Evolution of functional diversity in the cupin superfamily.

The cupin superfamily of proteins is among the most functionally diverse of any described to date. It was named on the basis of the conserved beta-barrel fold ('cupa' is the Latin term for a small barrel), and comprises both enzymatic and non-enzymatic members, which have either one or two cupin domains. Within the conserved tertiary structure, the variety of biochemical function is provided by minor variation of the residues in the active site and the identity of the bound metal ion. This review discusses the advantages of this particular scaffold and provides an evolutionary analysis of 18 different subclasses within the cupin superfamily.

CM101 treatment overrides tumor-induced immunoprivilege leading to apoptosis.

CM101, a bacterial polysaccharide exotoxin produced by group B Streptococcus (GBS), also referred to as GBS toxin, has been shown to target pathological neovasculature and activate complement (C3), thereby inducing neovascularitis, infiltration of inflammatory cells, inhibition of tumor growth, and apoptosis in murine tumor models. Data from refractory cancer patients in a Phase I clinical trial with CM101 indicated a similar mechanism of tumor-targeted inflammation. To further our understanding of the mechanism of action of CM101 as an antitumor agent, we examined the role of the inflammatory response in inducing tumor apoptosis in a normal mouse and tumor-bearing mouse model. The i.v. infusion of CM101 into B16BL-6 melanoma tumor-bearing mice elevated p53 mRNA in circulating leukocytes as measured by reverse transcription-PCR, and immunohistochemistry demonstrated infiltration and sequestration of leukocytes. Whole tumor lysates from excised tumors exhibited an increase in binding to the murine p21(Waf1/Cip1) derived p53 DNA binding sequence compared with control whole tumor lysates, in which minimal or no DNA binding was observed. CM101 infusion led to elevated levels of Fas protein within the tumors as well as a decrease in the expression of fas ligand (fasL). Furthermore, tumors were apoptotic as determined by terminal deoxynucleotidyl transferase-mediated nick end labeling and DNA fragmentation assays. Collectively, these data suggest that CM101 up-regulates p53 in tumor-infiltrating leukocytes, initiating a loss of tumor immunoprivilege and consequently rendering the tumor sensitive to Fas/fasL-mediated apoptosis. CM101 induced loss of tumor immunoprivilege through changes in the expression of leukocyte p53, tumor Fas and fasL coupled with neovascularitis and leukocyte infiltration, constitutes a plausible molecular pathway for tumor reduction observed in cancer patients.

Chromosome diminution in Ascaris suum. Two-fold increase of nucleosomal histone to DNA ratios during development.

The swine intestinal nematode, Ascaris suum, eliminates chromatin material from its primordial somatic cells during early embryogenesis. A technique for isolation of nuclei from pre- and post-diminution stage embryos has been developed and these isolated nuclei were used in investigations of nuclear events during diminution. The amount of DNA per nucleus determined by diphenylamine assays and isotope dilutions was 0.66 pg and 0.29 pg in pre- and post-diminution nuclei, respectively. Thus, A. suum loses 56% of its nuclear DNA during diminution. The loss of nuclear DNA enabled in vivo examination of histone to DNA ratios as a function of changes in DNA quantities. Ascaris histones were identified by acid extractability and tryptic fingerprint comparison with rat liver histones. Measurement of histone quantities was accomplished using linearity of Coomassie blue binding to histones separated in dodecyl sulfate gels. Ascaris nucleosomal histones levels were relatively constant in pre- and post-diminution nuclei. However, nucleosomal histone to DNA ratios approximately doubled during diminution.

Phase I study of the antineovascularization drug CM101.

CM101 is a bacterial polysaccharide that induces neovascular inflammation in malignant tumors. Fifteen patients with refractory malignancies received CM101 i.v. by a 15-min infusion every other day, three times in 1 week, at doses ranging from 1 unit (7.5 microgram)/kg to 5 units/kg. Serum was analyzed for anti-CM101 IgG and IgM weekly. Plasma levels of inflammatory cytokines, including tumor necrosis factor alpha, interleukin 8, interleukin 10, MIP-1alpha, and soluble E-selectin, were analyzed from -15 min to 12 h during each treatment. Dose-limiting toxicities, including grade IV dyspnea and arrhythmia, were encountered at the 5-unit/kg level. Toxicities occurred primarily within the first 12 h after therapy and included mild-to-moderate fever and chills, nausea, cough, headache, facial flushing, dyspnea, myalgias, and acute tumor-related pain. No patient developed detectable antibodies to CM101. All patients experienced marked time- and dose-dependent elevations in all cytokines studied. Three patients experienced tumor shrinkage. The results show that CM101 can be safely administered at doses that produce evidence for severe, and possibly tumor-specific, inflammation. Further study is necessary to better characterize the mechanism of action and determine the optimal dose and schedule of this new agent.

