Salivary glands of the cat: A histochemical study

Synopsis The submandibular, sublingual and parotid glands of the cat have been studied. Mucosubstance histochemistry demonstrated acidic mucosubstances with varying properties in the acini. Thiamine pyrophosphatase and nucleoside diphosphatase reaction products were seen with a Golgi-like appearance in acinar cells. Granules of acid phosphatase, -glucuronidase and E600-resistant esterase reaction products, presumably representing lysosomal enzyme activities, were seen in acinar and ductal cells. Diffuse acid phosphatase and -glucuronidase reaction products were seen in central cells of the submandibular acini, and diffuse non-specific esterase reaction product was seen in acinar and ductal cells. Arylamidase reaction product was associated with some acinar cells. Reaction product from a peroxidase technique was seen in demilunar cells of the submandibular acini, in parts of the sublingual acini, in parotid acini, and in ductal cells. Cytochrome oxidase and succinate dehydrogenase reaction products were seen most strongly in striated ducts, whereas NADH- and NADPH-diaphorase reaction products were seen at a high level throughout the ducts.     

Histochemical phenotypes of von Ebner's gland of ferret and their functional implications

Von Ebner's gland of ferret was examined by means of light microscopy, protein, mucosubstance and enzyme histochemistry, and neurohistology. Acinar cells were replete with granules containing neutral mucosubstances and disulphides, and showed strong diffuse acid phosphatase activity and weak granular staining for peroxidase. Staining for cytochrome oxidase, succinate dehydrogenase, and NADH and NAD(P)H dehydrogenases was also seen. Basolateral plasmalemma of acinar cells showed weak, ouabain-sensitive Na⁺,K⁺-ATPase activity. Ductal cells were of a simple appearance, contained thiols and showed variable staining for acid phosphatase, dehydrogenases and cytochrome oxidase. Variable amounts of -glucuronidase reaction product were localized in the glandular parenchyma, being marked in atrophic areas. Prominent stellate myoepithelial cells embracing acini and also basal ductal cells were demonstrated by alkaline phosphatase. Thiamine pyrophosphatase reaction product was concentrated in blood vessels around parenchyma, with little Golgi-like staining in acinar cells. Acetylcholinesterase activity was associated with an extensive network of nerve fibres embracing parenchyma, whereas catecholamine fluorescence was not seen. The results suggest that the acini of von Ebner's gland of ferret synthesise neutral secretory glycoproteins and peroxidase. Water mobilization is inconspicuous. Lysosomal activities feature in the parenchyma, possibly a consequence of processing secretory products in acini, absorption in ducts and/or adaptation atrophy. The gland receives a rich cholinergic-type innervation, and has extensive myoepithelial and microvascularbreak networks.     

Some observations on the phosphatase cytochemistry of the submandibular gland of cat

Synopsis The localization of reaction product arising from incubation with thiamine pyrophosphate at pH 7.2 and -glycerophosphate or nitrophenylphosphate at pH 5 has been examined in central and demilunar acinar cells, myoepithelial cells, and cells of the striated ducts. Thiamine pyrophosphate reaction product was seen in the inner saccules of the Golgi apparatuses of the different types of cell. -Glycerophosphate and nitrophenylphosphate reaction products were seen in lysosomes, and in GERL-like structures in all except the cells of the striated ducts, in which the reaction products were observed in the Golgi apparatus. In central cells, the latter two reaction products were also seen in secretory granules and the cytosol.     

Electron microscopic localization of 5′-nucleotidase in rat salivary glands

Summary The localization of 5-nucleotidase in rat parotid and submandibular glands was investigated at the electron microscope level by an immunohistochemical technique using a highly specific antibody, and the results were compared with those obtained using the newley developed cerium method for enzyme histochemistry. Both methods demonstrated that 5-nucleotidase is located on the external surface of the luminal plasma membranes of acinar cells as well as on intercalated and striated ductal cells. In the basolateral membranes of these cells, the portions adjacent to myoepithelial cells exhibited intense reaction products, but the other areas of plasma membranes contained only trace amounts of the reaction products. Both cerium-based enzyme histochemistry and immunohistochemistry showed that myoepithelial cells retain the enzyme on their plasma membranes. Neither method produced reaction products in the intracytoplasmic structure of constitutive cells of the salivary glands. We discuss the usefulness of the cerium-ion method for the demonstration of 5-nucleotidase activity and compare it with the traditional lead-ion method.     

