Search

Acinetobacter baumannii-induced nosocomial pneumonia has become a serious clinical problem because of high antibiotic resistance rates. Antimicrobial peptides (AMP) are an ideal alternative strategy due to their broad-spectrum of antimicrobial activity and low incidence of bacterial resistance. However, their application is limited by toxicity and stability in vivo. The present study used a mouse model to directly identify potential AMPs effective for treatment of A. baumannii-induced pneumonia. Fifty-eight AMPs were screened and two identified (SMAP-29 and TP4) to have prophylactic effects which prevented the death of mice with pneumonia. Furthermore, two TP4 derivatives (dN4 and dC4) were found to have therapeutic activity in pneumonia mouse models by peritoneal or intravenous administration. Both dN4 and dC4 also inhibited and/or eliminated A. baumannii biofilms at higher doses. Taken together, these data suggest the AMP derivatives dN4 and dC4 represent a potential treatment strategy for A. baumannii-induced pneumonia.

Several antimicrobial peptides (AMPs) have been developed for the treatment of infections caused by antibiotic-resistant microbes, but their applications are primarily limited to topical infections because in circulation they are bound and inhibited by serum proteins. Here we have found that some AMPs, such as TP4 from fish tilapia, and drugs, such as antipyretic ibuprofen, were bound by bovine serum albumin only in complex with α1-antitrypsin which is linked by disulfide bond. They existed in dimeric complex (2 albumin -2 α1-antitrypsin) in the bovine serum only at fetal stage, but not after birth. The hydrophobic residues of TP4 were responsible for its binding to the complex. Since bovine serum is a major supplement in most cell culture media, therefore the existence and depletion of active albumin/α1-antitrypsin complex are very important for the assay and production of biomolecules.

Antimicrobial peptides (AMPs) have been developed for the treatment of bacterial infections, but their applications are limited to topical infections since they are sequestered and inhibited in serum. Here we have discovered that the inhibition of AMPs by human serum was mediated through high-density lipoproteins (HDL) which are known to remove cholesterol from peripheral tissues. The susceptibility of AMPs to HDL varied depending on the degree of hydrophobicity of AMPs and their binding affinities to HDL. The phospholipids, such as phosphatidylcholine, of HDL were essential for AMP-binding. The dynamic binding interactions between AMPs and HDL were mediated through the hydrophobic interactions rather than by ionic strength. Interestingly, some AMPs, such as SMAP29, dissociated from the AMP-HDL complex and translocated to bacteria upon contact, while some AMPs, such as LL37, remained in complex with HDL. These results suggest that HDL binds AMPs and facilitates the translocation of them to the bacteria.

Medical devices are widely used in modern medicine, but the high prevalence of biomaterial-associated infections still presents a major problem. Especially problematic is the formation of biofilms that are tolerant to most antibiotics. In this report, antimicrobial peptides (AMPs) were driven into an amphipathic structure by anionic surfactant. To increase the coating efficacy and spectrum of antimicrobial activity, the AMPs were coated simultaneously with antibiotic, Polymyxin B, by surfactant onto polystyrene, silicone, polyurethane, and titanium which are commonly used with biomedical devices. These coated antimicrobials stably adhered to the substrate and were gradually released into urine and serum. They exhibited high bactericidal activity, but low cytotoxicity and hemolytic activity. Most importantly, the antimicrobials coated onto silicone tubing inhibited the planktonic growth of E. coli in mouse urine and also markedly prevented bacterial adherence to the bladder and the silicone tubing implanted in the bladder. These results provide a promising approach to circumvent catheter-associated infections due to bacterial adherence.

Medical devices are widely used in modern medicine, but their utilities are often limited by the biofilm formation of bacteria that are tolerant to most antibiotics. In this report, antimicrobial peptides (AMPs) were coated onto biomaterials by the aid of surfactant through hydrophobic interactions. To increase the coating efficiency, stability of AMPs in body fluids and spectrum of antimicrobial activity, pairs of AMPs were coated simultaneously onto various substrates, such as silicone, polyurethane and titanium, which are commonly used components of biomedical devices. These coated AMPs exhibited very low cytotoxicity and hemolytic activities because they were gradually released into urine or serum. The AMP pairs, such as T9W + SAAP159 and T9W + RRIKA, coated onto the silicone discs were able to inhibit in vitro bacterial adherence in urine. Most importantly, AMP pairs coated onto the silicone tubing by surfactant SDBS could prevent bacterial adherence to mouse bladder and the silicone tubing implanted within it. These results provide a promising approach towards circumventing urinary catheter-associated infections caused by bacterial adherence.

