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B Lane

Publications and source records attributed to B Lane.

At least 109 records · Page 6Linked to original sources
PubMed1987

Metastatic mouse melanoma cells release collagen-gelatin degrading metalloproteinases as components of shed membrane vesicles.

The purpose of this study has been to compare collagen-gelatin degrading enzymes isolated from cancer cell organelles and cytosol to the metalloproteinases released by cancer cells. To this end, metastatic mouse melanoma cell organelles were isolated by sucrose density gradient centrifugation and metalloproteinases were assayed using native and denatured [methyl-3H]collagen substrates. Solubilized proteinases were purified by ammonium sulfate precipitation, anion exchange, concanavalin A affinity and gel-filtration column chromatographic procedures and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The conclusions were as follows: malignant melanoma cells have a metalloproteinase (Mr = 59,000) which is shed from cells into conditioned medium as a component of intact membrane vesicles rather than as a soluble enzyme; storage of tumor-conditioned medium leads to the generation of autoactivated soluble metalloproteinases of lower molecular weight; purification of these metalloproteinase species yielded variant collagenases that have considerable gelatinolytic activity and a cleavage preference site for the Gly-Ile bond in a collagen-like synthetic octapeptide substrate which is typical for collagenase-type metalloproteinases. It is proposed that localization of potent proteinases to the surface of cancer cells facilitates the local breakdown of connective tissues during the invasive process.

Animals↗
PubMed1987

Enrichment of collagen and gelatin degrading activities in the plasma membranes of human cancer cells.

Interactions between connective tissue substrates and proteinases localized to the surface of cancer cells are implicated in cancer invasion. In this report we have compared the enrichment of collagen and gelatin degrading activities and cysteine proteinase(s) in well-characterized (enzyme markers and electron microscopy) subcellular membrane fractions isolated from human small cell lung cancer lines (NCI-H69 and NCI-H82) and the RWP-1 pancreatic cancer line. With each cell line collagenolytic, gelatinolytic, and cysteine proteinase activities were enriched 5- to 128-fold in the plasma membrane fractions with differences noted between microvilli versus smooth membrane profiles. Incubation of tumor plasma membranes with methyl-3H-labeled collagen resulted in extensive degradation of the gamma, beta, alpha 1, and alpha 2 chains, suggesting the combined action of metalloproteinases. Treatment of tumor plasma membranes with the chaotropic agent, 2 M KCl, did not diminish membrane collagen- or gelatin-degrading activity, but extensively leached out the cysteine proteinase, suggesting that the latter enzyme is not an integral membrane protein. Enzyme inhibitors specific for metalloproteinases and cysteine proteinase were used to corroborate enzymatic classification. In conclusion, we have demonstrated variations in the localization of proteinases in the plasma membrane domains of different human cancer cells.

Cell Membrane↗
PubMed1987

Blunt injury of the neck of the pancreas: a report of nine patients.

Nine patients with injury of the neck of the pancreas following blunt abdominal injury are reported. Eight were sustained in road traffic accidents and only two victims had used seatbelts. Serious associated injuries were present in four patients and the pancreatic injury was missed in two patients. The diagnosis of this injury was made at laparotomy in six, while one patient had the diagnosis confirmed at endoscopic retrograde pancreatography (ERP). The neck of the pancreas was the site of injury in all nine cases. Pancreatic drainage (3), suture repair (1) and distal resection with splenectomy (2) were the operative procedures performed. Pseudocyst of the pancreas (2), pancreatic fistulas (2), pancreatitis (1) and ascites (1), which necessitated a further laparotomy, accounted for postoperative morbidity. There was one postoperative death from haemorrhage due to stress ulceration. This study emphasizes that the neck of the pancreas is at special risk following blunt abdominal injury.

Accidents, Traffic↗
PubMed1987

Progestin-dependent effect of forskolin on human endometrial aromatase activity.