Morphometric and fine-structural studies of rat pancreatic acinar cells during early postnatal life

Summary Pancreatic acinar cells of rats obtained at 1,2, 3, 5, 7 and 14 days of age were examined using fine structural and morphometric techniques. From 5 days of age onwards, the acinar cells were analysed twice per day, at 20.00 h and 08.00 h.The present study demonstrates changes in the average volume of the cell, nucleus and cytoplasm, and volume densities of various cytoplasmic organelles during the first two weeks after birth. During early postnatal life, the volume density of rER increases, whereas that of zymogen granules decreases. From 5 days of age onwards, the volume densities of these two organelles differ significantly at 20.00 h and 08.00 h. During the first 2–3 days after birth, inclusion body-like structures appear in the cytoplasm of acinar cells; they contain aggregated zymogen granules and, sometimes, amorphous structures or cytoplasmic organelles. These structures also occur in interstitial cells and cells located in the intercalated region between acinar and ductal epithelial cells. Serum level of -amylase activity is high at birth, compared with other stages during the first three weeks. Degenerating acinar cells and cell debris can be seen in the acinar and ductal lumina during these stages, a feature suggesting holocrine secretion. Cellular polarity appears to be incomplete during the first two or three days after birth.     

Localisation of esteroproteases in ‘resting’ salivary glands from different species and the effects of the organophosphorus inhibitor E600

Summary The distribution of reaction sites for esteroproteases has been assessed in resting salivary glands from rats, guinea-pigs, cats, dogs and rabbits using the new substrate N-acetyl-l-methionine -naphthyl ester (Lexow et al. 1979). Two main types of activity were observed in the parenchyma. 1) Condensed staining was present on the luminal sides of certain ducts in some, but not all, glands, and this type of staining was always most conspicuous in the submandibular glands of each species. It is likely that this periluminal esteroprotease activity arises from secretory enzymes that will pass into the saliva. Their greater presence in submandibular glands may have association with specialised licking activities. 2) Variable diffuse cytoplasmic staining was present in certain acinar cells from some, but not all, glands. This was most pronounced in the mucous cells of zygomatic glands from dogs. It is considered that the enzymes demonstrated in acinar cells are involved in processing secretory products rather than in being secretory themselves. The possibility that this includes signal peptidase is discussed. Ductal activity in rats, cats and guinea-pigs had some relationship with sites of tryptophan staining but not in rabbits or dogs. Any relationship with acinar staining was less evident. Mast cells, showing strong esteroprotease staining, were present in variable numbers in the different glands. Preliminary attempts to qualify the enzymes being demonstrated were made by using the inhibitor E600. This substance inhibited all activity in acinar cells from each species, and in rabbits and dogs it also inhibited the ductal activity as well. However, in rats and guinea-pigs it had little or no effect on the ductal activity and in cats it had only a small inhibitory effect on the ductal activity. E600 had no obvious inhibitory effects on mast cell activity.We are indebted to Professor O. von Deimling for the substrate, N-acetyl-l-methionine -naphthyl ester, used in this investigation     

Establishment and characterization of a new,spontaneously immortalized,pancreatic ductal cell line from the Syrian golden hamster

