Health care concerns worldwide.
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Education, Medical↗
Biomedical subjects
R Brandt
Publications and source records attributed to R Brandt.
Interspecies scaling of bosentan, a new endothelin receptor antagonist and integration of in vitro data into allometric scaling.
PURPOSE: The goal of this study was to find a rational and reliable method of using animal data to predict the clearance of metabolised drugs in humans. METHODS: One such approach is to use in vitro liver models (e.g. hepatocytes and microsomes) to determine the relative capacities of the various animal species and humans to metabolise the test compound. These data can then be combined with the in vivo clearances in animals, to calculate the in vivo clearance in humans using allometric scaling techniques. In this study, this approach was evaluated with a new endothelin receptor antagonist, bosentan, which is eliminated mainly through metabolism and is characterized by very large interspecies differences in clearance. Therefore, this compound provided a stringent test of our new extrapolation method for allometric scaling. RESULTS: The results obtained with bosentan showed that adjusting the in vivo clearance in the different animal species for the relative rates of metabolism in vitro gave a far better prediction of human clearance than an empirical correcting factor (brain weight). CONCLUSIONS: This approach provided a more rational basis for predicting the clearance of metabolised compounds in humans.
Animals↗
Phosphorylation of native and truncated isoforms of protein tau by the double-stranded DNA-dependent protein kinase (DNA-PK) shows that the primary phosphorylation sites are localized between amino acid residues 212-231 of the longest tau.
Alzheimer's disease (AD) is pathologically characterized by the appearance of neurofibrillary tangles (NFT), senile plaques, and loss of subpopulation of neuronal cells. The NFT is composed of paired helical filaments (PHT) with extensively modified protein T as its primary constituent. Previously we had reported on the hyperphosphorylation of T by a double-stranded-DNA-stimulated protein kinase (DNA-PK). In this communication, we have compared the DNA-PK mediated phosphorylation of native and truncated TS with that catalyzed by the cAMP-dependent protein kinase (PKA). In addition, we have attempted to map the primary site(s) of phosphorylation of T by DNA-PK. Our results suggest that DNA-PK phosphorylates T at sites substantially different from those targeted by PKA. Furthermore, we show that the primary phosphorylation sites lie between amino acid residues 212-231 (using numbering system for the longest T, which is the isoform with four "repeats" and a 58 amino acid "insert" at the carboxyl- and amino-termini, respectively, of the protein molecule).
Alzheimer Disease↗
Animal pharmacokinetics and interspecies scaling from animals to man of lamifiban, a new platelet aggregation inhibitor.
Relating pharmacokinetic information obtained in animal species to man (interspecies scaling) can play an important role in enabling understanding of the differences and similarities between species, and helping to predict the kinetic profile of a new compound in man. Interspecies scaling techniques have been applied to lamifiban (Ro 44-9883), a new selective and potent nonpeptidic inhibitor of human glycoprotein IIb-IIIa intended for use in clinical treatment of, for example, acute coronary syndrome. The pharmacokinetic profile of lamifiban in man was predicted from animal data (in rats, dogs and cynomolgus monkeys) by using allometric scaling and concentration-time transformations. These extrapolations for lamifiban were performed prospectively, to help design the first pharmacokinetic studies in man. The approach based on equivalent time was preferred for our prospective predictions, in view of the high values found for the allometric exponents. Using allometric scaling, clearance (CL), half-life (t1/2) and volume of distribution (Vd) were overestimated by approximately two- to fourfold. Compared with allometric scaling, the transformation based on equivalent time improved the prediction for all human pharmacokinetic parameters. For t1/2 and CL, the observed values for man were within the range predicted from the various animal species. Of the individual animal species, the cynomolgus monkey gave the most reliable predictions of these two parameters, as well as accurately predicting the Vd value.
Acetates↗
Vitamin E ameliorates renal injury in an experimental model of immunoglobulin A nephropathy.
