Colorectal cancer tissue-originated spheroids reveal tumor intrinsic signaling pathways and mimic patient clinical chemotherapeutic response as a rapid and valid model.

Locally advanced colorectal cancer requires preoperative chemotherapy to reduce local recurrence and metastasis rates, but it remains difficult to predict the tumor will be sensitive to which treatments. The patient-derived organoids (PDOs) are considered an effective platform for predicting tumor drug responses in precision oncology. However, it has the limitation of being time-consuming in practical applications, especially in neoadjuvant treatment. Here we used cancer tissue-originated spheroids (CTOS) method to establish organoids from a heterogeneous population of colorectal cancer specimens, and evaluated the capacity of CTOS to predict clinical drug responses. By analyzing the relationship of the activities of drug-treated CTOS, drug targets and target-related pathways, tumor intrinsic effective-target-related pathways can be identified. These pathways were highly matched to the abnormal pathways indicated by whole-exome sequencing. Based on this, we used half effective concentration gradients to classify CTOS as sensitive or resistant to chemotherapy regimens within a week, for predicting neoadjuvant treatment outcomes for colorectal cancer patients. The drug sensitivity test results are highly matched to the clinical responses to treatment in individual patients. Thus, our data suggested that CTOS models can be effectively screened ex vivo to identify pathways sensitive to chemotherapies. These data also supported organoid research for personalized clinical medication guidance immediately after diagnosis in patients with advanced colorectal cancer.

High ligation versus low ligation of the inferior mesenteric artery in laparoscopic rectal cancer surgery: a retrospective study on surgical and long-term outcome.

BACKGROUND: In laparoscopic low anterior resection for rectal cancer surgery, there has been controversy to whether the inferior mesenteric artery (IMA) should be ligated at the origin of its aorta (high ligation (HL)) or below the branches of the left colonic artery (LCA) (low ligation (LL)). This study was intended to clarify oncological outcome and long-term prognosis of retrospective analysis. METHODS: Analyzed the cases who underwent laparoscopic low anterior resection (LAR) in Shanghai Ruijin Hospital from January 2015 to December 2016, 357patients scheduled into 2 groups according to the level of IMA ligation: HL (n = 247) versus LL (n = 110). RESULTS: The primary endpoint is long-term outcomes, and the secondary endpoint is the incidence rate of major postoperative complications. There were no significant differences in 5-year overall survival (P = 0.92) and 5-year disease-free survival (P = 0.41). There were no differences between the clinical baseline levels in each group. The incidence of low anterior resection syndrome (LARS) in the two groups was statistically significant (P = 0.037). No significant differences were observed in operative time (P = 0.092) and intraoperative blood loss (P = 0.118). In the HL group, 6 cases (2.4%) had additional colonic excision due to poor anastomotic blood supply; none of the colonic anastomosis in the low ligation group had ischemic manifestations, and length from the proximal margin (P = 0.076), length from the distal margin (P = 0.184), the total number of lymph nodes excised (P = 0.065), and anastomotic leakage incidence (P = 0.33). CONCLUSION: Low ligation of the IMA which reserved LCA with vascular root lymph node dissection in laparoscopic low anterior resection for rectal cancer surgery may help protect the blood supply of the anastomosis, and will not increase postoperative complications while enhance recovery, without compromising radical excision and long-term prognosis.

Quantitative dynamic contrast-enhanced parameters and intravoxel incoherent motion facilitate the prediction of TP53 status and risk stratification of early-stage endometrial carcinoma.