Spontaneously immortal pancreatic cell lines are not available. By use of a defined culture medium, such a line (TAKA-1) was established from the Syrian golden hamster. Cytological, cytogenetic, molecular biological, enzymatic and receptor patterns as well as antigenicity were studied and were compared with those of the normal hamster pancreatic ductal cells in vivo. TAKA-1 cells grew exponentially in a monolayer on collagen gel in a defined medium but did not proliferate in soft agar. Ultrastructurally, the cells closely resembled the normal hamster pancreatic ductal cells. Similarities and dissimilarities were found between the normal ductal cells and TAKA-1 cells. Similarities included the presence of cytokeratin, carbonic anhydrase and some tumor-associated antigens. However, unlike the normal ductal cells, TAKA-1 cells expressed blood group A angigen and anti-vimentin, showed affinity to selected lectins, and an abnormality of chromosome 3, which is suggested to be associated with immortality. Moreover, unlike the hamster pancreatic ductal cancer cells but like the normal hamster pancreatic ductal cells, TAKA-1 cells did not have a c-Ki-ras mutation. EGF, TGF- and secretin, but not CCK or GRP, bound to the TAKA-1 cells. TAKA-1 cells produced TGF-, and their growth was stimulated by exogenous EGF in serum-free medium. This cell line presents a suitable model for biologic and pathologic study of the hamster pancreatic ductal cells in vitro.     

Cytochemical studies on the localization of 5′-nucleotidase in the acinar cells of the rat salivary glands

Summary The cytochemical localization of 5-nucleotidase (5-AMPase), and its validity, were investigated in parotid and submandibular acinar cells of a rat. Biochemical determinations showed that adequate treatment with glutaraldehyde could minimize the loss of enzymatic activity, and that 5-AMPase and non-specific alkaline phosphatase (-GPase) possessed different pH optima.The cytochemical distribution of the reaction products from the 5-AMPase activity was distinct from those of -GPase. 5-AMPase activity was localized on the surface membranes of acinar, ductal and myoepithelial cells of both salivary glands. -GPase activity was evenly distributed on the entire plasma membranes of myoepithelial cells and on the basal plasmalemma of acinar cells. The reaction products, which appeared on the luminal and lateral plasma membranes of the acinar cells, were presumed to reflect the presence of 5-AMPase, while those on the myoepithelial surface and basal plasma membranes of the acinar cells demonstrated both 5-AMPase and -GPase.The results indicate that 5-AMPase activity can be utilized as a reliable marker enzyme of plasma membranes in the salivary acinar cells.     

Akt (protein kinase B) isoform phosphorylation and signaling downstream of mTOR (mammalian target of rapamycin) in denervated atrophic and hypertrophic mouse skeletal muscle

Background

The present study examines the hypothesis that Akt (protein kinase B)/mTOR (mammalian target of rapamycin) signaling is increased in hypertrophic and decreased in atrophic denervated muscle. Protein expression and phosphorylation of Akt1, Akt2, glycogen synthase kinase-3beta (GSK-3beta), eukaryotic initiation factor 4E binding protein 1 (4EBP1), 70?kD ribosomal protein S6 kinase (p70S6K1) and ribosomal protein S6 (rpS6) were examined in six-days denervated mouse anterior tibial (atrophic) and hemidiaphragm (hypertrophic) muscles.

Results

In denervated hypertrophic muscle expression of total Akt1, Akt2, GSK-3beta, p70S6K1 and rpS6 proteins increased 2?C10 fold whereas total 4EBP1 protein remained unaltered. In denervated atrophic muscle Akt1 and Akt2 total protein increased 2?C16 fold. A small increase in expression of total rpS6 protein was also observed with no apparent changes in levels of total GSK-3beta, 4EBP1 or p70S6K1 proteins. The level of phosphorylated proteins increased 3?C13 fold for all the proteins in hypertrophic denervated muscle. No significant changes in phosphorylated Akt1 or GSK-3beta were detected in atrophic denervated muscle. The phosphorylation levels of Akt2, 4EBP1, p70S6K1 and rpS6 were increased 2?C18 fold in atrophic denervated muscle.