Functional studies on the anti-pathoangiogenic properties of CM101.

Group B streptococcus (GBS) isolated from human neonates diagnosed with sepsis and respiratory distress produces a polysaccharide exotoxin (CM101) which has been previously described as GBS toxin. CM101 infused i.v. into tumor-bearing mice causes rapid tumor neovascularitis, infiltration of inflammatory cells, inhibition of tumor growth and tumor apoptosis. CM101 has successfully completed phase I studies in refractory cancer patients with very encouraging results. We have now demonstrated a mechanism of action for CM101. Using a normal mouse tumor model, we have examined tumor and normal tissues which were harvested at 0, 5, 15, 30 and 60min post-infusion of either CM101 or dextran. We present evidence that CM101 is rapidly (within the first 5min) bound to the tumor neovasculature. Complement is activated by the alternative pathway (C3) and leukocytes start to infiltrate the tumor within the first 5min. Through RT-PCR and immunohistochemical techniques, we demonstrate that proinflammatory cytokines, interleukin-6 and tumor necrosis factor (TNF)-alpha, are up-regulated in infiltrating leukocytes and TNF receptor 2 is up- regulated in the targeted tumor neovasculature. Combined, these events constitute possible explanations for the observed pathophysiology of tumor ablation.

Cloning of a Schistosoma japonicum gene encoding a major immunogen recognized by hyperinfected rabbits.

A library of randomly sheared Schistosoma japonicum genomic DNA fragments was constructed in the bacteriophage expression vector lambda gt11. A portion of the library was screened with sera collected from rabbits 8 weeks after they were infected with 1000 cercariae. Four clones whose recombinant gene products react with the rabbit sera were purified to homogeneity. Clone SjIR-12A was chosen for detailed study because of its very intense reaction with the rabbit sera. SjIR-12A was found to encode part of a 70 kDa protein (Sj70) that is present in both soluble egg antigen (SEA) and soluble worm antigen preparations (SWAP). Western blot analysis suggests that Sj70 is the only SWAP component that is strongly immunoreactive with the rabbit sera. Rabbit antibodies that react with the SjIR-12A fusion protein were immunoaffinity purified and used to localize immunoreactive product to the nervous tissue of male and female adult worms, the dorsal and lateral tegument of male adult worms, and in eggs to the miracidial tegument and the area between the eggshell and miracidium. Southern hybridization analysis suggests there are approximately four copies of the Sj70 gene per haploid genome.

Characterization of a large gene family in Schistosoma japonicum that encodes an immunogenic miracidial antigen.

Three of eleven clones isolated from a genomic expression library of Schistosoma japonicum DNA using chronically infected human sera also react with chronically infected mouse sera. Characterization of these three clones showed that they contain different members of the same gene family. One clone contains two members of the gene family approximately 2 kb apart and in opposite orientation to each other. DNA sequence homologies between pairs of genes range from 98% to 99.5%. Southern hybridization results indicate there are approximately 40 copies of these genes per haploid genome. Sera from mice immunized with purified fusion protein detected immunoreactive products in the central ganglion and ciliated epidermal cells of miracidia.

Antisera against Periplaneta americana Cu,Zn-superoxide dismutase (SOD): separation of the neurohormone bursicon from SOD, and immunodetection of SOD in the central nervous system.

