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Biomedical subjects

K G Bensch

Publications and source records attributed to K G Bensch.

At least 19 recordsLinked to original sources
PubMed1992

Histamine-modulated transdifferentiation of dermal microvascular endothelial cells.

Homeostatic and inflammatory functions of skin microvessels are tightly regulated by vasoactive amines. Following stimulation with histamine, dermal microvascular endothelial cells (MEC) undergo a rapid change in phenotype (transdifferentiation) and subsequently exhibit an enhanced rate of growth. To elucidate mechanisms regulating MEC transdifferentiation, this study investigated the functional relationships among vimentin, Ca2+, and protein kinase C (PKC) in histamine-modulated dermal MEC in vitro. Distribution of vimentin and PKC in foreskin-derived MEC cultivated in a modified Iscove's medium was assessed with immunocytochemistry. Calcium ion kinetics in histamine-treated MEC were analyzed using the Ca2+ probe Fluo-3 in conjunction with interactive laser cytometry. Histamine, acting through H-1 receptors, produces a rapid (less than 100 ms) and differential elevation of free calcium in each of three cytological compartments defined by the vimentin cytoskeleton in epithelial MEC. A distinctive compartmentalized and nonuniform distribution of PKC precisely coincides with that observed for free-Ca2+ released in response to histamine. The studies reveal that histamine modulation of the MEC phenotype is associated with a rapid patterned reorganization of the vimentin skeleton. It is hypothesized that histamine induces vimentin post-translational modifications by activating a spatially localized interaction among cytoplasmic free Ca2+, PKC, and the vimentin matrix. The results further suggest that vimentin, in addition to its structural role, may participate in signal transduction and gene regulation processes in effecting MEC transdifferentiation.

Calcium↗
PubMed1991

Microvessel endothelial cell transdifferentiation: phenotypic characterization.

Human dermal microvessel endothelial cells (MEC) have two basic functions: maintenance of tissue homeostasis and facilitation of inflammatory responses. The former requires that the endothelium expresses traits of an epithelium, while inflammatory reactions are associated with intimal disruption. Acute inflammation transiently alters endothelium, whereas chronic inflammation may result in vessel reorganization and MEC mesenchymalization. Foreskin MEC in vitro undergo a similar epithelial-mesenchymal modulation. In the presence of cAMP, cultivated dermal MEC exhibit the structural and functional characteristics of an epithelium. MEC grown in cAMP-deficient medium initially have a "transitional" configuration and are subsequently transformed into mesenchymal cells. If cAMP is replaced by histamine, MEC maintain a stable intermediate transitional configuration. Transitional MEC refed cAMP-supplemented medium revert to an epithelial phenotype, whereas parallel cultures fed cAMP-deficient medium are transformed into mesenchymal cells. Phenotypic modulation can be induced without cell division and thus provides a unique example of direct transdifferentiation. Our data furthermore suggest that this transdifferentiation results in the acquisition of properties usually attributed to cells of the reticuloendothelial system.

Cell Differentiation↗
PubMed1991

Expression of bovine superoxide dismutase in Drosophila melanogaster augments resistance of oxidative stress.

Superoxide dismutases (SOD) play a major role in the intracellular defense against oxygen radical damage to aerobic cells. In eucaryotes, the cytoplasmic form of the enzyme is a 32-kDa dimer containing two copper and two zinc atoms (CuZn SOD) that catalyzes the dismutation of the superoxide anion (O2-) to H2O2 and O2. Superoxide-mediated damage has been implicated in a number of biological processes, including aging and cancer; however, it is not certain whether endogenously elevated levels of SOD will reduce the pathological events resulting from such damage. To understand the in vivo relationship between an efficient dismutation of O2- and oxidative injury to biological structures, we generated transgenic strains of Drosophila melanogaster overproducing CuZn SOD. This was achieved by microinjecting Drosophila embryos with P-elements containing bovine CuZn SOD cDNA under the control of the Drosophila actin 5c gene promoter. Adult flies of the resulting transformed lines which expressed both mammalian and Drosophila CuZn SOD were then used as a novel model for evaluating the role of oxygen radicals in aging. Our data show that expression of enzymatically active bovine SOD in Drosophila flies confers resistance to paraquat, an O2(-)-generating compound. This is consistent with data on adult mortality, because there was a slight but significant increase in the mean lifespan of several of the transgenic lines. The highest level of expression of the active enzyme in adults was 1.60 times the normal value. Higher levels may have led to the formation of toxic levels of H2O2 during development, since flies that died during the process of eclosion showed an unusual accumulation of lipofuscin (age pigment) in some of their cells. In conclusion, our data show that free-radical detoxification has a minor by positive effect on mean longevity for several strains.

