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PubMed2005

In vitro analysis of the digestive enzymes amylase and alpha-glucosidase in the midguts of Locusta migratoria L. in response to the myosuppressin, SchistoFLRFamide.

We have investigated the effect of the locust myosuppressin, SchistoFLRFamide, on the activity of amylase and alpha-glucosidase in the midgut of 2-week old male locusts. Total enzyme activity in the lumen contents and tissue extracts of midguts responds to SchistoFLRFamide in a dose-dependent manner that appears to vary with the feeding state of the locust and duration of exposure to the peptide. Starvation for 24h prior to assessment alters the distribution of enzyme activity between the midgut lumen contents and tissue extracts in response to SchistoFLRFamide when compared with fed locusts. Duration of exposure to SchistoFLRFamide also alters the distribution of total amylase and alpha-glucosidase activity; as duration of exposure increases, lower concentrations of SchistoFLRFamide increase total enzyme activity in the lumen contents while decreasing total enzyme activity in the tissue extracts. We suggest that the minimum amino acid sequence in SchistoFLRFamide necessary to increase both amylase and alpha-glucosidase activity is DHVFLRFamide. We have determined that two other peptides endogenous to the locust, AFIRFamide and GQERNFLRFamide, increase amylase and alpha-glucosidase activity in midgut lumen contents.

Amylases↗
PubMed2003

Human kallikrein 5: a potential novel serum biomarker for breast and ovarian cancer.

The kallikrein family is a group of 15 serine protease genes clustered on chromosome 19q13.4. Human kallikrein (hK) gene 5 (KLK5) is a member of this family and encodes for a secreted serine protease (hK5). KLK5 was shown to be differentially expressed at the mRNA level in breast and ovarian cancer. Until now, detection of hK5 protein in either biological fluids or tissues has not been described due to lack of suitable reagents and methods. The aim of this study was to develop immunological reagents and a sensitive and specific fluorometric immunoassay (ELISA) for hK5, to examine the presence of hK5 in human tissues and biological fluids, and to study the possible clinical utility of hK5 as a biomarker for endocrine-related malignancies. Recombinant hK5 protein was produced and purified using a Pichia pastoris yeast expression system. The protein was used as an immunogen to generate mouse and rabbit polyclonal anti-hK5 antibodies. A sandwich-type microplate immunoassay (ELISA) was developed using these antibodies, coupled with a time-resolved fluorometric detection technique. The ELISA assay was then used to measure hK5 in various biological fluids, tissue extracts, and serum samples from normal individuals and patients with various malignancies. The hK5 ELISA immunoassay has a lower detection limit of 0.1 micro g/liter, is specific for hK5, and has no cross-reactivity with other homologous kallikreins. The dynamic range is 0.1-25 micro g/liter, and within-run and between-run coefficients of variation within this range are <10%. hK5 is found in many tissues, with the highest expression levels seen in the skin, breast, salivary gland, and esophagus. hK5 is present at relatively high levels in milk of lactating women. Whereas the levels of hK5 are almost undetectable in serum of normal individuals (male and female) and patients with diverse malignancies, higher concentrations were found in a proportion of patients with ovarian (69%) and breast (49%) cancer. High levels were also detected in ascites fluid from metastatic ovarian cancer patients and in ovarian cancer tissue extracts. In conclusion, we report development of the first immunofluorometric assay for hK5 and describe the distribution of hK5 in biological fluids and tissue extracts. Our preliminary data indicate that hK5 is a potential biomarker in patients with ovarian and breast cancer.

Adult↗
PubMed1983

Natriuretic activity of extracts obtained from hearts of different species and from various rat tissues.

Heart tissue extracts from different species as well as extracts obtained from various rat tissues were tested for natriuretic activity in the nondiuretic bioassay rat. Natriuretic activity was found restricted to heart tissues. Atrial but not ventricular extracts of mammals and both atrial and ventricular extracts in the frog displayed natriuretic activity. Specific natriuretic activity values were highest in the atria of small rodents. It is concluded that the distribution and relative content of natriuretic activity of cardiac tissues from different species approximately parallels the distribution and relative abundance of specific heart granules.