BACKGROUND: The aim of the study was to investigate the value of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and intravoxel incoherent motion (IVIM) in differentiating TP53-mutant from wild type, low-risk from non-low-risk early-stage endometrial carcinoma (EC). PATIENTS AND METHODS: A total of 74 EC patients underwent pelvic MRI. Parameters volume transfer constant (Ktrans), rate transfer constant (Kep), the volume of extravascular extracellular space per unit volume of tissue (Ve), true diffusion coefficient (D), pseudo-diffusion coefficient (D*), and microvascular volume fraction (f) were compared. The combination of parameters was investigated by logistic regression and evaluated by bootstrap (1000 samples), receiver operating characteristic (ROC) curves, calibration curves, and decision curve analysis (DCA). RESULTS: In the TP53-mutant group, Ktrans and Kep were higher and D was lower than in the TP53-wild group; Ktrans, Ve, f, and D were lower in the non-low-risk group than in the low-risk group (all P < 0.05). In the identification of TP53-mutant and TP53-wild early-stage EC, Ktrans and D were independent predictors, and the combination of them had an optimal diagnostic efficacy (AUC, 0.867; sensitivity, 92.00%; specificity, 80.95%), which was significantly better than D (Z = 2.169, P = 0.030) and Ktrans (Z = 2.572, P = 0.010). In the identification of low-risk and non-low-risk early-stage EC, Ktrans, Ve, and f were independent predictors, and the combination of them had an optimal diagnostic efficacy (AUC, 0.947; sensitivity, 83.33%; specificity, 93.18%), which was significantly better than D (Z = 3.113, P = 0.002), f (Z = 4.317, P < 0.001), Ktrans (Z = 2.713, P = 0.007), and Ve (Z = 3.175, P = 0.002). The calibration curves showed that the above two combinations of independent predictors, both have good consistency, and DCA showed that these combinations were reliable clinical prediction tools. CONCLUSIONS: Both DCE-MRI and IVIM facilitate the prediction of TP53 status and risk stratification in early-stage EC. Compare with each single parameter, the combination of independent predictors provided better predictive power and may serve as a superior imaging marker.

Use of biexponential and stretched exponential models of intravoxel incoherent motion and dynamic contrast-enhanced magnetic resonance imaging to assess the proliferation of endometrial carcinoma.

Background: It is important to assess the proliferation of endometrial carcinoma (EC) noninvasively using imaging methods. This prospective diagnostic study investigated the value of biexponential and stretched exponential models of intravoxel incoherent motion (IVIM) and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in predicting the Ki-67 status of EC. Methods: In all, 70 patients with EC underwent pelvic MRI. The diffusion coefficient (D), pseudo diffusion coefficient (D*), perfusion fraction (f), distributed diffusion coefficient (DDC), water molecular diffusion heterogeneity index (α), volume transfer constant (Ktrans), rate transfer constant (Kep), and volume of extravascular extracellular space per unit volume of tissue (Ve) were compared. The area under the receiver operating characteristic (ROC) curve (AUC) was used to quantify diagnostic efficacy. Multivariate logistic regression and bootstrap (1,000 samples) analyses were used to establish and evaluate, respectively, the optimal model to predict Ki-67 status. Results: D, Ktrans, and Kep were lower while α was higher in the high-proliferation group as compared with low-proliferation group (all P values<0.05). D and Kep were independent predictors of Ki-67 status in EC, and the combination of these parameters had optimal diagnostic efficacy (AUC =0.920; sensitivity 85.71%; specificity 89.29%), which was significantly better than that of D (AUC =0.753; Z=2.874; P=0.004), α (AUC =0.715; Z=3.505; P=0.001), Ktrans (AUC =0.808; Z=2.741; P=0.006), and Kep (AUC =0.832; Z=2.147; P=0.032) alone. The validation model showed good accuracy (AUC =0.882; 95% confidence interval 0.861-0.897) and consistency (C-statistic =0.902). D, Kep, Ktrans, and α showed a slightly negative (r=-0.271), moderately negative (r=-0.534), slightly negative (r=-0.409), and slightly positive (r=0.488) correlation with the Ki-67 index, respectively (all P values <0.05). Conclusions: IVIM- and DCE-MRI-derived parameters, including D, α, Ktrans, and Kep, were associated with Ki-67 status in EC, and the combination of D and Kep may serve as a superior imaging marker for the identification of low- and high-proliferation EC.