We have previously demonstrated that the aromatase activity in human endometrial stromal cells is stimulated by progestin and enhanced by oestrogen. In this study, we have investigated the effect of forskolin (Fk), an agent that stimulates the hormone-sensitive adenylate cyclase in mammalian cells, on the intracellular cAMP content and aromatase activity in endometrial stromal cells in primary culture. Stromal cells were isolated from proliferative and secretory endometria and were individually cultured in nutrient medium or medium supplemented with medroxyprogesterone acetate (MPA), oestradiol (E2) and Fk, separately or in combination. The intracellular cAMP content of stromal cells was increased after incubation with Fk. Stromal cells treated with Fk alone or FK and MPA for 1-3 days sustained the elevated intracellular cAMP content but 90% of this nucleotide was released to the medium. Aromatase activity was either not affected or was increased up to 5-fold over the control by Fk alone. Forskolin exerted a synergistic effect upon aromatase activity in the presence of progestin or progestin and oestradiol. Sequential incubation of the stromal cells with MPA and then Fk indicated that the additional increase in aromatase activity caused by Fk occurred after 24 h of incubation. These results demonstrate that intracellular cAMP exerts a stimulatory effect on aromatase activity in progestin-conditioned stromal cells. We also investigated whether the endometrial aromatase activity changes in vivo during the reproductive cycle by measuring the activities in endometrial specimens obtained from women during their reproductive age.(ABSTRACT TRUNCATED AT 250 WORDS)

4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone↗
PubMed1987

The protective effect of calcium inhibitors and of captopril on the renal microcirculation during reperfusion.

Reperfusion injury is increasingly recognized as a key factor in the development of posttransplant acute tubular necrosis. Previous studies have shown that addition of the calmodulin inhibitor trifluoperazine (TFP) to Collins' flush solution protected the cortical microcirculatory integrity and dramatically improved renal viability after transplantation. The present report describes the protective effect(s) of TFP in the course of reperfusion injury. Twenty mongrel dogs underwent bilateral nephrectomy; in each instance, the left kidney was flushed immediately with 250 ml of cold Collins' solution, and the right kidney was flushed with the same solution containing TFP, 5 mg/L. After 48 and 72 hr of preservation, each kidney was connected through silastic shunts to the femoral vessels of another dog. The mean renal blood flow (RBF) immediately after reperfusion was 2.2 ml/g/min and 1.7 ml/g/min in the left and right kidneys, respectively, and was similar to mean RBF measurements prior to nephrectomy. After 15 min of reperfusion, there was a sharp decrease in mean RBF in the Collins' flushed kidneys, which persisted after 60 min of reperfusion (0.37 ml/g/min). In contrast, there was only a mild decrease in mean RBF in the TFP-flushed kidneys (1.27 ml/g/min). A partial explanation for the favorable effect of TFP may be related to the inability of the ischemic cell to handle the increased calcium load associated with reperfusion (calcium paradox). In a test of this possibility, 0.5 mg/kg of verapamil, a calcium channel blocker, was infused during reperfusion. No beneficial effects of this drug were noted in either Collins' or TFP-flushed kidneys (n = 10). However, when 1.25 mg/kg of captopril, an angiotensin-converting enzyme inhibitor, was infused at the time of reperfusion, a dramatic amelioration of the reperfusion injury occurred in the Collins' flushed kidneys (1.2 ml/g/min) (n = 10). Taken together, these data suggest that the damage to cold-preserved kidneys flushed with Collins' solution alone may occur at the time of actual reperfusion. Such reperfusion damage is ameliorated by TFP and captopril. The known relationship between calcium and the effect of angiotensin on the vascular smooth muscle cell may explain in part the protective role of calcium inhibitors placed in preserved kidneys prior to reperfusion.

Angiotensin II↗
PubMed1986

Proton transport and membrane shuttling in turtle bladder epithelium.

Proton secretion in the urinary bladder of the fresh-water turtle is mediated by proton pumps located in the apical membrane of carbonic-anhydrase (CA)-rich cells. It has been proposed that the rate of proton transport is regulated by endocytotic and exocytotic fusion processes which alter the apical membrane area, and hence number of exposed pumps. Three techniques were used to study this process. Analyses of transepithelial impedance provided estimates of transport-associated changes in net membrane area, as well as other electrical parameters. Electron microscopy allowed visualization of the endocytotic vesicles thought to be involved in the process. Finally, uptake of a fluorescent fluid-phase marker provided measurements of the rates of endocytosis. We report the following: endocytotic and exocytotic processes occur primarily in the CA-rich cells; inhibition of proton transport resulting from 0.5 mM acetazolamide (AZ) results in a decrease in the apical membrane area of approximately 0.47 cm2/cm2 tissue; the apical membrane specific conductance of the CA-rich cells is approximately 220 microS/microF, and possibly represents a Cl- conductance that may function in counter-ion flow; the decline in transport following AZ is not directly proportional to the decline in apical membrane area, suggesting that changes in pump kinetics are also involved in the regulation of transport; the CA-rich cells exhibit a high rate of constitutive pinocytosis, and hence membrane shuttling, which appears to be independent of the rate of transport; AZ induces a transient increase in the rates of endocytosis and shuttling; and the transport-associated changes in apical membrane area may reflect an effect of AZ on a regulated endocytotic pathway which is distinct from the pinocytotic process.

