine IGF-I secretion on cell responsiveness, a cell line that secretes IGF-I was tested. SV40-lGF-I cells also registered concentration-dependent increases in ECAR; however, unlike the parental cell line, signal attenuation upon repeat challenges was not evident. Furthermore, SV40-IGF-I cells did not proliferate in response to IGF-I. We investigated if lack of proliferation was due to differences in the protocols of the assays ([3H]thymidine incorporation and microphysiometry). We identified three key differences in the protocols: surface substrate, cell density, and fluid residence time. We found no increase in [3H]thymidine incorporation for cells on either tissue-culture plastic or polycarbonate transwells. Control levels of [3H]thymidine incorporation were cell-density-dependent, but IGF-I did not increase proliferation at any density studied. Finally, we investigated IGF-I stimulation for cells under microphysiometer flow conditions and found no proliferative response to IGF-I. We found that the cells do respond to IGF-I with increased amino acid uptake. These data suggest that IGF-I signaling is operational in the SV40-IGF-I cells, but the transduction pathway for IGF-I-induced proliferation is compromised, despite the fact that these cells respond to fetal bovine serum with increased growth. Ongoing studies are focused on identifying which elements in the signaling cascade are altered by autocrine secretion of IGF-I.

Amino Acids↗
PubMed2000

A simple assay for evaluating inhibitors of proteoglycan-ligand binding.

Proteoglycans, once thought to primarily serve as structural components of extracellular matrix, are now being focused on for their role in tissue and cell regulation, particularly angiogenesis. Many growth factors, notably the fibroblast growth family (FGF) which now numbers 19 members, bind to heparin and heparan sulfate proteoglycans and this binding has been shown to have a significant impact on the availability and activity of these growth factors. Proteoglycans can serve as both temporal and spatial regulators and effective inhibitor design may depend on disruption of these interactions. We have developed a simple assay for evaluating small inhibitors of proteoglycan-ligand binding. The assay is based on cell-free incubation of the reactants and filtration across a cationic membrane. Conditions were established that allow one to semiquantitatively determine binding constants for both direct proteoglycan as well as soluble inhibitor affinity. The assay has been demonstrated using a model heparan sulfate proteoglycan preparation (perlecan from cultured bovine endothelial cells) and FGF-2. Protamine sulfate, sucrose octasulfate, and heparin were analyzed as model inhibitor molecules. This type of assay may have wide application as a fast and easy screening tool for small potential agonists and antagonists of proteoglycan-protein interactions.

Animals↗
PubMed2000

Real-time detection of insulin-like growth factor-1 stimulation of the MAC-T bovine mammary epithelial cell line.

Binding of growth factors by cell-surface receptors is an essential means by which cells regulate normal tissue growth and differentiation. Exposure to growth factors is often transient, and our goal was to determine whether short-term exposure to insulin-like growth factor-1 (IGF-1) would lead to activation, assayed as cell proliferation, of mammary epithelial cells. The MAC-T cell line is an immortalized bovine mammary epithelial cell line, chosen as our model mammary cell line because of its known sensitivity to IGF-1. Using the Cytosensor Microphysiometer System, a biosensor capable of measuring extracellular acidification, we were able to measure activation of the cells owing to IGF-1 addition in real time and found that peak acidification occurred in only 14 min. We show that this rapid response to IGF-1 is dose dependent and specific for IGF-1. A significant increase in [3H]thymidine incorporation by cells after a similar short-term exposure to IGF-1 suggests that the measured increase in extracellular acidification following IGF-1 addition is physiologically relevant. This technology offers a new, novel, and rapid means for the study of IGF-1 activity, as well as the screening of IGF-1 inhibitors, in mammary epithelial cells.

Animals↗
PubMed2000

Microphysiometry to evaluate real-time response of mammary epithelial cells to IGF-I.