Objective: The mechanisms by which bariatric surgeries, including gastric bypass (GB) and sleeve gastrectomy (SG), achieve remission of type 2 diabetes mellitus (T2DM) and sustained weight reduction are unknown. We hypothesized that the novel anorexic hormone nesfatin-1 and another new hormone obestatin might contribute to the marked improvement in glycemic homeostasis and weight loss in diabetics after GB and SG. Methods: A hospital-based, prospective study was conducted. Overnight fasting plasma concentrations of nesfatin-1 and obestatin were analyzed in T2DM patients before surgery, and at 3 and 12 months after laparoscopic GB (n =12) and SG (n = 6). Results: At 12 months, reductions of body mass index (BMI), fasting blood glucose, and glycated hemoglobin were similar between GB and SG groups (P all > 0.05). Plasma nesfatin-1 levels in patients undergoing GB or SG significantly decreased after surgeries (P both < 0.05). In contrast, plasma obestatin concentrations significantly increased in patients after SG (P < 0.05) but without any alteration after GB. The alterations of plasma nesfatin-1 were significantly and negatively associated with the reduction of fasting blood glucose (P < 0.05) at 12 months after GB and SG. In the SG group, the reduction of nesfatin-1 significantly and positively correlated with the decrease of BMI (P < 0.05). Conclusions: GB and SG produce differential influences with regards to circulating nesfatin-1 and obestatin levels in non-morbidly obese, T2DM patients. Circulating nesfatin-1 may modulate glucose homeostasis in two surgical procedures, and participate in regulating body weight in SG.

Granulysin, a cationic protein expressed by human natural killer cells and cytotoxic T lymphocytes, is a mediator for drug-induced Stevens-Johnson syndrome and graft-versus-host disease. Some 15 kDa granulysin are processed into 9 kDa forms and sequestered in cytolytic granules, while others are constitutively secreted into body fluids. Both 9 and 15 kDa granulysin have been shown to be a serum marker for cell-mediated immunity. Furthermore, 15 kDa is able to activate monocyte differentiation. However, its antimicrobial properties have not been clearly addressed. Here, we report a novel method to prepare both the soluble 9 and 15 kDa granulysin and show that the 15 kDa form is more effective than the 9 kDa form in exerting specific antimicrobial activity against Pseudomonas aeruginosa within a range of few micromolars. We also show that the 15 kDa granulysin is able to hyperpolarize the membrane potential and increase membrane permeability of treated bacteria. Interestingly, the bactericidal activity and membrane permeability of the granulysins were markedly reduced at lower pH (pH 5.4) as a result of probable increase in hydrophobicity of the granulysins. Additionally, we’ve also shown the granulysin to inhibit biofilm formation by P. aeruginosa. These results suggest that the 15 kDa granulysin exhibits a novel mechanism in bacteria killing in a way that’s different from most antimicrobial peptides. Our novel granulysin preparation methodology will be useful for further study of action mechanisms of other antimicrobial, cytotoxic and immunomodulating properties in granulysin-mediated diseases.

Cationic antimicrobial peptides/proteins (AMPs) are important components of the host innate defense mechanisms against invading microorganisms. Here we demonstrate that OprI (outer membrane protein I) of Pseudomonas aeruginosa is responsible for its susceptibility to human ribonuclease 7 (hRNase 7) and alpha-helical cationic AMPs, instead of surface lipopolysaccharide, which is the initial binding site of cationic AMPs. The antimicrobial activities of hRNase 7 and alpha-helical cationic AMPs against P. aeruginosa were inhibited by the addition of exogenous OprI or anti-OprI antibody. On modification and internalization of OprI by hRNase 7 into cytosol, the bacterial membrane became permeable to metabolites. The lipoprotein was predicted to consist of an extended loop at the N terminus for hRNase 7/lipopolysaccharide binding, a trimeric alpha-helix, and a lysine residue at the C terminus for cell wall anchoring. Our findings highlight a novel mechanism of antimicrobial activity and document a previously unexplored target of alpha-helical cationic AMPs, which may be used for screening drugs to treat antibiotic-resistant bacterial infection.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening adverse drug reactions characterized by massive epidermal necrosis, in which the specific danger signals involved remain unclear. Here we show that blister cells from skin lesions of SJS-TEN primarily consist of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, and both blister fluids and cells were cytotoxic. Gene expression profiling identified granulysin as the most highly expressed cytotoxic molecule, confirmed by quantitative PCR and immunohistochemistry. Granulysin concentrations in the blister fluids were two to four orders of magnitude higher than perforin, granzyme B or soluble Fas ligand concentrations, and depleting granulysin reduced the cytotoxicity. Granulysin in the blister fluids was a 15-kDa secretory form, and injection of it into mouse skin resulted in features mimicking SJS-TEN. Our findings demonstrate that secretory granulysin is a key molecule responsible for the disseminated keratinocyte death in SJS-TEN and highlight a mechanism for CTL- or NK cell—mediated cytotoxicity that does not require direct cellular contact. Adverse drug reactions (ADRs) account for 6–7% of all hospital admissions and remain a major clinical problem 1 . Among them, SJS