Conclusions

The results are consistent with increased Akt/mTOR signaling in hypertrophic skeletal muscle. Decreased levels of phosphorylated Akt (S473/S474) were not observed in denervated atrophic muscle and results downstream of mTOR indicate increased protein synthesis in denervated atrophic anterior tibial muscle as well as in denervated hypertrophic hemidiaphragm muscle. Increased protein degradation, rather than decreased protein synthesis, is likely to be responsible for the loss of muscle mass in denervated atrophic muscles.     

Downregulation of TRAF2 Mediates NIK-Induced Pancreatic Cancer Cell Proliferation and Tumorigenicity

Background

Increased levels of NF-κB are hallmarks of pancreatic ductal adenocarcinoma (PDAC) and both classical and alternative NF-κB activation pathways have been implicated.

Methodology/Principal Findings

Here we show that activation of the alternative pathway is a source for the high basal NF-κB activity in PDAC cell lines. Increased activity of the p52/RelB NF-κB complex is mediated through stabilization and activation of NF-κB-inducing kinase (NIK). We identify proteasomal downregulation of TNF receptor-associated factor 2 (TRAF2) as a mechanism by which levels of active NIK are increased in PDAC cell lines. Such upregulation of NIK expression and activity levels relays to increased proliferation and anchorage-independent growth, but not migration or survival of PDAC cells.

Conclusions/Significance

Rapid growth is one characteristic of pancreatic cancer. Our data indicates that the TRAF2/NIK/NF-κB2 pathway regulates PDAC cell tumorigenicity and could be a valuable target for therapy of this cancer.     

Mitogen-activated protein kinase up-regulation and activation during rat parotid gland atrophy and regeneration: role of epidermal growth factor and beta2-adrenergic receptors

Abstract The rat secretory ductal obstruction model has been widely used to assess salivary gland injury, growth, and differentiation. In this study, a novel ductal obstruction and release procedure was used to explore the signaling pathways leading to salivary gland regeneration. Rats underwent bilateral parotid ductal obstruction in which the duct was occluded against a plastic disk subcutaneously and released by external ligature removal. This ductal obstruction/release procedure was validated to produce glandular atrophy and regeneration with histological analysis and periodic acid-Schiff staining. Immunoblot analysis indicated that during ductal obstruction and the early post-release period (day 7), expression of immunoreactive proliferating cell nuclear antigen and vimentin was increased in the parotid glands compared with sham-operated animals. Immunohistochemical staining and immunoblots revealed up-regulation of the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated receptor kinase (ERK)1/2, and p38 during the atrophic and regeneration phases of ductal obstruction/release. Similarly, increases in activated, i.e., phosphorylated, ERK1/2 (pERK1/2) and p38 (phospho-p38) were demonstrable in both ductal and recovering acinar cells, with pERKs expressed predominantly in the nuclei and phospho-p38 distributed throughout the cells. Furthermore, levels of epidermal growth factor (EGF) receptor and β2-adrenergic receptor (β2-AR) were elevated in the ligated glands and at day 7 post-release; β1-AR levels did not change over the same time period. These results support the view that cell proliferation is involved in duct ligation-induced atrophy of the rat parotid gland and gland recovery upon ligature removal. Up-regulation of ERKs and p38, and the activation of these MAPKs by up-regulated EGF and β2-ARs, may be important signaling components underlying glandular atrophy and subsequent regeneration.     

The cell-mediated immune reaction in the cutaneous lesion of Chromoblastomycosis and their correlation with different clinical forms of the disease

Chromoblastomycosis is a fungal infection caused by dematiaceous fungi inducing skin lesions of difficult treatment and of frequent recurrence. The objective of the present investigation was to characterize cell-mediated tissue reactions in the skin in cases of Chromoblastomycosis using histopathology and immunocytochemistry methods and to correlate them with different clinical forms of Chromoblastomycosis. Biopsies from 19 patients were stained with HE and Giemsa, and serial sections were immunohistochemically stained using CD45RO, CD20, CD4, CD8, CD68, CD1a, CD34, IL4, IL10, TNF- and IFN- antibodies. A quantitative and semi quantitative analysis of the cell subsets and cytokines in the inflammatory infiltrates was performed by counting ten high-power fields (400×). The cutaneous lesion presented as verrucous plaque (n = 15) or erythematous atrophic plaque (n = 4). We observed two types of tissue reaction: A) a granulomatous reaction with a suppurative granuloma with several fungi cells in the cutaneous lesion presenting as verrucous plaque; B) a granulomatous reaction with a tuberculoid granuloma with few fungi cells in the cutaneous lesion presenting as atrophic plaque. The data obtained suggest that patients with lesion presented as verrucous plaque have a type Th2 immunological response, while patients with lesion presented as erythematous atrophic plaque have a type Th1 response.This revised version was published online in October 2005 with corrections to the Cover Date.     