Dysbiosis of human tumor microbiome and aberrant residence of Actinomyces in tumor-associated fibroblasts in young-onset colorectal cancer.

Colorectal cancer (CRC) is the third most common form of cancer, and the incidence of sporadic young-onset colorectal cancer (yCRC) has been increasing. Microbiota residing in the tumor microenvironment are emerging tumor components. The colonic microbiome differs between patients with CRC and healthy controls; however, few studies have investigated the role of the tumor microbiota in disease diagnosis and tumorigenesis of yCRC. We performed 16S rRNA sequencing analysis to identify the microbiome in CRC and found that tumor microbial diversity decreased in yCRC. Proteobacteria and Firmicutes were the most abundant phyla in all CRC samples, and Actinomyces and Schaalia cardiffensis were the key microbiota in the yCRC group. Correlation analysis revealed that Actinomyces co-occurred with various pro-tumor microbial taxa, including Bacteroidia, Gammaproteobacteria, and Pseudomonas. An independent cohort was used to validate the results. The Actinomyces in CRC was co-localized with cancer-associated fibroblasts and activated the TLR2/NF-κB pathway and reduces CD8+ T lymphocyte infiltration in CRC microenvironment. This study suggests that tumoral microbiota plays an important role in promoting tumorigenesis and therefore has potential as a promising non-invasive tool and intervention target for anti-tumor therapy.

CCL7 and TGF-ß secreted by MSCs play opposite roles in regulating CRC metastasis in a KLF5/CXCL5-dependent manner.

CXCL5 is overexpressed in colorectal cancer (CRC) and promotes distant metastasis and angiogenesis of tumors; however, the underlying mechanism that mediates CXCL5 overexpression in CRC remains unclear. Here, we successfully extracted and identified primary mesenchymal stromal cells (MSCs) and verified the promoting effects of tumor-associated MSCs on CRC proliferation and metastasis in vivo and in vitro. We found that MSCs not only promoted the expression of CXCL5 by secreting CCL7 but also secreted TGF-ß to inhibit this process. After secretion, CCL7/CCR1 activated downstream CBP/P300 to acetylate KLF5 to promote CXCL5 transcription, while TGF-ß reversed the effect of KLF5 on transcription activation by regulating SMAD4. Taken together, our results indicate that MSCs in the tumor microenvironment promoted the progression and metastasis of CRC and regulated the expression of CXCL5 in CRC cells by secreting CCL7 and TGF-ß. KLF5 is the key site of these processes and plays a dual role in CXCL5 regulation. MSCs and their secreted factors may serve as potential therapeutic targets in the tumor environment.

Lipidomics reveals association of circulating lipids with body mass index and outcomes in IgA nephropathy patients.

IgA nephropathy (IgAN) is a leading cause of chronic kidney disease (CKD), which are commonly accompanied by dyslipidemia. Obesity is also associated with dyslipidemia and risk of CKD, but the relation of the dyslipidemia patterns with obesity and disease progression in IgAN patients remains unknown. Traditional Chinese medicine (TCM) and the combined treatment with corticosteroids and TCM have been shown to be of benefit for IgAN patients, but predictive markers for guiding these treatments are lacking. Here, we quantified 545 lipid species in the plasma from 196 participants, including 140 IgAN patients and 56 healthy volunteers, and revealed an altered plasma lipidome in IgAN patients as compared to healthy participants. Association analysis showed that a sub-group of glycerides, particularly triacylglycerols (TGs) containing docosahexaenoic acid, were positively associated with high body mass index (BMI) in under- or normal weight IgAN patients, while several free fatty acids and sphingomyelins were positively associated with high BMI in overweight or obese IgAN patients. Further, our study suggested that elevated levels of eight lipids, mainly TG species containing linolenic acid, were independent risk factors for IgAN progression and also reported the prospective association of circulating lipids with treatment outcomes in IgAN. Taken together, our findings may not only help to achieve precision medicine but also provide a knowledge base for dietary intervention in the treatment of IgAN.