Antimicrobial peptides (AMPs) are important components of the host innate defense mechanism against invading pathogens. Our previous studies have shown that the outer membrane protein, OprI from Pseudomonas aeruginosa or its homologue, plays a vital role in the susceptibility of Gram-negative bacteria to cationic α-helical AMPs (Y. M. Lin, S. J. Wu, T. W. Chang, C. F. Wang, C. S. Suen, M. J. Hwang, M. D. Chang, Y. T. Chen, Y. D. Liao, J Biol Chem 285:8985–8994, 2010, http://dx.doi.org/10.1074/jbc.M109.078725 ; T. W. Chang, Y. M. Lin, C. F. Wang, Y. D. Liao, J Biol Chem 287: 418–428, 2012, http://dx.doi.org/10.1074/jbc.M111.290361 ). Here, we obtained two forms of recombinant OprI: rOprI-F, a hexamer composed of three disulfide-bridged dimers, was active in AMP binding, while rOprI-R, a trimer, was not. All the subunits predominantly consisted of α-helices and exhibited rigid structures with a melting point centered around 76°C. Interestingly, OprI tagged with Escherichia coli signal peptide was expressed in a hexamer, which was anchored on the surface of E. coli , possibly through lipid acids added at the N terminus of OprI and involved in the binding and susceptibility to AMP as native P. aeruginosa OprI. Deletion and mutation studies showed that Cys1 and Asp27 played a key role in hexamer formation and AMP binding, respectively. The increase of OprI hydrophobicity upon AMP binding revealed that it undergoes conformational changes for membrane fusion. Our results showed that OprI on bacterial surfaces is responsible for the recruitment and susceptibility to amphipathic α-helical AMPs and may be used to screen antimicrobials.

Onconase, a cytotoxic ribonuclease from Rana pipiens, possesses pyroglutamate (Pyr) at the N-terminus and has a substrate preference for uridine-guanine (UG). To identify residues responsible for onconase's cytotoxicity, we cloned the rpr gene from genomic DNA and expressed it in Escherichia coli BL21(DE3). The recombinant onconase with Met at the N-terminus had reduced thermostability, catalytic activity and antigenicity. Therefore, we developed two methods to produce onconase without Met. One relied on the endogeneous E.coli methionine aminopeptidase and the other relied on the cleavage of a pelB signal peptide. The Pyr1 substitutional variants maintained similar secondary structures to wild-type onconase, but with less thermostability and specific catalytic activity for the innate substrate UG. However, the non-specific catalytic activity for total RNAs varied depending on the relaxation of base specificity. Pyr1 promoted the structural integrity by forming a hydrogen bond network through Lys9 in alpha1 and Val96 in beta6, and participated in catalytic activity by hydrogen bonds to Lys9 and P(1) catalytic phosphate. Residues Thr35 and Asp67 determined B(1) base specificity, and Glu91 determined B(2) base specificity. The cytotoxicity of onconase is largely determined by structural integrity and specific catalytic activity for UG through Pyr1, rather than non-specific activity for total RNAs.