IgA nephropathy is one of the most common forms of glomerular disease. Nearly 25% of affected patients progress to end-stage renal disease over a 20-25-y follow-up period. IgA-containing immune complexes stimulate oxygen-free radical production by mesangial cells in vitro. The excessive oxidant stress may mediate glomerular injury in this disorder. Therefore, we studied whether dietary supplementation with the antioxidant agent, vitamin E, attenuates renal disease in an experimental model of incipient IgA nephropathy with mild kidney inflammation. IgA nephropathy was induced in male Lewis rats by oral immunization with 0.1% bovine gamma-globulin (BGG)-containing drinking water for 8 wk. At the completion of this period, animals received BGG, 1 mg/dose i.v., on three successive days. Experimental rats (n = 10) received a specially formulated diet containing 100 IU of vitamin E/kg of chow, whereas control animals (n = 10) were fed chow containing 30 IU of vitamin/kg of chow. The BGG immunization regimen induced mesangial IgA deposition in all rats. Vitamin E supplementation resulted in a nearly 5-fold increase in the serum vitamin E concentration. Vitamin E-treated rats gained more weight and had a lower incidence of hematuria, 20% versus 80% (p < 0.03). Moreover, proteinuria was decreased by 50%, and reduced renal plasma flow was restored to normal, compared with untreated rats with IgA nephropathy. Glomerular hypertrophy occurred in animals with IgA nephropathy, but less so in those receiving vitamin E supplementation. Renal cortical malondialdehyde content was reduced from 1.55 +/- 0.10 to 1.22 +/- 0.09 nmol/mg of protein (p < 0.01) in rats fed the vitamin E-enriched diet. Finally, renal transforming growth factor-beta 1 gene expression was reduced by 34% in rats with IgA nephropathy receiving vitamin E treatment (p < 0.05). We conclude that experimental IgA nephropathy is associated with increased renal oxidant injury. Dietary treatment with the antioxidant agent, vitamin E, attenuated renal functional and structural changes in this experimental glomerulopathy. These studies support the importance of clinical trials for the evaluation of the efficacy of antioxidant therapy in patients with IgA nephropathy.
Animals↗
Expression of fructosyllysine receptors on human monocytes and monocyte-like cell lines.
Short-term exposure of human serum albumin to glucose in vitro results in the formation of fructosyllysine residues. Using short-term glucose-modified albumin the interactions with human monocytes and with the human monocyte-like cells U937, MonoMac 6, HL60, and THP1 were studied. Short-term glycated albumin was specifically bound by monocytes, U937 and MonoMac 6 cells, but not by HL60 and THP1 cells. This specific binding of short-term glycated albumin was inhibited by fructosyllysine, but not by hexitollysine. Short-term glycated albumin did not compete for binding of albumin, modified by advanced glycation end products. Scatchard analysis of the binding data indicated that there are 10,000 binding sites per cell in monocytes or U937 cells and 2000 sites per cell on MonoMac 6 cells with affinity constants of 9 x 10(6) M-1. Specific binding of short-term glycated albumin to human monocytes was observed in 29% of the 101 human subjects investigated. Ligand-receptor cross-linking and ligand blotting experiments revealed two binding proteins of 100 to 110 and 190 kDa in SDS-PAGE after membrane protein solubilization of U937 and MonoMac 6 cells. The binding of short-term glycated albumin to MonoMac 6 cells induced the production of the cytokines IL-1 and TNF.
Binding Sites↗
Purification and partial amino acid sequencing of a fructosyllysine-specific binding protein from cell membranes of the monocyte-like cell line U937.
The ability of short-term in vitro glycated albumin to react with monocytes or the monocyte-like cells U937 is due to the Amadori adduct fructosyllysine. Two binding proteins of about 100 and 200 kDa have been previously described to interact specifically with the monocyte-like cell line U937. Detergent extracts from U937 cell membranes were used to purify the 100kDa protein by ion exchange chromatography, fructosyllysine-Sepharose affinity chromatography and SDS-PAGE. Six amino acids from the N-terminal end and two peptide sequences of 14 and 15 amino acids were identical with the N-terminus and the positions 349 to 362 and 610 to 624 of the major nuclear protein nucleolin. However, ligand blotting experiments with nuclear extracts from U937 and RIN cells showed no binding of glycated albumin with nucleolin. The reported amino acid sequences of the 100kDa fructosyllysine specific binding protein do not show any homologies with AGE-receptors. This receptor protein as a nucleolin-like polypeptide belongs to the superfamily of RNA-binding proteins.
Amino Acid Sequence↗
Regulation of Dictyostelium adenylylcyclases by morphogen-induced modulation of cytosolic pH or Ca2+ levels.