Traditional Chinese Medicine as an adjunct therapy in the treatment of idiopathic membranous nephropathy: A systematic review and meta-analysis.

BACKGROUND: Idiopathic membranous nephropathy (IMN) is one of the most common causes of nephrotic syndrome in adults involving multiple targets and factors. The effect of conservative nonimmunosuppressive or immunosuppressive therapies is unsatisfactory and with many side effects. Traditional Chinese medicine (TCM) can regulate immune function and improve kidney function. PURPOSE: To evaluate the total effective rate, curative rate, recurrence rate and adverse events of TCM alone or TCM as an adjunctive therapy for IMN. METHODS: Randomized controlled trials (RCT) comparing either TCM alone or the combination of TCM to western medicine (WM) therapies for patients with IMN were retrieved by searching English and Chinese database. Risk of bias summary was used to assess the methodological quality of eligible studies. Dichotomous data were presented using odds ratios (OR). The primary outcome measure was the total effective rate. Secondary outcomes included curative rate, recurrence rate and adverse events. RESULTS: 29 RCTs involving 1883 participants met the inclusion criteria. There was no statistically significant difference between the therapy of TCM alone and WM on the total effective rates (OR: 2.00; 95% CI: 0.80-4.98; P = 0.14) and curative rate (OR: 1.66; 95%CI: 0.66-4.22; p = 0.28). However, compared to basic treatment or immunosuppressive therapies alone, results showed that TCM as an adjunctive therapy had beneficial effects on the total effective rate (OR: 2.59; 95% CI: 1.38-4.86; P = 0.003 and OR: 3.01; 95% CI: 2.25-4.04; P < 0.00001) and curative rate (OR: 3.01; 95%CI: 1.24-7.28; p = 0.01 and OR: 1.73; 95%CI: 1.10-2.71; p = 0.02). In addition, the combination of TCM treatment could reduce the recurrence rate (OR: 0.28; 95% CI: 0.12-0.68; P = 0.004) and adverse reactions (OR: 0.38; 95% CI: 0.27-0.54; p < 0.00001). CONCLUSION: The results indicate that TCM is well-tolerated for the treatment of IMN. However, there remains a need for large-scale and high-quality trials.

The Efficacy and Mechanism of Chinese Herbal Medicine on Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is the most common microvascular complication of diabetes and is one of the main causes of end-stage renal disease (ESRD) in many countries. The pathological features of DKD are the hypertrophy of mesangial cells, apoptosis of podocytes, glomerular basement membrane (GBM) thickening, accumulation of extracellular matrix (ECM), glomerular sclerosis, and tubulointerstitial fibrosis. The etiology of DKD is very complicated and many factors are involved, such as genetic factors, hyperglycemia, hypertension, hyperlipidemia, abnormalities of renal hemodynamics, and metabolism of vasoactive substances. Although some achievements have been made in the exploration of the pathogenesis of DKD, the currently available clinical treatment methods are still not completely effective in preventing the progress of DKD to ESRD. CHM composed of natural products has traditionally been used for symptom relief, which may offer new insights into therapeutic development of DKD. We will summarize the progress of Chinese herbal medicine (CHM) in the treatment of DKD from two aspects. In clinical trials, the Chinese herbal formulas were efficacy and safety confirmed by the randomized controlled trials. In terms of experimental research, studies provided evidence for the efficacy of CHM from the perspectives of balancing metabolic disorders, reducing inflammatory response and oxidative stress, antifibrosis, protecting renal innate cells, and regulating microRNA and metabolism. CHM consisting of different ingredients may play a role in synergistic interactions and multiple target points in the treatment of DKD.

Research on ribosome-inactivating proteins from angiospermae to gymnospermae and cryptogamia.