Cytotoxic ribonucleases with antitumor activity are mainly found in the oocytes and early embryos of frogs. Native RC-RNase 4 (RNase 4), consisting of 106 residues linked with four disulfide bridges, is a cytotoxic ribonuclease isolated from oocytes of bullfrog Rana catesbeiana . RNase 4 belongs to the bovine pancreatic ribonuclease (RNase A) superfamily. Recombinant RC-RNase 4 (rRNase 4), which contains an additional Met residue and glutamine instead of pyroglutamate at the N terminus, was found to possess less catalytic and cytotoxic activities than RNase 4. Equilibrium thermal and guanidine-HCl denaturation CD measurements revealed that RNase 4 is more thermally and chemically stable than rRNase 4. However, CD and NMR data showed that there is no gross conformational change between native and recombinant RNase 4. The NMR solution structure of rRNase 4 was determined to comprise three α-helices and two sets of antiparallel β-sheets. Superimposition of each structure with the mean structure yielded an average root mean square deviation (RMSD) of 0.72(±0.14) A for the backbone atoms, and 1.42(±0.19) A for the heavy atoms in residues 3–105. A comparison of the 3D structure of rRNase 4 with the structurally and functionally related cytotoxic ribonuclease, onconase (ONC), showed that the two H-bonds in the N-terminal pyroglutamate of ONC were not present at the corresponding glutamine residue of rRNase 4. We suggest that the loss of these two H-bonds is one of the key factors responsible for the reductions of the conformational stability, catalytic and cytotoxic activities in rRNase 4. Furthermore, the differences of side-chain conformations of subsite residues among RNase A, ONC and rRNase 4 are related to their distinct catalytic activities and base preferences.

The Rana catesbeiana (bullfrog) ribonucleases, which belong to the RNase A superfamily, exert cytotoxicity toward tumor cells. RC-RNase, the most active among frog ribonucleases, has a unique base preference for pyrimidine-guanine rather than pyrimidine-adenine in RNase A. Residues of RC-RNase involved in base specificity and catalytic activity were determined by site-directed mutagenesis, k(cat)/K(m) analysis toward dinucleotides, and cleavage site analysis of RNA substrate. The results show that Pyr-1 (N-terminal pyroglutamate), Lys-9, and Asn-38 along with His-10, Lys-35, and His-103 are involved in catalytic activity, whereas Pyr-1, Thr-39, Thr-70, Lys-95, and Glu-97 are involved in base specificity. The cytotoxicity of RC-RNase is correlated, but not proportional to, its catalytic activity. The crystal structure of the RC-RNase.d(ACGA) complex was determined at 1.80 A resolution. Residues Lys-9, His-10, Lys-35, and His-103 interacted directly with catalytic phosphate at the P(1) site, and Lys-9 was stabilized by hydrogen bonds contributed by Pyr-1, Tyr-28, and Asn-38. Thr-70 acts as a hydrogen bond donor for cytosine through Thr-39 and determines B(1) base specificity. Interestingly, Pyr-1 along with Lys-95 and Glu-97 form four hydrogen bonds with guanine at B(2) site and determine B(2) base specificity.

Cytotoxic ribonucleases found in the oocytes and early embryos of frogs with antitumor activity are well-documented. RC-RNase 2, a cytotoxic ribonuclease isolated from oocytes of bullfrog Rana catesbeiana, consists of 105 residues linked with 4 disulfide bridges and belongs to the bovine pancreatic ribonuclease (RNase A) superfamily. Among the RC-RNases, the base preference for RNase 2 is UpG but CpG for RC-RNase 4; while RC-RNase possesses the base specificity of both UpG and CpG. Interestingly, RC-RNase 2 or 4 has much lower catalytic activity but only three-fold less cytotoxicity than RC-RNase. Here, we report the NMR solution structure of rRC-RNase 2, comprising three alpha-helices and two sets of antiparallel beta-sheets. The differences of side-chain conformations of subsite residues among RNase A, RC-RNase, RC-RNase 4 and rRNase 2 are related to their distinct catalytic activities and base preferences. Furthermore, the substrate-related residues in the base specificity among native RC-RNases are derived using the chemical shift perturbation on ligand binding.

Multiple ribonucleases are widely found in living organisms, but the function and regulation of individual ribonucleases are still not clear. In the present study, we found that one oocytic ribonuclease, RC-RNase, is developmentally expressed in the liver and stored in the oocyte of the bullfrog, while another ribonuclease, RC-RNase L1, is constitutively expressed and retained in the liver at all stages. In females, the expression of RC-RNase increased with the degree of maturity and the concentration of plasma estradiol during oogenesis. In males, the RC-RNase gene was activated in the liver and the newly synthesized protein was secreted into plasma if estradiol was administered. To investigate the mechanism of estrogen-mediated activation of ribonuclease expression, we cloned the RC-RNase promoter and analyzed the putative transcription factor binding sites, e.g. TATA box, ERE, AP1 and CAAT box. Using luciferase as a reporter gene, we found that an estrogen response element in the promoter of RC-RNase was essential for both basic transcription and estradiol-mediated gene activation in estrogen receptor-positive MCF7 cells. These results support the hypothesis that RC-RNase is synthesized in the liver upon stimulation by estradiol during oogenesis, then secreted into the bloodstream and stored in oocytes for embryonic development.