Animals↗
PubMed2003

An integrated metabonomic investigation of acetaminophen toxicity in the mouse using NMR spectroscopy.

An integrated metabonomics study using high-resolution 1H NMR spectroscopy has been applied to investigate the biochemical composition of intact liver tissue (using magic angle spinning), liver tissue extracts, and blood plasma samples obtained from control and acetaminophen-treated mice. Principal components analysis was used to visualize similarities and differences in biochemical profiles. The time- and dose-dependent biochemical effects of acetaminophen were related to the drug toxicity, as determined using histopathology. Metabolic effects in intact liver tissue and lipid soluble liver tissue extracts from animals treated with the high dose level of acetaminophen included an increase in lipid triglycerides and monounsaturated fatty acids together with a decrease in polyunsaturated fatty acids, indicating mitochondrial malfunction with concomitant compensatory increase of peroxisomal activity. In addition, a depletion of phospholipids was observed in treated liver tissue, which suggested an inhibition of enzymes involved in phospholipid synthesis. There was also a depletion in the levels of liver glucose and glycogen. In addition, the aqueous soluble liver tissue extracts from high dose animals also revealed an increase in lactate, alanine, and other amino acids, together with a decrease in glucose. Plasma spectra showed increases in glucose, acetate, pyruvate, and lactate. These observations all provide evidence for an increased rate of glycolysis. These findings could indicate a mitochondrial inability to use pyruvate in the citric acid cycle and also reveal the impairment of fatty acid beta-oxidation in liver mitochondria of such treated mice.

Acetaminophen↗
PubMed2001

Alveolar albumin leakage during large tidal volume ventilation and surfactant dysfunction.

Detergent given as an aerosol and large tidal volume ventilation (LTVV) have been observed, by us, to promote lung injury by an additive effect on alveolocapillary barrier function. The surfactant system may be further damaged if protein leakage occurs into the alveoli. The aim was to study the effect of detergent and LTVV on the alveolar leakage of albumin and also the effect of detergent on surface activity of lung washings and lung tissue extracts. Technetium-99m-labelled human serum albumin was given intravenously. The alveolar leakage of albumin was measured after perturbing the surfactant system with the detergent dioctyl sodium sulfosuccinate either singly or in combination with LTVV. Four groups of rabbits were studied after 3 h of experimental ventilation. Surface tension measurements of tissue extracts, lung mechanics and gas exchange did not show any differences between groups. Wet lung weight and albumin leakage were significantly increased in the two groups subjected to LTVV compared with groups given normal tidal volume ventilation. Low doses of detergent did not affect surface activity of lung tissue extracts or alveolar leakage of albumin. LTVV increased alveolar leakage of albumin and produced oedema. No additive effect was seen when detergent and LTVV were combined.

Albumins↗
PubMed2001

The influence of L-arginine on heart rate and tissue oxygen extraction in haemorrhaged rabbits.

Several studies have already indicated some beneficial effects of L-arginine in haemorrhaged rats. The aim of our study was to assess whether intravenous bolus injection of L-arginine could improve some cardiovascular and metabolic parameters in anaesthetized haemorrhaged rabbits (intermittent bleeding; 40% of the estimated blood volume for 15 min). I.v. bolus injection of L-arginine ( 300 mg kg(-1)--L-Arg(300)) increased heart rate (app. 10%) and decreased venous haemoglobin saturation with oxygen (sO(2)) (app. 23%) 60 min after the cessation of bleeding, without changes in arterial pressure. D-arginine (300 mg kg(-1)i.v. bolus-D-Arg(300)) produced similar, but insignificant haemodynamic and metabolic changes. In addition, no difference was found between the effects of the L- and D-isomers. Accordingly, L-arginine produces beneficial effects on the heart rate and tissue oxygen extraction in haemorrhaged rabbits. However, such changes do not appear to be stereospecific.

Acid-Base Equilibrium↗
PubMed1993

Partial characterization of a novel oestrogen-induced protein in the rat adenohypophysis.