Animals↗
PubMed1986

Analysis of the cell membrane proteolytic enzymes of the B16, F1, F10, and BL6 melanoma and their role in target cell destruction.

The tumor-induced red blood cell (RBC) cytolysis assay has been used to demonstrate that three B16 melanoma sublines, the F1, F10, and BL6, cause the cytolysis of normal red blood cells in vitro. RBC cytolysis was inhibited for all three sublines by metalloprotease inhibitors. Cell membrane preparations have been prepared for all three sublines and tumor cell membrane-induced RBC cytolysis was also shown to be inhibited by metalloprotease inhibitors. The F10 and BL6 sublines were shown to have cell membrane-bound proteases. The BL6 subline has a cell membrane enriched in an enzyme with a trypsin-like arginine specificity. The trypsin-like protease may have a metal dependence. The BL6 subline has a collagenolytic cell membrane enzymes and a chymotrypsin-like cell membrane enzyme. B16 cell membrane enzymes may be responsible for RBC cytolysis in vitro in a process requiring divalent cations.

Animals↗
PubMed1985

Evidence that the [3H]estradiol-binding protein in pancreas is localized in exocrine cells.

Extracts of rat pancreas contain significant amounts of an [3H]estradiol-binding protein. The amount of steroid-binding activity that could be measured varied considerably depending on the tonicity of the homogenizing medium. High speed supernatants of homogenates initially prepared in isotonic buffer contained about 10% of the binding activity as homogenates prepared in hypotonic buffer. Extraction with hypotonic buffer of pellets obtained by the isotonic procedure yielded most of the remaining [3H]estradiol-binding activity. In an attempt to avoid errors resulting from incomplete homogenization and to detect possible changes in intracellular distribution of [3H]estradiol-binding activity, pancreata were initially homogenized in isotonic buffer and centrifuged at high speed (100,000 X g; 1 hr). The pellet was then extracted with hypotonic buffer and centrifuged again at high speed, and both supernatants were analyzed for [3H]estradiol-binding and amylase activities. Two or 14 days after treatment of male rats with streptozotocin, no apparent decline or redistribution of [3H]estradiol-binding activity to the cytosol was noted despite extensive alteration of beta-islet cells, as determined by electron microscopic examination of sections of these pancreata and significant loss of insulin, as measured by RIA. Amylase activity was unaffected 2 days after streptozotocin treatment, but was depressed to about 1% of control levels at 14 days. Administration of insulin to the latter group of animals resulted in return of amylase to normal levels and a modest increase (approximately 50%) in [3H]estradiol-binding activity. Since amylase levels remained unchanged 2 days after streptozotocin treatment, during which time beta-islet cells were irreversibly altered, and amylase activity was restored to normal levels by insulin treatment after its depletion in chronically treated animals, it follows that neither amylase nor the [3H]estradiol-binding protein could have been associated with beta-islet cells. This was consistent with the observation that M cells (a tumor line of beta-cells only) and 14B cells (a cloned variant of this insulinoma) had neither detectable amounts of amylase nor [3H]estradiol-binding activity. To determine whether estrogen-binding activity was associated with any other type of islet cell, islets of Langerhans were isolated by the sedimentation procedure of Lacy and Kostianovsky. In this procedure, several washing steps are employed to separate the suspended acinar cells from the denser islets that sediment rapidly. During this isolation procedure, the cells from each wash were analyzed for protein, [3H]estradiol-binding protein, and amylase a

Amylases↗
PubMed1985

Renal pathology of prenatally diagnosed nephrosis.

Congenital Finnish nephrosis is a rare autosomal-recessive disorder, usually fatal at an early age. The disease is prenatally detected through elevation of alpha fetoprotein in the amniotic fluid of pregnancies at risk. This originates from fetal proteinuria. Maternal serum alpha fetoprotein reflects amniotic fluid levels. We describe a case of congenital nephrosis diagnosed through maternal serum screening in a low-risk population. The characteristic histology of congenital nephrosis is demonstrated, and evidence of proteinuria by electron microscopy, light microscopy, and immunofluorescence is presented.