Rana catesbeiana ribonuclease (RC-RNase) is a pyrimidine-guanine sequence-specific ribonuclease found in R. catesbeiana (bullfrog) oocytes. It possesses both ribonuclease activity and cytotoxicity against tumor cells. We report here for the first time the cloning of RC-RNase cDNA from liver rather than from oocytes where RC-RNase is stored. An internal fragment of cDNA was obtained by reverse transcription-PCR using deduced oligonucleotides as primers. Full-length cDNA was obtained by 5′- and 3′-RACE technique. The cDNA clone, named rcr gene, contained a 5′-untranslated region, a putative signal peptide (22 amino acids), a mature protein (111 amino acids), a 3′-untranslated region, and a polyadenylation site. The cDNA which encoded the mature protein was fused upstream with a modified pelB signal peptide DNA and inserted into pET11d for expression in Escherichia coli strain BL21(DE3). The secretory RC-RNase in the culture medium was enzymatically active and was purified to homogeneity. The recombinant RC-RNase had the same amino acid sequence, specific activity, substrate specificity, antigenicity, and cytotoxicity as that of native RC-RNase from frog oocytes. Amino acid residues His-10, Lys-35, and His-103 are involved in RC-RNase catalytic activity. Ribonucleolytic activity was involved in and may be essential for RC-RNase cytotoxicity. DNA sequence analysis showed that RC-RNase had approximately 45% identity to that of RNase superfamily genes. This indicates that RC-RNase is a distinct ribonuclease gene in the RNase superfamily.

Obestatin, which is a putative 23-amino-acid peptide, is derived from the C-terminal part of the mammalian preproghrelin gene. Nesfatin-1 mRNA is co-expressed with ghrelin in gastric endocrine X/A-like cells; therefore, nesfatin-1 may also interact with preproghrelin gene products in the stomach. In this study, we investigated the impact of obestatin on the plasma levels of acyl ghrelin, des-acyl ghrelin and nesfatin-1, and on the gastric emptying of a solid nutrient meal 2 h after an intracerebroventricular (ICV) injection in conscious, fasted rats. The rats were implanted with ICV catheters. Plasma levels of acyl ghrelin, des-acyl ghrelin and nesfatin-1, expected to be co-expressed with obestatin, were measured, whereas the human/rat corticotropin-releasing factor (h/rCRF) was applied as an inhibitor of gastric emptying. The ICV administration of obestatin (0.1, 0.3 and 1.0 nmol/rat) did not modify the plasma acyl ghrelin and des-acyl ghrelin levels, the acyl ghrelin/des-acyl ghrelin ratio and nesfatin-1 concentrations. The ICV acute administration of obestatin had no influence on the 2-h rate of gastric emptying of a solid nutrient meal, but the ICV h/rCRF injection delayed it. The weight of food ingested 1 h before ICV injection significantly, but negatively correlated with the gastric emptying of a solid nutrient meal. Our study indicates that the ICV injection of obestatin does not change the 2-h rate of gastric emptying of a solid nutrient meal and the relatively weak interrelationships between ghrelin gene products and nesfatin-1. However, the weight of the ingested food negatively affects the gastric emptying of a solid nutrient meal in conscious, fasted rats.

Antimicrobial peptides/proteins (AMPs) are important components of the host innate defense mechanisms. Here we demonstrate that the outer membrane lipoprotein, Lpp, of Enterobacteriaceae interacts with and promotes susceptibility to the bactericidal activities of AMPs. The oligomeric Lpp was specifically recognized by several cationic α-helical AMPs, including SMAP-29, CAP-18, and LL-37; AMP-mediated bactericidal activities were blocked by anti-Lpp antibody blocking. Blebbing of the outer membrane and increase in membrane permeability occurred in association with the coordinate internalization of Lpp and AMP. Interestingly, the specific binding of AMP to Lpp was resistant to divalent cations and salts, which were able to inhibit the bactericidal activities of some AMPs. Furthermore, using His-tagged Lpp as a ligand, we retrieved several characterized AMPs, including SMAP-29 and hRNase 7, from a peptide library containing crude mammalian cell lysates. Overall, this study explores a new mechanism and target of antimicrobial activity and provides a novel method for screening of antimicrobials for use against drug-resistant bacteria.