Chilling,oxidative stress and antioxidant responses inArabidopsis thaliana callus

Chilling ofArabidopsis thaliana (L.) Heynh. callus tissue to 4 °C led to conditions of oxidative stress, as indicated by increased levels of the products of peroxidative damage to cell membranes. Cellular H₂O₂ was also observed to increase initially upon chilling but by day 8 cellular levels had declined to below control levels. Although levels of catalase activity remained similar to those in unchilled tissue, activity of ascorbate peroxidase increased between days 4 and 8 of chilling to 4 °C. In callus held at 23 °C, levels of reduced glutathione remained static whereas they rose in callus held at 4 °C. Levels of oxidised glutathione were initially low but increased significantly by day 4 in the chilled callus. At 23 °C, however, levels of oxidised glutathione remained low. Between days 1 and 3 at 4 °C, levels of glutathione reductase activity increased but by day 8 glutathione reductase activity was similar to that in cells held at 23 °C. Exposure of callus to abscisic acid at 23 °C also led to increased activities of ascorbate peroxidase and glutathione reductase.Abbreviations ABA abscisic acid - GSH reduced glutathione - GSSG oxidised glutathione - TTC 2,35-triphenyltetrazolium chloride This work is supported by a grant from the Biotechnology and Biological Sciences Research Council.     

HMGB1/autophagy pathway mediates the atrophic effect of TGF-β1 in denervated skeletal muscle

Background

Transforming growth factor beta 1 (TGF-β1) is a classical modulator of skeletal muscle and regulates several processes, such as myogenesis, regeneration and muscle function in skeletal muscle diseases. Skeletal muscle atrophy, characterized by the loss of muscle strength and mass, is one of the pathological conditions regulated by TGF-β1, but the underlying mechanism involved in the atrophic effects of TGF-β1 is not fully understood.

Methods

Mice sciatic nerve transection model was created and gastrocnemius were analysed by western blot, immunofluorescence staining and fibre diameter quantification after 2 weeks. Exogenous TGF-β1 was administrated and high-mobility group box-1 (HMGB1), autophagy were blocked by siRNA and chloroquine (CQ) respectively to explore the mechanism of the atrophic effect of TGF-β1 in denervated muscle. Similar methods were performed in C2C12 cells.

Results

We found that TGF-β1 was induced in denervated muscle and it could promote atrophy of skeletal muscle both in vivo and in vitro, up-regulated HMGB1 and increased autophagy activity were also detected in denervated muscle and were further promoted by exogenous TGF-β1. The atrophic effect of TGF-β1 could be inhibited when HMGB1/autophagy pathway was blocked.

Conclusions

Thus, our data revealed that TGF-β1 is a vital regulatory factor in denervated skeletal muscle in which HMGB1/ autophagy pathway mediates the atrophic effect of TGF-β1. Our findings confirmed a new pathway in denervation-induced skeletal muscle atrophy and it may be a novel therapeutic target for patients with muscle atrophy after peripheral nerve injury.