Animals↗
PubMed1990

Cyclic adenosine monophosphate levels and the function of skin microvascular endothelial cells.

The maintenance of the normal epithelioid morphology of human dermal microvascular endothelial cells (MEC) grown in vitro depends strongly on the presence of factors that increase intracellular levels of cyclic AMP. Complete removal of dibutyryl cAMP and isobutylmethylxanthine (IMX) from the growth medium results in a progressive transition from an epithelioid to a spindle-shaped cell line. This transition cannot be reversed by the readdition of dibutyryl cAMP and IMX to the growth medium or by addition of agonists that increase cAMP levels. Spindle-shaped MEC lose the ability to express Factor VIII rAG and DR antigens and to bind peripheral blood mononuclear leukocyte (PBML). Ultrastructural analyses of transitional cells and spindle-shaped cells show decreased numbers of Weibel-Palade bodies in transitional cells and their complete absence in spindle-shaped cells. Interferon-gamma alters several functional properties of both epithelioid and spindle-shaped cells. In the absence of dibutyryl cAMP it accelerates the transition from epithelial to spindle-shaped cells, whereas in the presence of cyclic AMP interferon-gamma increases the binding of PBMLs to both epithelioid and spindle-shaped MEC and the endocytic activity of the endothelial cells. These results suggest that cyclic AMP is an important second messenger in the maintenance of several key functions of microvascular endothelial cells. Factors that influence the levels of this messenger in vivo can be expected to influence the angiogenic and immunologic functions of the microvasculature.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Ascorbic acid uptake by young and aged guinea pig lenses.

The dynamics of in vitro uptake of [14C]-ascorbic acid into intact lenses of young (50 days old) and old (730 days old) guinea pigs was evaluated in this study. Two-dimensional protein gel analysis of [35S]-methionine labeled proteins provided evidence that the isolated lenses were viable throughout the culture period. It was found that the in vitro uptake of ascorbate into these lenses follows similar saturation kinetics for lenses from both age groups. Moreover, the linear uptake rate was identical. Ascorbate transport was inhibited by phloretin, p-chloromercuribenzoate, insulin, glucose and ouabain; however, no inhibition was observed with 2,4-dinitrophenol or NaF. These results suggest that the lenticular ascorbate concentration is regulated by a facilitated diffusion process and it not energy-dependent. Data on ascorbate transport into lenses from aged guinea pigs provide strong evidence that the abnormally low ascorbic acid concentrations in lenses of old mammals is not likely the result of a decreased lenticular uptake ability.

Aging↗
PubMed1989

Preparation of antibodies against xanthine oxidase from human milk.

1. Human xanthine oxidase [XO; EC 1.2.3.2.] was isolated by a non-proteolytic method from fresh human milk. Final purification of the protein was achieved by hydroxyapatite chromatography. Most (less than 95%) of the enzyme was released in the 0.40 M phosphate fraction at pH 6.8. 2. The specific activity of this preparation was found to be 0.047 microM min-1 mg-1 with xanthine as substrate. 3. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) separated two subunits, each with a mol. wt approximately 122 kDa. 4. On non-denaturing acrylamide gels both of these subunits exhibited oxidase-like activity with xanthine as substrate in the presence of nitroblue tetrazolium and molecular oxygen. 5. Immunoconjugates of XO were prepared by the keyhole limpet hemocyanin (KLH)- and glutaraldehyde-crosslinking techniques. 6. Polyclonal antibodies to XO were raised by i.m. injection of these conjugates into female New Zealand rabbits. 7. Western blot analysis using the semi-dry technique was employed to confirm the specificity of the antibody.