RC-RNases are ribonucleases from Rana catesbeiana oocytes with pyrimidine-guanine sequence specificity. They also possess cell cytotoxicity and lectin activity. Protein crystals of three RC-RNase isozymes, RC-RNase 3, RC-RNase 4 and RC-RNase 6, were grown in various crystal systems under different conditions. Crystals of RC-RNase3 belong to the orthorhombic C222(1) space group, with unit-cell parameters a = 66.66, b = 97.38, c = 85.74 A. Crystals of RC-RNase 4 belong to the trigonal space group P3(1) or P3(2), with unit-cell parameters a = b = 32.22, c = 92.12 A. Crystals of RC-RNase 6 complexed with cytidylyl 2'-5' guanosine belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 61.80, c = 65.96 A.

Many proteins and bioactive peptides contain an N-terminal pyroglutamate residue (Pyr1). This residue reduces the susceptibility of the protein to aminopeptidases and often has important functional roles. The antitumor ribonuclease RC-RNase 3 (RNase 3) from oocytes of Rana catesbeiana (bullfrog) is one such protein. We have produced recombinant RNase 3 containing the N-terminal Pyr1 (pRNase 3) and found it to be indistinguishable from the native RNase 3 by mass spectrometry and a variety of other biochemical and immunological criteria. We demonstrated by NMR analysis that the Pyr1 of pRNase 3 forms hydrogen bonds with Lys9 and Ile96 and stabilizes the N-terminal alpha-helix in a rigid conformation. In contrast, the N-terminal alpha-helix becomes flexible and the pKa values of the catalytic residues His10 and His97 altered when Pyr1 formation is blocked by an extra methionine at the N terminus in the recombinant mqRNase 3. Thus, our results provide a mechanistic explanation on the essential role of Pyr1 in maintaining the structural integrity, especially at the N-terminal alpha-helix, and in providing the proper environment for the ionization of His10 and His97 residues for catalysis and cytotoxicity against HeLa cells.

The removal of N-terminal translation initiator Met by methionine aminopeptidase (MetAP) is often crucial for the function and stability of proteins. On the basis of crystal structure and sequence alignment of MetAPs, we have engineered Escherichia coli MetAP by the mutation of three residues, Y168G, M206T, Q233G, in the substrate-binding pocket. Our engineered MetAPs are able to remove the Met from bulky or acidic penultimate residues, such as Met, His, Asp, Asn, Glu, Gln, Leu, Ile, Tyr, and Trp, as well as from small residues. The penultimate residue, the second residue after Met, was further removed if the antepenultimate residue, the third residue after Met, was small. By the coexpression of engineered MetAP in E. coli through the same or a separate vector, we have successfully produced recombinant proteins possessing an innate N terminus, such as onconase, an antitumor ribonuclease from the frog Rana pipiens. The N-terminal pyroglutamate of recombinant onconase is critical for its structural integrity, catalytic activity, and cyto-toxicity. On the basis of N-terminal sequence information in the protein database, 85%–90% of recombinant proteins should be produced in authentic form by our engineered MetAPs.

We present sequences of five novel RNase A superfamily ribonuclease genes of the bullfrog, Rana catesbeiana. All five genes encode ribonucleases that are similar to Onconase, a cytotoxic ribonuclease isolated from oocytes of R. pipiens. With amino acid sequence data from 14 ribonucleases from three Rana species (R. catesbeiana, R. japonica, and R. pipiens), we have con- structed bootstrap-supported phylogenetic trees that re- organize these ribonucleases into five distinct lineages— the pancreatic ribonucleases (RNases 1), the eosinophil- associated ribonucleases (RNases 2, 3, and 6), the ribonucleases 4, the angiogenins (RNases 5) and the Rana ribonucleases—with the Rana ribonucleases no more closely related to the angiogenins than they are to any of the other ribonuclease lineages shown. Further phylogenetic analysis suggests the division of the Rana ribonucleases into two subclusters (A and B), with posi- tive (Darwinian) selection (dN/dS > 1.0) and an elevated rate of radical nonsynonymous substitution (dR) contrib- uting to the rapid diversification of ribonucleases within each cluster. This pattern of evolution—rapid diversifi- cation via positive selection among sequences of a mul- tigene cluster—bears striking resemblance to what we have described for the eosinophil-associated ribonucle- ase genes of the rodent Mus musculus,a finding that may have implications with respect the physiologic function of this unique family of proteins.