     

Metabolism of free and membrane-bound ribosomes during aging of Jerusalem artichoke tuber slices

Summary A biochemical and cytochemical study has been made of the distribution of -glycerophosphatase (EC 3.1.3.2) activity in mature and differentiating phloem cells of Nicotiana tabacum L. and the pH dependence and kinetics of -glycerophosphate hydrolysis of homogenates of fresh leaf midveins and midveins fixed in formaldehyde-gluteraldehyde. -glycerophosphatase showed two peaks of activity at pH 5.5 and 6.2. Enzyme saturation kinetics were exhibited by both fresh and fixed tissue homogenates. At a substrate concentration of 2 mM, 65% of the enzyme activity survived fixation. Specimens for cytochemical localization were incubated with 2 mM -glycerophosphate at pH 5.5 and 6.2. Specimens showed consistent patterns of reaction product deposition. Little or no reaction product was deposited in controls incubated without substrate or with substrate plus 0.01 M fluoride. -glycerophosphatase activity in the phloem and xylem is considerably higher than in surrounding tissue. Dense localization of reaction product was demonstrated on the vacuolar membranes, the inner membranes of mitochondria, and the dictysomes of phloem parenchyma and companion cells. The plasma membrane and endoplasmic reticulum cisternae of these cells were usually free of reaction products. Enzyme activity in mature sieve elements was associated with the parietal and stacked systems of endoplasmic reticulum and with the P-protein. There was inconsistency of staining of P-protein in mature sieve elements although the association of reaction products with the P-protein appeared to show a correlation with maturity and dispersal. The P-protein bodies of differentiating sieve elements showed no reaction product deposition. The distribution of -glycerophosphatase activity has been compared with that previously recorded for ATPase activity in the phloem of Nicotiana tabacum.     

Nucleosides 9: Design and synthesis of new 8-nitro and 8-amino xanthine nucleosides of expected biological activity

The coupling reaction of 1,3-dimethylxanthine (theophylline), 3-benzylxanthine and 3-benzyl-1-methylxanthine with 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose afforded the corresponding protected nucleosides, respectively. Nitration of each of the theophylline and 3-benzy-1-methyllxanthine protected nucleosides yielded the corresponding 8-nitronucleosides derivatives, which were reduced to give the corresponding 8-aminonucleoside derivatives.

Debenzoylation of protected nucleosides formed by using methanolic sodium methoxide afforded the corresponding free N-nucleosides, respectively. The structures of products have been elucidated and reported and also some of the products were screened for their antimicrobial activity. Some of tested products showed moderate activity.     

Neuronal and oligodendrocytic response to cortical injury: Ultrastructural and cytochemical changes

Summary A needle wound was made in the adult rat cerebral cortex. Responses of neurons and oligodendrocytes at the site of injury were followed over a period of 450 days and correlations made between morphological and enzyme cytochemical changes to clarify some phenomena previously unresolved.Evidence from acid phosphatase activity in degenerating neurons showed no increase in the number of cytochemically stained lysosomal profiles nor changes in the subcellular localization of the acid phosphatase reaction product. Our observations indicated that the majority of dying neurons were not digested by their own acid phosphatase autodigestion but by the process of heterodigestion. The time-course study revealed that not all the traumatized neurons were eliminated but some persisted permanently in an attenuated atrophic state. The atrophic neurons were small in size with low cytoplasmic-nuclear ratios and exhibited low levels of glucose-6-phosphatase and cytochrome oxidase activities. The acid phosphatase activity was slightly increased as evidenced by cytochemically stained hypertrophic Golgi cisternae and a slight increase in the number of lysosomes. The low level of enzyme activities, concerned with carbohydrate metabolism reflected the low metabolic activity in atrophic neurons whilst an increase in Golgi-lysosomal enzyme activity suggested some anabolic process necessary for their survival.Oligodendrocytes displayed only minor changes in morphology, and their glucose-6-phosphatase and cytochrome oxidase activities were normal, suggesting that these cells have little or no involvement in the repair of a cerebral wound. The absence of significant changes in lysosomal acid phosphatase activity indicated a minimal role, if any, of oligodendrocytes in the process of phagocytosis.     