Animals↗
PubMed1988

Aging results in an unusual expression of Drosophila heat shock proteins.

We used high-resolution two-dimensional polyacrylamide gel electrophoresis to evaluate the effect of aging on the heat shock response in Drosophila melanogaster. Although the aging process is not well understood at the molecular level, recent observations suggest that quantitative changes in gene expression occur as these fruit flies approach senescence. Such genetic alterations are in accord with our present data, which clearly show marked differences in the synthesis of heat shock proteins between young and old fruit flies. In 10-day-old flies, a heat shock of 20 min results in the expression of 14 new proteins as detectable by two-dimensional electrophoresis of [35S]methionine-labeled polypeptides, whereas identical treatment of 45-day-old flies leads to the expression of at least 50 new or highly up-regulated proteins. In addition, there is also a concomitant increase in the rate of synthesis of a number of the normal proteins in the older animals. Microdensitometric determinations of the low molecular weight heat shock polypeptides on autoradiographs of five age groups revealed that their maximum expression occurs at 47 days for a population of flies with a mean life span of 33.7 days. Moreover, a heat shock effect similar to that observed in senescent flies occurs in young flies fed canavanine, an arginine analogue, before heat shock.

Aging↗
PubMed1987

Expression of opioid peptides in tumors.

We looked for opioid peptides and their precursors in 108 tumors of both neuroendocrine and nonneuroendocrine origin, using a monoclonal "pan-opioid" antibody, 3-E7, which recognizes the tetrapeptide Tyr-Gly-Gly-Phe (the sequence responsible for pharmacologic activity in all known opioid peptides), in conjunction with polyclonal antibodies directed against representative peptides of each of the three precursors (alpha-endorphin, [met]enkephalin-Arg-Gly-Leu, and dynorphin B). Using the avidin-biotin immunoperoxidase technique, we observed consistent cytoplasmic immunoreactivity (at least focally) in all of 15 adrenal pheochromocytomas, all of 6 thyroid medullary carcinomas, and all of 5 pituitary adenomas. Opioid staining was also observed in parathyroid adenomas (8 of 9), pancreatic islet-cell tumors (7 of 10), carcinoid tumors from various sites (18 of 26), and paragangliomas (1 of 2). There was no immunoreactivity in pulmonary small-cell carcinomas, Merkel-cell tumors of skin, neuroblastomas, or any of the non-neuroendocrine tumors examined. The expression of alpha-endorphin, [met]enkephalin-Arg-Gly-Leu, and dynorphin B varied from tumor to tumor; however, positive staining with the "pan-opioid" antibody was found in each tumor containing at least one of the three precursors. Opioid peptide immunoreactivity was also detected in non-neoplastic cells of the adrenal medulla, pancreatic islets, pituitary, intestinal and bronchial mucosa, and intestinal myenteric plexuses. We conclude that opioid expression within tumors is most likely due to enhanced expression of a normal cell product and that opioid peptides are useful markers of neuroendocrine differentiation in many tumors.

Antibodies, Monoclonal↗
PubMed1987

pH-independent HIV entry into CD4-positive T cells via virus envelope fusion to the plasma membrane.