The Cytosensor Microphysiometer, a biosensor developed by Molecular Devices, was used to assay rapid binding activity of IGF-I for bovine mammary epithelial cell lines. Insulin-like growth factor-I (IGF-I) is a potent mitogen for mammary epithelial cells and has been implicated in breast cancer cell proliferation, a leading cause of cancer death of women in the U.S. today. IGF-I acts by binding to cell surface receptors. We are interested in how autocrine secretion might alter the activity and regulation of IGF-I. Real-time changes in excretion of protons, which we assert results from IGF-I binding, are detected by the Cytosensor Microphysiometer and can be correlated with cellular activity. We present IGF-I dose-dependent responses as well as correlated binding data to detect cell surface receptor concentration and thymidine incorporation results to determine cell proliferation following IGF-I stimulation. We examine the effect of insulin-like binding protein-3 (IGFBP-3) both in the presence and absence of IGF-I. We believe comparison of autocrine and paracrine environments for IGF-I stimulation, and the components contributing to the binding of IGF-I to the cell membrane receptor may provide pertinent information for the development of intervention schemes to slow or interrupt IGF-I binding to tumor cells and therefore cancer growth.

Animals↗
PubMed1997

A new method for straightening DNA molecules for optical restriction mapping.

We have developed an improved method of straightening DNA molecules for use in optical restriction mapping. The DNA was straightened on 3-aminopropyltriethoxysilane-coated glass slides using surface tension generated by a moving meniscus. In our method the meniscus motion was controlled mechanically, which provides advantages of speed and uniformity of the straightened molecules. Variation in the affinity of the silanized surfaces for DNA was compensated by precoating the slide with single-stranded non-target blocking DNA. A small amount of MgCl2 added to the DNA suspension increased the DNA-surface affinity and was necessary for efficient restriction enzyme digestion of the straightened surface-bound DNA. By adjusting the amounts of blocking DNA and MgCl2, we prepared slides that contained many straight parallel DNA molecules. Straightened lambda phage DNA (48 kb) bound to a slide surface was digested by EcoRI restriction endonuclease, and the resulting restriction fragments were imaged by fluorescence microscopy using a CCD camera. The observed fragment lengths showed excellent agreement with their predicted lengths.

Bacteriophage lambda↗
PubMed1993

Causes and costs of injuries in multiple trauma patients requiring extrication from motor vehicle crashes.

Prospective and contemporaneous medical and economic cost studies of 144 victims of motor vehicle crashes admitted to a regional level I trauma center with multiple injuries (ISS > or = 16) revealed 122 non-ejected patients, of whom 102 required extrication (EXTRIC) from the vehicle for physical or medical reasons and 20 who did not (N group). There were no differences in age (EXTRIC, 34 +/- 17 years; N, 41 +/- 24 years), type of crash (Frontal: 57% EXTRIC, 60% N; Lateral: 32% EXTRIC, 35% N) restraint use (35% EXTRIC, 35% N), or mortality (29% EXTRIC, 30% N). However, the estimated maximum speed before the crash was higher in EXTRIC patients (50 +/- 16 mph vs. 46 +/- 18 mph N, p < 0.04), as was the change in velocity (delta V) on impact (EXTRIC 30 +/- 15 mph; N, 24 +/- 8 mph, p < 0.01). Brain injuries (51% EXTRIC vs. 35% N) and lower extremity injuries were more numerous in EXTRIC patients (59% vs. 20% N, p < 0.003) and the number of splenic, lower extremity, and pelvic injuries associated with shock was greater in EXTRIC patients, p < 0.02; as were postinjury complications. As a result, operating room costs from orthopedic and plastic surgery increased professional charges in the EXTRIC group versus the N group ($20,000, EXTRIC; $17,000, N) and critical care costs ($13,000, EXTRIC; $4,000, N) with total costs of $72,000 and $77,000, respectively. The lower extremity injuries in EXTRIC patients were primarily a result of body part contacts with intrusions (CIs) of the car occupant compartment structures [73% with vs. 24% without (p < 0.0001)]. In lateral MVCs, brain injuries were also more commonly associated with CIs of the side window frame or A pillar (72% CI vs. 25% no CI; p < 0.035); but as a whole in MVCs in which extrication was necessary, lower extremity injuries from instrument panel or toepan CIs appeared more frequent than those resulting from contacts only (p < 0.0001). In EXTRIC patients, 69% of those in shock had CI injuries, and 80% of the deaths in the EXTRIC group were associated with CI injury. These data suggest that measures designed to prevent CIs by strengthening car passenger compartment structures may reduce the incidence of severe brain and lower extremity injuries and may reduce the need for extrication after MVCs.