In an effort to characterize the insect molting hormone bursicon from the cockroach, Periplaneta americana, amino acid sequences with high identity of Cu,Zn-superoxide dismutase (SOD) of Drosophila virilis were identified. Antisera against a conserved region of SOD, and a sequence unique to Periplaneta SOD were produced and used to test whether bursicon might be a form of SOD. Western blots of one- and two-dimensional gels revealed that the dimeric form of SOD and bursicon have a similar molecular mass (30 kDa). The two proteins can be separated, however, according to their different isoelectric points. Bursicon is identified in two-dimensional gels by elution from four unique spots not labeled by the anti-SOD antisera. In sections of Periplaneta nerve cords the antisera labeled glial material surrounding neuronal somata close to the neural sheath. Bursicon, however, is contained in unique cell pairs in the ganglia of the ventral nerve cord. These neurons were labeled with new antisera produced against novel sequences of one of the four above-mentioned bursicon active spots. The results show unequivocally that SOD and bursicon are distinctly different proteins. Furthermore, the anti-SOD antisera provided a tool to isolate and sequence bursicon.

Detection of a circulating antigen in human schistosomiasis japonica using a monoclonal antibody.

A double antibody enzyme-linked immunosorbent assay (sandwich ELISA) was used for the detection of a circulating antigen from human schistosomiasis japonica infections. This assay involves the use of polyclonal rabbit Schistosoma japonicum soluble egg antigen (SEA) antiserum to bind circulating antigen and a monoclonal antibody (MabH4) to identify and quantify this antigen. Sera from 108 S. japonicum-infected patients (acute and chronic) were tested. Sera from 93 of 95 patients with chronic infection were positive for this antigen; sera from 12 of 13 patients with acute infections were also positive. Antigen was not detectable in control human sera. Sera from 35 chronic schistosomiasis patients were collected 6-12 months after praziquantel treatment. Circulating antigen was not detectable in the sera of 33 of these patients and was dramatically reduced in 2. This ELISA system may prove valuable in differentiating past and current infections.

The effect of praziquantel on the circulating antigen SJ 70 in murine schistosomiasis japonica.

A circulating schistosome 70 kDa antigen (SJ 70) has been detected in the sera of mice infected with Schistosoma japonicum. For the detection of this antigen, a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was employed. SJ 70 is first detected in the serum of S. japonicum-infected mice 4 weeks after infection. Antigen levels rise rapidly, plateau by 7 weeks after infection, and remain relatively unchanged for at least another 9 weeks. In mice infected with S. japonicum for 7 weeks and then treated with praziquantel (100 mg/kg body weight), there is a significant decrease in serum antigen levels within 2 weeks after treatment, and an almost complete disappearance of antigen from the serum 3-4 weeks after treatment.

Modulation of Schistosoma japonicum pulmonary egg granulomas with monoclonal antibodies.

We have examined the ability of various Schistosoma japonicum egg antigens to sensitize mice for subsequent secondary pulmonary egg granuloma formation. Further, we produced monoclonal antibodies (Mabs) specific for egg antigens and evaluated their abilities to modulate pulmonary granuloma formation and inhibit soluble egg antigen (SEA)-stimulated lymphocyte blastogenesis. A homogeneous, 140 Kd egg glycoprotein, SJe; 140-GP was nearly as effective as intact eggs in sensitizing for egg-focused pulmonary granuloma formation, while SEA, or the heterogeneous immunoaffinity (IA)-purified C10 antigens were ineffective. The in vivo administration of chronic infection serum (CIS) or Mabs reactive with SJe; 140-GP, to egg-sensitized/egg-challenged mice, modulated pulmonary granuloma formation. Two of these modulatory SJe; 140-GP-specific Mabs (1 A1-1 or 14B3-8), an IgG1, and an IgG3, respectively, did not alter the responses of SEA-stimulated lymph node cells from mice with acute or chronic schistosomiasis japonica. In contrast, another SJe; 140-GP-specific IgG3 Mab (7A6-5), CIS, immunoaffinity purified anti-SEA from CIS, or the non-SEA-binding fraction of CIS, all resulted in dose-related inhibition of SEA-induced proliferation. These data confirm the antibody-driven nature of some immunoregulatory networks in murine schistosomiasis japonica, and extend these observations to include certain Mabs. The immunogenic nature of SJe; 140-GP, and the modulatory and inhibitory activities of Mabs specific for this glycoprotein, indicate it may play a central role in granuloma formation and modulation in this infection.

Effects of anti-schistosomal chemotherapy on immune responses, protection and immunity. I. Changes in cellular and humoral responses.

