Inhibition of Notch activity promotes pancreatic cytokeratin 5-positive cell differentiation to beta cells and improves glucose homeostasis following acute pancreatitis

Some individuals develop prediabetes and/or diabetes following acute pancreatitis (AP). AP-induced beta-cell injury and the limited regenerative capacity of beta cells might account for pancreatic endocrine insufficiency. Previously, we found that only a few pancreatic cytokeratin 5 positive (Krt5⁺) cells differentiated into beta cells in the murine AP model, which was insufficient to maintain glucose homeostasis. Notch signaling determines pancreatic progenitor differentiation in pancreas development. This study aimed to examine whether Notch signaling inhibition could promote pancreatic Krt5⁺ cell differentiation into beta cells and improve glucose homeostasis following AP. Pancreatic tissues from patients with acute necrotizing pancreatitis (ANP) were used to evaluate beta-cell injury, Krt5⁺ cell activation and differentiation, and Notch activity. The murine AP model was induced by cerulein, and the effect of Notch inhibition on Krt5⁺ cell differentiation was evaluated both in vivo and in vitro. The results demonstrated beta-cell loss in ANP patients and AP mice. Krt5⁺ cells were activated in ANP pancreases along with persistently elevated Notch activity, which resulted in the formation of massive duct-like structures. AP mice that received Notch inhibitor showed that impaired glucose tolerance was reversed 7 and 15 days following AP, and increased numbers of newborn small islets due to increased differentiation of Krt5⁺ cells to beta cells to some extent. In addition, Krt5⁺ cells isolated from AP mice showed increased differentiation to beta cells by Notch inhibition. Collectively, these findings suggest that beta-cell loss contributes to pancreatic endocrine insufficiency following AP, and inhibition of Notch activity promotes pancreatic Krt5⁺ cell differentiation to beta cells and improves glucose homeostasis. The findings from this study may shed light on the potential treatment of prediabetes/diabetes following AP.Subject terms: Endocrine system and metabolic diseases, Pancreatitis     

Immunoregulation by natural killer cells

Polyinosinic-polycytidilic acid (poly (I:C], a synthetic analog of viral double-stranded RNA (dsRNA), activates natural killer (NK) cells and inhibits induction or promotes termination of the primary IgM response in vivo. Suppression of responses was reproduced in vivo by interferons (IFN) which activate NK cells and in vitro by cells enriched for NK cells. The likelihood that NK cells may be involved in the normal regulation of IgM responses is supported by the following observations: immunization itself induces NK activity at times appropriate to account for termination, NK cells activated by immunization suppress in vitro, mice with high NK activity induced by immunization with one antigen have reduced responses to immunization with a second antigen, and mice with induced loss of NK activity fail to down-regulate IgM antibody responses normally.     

Type beta transforming growth factor (TGF beta) regulation of alkaline phosphatase expression and other phenotype-related mRNAs in osteoblastic rat osteosarcoma cells

TGF beta 1 from porcine platelets increased alkaline phosphatase (AP) activity in the rat osteoblastic cell line ROS 17/2.8 about three-fold. This effect was dose-dependent with an ED50 of about approximately 0.2 ng/ml and was larger during logarithmic growth than at confluence. TGF beta 1 inhibited cell growth by about 30% with similar dose dependence. Thirty min exposure to TGF beta 1 was sufficient to increase AP activity 3 days later by about two-fold but did not affect cell growth, suggesting dissociation between effects on proliferation and differentiation. The rise in AP activity started 6 h after TGF beta 1 addition and was blocked by cycloheximide and actinomycin D. TGF beta 1 also increased AP mRNA by two- to three-fold and this effect was not blocked by cycloheximide. The half-life of AP mRNA, estimated following the addition of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole was about ten h in both control and TGF beta 1-treated cells. The mRNAs for type I procollagen and osteonectin were also increased by TGF beta 1 but fibronectin mRNA was decreased. TGF beta 2 effects on AP and cell growth were similar to those of TGF beta 1, except for lack of activity following transient exposure. At saturating concentrations, TGF beta 2 (2 ng/ml) or dexamethasone (10(-7) M), which has similar effects on these cells, did not further augment the effects of TGF beta 1 (at 2 ng/ml). Above findings suggest that TGF beta promotes osteoblastic differentiation in rat osteosarcoma cells at least in part by acting at the pretranslational level.     

Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate.

BACKGROUND: The alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF((R))-97 for enzyme-labeled fluorescence) has been found useful for the histochemical detection of endogenous AP activity and AP-tagged proteins and oligonucleotide probes. In this study, we evaluated its effectiveness at detecting endogenous AP activity by flow cytometry. METHODS: The ELF-97 phosphatase substrate was used to detect endogenous AP activity in UMR-106 rat osteosarcoma cells and primary cultures of chick chondrocytes. Cells were labeled with the ELF-97 reagent and analyzed by flow cytometry using an argon ultraviolet (UV) laser. For comparison purposes, cells were also assayed for AP using a Fast Red Violet LB azo dye assay previously described for use in detecting AP activity by flow cytometry. RESULTS: The ELF-97 phosphatase substrate effectively detected endogenous AP activity in UMR-106 cells, with over 95% of the resulting fluorescent signal resulting from AP-specific activity (as determined by levamisole inhibition of AP activity). In contrast, less than 70% of the fluorescent signal from the Fast Red Violet LB (FRV) assay was AP-dependent, reflecting the high intrinsic fluorescence of the unreacted components. The ELF-97 phosphatase assay was also able to detect very low AP activity in chick chondrocytes that was undetectable by the azo dye method. CONCLUSIONS: The ELF-97 phosphatase assay was able to detect endogenous AP activity in fixed mammalian and avian cells by flow cytometry with superior sensitivity to previously described assays. This work also shows the applicability of ELF-97 to flow cytometry, supplementing its previously demonstrated histochemical applications.     

The effects of epidermal growth factor and the state of confluence on enzymatic activities of cultured rat liver epithelial cells

In cultured normal rat liver epithelial cells, the specific activity and/or isozyme expression of NADH-diaphorase (NADH-D), pyruvate kinase (PK), glucose-6 phosphate dehydrogenase (G6PD), gamma-glutamyl transpeptidase (GGT), and alkaline phosphatase (AP) were markedly dependent on the growth state of the cultures. Proliferating, preconfluent cells had higher specific activities of PK, NADH-D, and G6PD but lower activities of GGT and AP than did the more stationary confluent cells. Addition of epidermal growth factor [EGF] to the media of proliferating cells enhanced the specific activities of PK, NADH-D, G6PD, GGT, and lactate dehydrogenase (LDH) of these cells, but the specific activity of AP was markedly depressed. The increase in activity of PK and GGT by EGF appeared to involve new protein synthesis, whereas the effect of EGF on AP appeared to involve the EGF-directed suppression of the synthesis of a form of AP that is produced exclusively by cells in confluent cultures. Furthermore, the preconfluent cells were more responsive to the action of EGF on AP than were confluent cells, i.e., the EGF-mediated decrease in AP activity was seen at lower concentration in preconfluent than in confluent cells. Paradoxically, confluent cells exhibited a two-to threefold higher capacity to bind [125 I]EGF because of an increase in surface receptor number. The results of this study indicate that enzymatic or other biochemical studies performed on cultured cells must take into account the growth-state of the cultures. EGF can modulate enzyme activity in growing and nongrowing cells; one effect of EGF is to maintain higher activity of glycolytic enzymes, suggesting that EGF or EGF-like factors may contribute to the high rate of glycolysis in certain neoplasms.     

Effect of antibiotic and vaccine therapies on the succinate dehydrogenase and phosphatase activity of liver cells of mice infected with Staphylococcus

Changes in the activity of succinate dehydrogenase (SDH), total and acid phosphatase (TP and AP) were studied in treatment of laboratory animals with rifampicin, lincomycin and with inactivated staphylococcal vaccine used alone or in combinations. It was shown that immunization of the animals with inactivated staphylococcal vaccine under conditions of experimental staphylococcal infection promoted stimulation of the enzyme activity. Rifampicin and lincomycin used for the treatment of such animals lowered the activity of the enzymes. The suppressing effect of the antibiotics increased with an increase in the period of their use. It should be noted that the inhibitory effect of rifampicin on the activity of SDH, TP and AP was less pronounced than that of lincomycin. The combined use of the vaccine and antibiotics for the treatment of the animals promoted an increase in the enzyme activity as compared to the use of the antibiotics alone. Sometimes the activity of SDH, TP and AP reached the control levels in such animals or the levels observed in the animals treated with the vaccine alone. Stimulation of the enzyme activity was more pronounced when the vaccine was used in combination with rifampicin.     