Adult↗
PubMed1985

Contrasting effects of gentamicin and mercuric chloride on urinary excretion of enzymes and phospholipids in the rat.

Gentamicin induces a phospholipiduria in the rat. We sought to determine whether the phospholipiduria reflected shedding of brush border membrane by measuring the urinary excretion of membrane marker enzymes, by defining the composition of the urinary phospholipids, and by examining the renal cortex and urinary sediment using transmission electron microscopy of rats injected with gentamicin sulfate (100 mg/kg of body weight for 1 to 6 days). The results were compared and contrasted with those of rats injected with mercuric chloride, a nephrotoxin known to cause selective injury to the brush border membrane. Gentamicin-injected rats exhibited a phospholipiduria that by the third day was 10-fold above the baseline level. In contrast the brush border membrane enzymes alanine aminopeptidase and gamma-glutamyl transferase and the lysosomal enzyme N-acetyl-beta-glucosaminidase increased less than 1-fold above baseline. Electron microscopy of the renal cortex revealed the presence of myeloid bodies within lysosomes of proximal tubule cells and lying free within the lumen. The brush border membrane was largely intact. The urine sediment was dominated by the presence of myeloid bodies; there was little evidence of brush border membrane fragments. The urinary phospholpids were enriched in phosphatidylinositiol, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. This pattern is similar to that of a lysosomal fraction enriched in myeloid bodies. Rats injected with mercuric chloride exhibited a phospholipiduria associated with sharp increases (7- to 15-fold) in the urinary excretion of brush border membrane enzymes, whereas N-acetyl-beta-glucosaminidase increased less than 3-fold. Electron microscopy revealed blebbing of the apical membrane with loss of microvilli of proximal tubular cells particularly evident along the pars recta. The urinary sediment contained microvilli and fragments of plasma membrane but no myeloid bodies. The urine contained large quantities of sphingomyelin. In the rat renal cortex the brush border membrane is the major source of this phospholipid. We conclude that the phospholipiduria induced by mercuric chloride is derived primarily from the shedding of brush border membrane fragments into the urine. In contrast, the urinary excretion of phospholipid induced by gentamicin reflects primarily the extrusion of the lysosomal myeloid body from proximal tubular cells. We speculate that the origin of the phospholipids making up the lysosomal myeloid body may be the endocytic vesicle that mediates the transport of aminoglycosides into the lysosomal system.

Aminopeptidases↗
PubMed1985

Diversity of human pancreatic cancer cell proteinases: role of cell membrane metalloproteinases in collagenolysis and cytolysis.

In this study we have examined the tissue-destructive proteinases of human pancreatic ductal cancer cell lines derived initially from xenogenic transplants. Cancer cell organelles were isolated following nitrogen cavitation using sucrose density gradient centrifugation. Serine, cysteine, and metalloproteinases were assayed using radiolabeled protein and synthetic substrates. Tumor-induced RBC lysis was quantitated by measuring the release of isotope from 59Fe-labeled RBCs co-cultivated with tumor cells or subcellular fractions. Enzyme inhibitors with specificity toward different classes of proteinases were used in the above assays to categorize the enzymes responsible for substrate degradation. Results indicated that intact pancreatic cancer cells (RWP-1 and RWP-2 cell lines), cell homogenate, and cytosol contain proteinases which were able to degrade [3H]collagen (type I) and [3H]gelatin and lyse normal RBCs. Cancer cell membrane fractions were enriched in collagenolytic, gelatinolytic, and cytolytic activities which could be abrogated by EDTA but not by inhibitors of serine or cysteine proteinases, which indicates that metalloproteinases are the active enzymes in these assays. Although plasminogen activator and cysteine proteinases were also enriched in the tumor cell membranes, these activities were not required for collagen degradation or cytolysis. We conclude that human cancer cell membrane proteinases are advantageously situated to facilitate damage to surrounding normal tissues.

Cell Line↗
PubMed1985

Effect of netilmicin on the phospholipid composition of subcellular fractions of rat renal cortex.