Inhibition of cAMP production and consequent inactivation of protein kinase A (PKA) by the putative morphogen ammonia has been suggested to block culmination and stalk cell differentiation in Dictyostelium. Since other weak bases mimic and weak acids act oppositely to ammonia, its effects were attributed to cytosolic or vesicular alkalinization; the latter resulting in impaired Ca2+ sequestration. We investigated whether weak bases and acids modulate the activity of the two Dictyostelium adenylylcyclases ACA and ACG in a manner consistent with their effects on development. It appeared that ammonia inhibits both ACG activity and ACA activation only transiently and does not significantly affect cAMP levels in slugs. Surprisingly, weak acids inhibit both ACA and ACG permanently, but do not affect secretion of cAMP as was suggested earlier. The effects of weak acids, which reduce cytosolic pH, are consistent with the pH dependence of ACA and ACG. In lysates, basal and GTP gamma S-stimulated ACA activity as well as ACG activity are optimal at pH 8 and are virtually absent below pH 7. ACG activity in cell lysates is completely insensitive to Ca2+, while GTP gamma S-stimulated ACA activity is maximally 50% reduced by supraphysiological Ca2+ concentrations. The observation that weak acids strongly inhibit ACA and ACG while promoting a PKA-dependent process such as stalk cell differentiation suggests that in Dictyostelium PKA can be activated in the absence of cAMP production.
Adenylyl Cyclases↗
The role of amphiregulin in breast cancer.
Amphiregulin (AR) is an epidermal growth factor (EGF)-related peptide that operates exclusively through the EGF receptor and that can bind to heparin. AR also possesses nuclear localization sequences in the extended NH2-terminal region suggesting an additional intracellular site of action. AR mRNA and protein expression have been detected in primary human mammary epithelial cell strains, nontransformed human mammary epithelial cell lines, several human breast cancer cell lines, and primary human breast carcinomas. The frequency and levels of AR protein expression are generally higher in invasive breast carcinomas than in ductal carcinomas in situ or in normal, noninvolved mammary epithelium. In addition, AR can function as an autocrine and/or juxtacrine growth factor in human mammary epithelial cells that have been transformed by an activated c-Ha-ras proto-oncogene or by overexpression of c-erb B-2. AR expression is also enhanced by mammotrophic hormones such as estrogens and other growth factors such as EGF.
Amino Acid Sequence↗
A new extrapolation method from animals to man: application to a metabolized compound, mofarotene.
Allometric scaling (a technique which uses data obtained in laboratory animals to predict human pharmacokinetics) works well for drugs that are cleared intact, but is less successful with extensively metabolised compounds. This paper describes a new method to improve the accuracy of such projections, by integrating metabolic data obtained in vitro (e.g. with liver microsomes or hepatocytes) into these calculations. The approach was used prospectively, to predict the clearance of mofarotene (Ro 40-8757) in humans from in vivo kinetic data obtained in mouse, rat and dog. This compound was selected to illustrate this approach because it is exclusively eliminated through metabolism. Without the metabolic correction or using empirical correcting factors, the values predicted for man were 2.7 and 0.6 ml/min/kg. This fell outside the range subsequently obtained in healthy volunteers dosed orally with 300 mg of mofarotene (7.5 +/- 4.0 ml/min/kg, n = 12). However, inclusion of the microsomal or hepatocyte data gave values of 5.1 and 4.2 ml/min/kg, respectively, illustrating that the integration of in vitro metabolic data improves the accuracy of kinetic extrapolations. In contrast to the existing empirical techniques, this approach offers a rational basis to predict clearance of metabolized compounds in human.
Animals↗
Epidermal growth factor-related peptides and their receptors in human malignancies.
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Animals↗
Interaction of tau with the neural plasma membrane mediated by tau's amino-terminal projection domain.
The neuronal microtubule-associated protein tau is required for the development of cell polarity in cultured neurons. Using PC12 cells that stably express tau and tau amino-terminal fragments, we report that tau interacts with the neural plasma membrane through its amino-terminal projection domain. In differentiated PC12 transfectants, tau is found in growth cone-like structures in a nonmicrotubule-dependent manner. In hippocampal neurons, tau is differentially extracted by detergent and enriched in the growth cone and the distal axon when membrane is left intact. In PC12 transfectants, overexpression of tau's amino-terminal fragment, but not of full-length tau, suppresses NGF-induced process formation. Our data suggest that tau's amino-terminal projection domain has an important role in neuritic development and establishes tau as a mediator of microtubule-plasma membrane interactions.