Ribonucleases with antitumor activity are mainly found in the oocytes and embryos of frogs, but the role of these ribonucleases in frog development is not clear. Moreover, most frog ribonuclease genes have not been cloned and characterized. In the present study, a group of ribonucleases were isolated from Rana catesbeiana (bullfrog). These ribonucleases in mature oocytes, namely RC-RNase, RC-RNase 2, RC-RNase 3, RC-RNase 4, RC-RNase 5 and RC-RNase 6, as well as liver-specific ribonuclease RC-RNase L1, were purified by column chromatographs and detected by zymogram assay and western blotting. Characterization of these purified ribonucleases revealed that they were highly conserved in amino acid sequence and had a pyroglutamate residue at their N-termini, but possessed different specific activities, base specificities and optimal pH values for their activities. These ribonucleases were cytotoxic to cervical carcinoma HeLa cells, but their cytotoxicities were not closely correlated to their enzymatic specific activities. Some other amino acid residues in addition to their catalytic residues were implicated to be involved in the cytotoxicity of the frog ribonucleases to tumor cells. Because the coding regions lack introns, the ribonuclease genes were cloned by PCR using genomic DNA as template. Their DNA sequences and amino acid sequences are homologous to those of mammalian ribonuclease superfamily, approximately 50 and approximately 25%, respectively.

RC-RNase is a pyrimidine-guanine sequence-specific ribonuclease and a sialic-acid-binding lectin purified from Rana catesbeiana (bullfrog) oocytes. This 111-amino acid protein exhibits cytotoxicity toward several tumor cell lines. In this paper we report the assignments of proton NMR resonances and the identification of the secondary structure deduced from NOE constraints, chemical shift index, 3JNH alpha and amide proton exchange rates. The protein was directly isolated from bullfrog oocytes; we were able to assign all but five of the amino acid backbone protons of the unlabeled protein by analyzing a large set of two-dimensional proton NMR spectra obtained at several temperatures and pH conditions. Our results indicate that the structure of RC-RNase is dominated by the presence of two triple-stranded antiparallel beta-sheets and three alpha-helices, similar to those of the pyrimidine family ribonucleases. Two sets of resonances were observed for 11 amide protons and 8 alpha-protons located in the loop-1 region, an alpha 2 helix, and three beta-strands, (beta 1, beta 3 and beta 4), suggesting the presence of nonlocalized multiple conformations for RC-RNase.

Rana catesbeiana ribonuclease (RC-RNase) is a pyrimidine-guanine sequence-specific ribonuclease found only in R. catesbeiana (bullfrog) oocytes, but not in other organs. The protein is localized in the yolk granules of oocytes but not in other organelles, as detected by immunohistochemistry. More than 99% of RC-RNase was found in the yolk granule pellet when a mild separation method was employed under physiological conditions. The ribonuclease was purified by precipitation of yolk granules, extraction of RC-RNase with 0.09 M NaCl, selective removal of impurities by Hepes buffer, and chromatographies on phosphocellulose and carboxymethyl cellulose columns. Three milligrams of RC-RNase was purified from a 1-g pellet of yolk granules prepared from 2 g of ovary tissue. Therefore, 150 milligrams of RC-RNase could be obtained from a mature female bullfrog (600 g in weight) which had 100 g of ovary tissue. The properties of RC-RNase isolated from yolk granules tested so far are identical to those of RC-RNase isolated from the cytosolic fraction and similar to those of a sialic acid-binding lectin from bullfrog oocytes. To investigate the possible role of RC-RNase in the regulation of cell growth and differentiation during embryogenesis, its cytotoxic activity against various cell lines was examined. The degradation of ribosomal RNA was found in RC-RNase-treated HeLa cells. However, both events were not found in RNase A-treated HeLa cells. Therefore, RC-RNase is proposed to have both ribonucleolytic and cytotoxic activity and a specific receptor on the tumor cell surface is suspected to be involved in the recognition and binding, and possibly entry of RC-RNase.

Rana catesbeiana ribonuclease (RC-RNase) is a pyrimidine guanine sequence-specific ribonuclease found only in R. catesbeiana (bullfrog) oocytes, not in other organs. An immunohistochemical assay showed that RC-RNase was present in the regular yolk granules, but not in forming yolk granules, yolk platelets, pigment granules, mitochondria clouds or the nucleus. The RC-RNase was restricted to the lateral amorphous area of the yolk granules, and was absent from the central area that has a vitellogenin crystal lattice. The RC-RNase was extracted from yolk granules by 0.5 M NaCl and purified by dialysis and affinity chromatography. Most of the RC-RNase (94%) was found in the yolk granules, the rest RC-RNase (6%) was found in the cytosol in the form of free RNase and latent RNase. The RC-RNase extracted from yolk granules was further analyzed by immunoprecipitation and RNase activity assay on an SDS/polyacrylamide gel. Our results suggest that the RC-RNase activity is regulated by both compartmentation and inhibitor binding.