In order to detect putative markers of prolactin-secreting pituitary tumours, adult rats were subjected to long-term oestrogenization with oestradiol benzoate (OE2) on a monthly basis. At 6 months, anterior pituitaries were dissected and incubated either as tissue fragments or as dispersed cells with a [35S]methionine mix for labelling. Proteins released into the incubation medium and from tissue extracts were further analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and fluorography. Oestrogen induced the appearance in the incubation medium of a protein (OE2 band) with an M(r) of 38,000 under reducing conditions, and high specific activity. Surprisingly, such a protein was not detected in tissue extracts. The OE2 band was detectable by 7 days after the first dose of oestrogen, and remained throughout 1 year of treatment. The tumour cell line GH3 showed a similar OE2 band which was further enhanced by oestrogens. The protein was observed similarly in both female and male pituitary donors, either intact or gonadectomized, and also in rats of different strains, suggesting that its appearance was independent of the strain of rat and gonadal status. Furthermore, the OE2 band was specific for pituitary cells and not produced by other oestrogenized tissues. No alteration in the rate of generation or the electrophoretic pattern of the OE2 band was observed when pituitary cells from oestrogenized rats were metabolically labelled while being incubated with tunicamycin. Furthermore, a system for glycan detection, adsorption to Concanavalin A or incubation with endoglycosidase F also failed to show a clear amount of glycosylation of the oestrogen-induced protein. Both immunoprecipitation experiments and time-limited proteolysis with V8 protease ruled out the possibility that the OE2 band could be structurally related to either GH or prolactin. In conclusion, oestrogens induce the generation of a new monocatenary protein with an apparent M(r) of 38,000, which has at least one intramolecular disulphide loop and is not glycosylated. The OE2 band was detected only in incubation medium and never in tissue extracts.

Adenoma↗
PubMed1997

Synthesis and decay of calmodulin-ubiquitin conjugates in cell-free extracts of various rabbit tissues.

Calmodulin is the natural substrate for ubiquitin-ligation by the enzyme ubiquitin-calmodulin ligase (uCaM-synthetase; EC 6.3.2.21). The activity of this ligase is regulated by the binding of the second messenger Ca2+ to the substrate calmodulin, which increases the activity ca. 10-fold. Up till now, two components of the ligase could be identified: uCaM Syn-F1 and uCaM Syn-F2, the first of which binds to ubiquitin and the second which binds to calmodulin. Since the physiological role of this enzyme is still unclear, this study was designed to examine whether the activity of uCaM-Synthetase in 40,000 x g tissue supernatants correlates with the calmodulin content in the various tissues. In reticulocytes, spleen, erythrocytes, testis and brain, which are rich in uCaM synthetase, the tissue contents calculated on the basis of activity measurements were between 4-80-fold higher than in red and white skeletal muscle. These activities did not correlate with the respective calmodulin contents of the tissues indicating that other factors were determining these enzyme levels. A second aim was to gain information on the role of the ATP-ubiquitin-dependent proteolytic pathway in those tissues displaying uCaM synthetase activity. In the reticulocyte system which contains the classical ATP-ubiquitin-dependent proteolytic pathway as measured with 125I-BSA, no ubiquitin-dependent degradation of calmodulin could be detected. We therefore examined the other tissues of the rabbit with the substrate 125I-BSA and succeeded in finding a ubiquitin-independent ATP-dependent proteolytic activity in every case but no ubiquitin-dependent activity. The ubiquitin-independent activity was highest in smooth muscle and red skeletal muscle being ca. 3-4-fold higher than in lung and testis. In 50% of the tissue crude extracts the time curve of calmodulin ubiquitylation progressed through a maximum indicating a dynamic steady state based on conjugate synthesis and decay. If a ubiquitylation pulse of 30 min was followed in liver crude extracts by the addition of EGTA, which specifically inhibits ubiquityl-calmodulin synthesis, a half-life of calmodulin-conjugate decay of 15-20 min is observed. A similar conjugate half-life of ca. 30 min was observed after addition of EDTA excluding that conjugate decay is due to an ATP-dependent proteolytic process. Studying the decay of purified ubiquitin-125I-BH-calmodulin conjugates in cell-free reticulocyte extracts led to the discovery of an ATP-independent isopeptidase activity which splits ubiquitin-calmodulin conjugates without leading to detectable calmodulin fragments. The rapid decay of ubiquitin-calmodulin conjugates in tissue extracts can therefore be plausibly explained by a ubiquityl-calmodulin splitting isopeptidase activity.