Amino Acid Sequence↗
The antimalarial drug halofantrine is bound mainly to low and high density lipoproteins in human serum.
1. The major serum proteins which bind halofantrine were identified by size exclusion chromatography. In addition, the binding affinity of halofantrine to human erythrocytes and serum proteins was measured by an erythrocyte partitioning technique. The influence of serum-drug binding on the distribution of halofantrine in whole blood was estimated by simulating several disease-related changes in the levels of the most important binding proteins. 2. The chromatographic resolution of serum preincubated with halofantrine allowed a quantitative analysis of binding to low density lipoproteins, high density lipoproteins, alpha 1-acid glycoprotein and albumin using the erythrocyte partitioning technique. Very low density lipoproteins did not bind halofantrine to a significant extent. 3. In whole blood halofantrine is bound to serum proteins (83%) and to erythrocytes (17%). Low density lipoproteins (affinity constant nKP = 44.4 l g-1) and high density lipoproteins (nKP = 14.4 l g-1) were the most important binding proteins in serum. alpha 1-acid glycoprotein (nKP = 4.39 l g-1) and albumin (nKP = 0.27 l g-1) had relatively low binding affinities. 4. The concentration of serum proteins influences both the fraction of unbound drug and the fraction of drug associated with the erythrocytes. Changes in serum protein concentrations often encountered in malaria are likely to increase both the unbound fraction and the fraction bound to the erythrocytes.
Animals↗
Radiology reports: how much descriptive detail is enough?
OBJECTIVE: The purpose of our study was to determine whether physicians prefer radiology reports with no description, a brief description, or a detailed description of the radiologic findings. We also examined the effects of various clinical circumstances and physician characteristics (e.g., specialty and number of years in practice) on these preferences. This study, which is limited to commonly encountered scenarios, is concerned solely with the description of imaging findings and not with other aspects of the reports, such as the diagnosis and technique. MATERIALS AND METHODS: A questionnaire was sent to the 100 physicians who most frequently refer patients to our practice for chest radiographs and abdominal sonograms. The questionnaire outlined six clinical scenarios. For each scenario the physician was asked to indicate the preferred radiology report from a set of three reports varying in descriptive detail. RESULTS: Seventy-seven physicians replied. For a normal chest radiograph in a patient without specific chest symptoms, the most popular report format indicated simply that the examination was normal. When there were clinical findings involving the chest or abnormal radiologic findings, the majority of referring physicians desired a description of the findings and the radiologic diagnosis. For abdominal sonograms the majority of physicians favored reports detailing the findings, even when the findings were normal. In all instances the preferences expressed were independent of the academic status, number of years in practice, number of reports read per day, and specialty of the referring physician. CONCLUSION: Our results show that preferences of referring physicians for the extent of description of imaging findings included in radiology reports depend on the clinical circumstances. In the specific clinical scenarios examined, physicians preferred that the report include a brief description of the findings, except for screening chest radiographs, for which "normal examination" was an acceptable report. The referring physicians' preferences regarding the amount of descriptive detail included in the report were independent of their specialty, academic status, and experience.
Abdomen↗
Sonographic assessment of infrarenal inferior vena caval dimensions.
The dimensions of the infrarenal inferior vena cava during quiet respiration, single leg lifting, and breath-holding were assessed using sonography in 156 patients. Sonographic assessment of infrarenal inferior vena caval dimensions was feasible in 69% of patients. Measurements during breath-holding were significantly greater than during quiet respiration (P < 0.001) and leg lifting (P < 0.005), although in approximately one quarter of the patients the mean calculated diameter was greatest during quiet respiration. we conclude that sonographic assessment of infrarenal inferior vena caval dimensions is feasible, but it should be performed during quiet respiration and breath-holding to allow for variation with different respiratory maneuvers.
Adult↗
Identification and biological characterization of an epidermal growth factor-related protein: cripto-1.