Ribosome-inactivating Proteins (RIPs) are a group of cytotoxin proteins that usually contain a RNA N-glycosidase domain, which irreversibly inactivates ribosome, thus inhibiting protein synthesis. During the past 14 years (1990-2004), the studies conducted in our laboratory had been focusing on the structure and enzymatic mechanism of several PIPs. Herein, we briefly described a summary of the studies conducted mainly in our laboratory on RIPs from angiospermae to gymnospermae and cryptogamia as follows. (1) Cinnamomin is a novel type II RIP isolated from mature seeds of camphor tree. Like ricin, it specifically removes the adenine at A4324 in rat liver 28S rRNA. We systematically studied this low-toxic RIP in term of its enzymatic mechanism, the primary and crystal structure and the nucleotide sequence of its gene, the genetic expression, and its physiological role in the seed cell and the toxicity to human cancer cells and insect larvae. The cleavage of supercoiled double-stranded DNA was its intrinsic property of cinnamomin A-chain, its N- and C-terminal regions were found to be required for deadenylation of rRNA and also necessary for deadenylation of supercoiled double-stranded circular DNA. These results strongly excluded the possibility that cleavage of supercoiled DNA was due to nuclease contamination. (2) Trichosanthin, an abortifacient protein, was purified from the Chinese medicinal herb, Tian-hua-fen, obtained from root tubers of Chinese trichosanthes plant. We proved that trichosanthin was a RNA N-glycosidase, inactivating eukaryotic ribosome by hydrolyzing the N-C glycosidic bond of the adenose at site 4324 in rat 28S rRNA, and inhibited protein synthesis in vitro. (3) A unique Biota orientalis RNase (RNase Bo) was extracted from the mature seeds of the cypress cypress tree (Oriental arborvita), which was gymnospermae plant. It cleaved only a specific phosphodiester bond between C4453 and A4454 of 28S RNA in rat ribosomes, producing a small RNA-fragment (S-fragment), thus inhibiting protein synthesis and belonging to RNase-like RIP, similar to α-sarcin, a special RIP. (4) Lamjapin, the first RIP purified from kelp, the marine cryptogamia algal plant, was shown to be the first single-chained RNA N-glycosidase from marine plant to date. It hydrolyzed rat ribosomal 28S RNA to produce meanly a rather smaller RNA, shorter than the diagnostic R-fragment under the restricted condition. The significance of existence of type I RIP in the lower marine algal plant was briefly discussed.

Fluorescence labeling of short RNA by oxidation at the 3'-end.

In RNA nanotechnology, construction of nanoparticles involves conjugation of functionalities, cross-linking of modules, labeling of RNA subunits, and chemical modification of nucleotides. Efficiency and sensitivity are important for the RNA labeling, which also can be used as probes in microarrays, Northern blotting, and gel-shift assays. Here, we describe a method for fluorescence labeling of short RNA at the 3'-end by oxidation. The 3'-terminus of in vitro-transcribed short RNA is oxidized by sodium periodate, and fluorescein-5-thiosemicarbazide is added after removal of excess oxidant. Purified short RNA with fluorescence is then applied for detection of RNA-protein interaction by gel-shift assay.

Low-density lipoprotein receptor-related protein 1 is an essential receptor for trichosanthin in 2 choriocarcinoma cell lines.

Type-I ribosome-inactivating protein-trichosanthin (TCS) exhibits selective cytotoxicity toward different types of cells. It is believed that the cytotoxicity results from the inhibition of ribosomes to decrease protein synthesis, thereby indicating that there are specific mechanisms for TCS entry into target cells to reach the ribosomes. Low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) is a large scavenger receptor that is responsible for the binding and endocytosis of diverse biological ligands on the cell surface. In this study, we demonstrated that 2 choriocarcinoma cell lines can significantly bind and internalize TCS. In contrast, Hela cell line displayed no obvious TCS binding and endocytosis. Furthermore LRP1 gene silencing in JAR and BeWo cell lines blocked TCS binding; TCS could also interact with LRP1.The results of our study established that LRP1 was a major receptor for phagocytosis of TCS in JAR and BeWo cell lines and might be the molecular basis of TCS abortificient and anti-choriocarcinoma activity.