Alkaline phosphatase as a reporter enzyme

This study examines the use of alkaline phosphatase (AP) as a reporter enzyme. We constructed a plasmid containing the cDNA which encodes the bone/liver/kidney rat AP under the control of the simian virus 40 (SV40) early promoter and used it to transfect Chinese hamster ovary, SV40-transformed African Green Monkey kidney 7, and rat osteosarcoma 25/1 mammalian cells. AP activity in these cells, measured three days later, was 40-400-fold above background. When AP and chloramphenicol acetyltransferase (CAT) plasmids were cotransfected, the detection of AP activity by a simple spectrophotometric assay was at least as sensitive as the detection of CAT activity using a radioactive substrate. Moreover, since mammalian AP is a membrane-bound ectoenzyme, transfected cells can be visualized by histochemical staining. This approach was used to estimate transfection efficiency. The convenient methods for AP detection should make it a useful reporter enzyme.     

Activity of alkaline phosphatase in rat skeletal muscle localized along the sarcolemma and endothelial cell membranes

Alkaline phosphatase (AP), a membrane-associated glycoprotein which enhances the hydrolysis of monophosphate esters at alkaline pH, is widely distributed in animal tissues. AP activity is increased in a variety of muscle disorders, i.e., myopathies and denervation. Established histochemical methods at the light microscopy level failed to demonstrate AP in skeletal muscles. In the present study we applied the Gomori lead nitrate method for ultrastructural examination of AP in rat gastrocnemius muscles and showed that the enzyme was linked to the sarcolemma of the striated muscle and to the membranes of endothelial cells in adjacent capillaries. In comparison with ATPase activity, AP activity was inhibited by both levamisole and a pH of 7.2, but not by ouabain. Hence, it appears that in skeletal muscles AP is active at a high pH and is bound to cell membranes.     

Identification of pulmonary surfactant that bears intestinal-type and tissue-nonspecific-type alkaline phosphatase in endotoxin-induced rat bronchoalveolar fluid

The characteristics of lipopolysaccharide (LPS)-induced alkaline phosphatase (AP) isozymes on the various pulmonary surfactant subtypes were investigated. We used continuous sucrose-gradient centrifugation to separate surfactant into subtypes. The density of each surfactant subtype isolated from LPS-instilled rats was greater than that of the subtypes from the control rats; and the proportion of light surfactant was lower, thereby decreasing the ratio of light to heavy surfactant. The results of an inhibition study revealed the main AP isozyme in bronchoalveolar fluid (BAF) to be tissue-nonspecific AP (TNAP), but some of the activity was characteristic of intestinal-type AP (IAP). IAP, in addition to TNAP and surfactant-associated protein A (SP-A), was detected on heavy surfactant, and LPS induced both APs. To examine the expression of IAP in the lungs, we prepared primers to detect the cDNAs of two types of rat IAP mRNA, IAP-I and -II, and amplified their cDNAs. LPS instillation induced IAP-I mRNA, but not IAP-II mRNA or TNAP mRNA. Immunohistochemical localization of IAP and TNAP revealed reaction products for both in type II cells. The present study thus demonstrated that, in rats, type II cells produce both IAP and TNAP and that these surfactants bearing AP isozymes are secreted into the alveolar space following induction by intratracheal instillation of LPS.     

Ultrastructural localization of alkaline phosphatase activity in the normal and osteochondrotic joint cartilage of growing pigs

The present study aimed to describe the ultrastructural localization of alkaline phosphatase (AP) activity in articular-epiphyseal growth cartilage of the commercial pig and the minipig of wild hog ancestry, comparing areas with a normal endochondral ossification with those where the calcification of the matrix is insufficient, as in osteochondrotic cartilage. Intense AP activity was primarily present in the cytoplasm, the plasmalemmae, the long cellular processes and the matrix vesicles budding off from proliferative and hypertrophic chondrocytes in those areas of cartilage where normal calcification appeared. In the osteochondrotic cartilage, the only detectable AP activity was restricted to a few morphologically viable hypertrophic cells in the surroundings of the lesion. The lack of AP activity could partially explain the insufficient calcification of the osteochondrotic cartilage.     