The human and mouse cripto-1 (CR-1) genes can code for proteins related in structure to epidermal growth factor (EGF). A specific 36-kDa immunoreactive protein was detected by Western blot analysis in human cell lines that express CR-1 mRNA but not in cell lines that fail to express this transcript. Immunoprecipitation of GEO colon carcinoma or mouse embryonal carcinoma cells detected 27-29-kDa and 24-kDa proteins, respectively. Cell lysates and conditioned medium that were prepared from several CHO clones and were expressing either a recombinant human or mouse CR-1 cDNA contained immunospecific 27-29-kDa and 24-kDa proteins, respectively. Monensin or tunicamycin treatment resulted in a shift of the 27-29-kDa human CR-1 protein to 24 kDa and 20 kDa, respectively. The 20-kDa protein was also observed after digestion of the 27-29-kDa human CR-1 protein with N-glycosidase F. Using two CR-1 synthetic refolded peptides that correspond to the EGF-like domain of the human CR-1 sequence or conditioned medium obtained from human CR-1 expressing CHO cells, growth stimulatory activity could be detected on non-transformed human mammary epithelial cells and on two human breast cancer cell lines. EGF receptor-blocking antibody did not inhibit the growth stimulatory action of the CR-1 protein. Likewise, the CR-1 refolded peptides or conditioned medium from the human CR-1-expressing CHO cells failed to inhibit the binding of 125I-EGF in an EGF-radioreceptor assay. These data demonstrate that the CR-1 is a glycoprotein that can function as a growth factor through an EGF receptor-independent pathway.
Amino Acid Sequence↗
Differential effect of phosphorylation and substrate modulation on tau's ability to promote microtubule growth and nucleation.
The neuronal microtubule-associated protein tau promotes microtubule assembly and has been implicated in the development of axonal morphology. To study the effect of phosphorylation and substrate modulation on tau's distinct activities to promote growth of existing microtubules and nucleation of new ones, we phosphorylated bacterially expressed human tau by cAMP-dependent protein kinase in the absence or presence of heparin, an acidic substrate modulator. We found that heparin increased phosphorylation of tau by a factor of more than 2 and produced tau bands with decreased electrophoretic mobility. We demonstrate that phosphorylation of tau in the absence or presence of heparin similarly reduced tau's activity to promote microtubule growth, whereas tau's activity to promote microtubules was suppressed much more after phosphorylation in the presence of heparin. Using recombinant tau fragments we showed that heparin-induced phosphorylation caused a specific shift in electrophoretic mobility indicative of a change in tau's conformation. By aminoterminal sequencing of a tau fragment starting at residue 154 we provide evidence that phosphorylation of serine 156 is responsible for this mobility shift and for the effect on tau's nucleation activity. We conclude that tau's activities to promote growth of existing microtubules and nucleation of new ones are differentially affected by the phosphorylation of specific tau residues. Regulation of the phosphorylation state by substrate modulation may play an important role in regulating tau's function.
Amino Acid Sequence↗
Amphiregulin as an autocrine growth factor for c-Ha-ras- and c-erbB-2-transformed human mammary epithelial cells.
Amphiregulin (AR), a member of the epidermal growth factor (EGF) family, was found to be as potent as EGF in stimulating the anchorage-dependent growth (ADG) of immortalized, nontransformed human mammary epithelial MCF-10A cells. MCF-10A cells transformed by either an activated human c-Ha-ras protooncogene (MCF-10A ras) or by overexpression of a nonactivated rat c-neu gene (MCF-10A neu) exhibited a 35% reduction in the response to AR in ADG when compared to MCF-10A cells, but AR was still as potent as EGF in these transformants. Exogenous AR exhibited only 15-20% of the activity of EGF in stimulating the anchorage-independent growth, a response that is normally dependent upon exogenous EGF, of the oncogene-transformed MCF-10A cells. MCF-10A cells express low levels of a 1.4-kb AR mRNA transcript, while MCF-10A ras and MCF-10A neu cells display a 15- to 30-fold increase in the levels of AR mRNA and endogenous AR protein as determined by Western blot analysis. Exogenous EGF was found to induced both the AR mRNA and protein in the MCF-10A parental and transformed cells. A 20-mer phosphorothioate antisense deoxyoligonucleotide complementary to the 5' sequence of AR mRNA was able to significantly reduce the levels of endogenous AR protein and to inhibit the EGF-stimulated ADG and anchorage-independent growth of MCF-10A ras and MCF-10A neu cells. These data suggest that AR may function as an EGF-dependent autocrine growth factor in mammary epithelial cells that have been transformed by either a point-mutated c-Ha-ras or c-neu.
Amphiregulin↗