Adenosine Triphosphate↗
PubMed1976

Transport and phosphorylation of D-galactose in renal cortical cells.

An improved analytical procedure for the extraction and determination of total, free and phosphorylated tissue sugar is described. This method, employing ZnSO4 plus Ba(OH)2 for the precipitation of sugar phosphates, yields values identical with those obtained by the more laborious separation of free and phosphorylated sugar by ion-exchange chromatography. Erroneous values for free sugar due to the action of a Zn2+ -activated phosphatase and/or the lability to acids of some sugar phosphates, are avoided. Using this technique for the sudy of transport and phosphorylation of D-galactose in rabbit renal cortical slices and tissue extracts, it was found: 1. The cellular uptake of D-galactose was associated with the appearance of both free and phosphorylated sugar whether or not external Na+ was present. At 1 mM sugar, galactose was accumulated in the cells against a modest concentration gradient of 1.445 +/- 0.097 (n = 17). Galactose phosphate appeared in the cells considerably faster than free sugar under conditions of net uptake as well as of steady-state exchange (pulse-labelling). 2. Increasing saline pH (6-8) increased the cellular levels of sugar phosphate without affecting the steady-state values of free sugar. With tissue extracts, increasing pH also stimulated the activity of galactokinase and the dephosphorylation of galactose 1-phosphate by a Zn2+ -activated phosphatase. 3. 0.5 mM phlorizin inhibited the tissue uptake of galactose and its subsequent oxidation to CO2 only to a minor degree (30 and 10%, respectively). The absence of external Na+ further depressed the phlorizin effect. Preincubation of the tissue with phlorizin and subsequent washing in part abolished the inhibitory effect. The data suggest that a major portion of the galactose uptake by the tissue proceeds by a mechanism with a low affinity for phlorizin. 4. Efflux studies showed that the wash-out of free galactose from slices was associated with a net decrease of both free and phosphorylated tissue sugar. 5. The above results suggest the possibility that phosphorylation may represent a step in the Na+ -independent, phloretin-sensitive transfer of D-galactose across the antiluminal cell membrane. The participation of intracellular galactokinase and a Zn2+ -activated alkaline phosphatase in the maintenance of the steady state of free and phosphorylated galactose in the cells has been demonstrated.

Animals↗
PubMed2005

Endostatin expression in pancreatic tissue is modulated by elastase.

Pancreatic tumours are scirrhous, avascular tumours, suggesting that they may produce angiogenesis inhibitors that suppress the growth of the vasculature to the tumour and metastases. We have sought evidence for the angiogenesis inhibitor, endostatin, in normal and cancerous pancreatic tissue. Using Western blotting, we found mature 20 kDa endostatin in cancer tissue but not in normal tissue. Several endostatin-related peptides of higher mol wt were present in both tissues. Extracts from normal tissue were able to degrade exogenous endostatin, whereas extracts from cancer were without effect. Although the exocrine pancreas secretes inactive proenzymes of trypsin, chymotrypsin and elastase, their possible role in this degradation was examined. The trypsin/chymotrypsin inhibitor, Glycine max, did not prevent the degradation of endostatin by normal pancreatic extracts but elastatinal, a specific inhibitor of elastase, reduced the rate of degradation. Extracts of pancreatic tumours did not express any detectable elastase activity, but an elastase (Km 1.1 mM) was expressed by extracts of normal pancreas. We conclude that endostatin is present and stable in pancreatic cancer tissues, which may explain their avascular nature, but that normal pancreatic tissue expresses enzymes, including elastase, which rapidly degrade endostatin. The stability of endostatin may have implications for its therapeutic use.

Angiogenesis Inhibitors↗
PubMed1989

Conjugated dienes detected in tissue lipid extracts by second derivative spectrophotometry.