The cellular and humoral immune responses of CF1 and C57BL/6 mice with schistosomiasis mansoni were evaluated before and after chemotherapeutic cure of their infections by praziquantel. Mice were infected for either 10 or 20 weeks prior to treatment and followed until 10 weeks after treatment. Peripheral blood eosinophilia, without concomitant general leukocytosis, was observed within 3 days of treatment and persisted for up to 4 weeks. By 6 and 10 weeks after treatment schistosomal-associated hepatosplenomegaly had greatly decreased. Delayed-type hypersensitivity to a soluble adult worm extract (SWAP) was modulated over 20 weeks of infection, and in C57BL/6 mice this modulation was alleviated by cure. In parallel studies of pulmonary egg granuloma formation, granuloma modulation was not effectively reversed. Antibodies against egg (SEA), cercarial (CAP) and adult worm (SWAP) extracts generally decreased by 10 weeks after chemotherapy of mice that were previously infected for 10 weeks. Mice infected for 20 weeks and then treated, generated increased levels of antibodies to SWAP and CAP by 10 weeks after treatment. Immunoglobulin isotypic analyses largely reflected the results of total antibody studies. These data demonstrate that the duration of infection prior to treatment is a determining factor in subsequent expression of immune reactivity, and provide the immunological background for experiments on resistance following chemotherapy of experimental murine schistosomiasis mansoni.

Detection of Trypanosoma cruzi with the polymerase chain reaction and in situ hybridization in infected murine cardiac tissue.

Chagas' disease is caused by the hemoflagellate protozoan Trypanosoma cruzi, which is predominantly found in South and Central America and Mexico. Although the parasite is present in the United States, confirmed cases of human disease are rare. The most serious manifestation of chronic Chagas' disease is a progressive inflammatory cardiomyopathy. However, T. cruzi has not been consistently demonstrated with histologic techniques in inflammatory cardiac lesions. In this study, we used both polymerase chain reaction (PCR) amplification of extracted DNA from hematoxylin and eosin-stained tissue scrapings, and in situ hybridization to detect the presence of T. cruzi in infected murine cardiac tissue sections. Three T. cruzi-specific DNA sequences were used: a 122-basepair (bp) sequence localized within the minicircle network (MCS), a 188-bp nuclear repetitive sequence (RS), and a 177-bp sequence within the open reading frame of a gene coding for a flagellar protein (FPS). We found that all three sequences are amplifiable from scrapings of murine cardiac tissue. The MCS and RS are detected at 0.167 and 0.24 amastigote DNA equivalents, while FPS is barely detected at 0.24 amastigote DNA equivalents. On the other hand, in situ hybridization with all three sequences allowed for the detection of T. cruzi amastigotes within the tissue. The MCS and FPS, however, consistently yielded a more intense signal. These results indicate that PCR and in situ hybridization may prove useful in establishing the prevalence of T. cruzi in human chagasic cardiomyopathy.

In vitro trypanocidal activity of tetraethylthiuram disulfide and sodium diethylamine-N-carbodithioate on Trypanosoma cruzi.

Tetraethylthiuram disulfide (disulfiram) (TETD) and sodium diethylamine-N-carbodithioate (DECD) were examined for their in vitro trypanocidal activity against Trypanosoma cruzi. Benznidazole (BNZ), the drug used clinically for the chemotherapy of Chagas' disease, was used as a positive control. Inhibition assays included evaluation against the epimastigote, trypomastigote, and amastigote forms. Tetraethylthiuram disulfide and its reductive metabolite DECD inhibited 64.6% and 69.7%, respectively, of epimastigotes at a concentration of 50 micrograms/ml after 72 hr and BNZ caused 69.1% inhibition at the same concentration. Both TETD and DECD were not as effective against tissue culture trypomastigotes as BNZ (TETD = 47.7%,; DECD = 46.1%; BNZ = 88.7%) at 50 micrograms/ml after 24 hr. However, TETD and DECD treatment of amastigote-infected 3T3 fibroblasts yielded 60 and 67% inhibition, respectively, as compared to BNZ with an inhibition of 62%. A possible mechanism of action of TETD and DECD is via interference with the essential metal metabolism of T. cruci. Since toxicity data for both TETD and DECD are available and both drugs are active against the parasite, these drugs are candidates for further study to determine if they are of potential clinical interest as a prophylactic or therapeutic agent against Chagas' disease.

