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.

Mutagenesis and kinetic studies of a plant cysteine proteinase with an unusual arrangement of acidic amino acids in and around the active site.

We report the cloning, overexpression, kinetic analysis, and modeling of the tertiary structure of an unusual plant cysteine proteinase. Ananain (EC 3.4.22.31), from Ananas comosus (pineapple) is distinguished from all other cysteine proteinases in the papain superfamily by having a unique combination of acidic amino acids. As well as lacking the acidic residue immediately preceding the active site histidine (position 158 in papain), it also lacks the extensive surface network of acidic residues that were postulated to compensate for the loss of charge at position 158 in mammalian cathepsins. Ananain has the fewest acidic residues, so far reported, of any plant cysteine proteinase, but two of the carboxyl residues (E50 and E35) postulated to have an enabling role in catalysis, the so-called "electrostatic switch", remain conserved. Comparisons of the kinetics of recombinant wild-type ananain with E50A and E35A mutants proves that these charged groups are not essential for catalysis. Hence this research does not confirm the presence of an electrostatic switch in this cysteine proteinase, and the role of acidic residues in the enhancement of catalytic competence in these enzymes is discussed in light of this new evidence.

Isolation and identification of the group B streptococcal toxin CM101 from infants with sepsis.

OBJECTIVE: To determine whether the group B streptococcal (GBS) polysaccharide exotoxin CM101, which induces a complement-activated cytokine-driven inflammatory response, is present in body fluids of infants with GBS disease. STUDY DESIGN: With a sandwich enzyme-linked immunosorbent assay, CM101 was measured in plasma, urine, and cerebrospinal fluid from newborn infants who were evaluated for possible infection and from older infants with culture-confirmed GBS disease. RESULTS: Urine from 11 newborn infants with culture-confirmed early-onset disease contained large amounts of CM101 (1.0 to 5.5 mg/48 h). Plasma concentrations were 62.6 +/- 10.5 microg/mL in these infants and were 69.0 +/- 21.2 microg/mL in 4 older infants with late-onset disease. Plasma CM101 concentrations did not correlate with indexes of illness severity, leukocyte counts, or interleukin-6 or interleukin-8 plasma concentrations. CM101 was present in cerebrospinal fluid of 5 infants with meningitis (8.4 +/- 1.6 microg/mL). CM101 was not found in control samples. CM101 isolated from urine had molecular weight and sugar composition similar to those obtained from GBS culture media, and they both elicited a comparable pathophysiologic response when infused intravenously in lambs. CONCLUSIONS: CM101 is present in infants with GBS disease, and it appears to be the same as CM101 obtained from GBS culture media.

Phytochrome regulation of phytochrome A mRNA levels in the model short-day-plant Pharbitis nil.

The exposure of dark-grown Pharbitis nil seedlings to continuous R induces a rapid decrease in PHYA mRNA abundance with a half-life of about 2 h. A 5 min R pulse also induces this decline, and the effect is partially reversible by subsequent FR irradiation, confirming that the regulation of expression is mediated via the Pfr form of a phytochrome. When de-etiolated seedlings are returned to darkness after a W photoperiod, PHYA mRNA slowly reaccumulates from 20% to 50% of the dark level within 24 h. The rate of reaccumulation is greatly accelerated by the removal of Pfr with a FR pulse, resulting in reaccumulation to 100% within approximately 11 h. Without FR irradiation PHYA mRNA expression remains fully repressed for at least 11 h after the end of the photoperiod, suggesting that the controlling Pfr is highly stable.

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.

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.

Ultrastructural effects of the chelating agent 1,10-phenanthroline on Trypanosoma cruzi epimastigotes in vitro.

The in vitro effects of the metal chelator 1,10-phenanthroline (OPHEN) on the ultrastructure of Trypanosoma cruzi epimastigotes were investigated. Epimastigotes treated with OPHEN display swelling and electron-dense deposits in the kinetoplast, mitochondrion, and cisternae of the endoplasmic reticulum. These morphological alterations are dose-dependent and first appear at an OPHEN concentration of 5.0 microg/ml. Analytical electron microscope examination indicates that the metallic portion of the electron-dense deposits is predominantly calcium.

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.

Cadmium-induced apoptosis in the urogenital organs of the male rat and its suppression by chelation.

Cadmium-induced apoptosis is shown to occur, in vivo, in several organs of the male Wistar rat urogenital system, 48 h after cadmium administration i.p. at a dose of 0.03 mmol/kg. Characteristic DNA fragmentation (as measured by an enzyme-linked immunosorbent-assay, ELISA) and histopathologically observed changes characteristic of apoptosis are found in the kidney, prostate, seminal vesicles, testes, and epididymis. TUNEL assay also demonstrates the apoptosis. Such changes are absent from bladder and vas deferens tissue. Timely administration of an appropriate chelating agent capable of reaching intracellular cadmium binding sites can suppress the processes leading to apoptosis. Administration of monoisomyl meso-2,3-dimercaptosuccinate (Mi-ADMS, 0.5 mmol/kg i.p.) to cadmium-treated rats is effective in greatly reducing typical histopathologic signs of apoptosis and the associated chromatin DNA fragmentation as revealed by ELISA when the antagonist is administered 1 h after cadmium. Administration of the chelating agent at law times results in greater degradation of DNA into oligonucleotides and more prominent histopathological evidence of apoptotic changes in the affected organs of the rat urogenital system. There is also a progressive increase in apoptotic changes indicated by TUNEL assay, as the antagonist is administered at progressively greater intervals after cadmium.

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.

Synthesis and in vitro trypanocidal activity of some novel iron chelating agents.