Ultrastructural cytochemical studies of plasma membrane phosphatase activities during the HeLa S3 cell cycle.

Alkaline phosphatase (AP), 5'-nucleotidase (5'N), Mg2+-activated ATPase (Mg-ATPase) and Ca2+-activated ATPase (Ca-ATPase) were studied in sychronized HeLa S3 cells with cytochemical methods and electron microscopy. It was found that AP activity, as determined by the deposition of lead phosphate reaction product (r.p.) was most active in mitotic (M), early and middle G1 cells, less active in late G1 and almost undetectable in S phase cells. Most AP enzyme activity was found to be associated with undulations (mainly microvilli) of the plasma membrane. Fluctuations and the redistribution of 5'N were also observed; the reaction for 5'N was positive in all phases of the cell cycle studied, it was strongest in M cells and in the majority of middle G1 cells. Mg-ATPase activity was present in the plasma membranes of cells throughout the cell cycle, but did not show noticeable fluctuations in activity and distribution. Ca-ATPase activity appeared in plasma membranes and in limited areas of cell nuclei but was evident only in S phase cells. The results of the present study confirm and extend previous biochemical observations and indicate that changes in membrane phosphate activities are associated with enzyme activity redistributions within the plasma membrane during the HeLa S3 cell cycle.     

Acid phosphatases: androgen dependent markers of rat prostate.

Our investigations on acid phosphatase (AP) were aimed at finding a biochemical assay marker for androgen actions in the rat prostrate. We quantitatively examined the effects of l-tartrate or formaldehyde on AP activity in tissue filtrates from nine adult male rat tissues, plasma and hemolysed red blood cells (HRBC). There was significant inhibition of AP activity in all instances with the exception of HRBC with tartrate. The prostate inhibition results were not different from those for seminal vesicles and adrenals but were different from the other tissues studied. Ten days following castration the inhibition by tartrate was less in all tissues studied except plasma and HRBC; the formaldehyde inhibition percentages were not altered.     

The cytolytic activity of pulmonary CD8+ lymphocytes, induced by infection with a vaccinia virus recombinant expressing the M2 protein of respiratory syncytial virus (RSV), correlates with resistance to RSV infection in mice.

Previous studies demonstrated that the pulmonary resistance to respiratory syncytial virus (RSV) challenge induced by immunization with a recombinant vaccinia virus expressing the M2 protein of RSV (vac-M2) was significantly greater 9 days after immunization than at 28 days and was mediated predominantly by CD8+ T cells. In this study, we have extended these findings and sought to determine whether the level of CD8+ cytotoxic T-lymphocyte (CTL) activity measured in vitro correlates with the resistance to RSV challenge in vivo. Three lines of evidence documented an association between the presence of pulmonary CTL activity and resistance to RSV challenge. First, vac-M2 immunization induced pulmonary CD8+ CTL activity and pulmonary resistance to RSV infection in BALB/c (H-2d) mice, whereas significant levels of pulmonary CTL activity and resistance to RSV infection were not seen in BALB.K (H-2k) or BALB.B (H-2b) mice. Second, pulmonary CD8+ CTL activity was not induced by infection with other vaccinia virus-RSV recombinants that did not induce resistance to RSV challenge. Third, the peak of pulmonary CTL activity correlated with the peak of resistance to RSV replication (day 6), with little resistance being observed 45 days after immunization. An accelerated clearance of virus was not observed when mice were challenged with RSV 45 days after immunization with vac-M2. The results indicate that resistance to RSV induced by immunization with vac-M2 is mainly mediated by primary pulmonary CTLs and that this resistance decreases to very low levels within 2 months following immunization. The implications for inclusion of CTL epitopes into RSV vaccines are discussed in the context of these observations.     