Polymerase chain reaction amplification of three different Trypanosoma cruzi DNA sequences from human chagasic cardiac tissue.

Chagas' disease is caused by the hemoflagellate protozoan Trypanosoma cruzi. The most common, serious manifestation of Chagas' disease is a progressive inflammatory cardiomyopathy, which occurs decades after primary infection. The inability to consistently demonstrate T. cruzi by histologic techniques in inflammatory cardiac lesions has suggested that the parasites' persistence may not be required for the pathology of the chronic phase. In this report we further analyze the persistence and localization of T. cruzi DNA in the hearts of seven patients with chronic chagasic cardiomyopathy, along with four indeterminate patients and seven control patients seronegative for T. cruzi infection. In the seven patients with chronic chagasic cardiomyopathy, we extracted DNA from selected inflammatory foci-positive (IFP) and inflammatory foci-negative (IFN) areas of' hematoxylin and eosin-stained cardiac tissue. We then used polymerase chain reaction methodology to amplify three different T. cruzi sequences (a minicircle sequence [MCS], a satellite repetitive sequence [RS], and, a low copy number sequence within the gene coding for a flagellar protein [FPS]). The MCS was detected in approximately 100% of both the IFP and IFN areas analyzed. The RS was detected in 37.5% and 23% of the IFP and IFN areas, respectively (difference not statistically significant; P > 0.10, degrees of freedom = 1, G test of independence = 1.9522). The FPS was rarely detected (2%), and was only present in DNA extracted from IFP areas. The MCS was also detected in most indeterminate cases (none of whom had inflammatory lesions) although with a markedly diminished amplification signal relative to cardiomyopathy cases. The MCS was not amplified from the cardiac tissues from seronegative controls. These results suggest that the quantity of T. cruzi DNA persisting in hearts of patients with Chagas' disease correlates with cardiomyopathy, but may not be preferentially associated with inflammatory foci.

In vivo studies of cadmium-induced apoptosis in testicular tissue of the rat and its modulation by a chelating agent.

In vivo CdCl2-induced apoptotic DNA fragmentation in the testes of the male Wistar rat has been demonstrated on agarose gel. Characteristic DNA migration patterns (laddering) provide evidence of apoptosis (programmed cell death) in testicular tissue of rats administered CdCl2 at a level of 0.03 mmol/kg 48 h previously. Evidence that administration of an appropriate cadmium chelating agent within the first 24 h can suppress some or all of the apoptotic changes in testicular DNA has also been obtained for the first time. A greater reduction in apoptosis is observed as the interval between the administration of the cadmium and that of the chelating agent is shortened. Administration of monoisoamyl meso-2,3-dimercaptosuccinate (Mi-ADMS) to male Wistar rats given CdCl2 is effective in the modulation of the typically apoptotic DNA fragmentation and associated histopathologic injury when the antagonist is given within approximately 1 h after the CdCl2 exposure. When the antagonist is given at later times there is a progressively more pronounced degradation of the DNA into oligonucleotides as seen in the typical electrophoretic DNA ladder pattern found with apoptosis. There is also a progressive increase in histopathological tissue changes as the antagonist is administered at progressively greater intervals after the cadmium.

Chelating agent inhibition of Trypanosoma cruzi epimastigotes in vitro.

A number of chelating agents and some of their derivatives are as effective as, or superior to, benznidazole, the compound currently in clinical use, in the suppression of the reproduction of epimastigotes of Trypanosoma cruzi, the protozoa that causes Chagas' disease. All compounds were examined at a culture concentration of 5 micrograms/mL. The most effective compounds included N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine, sodium diethylamine-N-carbodithioate, piperidine-N-carbodithioate and several of its analogs, a number of other carbodithioates with two nonpolar groups on the nitrogen, and tetraethylthiuram disulfide, a prodrug of sodium diethylamine-N-carbodithioate and widely used in the treatment of alcoholism. The introduction of additional ionic or nonionic polar groups on the chelating molecule generally results in a loss of tyrpanocidal activity. Common commercially available chelating agents which exhibited no activity included D-penicillamine, meso-2,3-dimercaptosuccinic acid, and triethylenetetramine tetrahydrochloride. Dose-response data on the culture indicated that some of these compounds exhibited inhibition of Trypanosoma cruzi epimastigotes at concentrations as low as 0.625 microgram/mL. It is proposed that the mechanism of action of these compounds is based on their ability to interfere with the essential metal metabolism at intracellular sites of the epimastigote involving iron, copper, or zinc. The results also indicate that a certain degree of hydrophobicity may be necessary for the groups attached to the literal metal-bonding structure if the compounds are to successfully inhibit the epimastigotes of Trypanosoma cruzi. The development of antiprotozoal drugs which are chelating agents specifically designed to selectively disrupt the essential metal metabolism of Trypanosoma cruzi should furnish a new generation of drugs which can be used in the treatment of Chagas' disease.