Accidents, Traffic↗
PubMed1990

Inpatient treatment for depressed children and adolescents: preliminary evaluations.

Reported are the results of two uncontrolled outcome studies that evaluate the effectiveness of inpatient psychiatric treatment of children and adolescents suffering from clinical depression. Study 1 employed a sample of 7 children and measured outcome with the Depression Self-rating Scale (DSRS), the Hopelessness Scale for Children (HSC), and the Global Asssessment of Functioning (GAF) scale, which were administered to each child upon admission and again at discharge. Inpatient treatment involved multiple interventions, including individual psychotherapy, medication, milieu therapy and token economy, and other procedures. At discharge, statistically significant improvements were found on the patients' GAF and HSC scores, but not on their DSRS scores. Study 2 used a sample of 15 adolescents, also admitted for clinical depression. Administered at each patient's admission and discharge, the Beck Depression Inventory (BDI), Generalized Contentment Scale (GCS), and Index of Self-esteem (ISE) were used to measure outcome. The multi-modal treatment program offered to the sample in Study 2 was similar to that offered the sample in Study 1. At discharge all three outcome measures reflected statistically significant improvements in the patients studies; therefore, these results provide addmtional support for the inpatient treatment of depressed children and adolescents.

Adolescent↗
PubMed1990

The gene for the alpha polypeptide of pyruvate dehydrogenase is X-linked in humans.

The chromosomal location of the gene for the alpha polypeptide of the pyruvate dehydrogenase (alpha E1), a major component of the pyruvate dehydrogenase complex, was determined by using a cloned cDNA for alpha E1. This 1-kb cDNA was isolated from a human liver lambda gt11 expression library with specific antibodies and included the coding (from amino acid 144 to the carboxy terminus) and the 3' untranslated regions. Southern blot analysis of the DNA from a panel of rodent-human hybrid cells showed that the absence or the presence of the major EcoRI fragment that hybridized with this cDNA probe was concordant with the presence of the Xq24-p22 region of the human X chromosome. The result of in situ hybridization with human metaphase chromosomes further mapped the alpha E1 gene to the Xp arm.

Antibodies↗
PubMed1986

Bovine abortion and death associated with consumption of aflatoxin-contaminated peanuts.

Approximately 12% of a herd of 68 crossbred cows aborted third-trimester fetuses after consuming moldy peanuts for 4 days. Further investigation revealed that less than 20% of the herd had access to this supplemental feed. Results of serum biochemical analysis indicated liver damage in the affected cows. All of these cows died within 8 days of aborting. The peanuts contained 77 micrograms aflatoxin B1/g, as determined by liquid chromatography. Tissues were submitted from 1 cow, and liver contained 5 ng aflatoxin B1/g. Results of other laboratory tests were negative for common toxins and abortifacients.

Abortion, Veterinary↗
PubMed1985

Interferon produced endogenously in response to CSF-1 augments the functional differentiation of progeny macrophages.

The role of endogenously produced interferon alpha/beta in the functional maturation of newly derived mononuclear phagocytes was investigated. Addition of highly specific anti-interferon alpha + beta antiserum to murine marrow cultures stimulated with colony-stimulating factor-1 (macrophage growth factor) markedly suppressed the capacity of resulting progeny mononuclear phagocytes to ingest opsonized sheep erythrocytes (EAIgG). This impairment was corrected either by direct addition of interferon alpha + beta at a concentration in excess of that neutralized by the antiserum or by the addition of lesser amounts of interferon (33 U/ml) following removal of the anti-interferon from the cultures. Conditioned media from control colony-stimulating factor-stimulated cultures similarly reversed the impairment of maturation resulting from 5 days of growth in the presence of anti-interferon. This enhancement of EAIgG ingestion reflected upon the interferon activity in the conditioned media and was neutralized by anti-interferon. Lastly, the endogenous interferon was found to enhance EAIgG ingestion by a majority of the mononuclear phagocyte progeny and not by a limited subpopulation.