CD4 functions as the cell-surface receptor for human immunodeficiency virus (HIV); however, the mechanism of virus entry into susceptible cells is unknown. To explore this question we used a human T lymphoblastic cell line (VB) expressing high levels of surface CD4. Neutralization of endosomal compartments (pH greater than 6.4) with lysosomotropic agents did not effectively inhibit HIV nucleocapsid entry into the cytoplasm, and virus treated at low pH (5.5) failed to induce rapid cell-to-cell fusion in uninfected cells. Electron microscopy of VB cells acutely exposed to HIV at neutral pH revealed direct fusion of the virus envelope with the plasma membrane within minutes at 4 degrees C. No endocytosed virions were visualized upon rewarming the HIV-exposed cells to 37 degrees C for as long as 60 min. These results indicate that HIV penetrates CD4-positive T cells via pH-independent membrane fusion.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1987

Characterization of opioid peptides in human thyroid medullary carcinoma.

A thyroid medullary carcinoma from a man with the multiple endocrine neoplasia syndrome Type IIB was examined for the presence of opioid peptides. The tumor contained peptides derived from all three opioid precursors: pro-opiomelanocortin (POMC), pro-dynorphin, and pro-enkephalin. The tissue concentrations of the various opioid peptides varied considerably. beta-Endorphin, a POMC-derived peptide, was present in concentrations between 9 to 12 pmoles/g tissue; 8 pmoles/g tissue of alpha-neo-endorphin, a pro-dynorphin-derived product, were seen, whereas the pro-enkephalin-associated peptides were present in much lower concentrations (0.6-2.1 pmoles/g tissue). Immunohistochemical studies showed scattered opioid-positive cells in the tumor tissue and in two other thyroid medullary carcinomas. These data demonstrate that malignant neuroendocrine tumors may contain peptides derived from all three families of the endogenous opioids.

Adult↗
PubMed1986

Age-dependent changes in proteins of Drosophila melanogaster.

Several molecular theories of aging postulate that there are age-dependent changes in gene expression and that these changes contribute to the reduction in the viability of senescent cells. High-resolution, semiautomated, quantitative two-dimensional gel electrophoresis of many soluble proteins was used to test this hypothesis in Drosophila. Two-dimensional protein gel patterns were analyzed for each of three age groups of [(35)S]methionine-labeled adult male Drosophila melanogaster, which, except for their spermatocytes, consist entirely of fixed postmitotic cells. Seven relatively abundant polypeptides expressed in middle-aged (28-day-old) flies were absent in both young(10-day-old) and old (44-day-old) flies. Quantitative analyses of an additional 100 polypeptides were carried out by computer-assisted microdensitometry of fluorograms of the gel preparations. These analyses revealed a significant age-related heterogeneity in the quantitative distribution of radiolabel in these proteins. The data indicate that the qualitative pattern of gene expression is identical in young and old flies, but that profound quantitative changes occur in the expression of proteins during the Drosophila life-span.

Aging↗
PubMed1986

Age dependent changes in the expression of Drosophila mitochondrial proteins.

A subset of 43 [35S]methionine labeled mitochondrial proteins, identified on two dimensional electrophoresis gels of whole body extracts of adult Drosophila, were compared between two age groups. These flies, which except for their spermatocytes consist entirely of postmitotic cells, were 6 days old and 38 days old, with the mean life span of this inbred strain being 32.5 days. Qualitative changes in any of these mitochondrial polypeptides were not found, but significant quantitative differences were observed. Quantitation of the 43 mitochondrial proteins was carried out by computer assisted microdensitometry of autoradiograms of the gel patterns. The total amount of isotope incorporated into the mitochondrial proteins of the senescent flies decreased to 71% of that of the young insects. This decrease was heterogeneously distributed among the 43 proteins; however, six remained unchanged and one protein (mol. wt = 75; pI = 7.4) was increased 2.4 times in the senescent flies. These data clearly provide evidence that the age-dependent changes in the expression of mitochondrial proteins are quantitative and not qualitative.

Aging↗
PubMed1986

Neurogenic hyperacute ascites in mice.