This report describes the syntheses and in vitro trypanocidal activity of a number of iron (III) chelators against epimastigotes of Trypanosoma cruzi. The compounds examined included a number of lipophilic N-alkyl derivatives of 2-ethyl- and 2-methyl-3-hydroxypyrid-4-ones, N,N'-bis(o-hydroxybenzyl)-(+/-)-trans-1,2-diaminocyclohexane, cyclotetrachromotropylene and four commercially available carboxy derivatives of pyridine, pyrazine, and pyarazole. Benznidazole, the drug clinically used in the treatment of Chagas' disease in humans, served as standard. All compounds were screened in vitro against Trypanosoma cruzi epimastigotes at 50 and 100 micrograms/ml for 72 h of exposure. At 100 micrograms/ml dosage, at least 4 compounds exhibited high epimastigote growth inhibition (65-69%) comparable to benznidazole (72%), whereas 9 compounds showed moderate to fair activity (53-64%) in the in vitro assay. At the lower concentration (50 micrograms/ml), the inhibitory activity of the best of these compounds was reduced significantly (39-48%) compared to the standard drug (59%). The activity of all the carboxylic acids remained in the lower range (4-25%). It is hypothesized that the enhanced activity of some of the compounds is due to their increased lipophilicity which enables them to successfully pass through the cellular membrane of Trypanosoma cruzi epimastigotes. The trypanocidal activities of the most effective compounds were significantly reduced when tested in the presence of added ferric ion.

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.

The in vitro effects of extracellular adenosine triphosphate on the ultrastructure of Trypanosoma cruzi epimastigotes.

Incubation for 24 h in culture medium containing 50 mM adenosine triphosphate (ATP) produces distinct alterations in the ultrastructure of Trypanosoma cruzi epimastigotes, most obvious of which is the formation of large membrane-bound vacuoles in the cytosol. These vacuoles become positive following exposure to the macromolecule horseradish peroxidase (HRP). After a 20-min chase in phosphate-buffered saline (PBS) the HRP-positive vacuoles begin to separate into discrete structures such that after a 60-min chase, obvious reservosomes are identifiable. It is hypothesized that extracellular ATP causes increased permeability of the epimastigote's plasma membrane, resulting in ionic fluxes that, in turn, interfere with the normal formation of reservosomes.

Inhibition of Trypanosoma cruzi epimastigotes in vitro by iron chelating agents.

The relative effectiveness of 20 iron chelating agents in suppressing the growth and multiplication of Trypanosoma cruzi epimastigotes has been examined in vitro. 1,2-Dimethyl-3-hydroxypyrid-4-one (L1) and several of its newly synthesised N-substituted analogs containing hydrophobic substituents were significantly more effective than deferoxamine, even though they possess only two donor sites for iron(III) while deferoxamine has six. Analogs with hydrophilic substituents were uniformly less active than L1 itself. Variations in effectiveness as the polarity of the compound is varied indicate that the ability to cross the cellular membrane is of critical importance in the determination of the in vitro trypanocidal activity of iron(III) chelating agents. A group of four tris(2-aminoethyl)amine based tris-imines were also screened, all of which had poor activity (0-28% inhibition). Among the other iron(III) chelating agents which showed a relatively high level of activity at 50 and 100 micrograms/ml were salicylhydroxamic acid (70 and 73% inhibition) and hydroxyurea (42 and 52% inhibition). N,N'-Di(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid and acetohydroxamic acid exhibited only slight activity at 50 and 100 micrograms/ml. The best of these iron(III) chelating agents were as effective against the epimastigote form at both 50 and 100 micrograms/ml (74-82% inhibition) as benznidazole (81% inhibition), the drug currently used in the clinic.

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.

Antibodies reactive to Trypanosoma cruzi epimastigotes or amastigotes express different idiotypic patterns if from patients with different clinical forms of Chagas' disease.

Antibodies (Abs) were purified from pooled sera of patients with either indeterminate (IND or I) or cardiac (CARD or C) Chagas' disease, on either epimastigote (EPI or E) or amastigote-enriched (AMAST or A) antigen (Ag) columns and their idiotypic (Id) expression examined. Anti-Id rabbit Abs were raised to the different preparations (E-IdI, E-IdC, A-IdI and A-IdC). Competitive ELISAs using anti-Ids were able to discriminate between IdI and IdC, disregarding Ag reactivity. E-IdI and A-IdI present different inhibitory abilities, as do E-IdC and A-IdC, but IdC always competes with IdI for anti-IdI comparably. In contrast, a 4-8-fold increase of IdI is required to compete in parallel with IdC for anti-IdC. Therefore, Ids from IND patients share only low levels of the Ids that are most characteristic of CARD patients. While some CARD Abs also express Ids in common with IND patients, these studies reveal that CARD Abs express some Ids that are characteristic to only CARD patients, and these Ids are present on Abs purified with either EPI or AMAST.

An ultrastructure study of the in vitro effects of L-leucine methyl ester and ammonium chloride on Trypanosoma cruzi epimastigotes.

Trypanosoma cruzi epimastigotes were subjected to the lysosomotropic agents L-leucine methyl ester and ammonium chloride to determine their effects on the ultrastructure of the parasite. The lysosomotropic agents applied to epimastigotes caused a time-dependent alteration in the morphology of the cells marked by a 5-fold increase in the number of lysosomes. Continued exposure to ammonium chloride caused slight disruption of the reservosomes. The amino acid ester, however, while causing the parasite to swell after prolonged exposure (e.g., 24 h), had little effect on the reservosomes, the kinetoplast, or even the mitochondrion. A specific inhibitor of cysteine proteinases provided some protection for lysosomes from the effects of the amino acid ester. Although it is agreed that reservosomes are similar to endosomes, no lysosomal fusion with the reservosomes was observed. Acid phosphatase activity was observed only in lysosomes.

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.