By studying lipid peroxidation induced by tetrachloromethane in rat liver microsomal PUFA, it has recently been shown that the primary products formed are conjugated diene hydroperoxides having either cis, trans (c,t) or trans,trans (t,t) stereochemistry. Both c,t and t,t hydroperoxidienes present distinct absorbances at 242 nm and 233 nm, respectively. The reaction is kinetically controlled in relation to the total H-atom donating ability of the cell environment. These results have been confirmed in vivo and in vitro experiments performed under different experimental conditions. The need for a precise and objective method to detect conjugated diene signals, the inherent difficulties with current techniques, and the availability of new spectrophotometric techniques have led us to devise a new method based on the second derivatization of the spectrum.

Alkadienes↗
PubMedDate not supplied

Vascular endothelial growth factor activating matrix metalloproteinase in ascitic fluid during peritoneal dissemination of ovarian cancer.

The role of vascular endothelial growth factor (VEGF) during peritoneal dissemination of ovarian carcinoma and the association with tumor microvessel density (MVD) and matrix metalloproteinase (MMP) activity was investigated. To this end, MVD, tumor tissue and ascitic fluid levels of VEGF, and MMP activity of ascitic fluid were examined in patients with ovarian cancer and benign ovarian tumor. The effect of ascites on cell growth, cell invasion activity and angiogenesis was investigated in vitro. Ascitic fluid and tumor tissue samples were obtained from 15 patients with benign ovarian tumor and 24 patients with ovarian carcinoma. Tissue extract and ascitic fluid levels of VEGF were measured using enzyme immunoassay. Tumor microvessels were detected immunohistochemically. MMP activity was measured by gelatin zymography. For the in vitro experiment, the SKOV-3 human ovarian carcinoma cell line was utilized. Cell growth was examined using MTT-assay, cell invasion activity was measured by Matrigel in vitro invasion assay, and neovascularization was assessed using an angiogenesis kit. VEGF levels in tissue extract and ascitic fluid, MVD, expression of active form MMP-2 in ascitic fluid and ascites volume were higher in ovarian cancer patients than in benign ovarian tumor patients. In addition, these were elevated in stage III and IV diseases compared to stage I and II diseases in ovarian cancer patients. MVD and expression of active form MMP-2 in ascitic fluid were closely correlated with VEGF level in tissue extracts, and MVD and ascites volume were closely correlated with VEGF level in ascitic fluid. Cell invasive activity and angiogenesis activity increased when cells were exposed to ascites. These increases were apparent when exposed to ascites obtained from ovarian cancer patients and were related to VEGF concentrations of ascitic fluid and expression of active form MMP-2 in ascitic fluid. The increased VEGF secreted from tumor cells is suggested to enhance tumor growth through angiogenesis, to produce ascites and to elevate ascitic VEGF concentrations and expression of active form MMP-2. The progression of peritoneal involvement may be induced by elevated VEGF and expression of active form MMP-2, followed by increased VEGF in the primary tumor. Control of VEGF in the primary tumor may become an effective strategy against peritoneal dissemination of ovarian carcinoma.

Adenoma↗
PubMed1987

Tissue oxygen extraction during hypovolemia: role of hemoglobin P50.

When the delivery of O2 to tissues (QO2 = blood flow X O2 content) falls below a critical threshold, tissue O2 uptake (VO2) becomes limited by QO2. The mechanism responsible for this extraction limitation is not understood but may involve molecular diffusion limitation as mean capillary PO2 drops below a critical minimum level in some capillaries. We tested this hypothesis by measuring the critical QO2 necessary to maintain VO2 independent of QO2 in anesthetized, paralyzed normal dogs (n = 7) and in a second group in which PO2 at 50% saturation of hemoglobin (P50) was reduced by exchange transfusion with low-P50 erythrocytes (n = 7). QO2 was reduced in stages by removing blood volume to reduce blood flow while VO2 was measured by spirometry at each step. To the extent that O2 extraction was limited by a critical capillary PO2, we reasoned that the onset of diffusion limitation should occur at a higher QO2 with low P50, since a lower end-capillary PO2 is required to achieve the same O2 extraction. The critical QO2 (7.8 +/- 1.2 ml X min-1 X kg-1) and extraction ratio (0.63 +/- 0.06) in dogs with reduced P50 were not different from controls. At the critical delivery, mixed venous PO2 was lower in low P50 (16.1 +/- 2.9 Torr) than controls (29.9 +/- 2.3 Torr). We concluded that diffusion limitation does not initiate the early fall in VO2 below the critical QO2 and offer an alternative model to explain the onset of supply dependency.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Thymosin alpha 1 is a native peptide in several tissues.