An electrophoretic analysis of Schistosoma mansoni soluble egg antigen preparation.

Schistosoma mansoni soluble egg antigen (SEA) has been examined electrophoretically. Sodium dodecyl sulfate (SDS) electrophoresis of SEA reveals an extremely heterogeneous protein composition. At least 18-20 distinct bands stain with Coomassie blue and at least 6 bands stain with periodic acid Schiff (PAS). Four of the PAS-positive bands stain only faintly with Coomassie blue. The estimated molecular weight range for these proteins is between 16,000 and 200,000 daltons. An acid soluble fraction was isolated from SEA which contained 5 of the 6 glycoproteins. An immunoelectrophoretic analysis of SEA reveals at least 5 distinct precipitin arcs when developed with serum from mice infected with S. mansoni for 16 weeks.

Properties of glycogen synthase and phosphorylase from Biomphalaria glabrata (mollusca).

Glycogen synthase and phosphorylase were characterized from the cephalopedal region of the snail, Biomphalaria glabrata. Glycogen synthase exhibited increases and decreases in its activity ratio (-G6P/+G6P) under conditions that are known to cause conversion of the two forms of the enzyme from mammalian systems, implying that the snail's synthase also possesses interconvertible forms. Each form had a distinct pH optimum, with the G6P-independent form (synthase alpha) exhibiting maximum activity at pH 7.4, whereas the G6P-dependent form (synthase beta) had optimal activity at pH 8.3. Both synthase alpha and beta displayed classical Michaelis-Menten kinetics for the substrates UDP-glucose and glycogen, and the beta form displayed sigmoidal kinetics for its modulator, G6P. Only UDP-glucose could function as a glucosyl donor in the synthase-catalyzed reaction. ADP, GDP, UDP, and ATP were all competitive inhibitors of synthase alpha, although at varying degrees of efficiency. Glycogen phosphorylase also demonstrated interconversion of its two forms (alpha and beta), as evidenced by changes in its activity ratio (-AMP/+AMP). AMP elicited hyperbolic kinetics from this enzyme. Concentrations of KF above 20 mM were found to inhibit glycogen synthase alpha while stimulating phosphorylase beta, thus causing erroneous activity ratios for both enzymes.

In vitro effects of mannan and cytochalasin B on the uptake of horseradish peroxidase and [14C]sucrose by Trypanosoma cruzi epimastigotes.

Ultrastructural and quantitative studies were conducted to determine the in vitro effects of mannan and cytochalasin B (CB) on the transport of horseradish peroxidase (HRP) and [14C]sucrose by epimastigotes of Trypanosoma cruzi (strain Y). Time-dependent changes in HRP uptake were observed in cells incubated with the actin inhibitor CB. After 60 min incubation in CB, HRP and sucrose uptakes were inhibited by 48 +/- 15.4% and 16.5 +/- 3.96%, respectively. Morphological changed included HRP reaction product on the cell surface and a reduction in the number of HRP-positive reservosomes when compared to controls. After 120 min incubation, no inhibition was measured for either molecule. However, electron microscopy revealed HRP reaction product on the surface of the cells and in the cytosol. Also, perturbation of the plasma membrane was evident, suggesting that CB compromised the integrity of the plasma membrane, allowing HRP and sucrose to diffuse into the cytosol, giving misleading quantitative results. Mannan displayed a concentration-dependent inhibitory effect on HRP uptake but had little effect on sucrose uptake. Electron microscopic analysis revealed no change in the number of reservosomes per cell but reduction in the amount of HRP in reservosomes concomitant with mannan concentration. These results suggest that T. cruzi epimastigotes transport HRP by receptor-mediated and fluid-phase pinocytosis.