Four groups of mice were subjected to controlled fatal head trauma and then evaluated for the presence of ascites (neurogenic hyperacute ascites, NHA). The animals died virtually instantaneously and without evidence of maintained pain or suffering. The volume of ascites was determined in one group of animals. Two of the traumatized groups were pretreated, one with the beta-blocker propranolol and the other with the alpha-blocker phentolamine. A fifth, non-traumatized, group which was killed with either inhalation served as a control group. Two more groups of non-traumatized mice were administered either the alpha-adrenergic agonist methoxamine hydrochloride or the beta-agonist isoethrane mesylate before killing by ether inhalation, and then evaluated for ascites. Transudative ascitic fluid was found in 87-100% of untreated traumatized mice and in no control animals. Pretreatment with phentolamine had no effect on the prevalence of ascites. Pretreatment with propranolol produced a significant decrease in the prevalence of ascites compared with trauma alone (P less than 0.001). Isoethrane (beta-agonist) administration caused ascites in 100% of the treated animals. Methoxamine (alpha-agonist) administration did not cause ascites. A previously undescribed consequence of acute brain trauma is described (NHA) which appears to be mediated by beta-sympathetic activity of central origin. NHA is inhibited by beta-blockade and can be simulated with beta-agonist administration.

Acute Disease↗
PubMed1986

Acromegaly and pheochromocytoma: a multiple endocrine syndrome caused by a plurihormonal adrenal medullary tumor.

A 42-yr-old man with congestive heart failure and diabetes mellitus was found to have acromegaly and a pheochromocytoma. Serum GH-releasing hormone (GHRH) levels were elevated (2.34 ng/dl; normal, less than 0.02 ng/dl), suggesting that the acromegaly was caused by ectopic secretion of GHRH. Postmortem examination revealed that the right adrenal gland contained a pheochromocytoma in which GHRH was demonstrated by immunohistochemical studies. Gel permeation chromatography combined with the use of two GHRH antisera showed that GHRH-(1-44)-NH2 was a predominant form of the hormone. When the RNA from the tumor was extracted and analyzed by Northern gel blotting, two mRNA species were identified, with transcripts corresponding to 1600 and 780 base pairs. The pituitary gland was enlarged, but no distinct adenoma was found. Diffuse and nodular hyperplasia of somatotrophs in some areas resembling adenoma was identified on histological examination. These findings indicate that GH excess accompanied by somatotroph hyperplasia and acromegaly were secondary to a pheochromocytoma which secreted not only catecholamines but also GHRH.

Acromegaly↗
PubMed1986

Invasion of reconstituted basement membrane matrix by metastatic human tumor cells.

A gel-like reconstituted basement membrane matrix containing type IV collagen, laminin, entactin, nidogen, and heparan sulfate proteoglycan was used to examine the invasive properties of human HT1080 fibrosarcoma cells. Within several hours after seeding, the tumor cells initiated a random migration, leaving behind channels etched in the surface of the matrix. Eventually the channels became interconnected into a complex network. As the tumor cells proliferated, the channels became filled until the surrounding matrix was gradually dissolved. Cells then migrated outward, forming the typical disorganized cell monolayer normally observed when fibrosarcoma cells are cultured on plastic surfaces. In contrast to the fibrosarcoma cells, normal skin fibroblasts, while able to attach to the matrix, exhibited minimal migration, tracking, and invasion during the same time period. When tumor cells were seeded onto thick layers of matrix, the cells ultimately invaded downward into the matrix, leaving behind open tunnels. At the front of the invading cells, long irregular pseudopodia projected in the direction of movement. Electron microscopy demonstrated these filopodial and lamellopodial projections to directly extend into the surrounding matrix, with focal clearing of the matrix in the immediate vicinity of these invading pseudopodia. Thus, tumor cell invasion of extracellular matrices, including basal lamina, may proceed by the formation of specialized pseudopodia that not only form adhesion contacts with the matrix but also provide an efficient mechanism for the focal hydrolysis of the matrix at the site of directed cell movement.

Basement Membrane↗