Failure to detect thymosin alpha 1 (T alpha 1) in tissue extracts prepared by procedures that prevent proteolytic activity has hitherto supported the suggestion that T alpha 1 is not a natural peptide, but the product of uncontrolled proteolysis of prothymosin alpha (ProT alpha), a polypeptide that includes T alpha 1 at its NH2 terminus. In this work, purification by isoelectric focusing of a product with the same isoelectric point as synthetic T alpha 1, and its further characterization, demonstrated that T alpha 1 is present as a native peptide in calf thymus and in several lymphoid and non-lymphoid rat tissues. T alpha 1 shows abnormal chromatographic behaviour which appears to be due to association with other components in tissue extracts. In all the tissues studied, T alpha 1 was present in higher concentration than ProT alpha (80-183 and 44-123 micrograms per gram of tissue, respectively). The ProT alpha/T alpha 1 ratio did not change when no measures were taken to prevent proteolysis during tissue homogenization.

Animals↗
PubMed1990

Heterogeneity of [3H]inositol 1,4,5-trisphosphate binding sites in adrenal-cortical membranes. Characterization and validation of a radioreceptor assay.

1. The characterization of a radioreceptor assay for determining Ins(1,4,5)P3 concentration in tissue extracts is described which utilizes the binding of [3H]Ins(1,4,5)P3 to an adrenal-cortex membrane fraction. 2. Analysis of [3H]Ins(1,4,5)P3 binding by isotope dilution demonstrated an apparent single population of binding sites (KD 3.65 +/- 0.18 nM, Bmax. 872 +/- 70 fmol/mg of protein). Specific binding of [3H]Ins(1,4,5)P3 was enhanced at alkaline pH values (maximum at pH 8.5), with complete loss of specific binding at pH less than 6. These binding sites displayed strict stereo- and positional specificity for Ins(1,4,5)P3, with L-Ins(1,4,5)P3, Ins(1,3,4)P3 and DL-Ins(1,3,4,5)P4 causing 50% displacement of specific [3H]Ins(1,4,5)P3 binding (IC50 values) at concentrations of 14 +/- 3 microM, 3.0 +/- 0.3 microM and 0.53 +/- 0.03 microM respectively. 3. Kinetic analysis of binding data, however, revealed a high-affinity [3H]Ins(1,4,5)P3 binding site (KD 0.052 nM) in addition to the lower-affinity site (KD 2.53 nM) already demonstrated in displacement studies. 4. It is shown that the presence of the high-affinity site can be exploited to increase the sensitivity of the [3H]Ins(1,4,5)P3 radioreceptor assay, allowing accurate detection of 20 fmol of Ins(1,4,5)P3 in 300 microliters of tissue extract. 5. Further validation of the specificity of the above assay for Ins(1,4,5)P3 was provided by incubating tissue extracts with either a 5-phosphatase or 3-kinase preparation. It was shown that identical loss occurred of both Ins(1,4,5)P3 mass and [3H]Ins(1,4,5)P3, added to parallel incubations. 6. The ability of the assay to measure basal and agonist-stimulated increases in Ins(1,4,5)P3 concentration has been demonstrated with rat cerebral cortex and bovine tracheal smooth-muscle slices and a range of cultured and isolated cell preparations.

Adrenal Cortex↗
PubMedDate not supplied

Distribution of galanin-like immunoreactivity in the pig, rat and human central nervous system.