The purpose of this study was to determine the subcellular site(s) of the renal cortical phospholipidosis induced by aminoglycosides. For this purpose we injected male Sprague-Dawley rats s.c. with netilmicin, containing tracer quantities of [3H]netilmicin, at 100 mg/kg/day for 2 days; control rats were injected with saline. Twenty-four hours after the second injection of drug the rats were sacrificed and the renal cortex was fractionated by differential ultracentrifugation and Percoll gradient density techniques to obtain purified lysosomes, mitochondria, microsomes, brush border membranes and basolateral membranes. The total phospholipid content of the renal cortex was 300 +/- 5 nmol/mg of protein in control rats and 340 +/- 5 nmol/mg of protein in netilmicin-injected rats. The total phospholipid content of the lysosomal fraction of netilmicin rats, which was enriched in myeloid bodies and [3H]netilmicin, was 91% greater than that of control rats and reflected significant increases of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol. This pattern is identical to that reported previously for the rat renal cortical phospholipidosis induced by aminoglycosides. The total phospholipid contents of the mitochondrial, microsomal, brush border membrane and basolateral membrane fractions of netilmicin-injected rats were higher by approximately 10% than the respective fractions of control rats and each fraction exhibited a significant increase of one or more of the four phospholipids elevated in the renal cortical homogenate and in the lysosomal fraction. The data indicate that the myeloid body is the primary source of the lysosomal phospholipidosis induced by netilmicin which provides support for the hypothesis that the lysosomal phospholipidosis is secondary to aminoglycoside-induced inhibition of phospholipid degradation. In addition the findings of increased phospholipid content and altered phospholipid composition of the other subcellular fractions raise the possibility that aminoglycoside antibiotics cause a more generalized disturbance of phospholipid metabolism characterized by altered synthesis as well as degradation in renal proximal tubular cells.

Acetylglucosaminidase↗
PubMed1985

Does indomethacin cause extension of intracranial hemorrhages: a preliminary study.

Thirty-six infants who had an intracranial hemorrhage (diagnosed by cranial ultrasound) within four days after delivery (mean age 2.4 +/- 0.9 (SD) days), were reexamined at three- to seven-day intervals for extension of their intracranial hemorrhage. Seventeen infants had a patent ductus arteriosus and were treated with indomethacin after the initial intracranial hemorrhage was diagnosed. The age for starting indomethacin was 3.8 +/- 1.1 days. Nineteen infants did not have a patent ductus arteriosus and did not receive indomethacin. Both the indomethacin-treated and nontreated groups were similar in birth weight, gestational age, Apgar scores, gender, incidence of respiratory distress, as well as the location and the degree of hemorrhage in the initial scans. Only one of 17 (6%) infants who received indomethacin v tow of 19 (11%) infants who did not receive it, had extension of their initial intracranial hemorrhage. Although indomethacin may alter platelet function, it does not appear to cause extension of a preexisting intracranial hemorrhage.

Cerebral Hemorrhage↗
PubMed1985

[3H]netilmicin binding constants and phospholipid composition of renal plasma membranes of normal and diabetic rats.

We examined the hypothesis that the decreased renal accumulation of aminoglycosides in rats with streptozotocin-induced diabetes mellitus is due to decreased membrane binding of drug consequent to reduced membrane content of the putative aminoglycoside receptor, phosphatidylinositol. Renal brush border membrane (BBM) and basolateral membrane (BLM) vesicles were prepared from normal and diabetic Sprague-Dawley rats by differential centrifugation and Percoll gradient techniques which yielded relatively pure membrane fractions as assessed by measurements of marker enzymes and by electron microscopy. Binding of [3H]netilmicin to plasma membranes was performed using a fast filtration technique. Scatchard analysis of the binding data indicated that netilmicin bound to a single class of receptors on BBM and BLM from normal rats with an affinity constant of 33 +/- 2 X 10(3)M-1 and 23 +/- 2 X 10(3)M-1, respectively. The maximal binding capacity of BLM (70 +/- 4 nmol/mg of protein) was significantly greater (P less than .01) than that of BBM (38 +/- 1 nmol/mg of protein). The affinity constants and maximal binding capacities of BBM and BLM from diabetic rats were not significantly different from those of normal rats. Moreover, 2 days of gentamicin injections at 100 mg/kg/day for 2 days had no appreciable effect on these binding parameters in either group. In control rats the total phospholipid content of BLM (785 +/- 19 nmol/mg of protein) was significantly greater (P less than .01) than that of BBM (592 +/- 19 nmol/mg of protein) and reflected significantly greater quantities of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