Ribonucleases are widely found in the tissues of living organisms, but the functions of individual ribonucleases are not clear. To facilitate characterization of individual ribonucleases, I have developed a rapid method to separate and identify each ribonuclease from a crude sample by gel electrophoresis instead of by time-consuming purification steps. The ribonucleases in a crude sample are first separated by RNA-cast SDS-polyacrylamide gel electrophoresis and then eluted from the gel after ethidium bromide staining. To determine the base specificity of each ribonuclease, a 5′ labelled oligonucleotide with known sequence is added to the enzyme eluate and the digested products are analyzed by denaturing gel electrophoresis. The base specificity of bovine pancreatic ribonuclease (RNase A), bullfrog oocyte-specific ribonuclease (RC-RNase), human serum ribonucleases and sweet potato leaf ribonucleases were determined by this method. Other properties of individual ribonucleases, e.g. substrate preference, may also be determined from crude samples by this method without further purification steps.

Cytotoxic ribonucleases with antitumor activity are mainly found in the oocytes and early embryos of frogs, and share sequence similarity with each other. They all belong to the RNase A superfamily but possess different cytotoxicities, base specificities and ribonucleolytic activities. RC-RNase 4, one of the five novel ribonucleases isolated from the oocytes of Rana catesbeiana, is a 106 amino acid protein with a pyroglutamate residue at the N-terminus (Liao et al., 2000). Phylogenetic analysis of RNase A superfamily reveals that RC-RNase 4 is very similar to onconase, a cytotoxic ribonuclease isolated from the oocytes of bullfrog Rana pipiens, which is now undergoing phase III clinical trials for cancer treatment (Juan et al., 1998). RC-RNase 4 shares 66.0% sequence identity with and its cytotoxicity resembles that of onconase. However, RC-RNase 4 has the base specificity of CpG whereas onconase has that of UpG. Thus, it is interesting to investigate the possibility of RC-RNase 4 as a potential agent for tumor therapy and to study its structural–functional relationship. In addition, among the newly identified frog ribonucleases, only RC-RNase 4 shows distinct CD data, which display two negative ellipticities at 212 and 229 nm when compared to one minimum (∼ 211 nm) observed for the other frog ribonucleases. The melting studies also reveal that RC-RNase 4 possesses higher thermostability. To elucidate RC-RNase 4’s similarities to onconase and its unique properties, we applied NMR

A pyrimidine-guanine sequence-specific ribonuclease (RC-RNase) was purified from Rana catesbeiana (bullfrog) oocytes by sequential phosphocellulose, Sephadex G75, heparin Sepharose CL 6B and CM-Sepharose CL 6B column chromatography. The purified enzyme with molecular weight of 13,000 daltons gave a single band on SDS-polyacrylamide gel. One CNBr-cleaved fragment has a sequence of NVLSTTRFQLNT/TRTSITPR, which is identical to residues 59-79 of a sialic acid binding lectin from R. catesbeiana eggs, and is 71% homologous to residues 60-80 of an RNase from R. catesbeaina liver. The RC-RNase preferentially cleaved RNA at pyrimidine residues with a 3' flanking guanine under various conditions. The sequence specificity of RC-RNase was further confirmed with dinucleotide as substrates, which were analyzed by thin layer chromatography after enzyme digestion. The values of kcat/km for pCpG, pUpG and pUpU were 2.66 x 10(7) M-1s-1, 2.50 x 10(7) M-1s-1 and 2.44 x 10(6) M-1s-1 respectively, however, those for other phosphorylated dinucleotides were less than 2% of pCpG and pUpG. As compared to single strand RNA, double strand RNA was relatively resistant to RC-RNase. Besides poly (A) and poly (G), most of synthetic homo- and heteropolynucleotides were also susceptible to RC-RNase. The RC-RNase was stable in the acidic (pH 2) and alkaline (pH 12) condition, but could be inactivated by heating to 80 degrees C for 15 min. No divalent cation was required for its activity. Furthermore, the enzyme activity could be enhanced by 2 M urea, and inhibited to 50% by 0.12 M NaCl or 0.02% SDS.