Animals↗
PubMed1984

Subunit interactions in human plasma fibronectin.

The fibronectin molecule was split chemically into its two constituent chains (mol. wt. 220,000) by mild reduction with dithiothreitol. However, physical properties (molecular weight and diffusion coefficient from light scattering, and elution in gel exclusion chromatography) remained those of intact fibronectin, except (reversibly) in the presence of denaturants which also change the conformation of non-reduced fibronectin to a more open form. Similarly, during digestion of fibronectin by plasmin to fragments of molecular weight less than 200,000, the light scattering intensity drops to roughly half in 30% glycerol but not in the absence of glycerol. These results suggest that the compact conformation of native fibronectin is stabilized by specific noncovalent contacts between constituent chains.

Dithiothreitol↗
PubMed1984

Evidence for an extracellular plasmin-dependent proteolytic system in mineralizing matrices.

Plasminogen activator, which specifically catalyzes the extracellular conversion of plasminogen to plasmin, was identified in the cell-free mineralizing matrices of enamel, dentin, cementum, and bone by a fibrinolytic overlay technique. The spatial separation in developing teeth of successive stages of dentinogenesis allowed us to identify predentin as a major site of plasminogen activator activity. In addition, plasminogen, the natural substrate for the activator, was demonstrated in predentin by immunohistochemical techniques. Extraction of human dentin/predentin with neutral demineralizing buffers solubilized the activator along with inhibitory components capable of blocking the activation of plasminogen. When resolved by polyacrylamide electrophoresis under dissociative conditions, however, the activator emerged in active form as two closely spaced bands at Mr 66,000 and 62,000. In the mineralizing enamel matrix of continuously forming rodent incisors, activator activity was limited to a 3-5 mm wide segment which marks the transition between "soft" and "chalky" enamel, whereas the entire overlying enamel organ was rich in activator activity at all developmental stages. This suggests that the activator is transported to the enamel matrix only in a narrow zone which coincides with the most rapidly mineralizing site. The coincident expression of plasminogen activator activity and mineral accretion suggests that plasmin-dependent proteolysis may play a role in the extracellular regulation of matrix mineralization.

Animals↗
PubMed1984

Techniques for collecting blood from collared peccaries, Dicotyles tajacu (L.).

Four methods are described for obtaining blood samples from the collared peccary. This animal lacks prominent superficial veins which makes the procedure of taking blood difficult for inexperienced persons. Large volumes of blood (greater than 20 ml) can be obtained easily via anterior vena cava venipuncture. Moderate amounts of blood (less than 20 ml) can be obtained from the orbital sinus. Lesser volumes of blood can be obtained from superficial veins located on the ear and the hind limb. The saphenous vein is distended easily due to its unique location across the cranial face of the tibia.

Animals↗
PubMed1983

Helix formation upon acidification of protein-dodecyl sulfate complexes.

The pH dependence of circular dichroism spectra has been studied for dodecyl sulfate complexes formed by 25 proteins and for a random copolypeptide of glutamic acid and alanine. The pH range covered is that in which titration of side-chain carboxyl groups is to be expected. Circular dichroism spectra signify an increase in helical content upon acidification, although in many cases the increase is quite small. For all but three of the proteins studied, the spectral changes are in reasonable agreement with those expected because helix propagation by glutamyl and aspartyl residues is enhanced when the state of the side-chain carboxyl changes from COO- to COOH. This simple explanation seriously underestimates conformational changes reported for gastrin, Kunitz trypsin inhibitor and tropomyosin. Changes in charge density appear to play an important role in these proteins.

Circular Dichroism↗