N-acetylcysteine prevents intra-acinar oxygen free radical production in pancreatic duct obstruction-induced acute pancreatitis

Although oxygen free radicals (OFR) are considered to be one of the pathophysiological mechanisms involved in acute pancreatitis (AP), the contribution of acinar cells to their production is not well established. The aim of the present study was to determine the effect of N-acetylcysteine (NAC) in the course of AP induced by pancreatic duct obstruction (PDO) in rats, directly analysing by flow cytometry the quantity of OFR generated in acinar cells. NAC (50 mg/kg) was administered 1 h before and 1 h after PDO. Measurements by flow cytometry of OFR generated in acinar cells were taken at different PDO times over 24 h, using dihydrorhodamine-123 as fluorescent dye. Histological studies of pancreas and measurements of neutrophil infiltration in the pancreas, pancreatic glutathione (GSH), malondialdehyde (MDA) levels, plasma amylase activity and hemoconcentration were carried out in order to assess the severity of AP at different stages. NAC effectively blunted GSH depletion at early AP stages and prevented OFR generation found in acinar cells as a consequence of AP induced by PDO. This attenuation of the redox state impairment reduced cellular oxidative damage, as reflected by less severe pancreatic lesions, normal pancreatic MDA levels, as well as diminished neutrophil infiltration in pancreas. Hyperamylasemia and hemoconcentration following AP induction were ameliorated by NAC administration at early stages, when oxidative stress seems to be critical in the development of pancreatitis. In conclusion, NAC reinforces the antioxidant defences in acinar cells, preventing OFR generation therefore attenuating oxidative damage and subsequently reducing the severity of PDO-induced AP at early stages of the disease.     

ALKALINE PHOSPHATASE ACTIVITY AS A FUNCTION OF INTERNAL PHOSPHORUS CONCENTRATION IN FRESHWATER PHYTOPLANKTON1

Single‐cell alkaline phosphatase (AP) activity is being increasingly used to characterize phosphorus (P) status of individual species of phytoplankton. As phytoplankton growth rates depend more directly on the internal rather than external P concentrations, we determine the AP activity in the two species of freshwater phytoplankton, Scenedesmus quadricauda (Turpin) Bréb. and Asterionella formosa Hassall, as a function of internal P concentration. AP activity strongly correlated with cellular P, increasing almost linearly with decreasing cellular P in both species. The dynamics of initial responses of AP activity to P limitation, as well as the final levels of AP activity, when cellular P approached minimum quotas, differed in two species. After P addition, cellular P concentrations increased rapidly, but AP activity remained high for several days. The lag in AP activity down‐regulation following an increase in cellular P made it difficult to infer current P status of cells under dynamic P conditions.     

Resistance to 6-thioguanine in spontaneously cycling and in mitogen-stimulated human peripheral lymphocytes

A study of the apurinic/apyrimidinic (AP) endonuclease activities of a mutant line of CHO cells, EM9, and its parental cell line, AA8, was undertaken to determine if the defective DNA repair exhibited by the mutant cell line after exposure to ethyl methanesulfonate was due to a defective AP endonuclease activity. Phosphocellulose chromatography of cell extracts resolved the AP endonuclease activities of both cell lines into two peaks as seen previously in mouse and human cells. No difference was found between the mutant and parental cell lines in the relative amount of AP endonuclease activity present in the two peaks.     

Changes in haematopoietic progenitor colony differentiation and proliferation and the production of different abzymes in EAE mice treated with DNA

Immunization of experimental autoimmune encephalomyelitis (EAE)‐prone C57BL/6 mice with MOG_35‐55 (a model used to study aspects of human multiple sclerosis) is known to lead to the production of various abzymes. The production of catalytic IgGs that can efficiently hydrolyse myelin basic protein (MBP), MOG and DNA is associated with changes in the profile of differentiation and level of proliferation of mice bone marrow haematopoietic stem cells (HSCs). As MOG simulates the production of abzymes with high DNase activity, we compared the effects of DNA and MOG immunization on EAE‐prone mice. In contrast to MOG, immunization with DNA leads to a suppression of proteinuria, a decrease in the concentrations of antibodies to MOG and DNA and a reduction in abzyme production. Immunization with DNA only resulted in a significant increase in DNase activity over 40 days where it became 122‐fold higher than before immunization, and fivefold higher when comparing to the maximal activity obtained after MOG treatment. DNA and MOG immunization had different effects on the differentiation profiles of HSCs, lymphocyte proliferation, and the level of apoptosis in bone marrow and other organs of mice. The data indicate that for C57BL/6 mice, DNA may have antagonistic effects with respect to MOG immunization. The usually fast immune response following MOG injection in C57BL/6 mice is strongly delayed after immunization with DNA, which is probably due to a rearrangement of the immune system following the response to DNA.