The distribution of galanin-like immunoreactivity in various regions of the central nervous system was assessed in three mammalian species, pig, rat, and human, by radioimmunoassay. Galanin concentrations were highest in the hypothalamus and pituitary region. In spinal cord, there was a rostrocaudal/dorsoventral gradient with highest levels observed in the sacral dorsal horn. Serial dilutions of porcine tissue extracts diluted parallel to the porcine standard curve, while the rat and human tissue extracts did not. In all tissues examined by high pressure liquid chromatography, the principal peak of immunoreactivity coeluted with the authentic porcine galanin standard and was decreased by trypsin cleavage. These results suggest a role for galanin in the central nervous system and support species differences in the structure of galanin.

Animals↗
PubMed1986

Alteration of acid phosphatase isoenzyme in a human prostatic cancer cell line.

The acid phosphatase (AcP) isoenzyme in a human prostatic cancer cell line was compared to that of prostatic tissue extract by electrophoresis. The major isoenzyme by prostatic tissue extract is the AcP isoenzyme 2, while only AcP isoenzyme 4 (AcP-4) was observed in the human prostatic cancer cell line. A monoclonal antibody specific to AcP-4 was used to investigate the ultrastructural distribution of AcP-4 in a prostatic cancer cell line. The peroxidase staining pattern indicates that AcP-4 is synthesized on bound ribosomes, discharged into the cisternae of rough endoplasmic reticulum, transported to the cisternae of Golgi apparatus for concentration and packaging, and transferred to the secretory vesicles for exocytosis. It is well known that synthesis and secretion of AcP-2 are the major characteristics of the highly differentiated prostatic epithelial cells. The present data demonstrate the loss of this specific function in the prostatic cancer cell line. Instead of AcP-2, the dedifferentiated cancer cell line synthesizes and secretes AcP-4, which is a common AcP isoenzyme of many nonprostatic tissues.

Acid Phosphatase↗
PubMed1983

Monoclonal antibodies to different epitopes on a prostate tumor-associated antigen. Implications for immunotherapy.

Mouse monoclonal antibodies alpha Pro3 and alpha Pro5 bind to different epitopes on an antigen (p54) of 54 kD reduced and 175 kD nonreduced MW. p54 antigen has been characterized previously with regard to tissue distribution using alpha Pro3 monoclonal antibody; the p54 antigen is present in substantially greater quantities in malignant prostatic tissue extracts than in benign prostatic and nonmalignant nonurogenital tissue extracts. In this report, we have established that alpha Pro5 and alpha Pro3 monoclonal antibodies exhibit same molecule-different epitope recognition. That both antibodies recognize the same molecular entity has been established by partial physiochemical characterization of the antigen recognized by the two antibodies and by sequential immunoprecipitation experiments. Different determinant recognition was established by lack of competitive surface binding between alpha Pro3 and alpha Pro5 to a prostatic carcinoma cell line. The p54 antigen can be labeled with glucosamine and immunoprecipitated from urea-solubilized membrane proteins; however, p54 cannot be detected by immunoprecipitation in a glycosylated form in spent culture medium removed from glucosamine-labeled cells. Experiments using indirect cellular immunoassays and directly radioiodinated monoclonal antibody have shown that both alpha Pro3 and alpha Pro5 form stable complexes with p54 antigen on the prostatic carcinoma cell surface. To the extent that modulation occurs upon interaction of p54 with alpha Pro3 and alpha Pro5; endocytosis of the immune complex appears to be the primary route of modulation. Furthermore, modulation by endocytosis is more intense when alpha Pro3 and alpha Pro5 are used in combination than when either monoclonal antibody is used alone. Although in vivo biologic behavior does not invariably correlate with in vitro behavior, careful in vitro analysis of monoclonal antibodies with respect to cell surface behavior, nevertheless, should precede in vivo evaluation. The data presented in this report indicate that preliminary in vitro analyses will expedite the effectiveness of in vivo immunotherapeutic trials; preliminary in vitro evaluations are absolutely essential if monoclonal-toxic agent (e.g., ricin A) conjugates, which must be internalized by the tumor cell to achieve cytotoxicity, are employed as immunotherapeutic agents.

Adenocarcinoma↗