Immunohistochemical study on the expression of c-Fos and c-Jun in human skin wounds

An immunohistochemical study on the temporal expression of c-Fos and c-Jun, both of which designate proto-oncogene products, was performed on 60 human skin wounds with different post-infliction intervals. In unwounded skin, c-Fos or c-Jun was immunolocalized at the nuclei of the epidermal cells in the basal layer, hair follicle cells and sweat gland cells. During the early inflammatory phase of wound healing, the nuclei of polymorphonuclear cells (probably neutrophils), mainly infiltrating at the wound site, were labeled with anti-c-Fos or -c-Jun antibody. As the wound age increased, the neutrophils had disappeared at the wound site, and both mononuclear cells (probably macrophages) and spindle-shaped fibroblastic cells, which expressed a c-Fos or c-Jun positive reaction in the nuclei, were mainly observed. Morphometrically, the distribution of the c-Fos-positive ratio was very similar to that of the c-Jun-positive ratio; the positive ratio was considerably increased in wound specimens with a post-infliction interval of 1 day, thus indicating the late inflammatory or proliferative phase. This study showed that c-Fos and c-Jun were closely involved in the inflammatory phase as well as the proliferative phase of the wound healing process.     

Studies on the histochemical “leucine aminopeptidase” reaction

Summary A correlative chemical and histochemical study on the leucyl--naphthyl-amide-splitting activity is presented.In the first part comparison is made between homogenates and smears of strain L cells and ELD ascites tumor cells. It was concluded that only the available enzyme activity could be visualized by histochemical means. The growing tumor cells appeared to have more activity available than the strain L cells. The intensity of the histochemical LNAse reaction bore no correlation to the total enzymatic activity extractable. Most of the available activity seemed to reside in lysosome-like structures. Cell damage by repeated freezing and thawing increased the amount of activity available to substrate interaction.In the second part a comparison is made between chemical and histochemical results in four types of progressively growing transplanted or induced mouse tumors. Chemical data showed the occurrence of a mixed pattern of LNA-splitting enzymes, among which the previously described metal-dependent group constituted the main component in the tumor cells. In all cases and types of tumors a positive histochemical LNAse reaction was noticed both in growing tumor cells as well as in different parts of the stromal compartment. The intracellular reaction in tumor cells seemed confined mainly to lysosomal-like bodies. A positive stromal reaction seemed associated with the destructive (cytolytic and collagenolytic) activity at the tumor periphery. The stromal LNAse reaction was probably due to enzymes partly different from the intracellular ones. Additional enzymatic terms, including a chymotrypsin-like and/or a carboxypeptidase activity, may originate from various host cells. These contributions may be greatly increased by accumulation of inflammatory host cells as previously noted by Hess and Mottet.     

Effects of the inhibitors of dynamics of cytoskeletal structures on the development of apoptosis induced by the tumor necrosis factor

Changes in cytoskeletal structures have been investigated during apoptosis of epithelial HeLa cells induced by tumor necrosis factor- (TNF-). Shape and surface cell activity were investigated by time-lapse video microscopy, and changes of the cytoskeletal structure were studied by immune fluorescent microscopy. Addition of TNF- to HeLa cell culture caused early disruption of the actin cytoskeleton and vinculin-containing focal contacts, keratin filaments, and microtubules. Rounding of cells, general blebbing, and nuclear fragmentation were observed at the terminal apoptotic stages. Actomyosin complex inhibitors, H7 and HA1077, suppressed blebbing (but not cell rounding) and activated the development of apoptosis. The latter suggests that in contrast to blebbing the general rounding does not depend on increased contractility of actomyosin cortex. These cytoskeletal inhibitors accelerated the development of apoptosis of HeLa cells and increased sensitivity of HeLa-Bcl-2 cells (transfected with DNA encoding antiapoptotic protein Bcl-2) to TNF-induced apoptosis. Damage of cytoskeletal structures significantly attenuated antiapoptotic activity of Bcl-2 in the HeLa-Bcl-2 cells. It is suggested that the stimulation of apoptosis by cytoskeletal inhibitors may be attributed to the altered distribution of cell organelles, especially, mitochondria.