The lower cytotoxicity of cinnamomin (a type II RIP) is due to its B-chain.

The cytotoxicity of intact cinnamomin (a type II ribosome-inactivating protein, RIP) and the RNA N-glycosidase activity of cinnamomin A-chain have been studied and compared with those of ricin. Cinnamomin A-chain exhibits a similar RNA N-glycosidase activity in inhibiting in vitro protein synthesis compared with that of ricin, whereas the cytotoxicity to BA/F3beta cells of intact cinnamomin is markedly lower than intact ricin. In order to demonstrate that it is the B-chains of the two RIPs that bear the difference in cytotoxicity, two hybrid RIPs are prepared from the purified A-/B-chains of cinnamomin and ricin by the disulfide exchange reaction. It has been found that hybrid RIP constructed from cinnamomin A-chain and ricin B-chain is more toxic to BA/F3beta cells than the native cinnamomin, and equivalent to the native ricin. However, the cytotoxicity to BA/F3beta cells of the hybrid RIP constructed from the ricin A-chain and cinnamomin B-chain is lower than ricin, equivalent to the native cinnamomin. Furthermore, the bound amounts of two B-chains on the cell surface are determined by the method of direct cellular ELISA and Scatchard analysis of the binding of the two B-chains indicates that cinnamomin and ricin share similar binding sites with different affinity.

Toxicity of two type II ribosome-inactivating proteins (cinnamomin and ricin) to domestic silkworm larvae.

Cinnamomin and ricin are two type II ribosome-inactivating proteins. They exhibited a different toxicity to domestic silkworm (Bombyx mori) larvae by oral feeding bioassay. The LC50 of ricin to the silkworm larvae at third instar was much lower than that of cinnamomin. When the isolated 80S ribosome from domestic silkworm pupae was treated separately with the reduced cinnamomin or the reduced ricin, a specific RNA fragment (R-fragment) was produced as characterized by 8 M urea-denatured polyacrylamide gel (3.5%) electrophoresis. The purified A-chains of both cinnamomin and ricin showed a slightly different RNA N-glycosidase activity to the domestic silkworm pupal ribosome. It was proposed that the difference of their toxicity to domestic silkworm larvae was not related to their A-chains but to the properties of their B-chains. It was also found that the vomit obtained from the midgut of domestic silkworm larvae could hydrolyze these two proteins apparently to a similar extent.

Cleavage of supercoiled circular double-stranded DNA induced by a eukaryotic cambialistic superoxide dismutase from Cinnamomum camphora.

A eukaryotic cambialistic superoxide dismutase (SOD) has been purified to homogeneity from mature seeds of the disease- and insect-resistant camphor tree (Cinnamomum camphora). Besides the known role of this SOD in protecting cells against oxidative stress, it can induce the cleavage of supercoiled double-stranded DNA into nicked and linear DNA. It can not cleave linear DNA or RNA, demonstrating there is no DNase or RNase in the purified cambialistic SOD. Furthermore, the SOD can linearize circular pGEM-4Z DNA that is relaxed by topoisomerase I. This result indicates that the DNA-cleaving activity requires substrates being topologically constrained. The supercoiled DNA-cleaving activity of the cambialistic SOD can be inhibited by either SOD inhibitor (azide) or catalase and hydroxyl radical scavengers (ethanol and mannitol). The chelator of iron, diethylenetriaminepentaacetic acid (DTPA), also inhibits the supercoiled DNA-cleaving activity. These results show that the dismutation activity is crucial for the supercoiled DNA cleavage. The modification of tryptophan residue of the cambialistic SOD with N-bromosuccinimide (NBS) shows that these two activities are structurally correlative. The reaction mechanism is proposed that the hydroxyl radical formed in a transition-metal-catalyzing Fenton-type reaction contributes to the DNA-cleaving activity. In addition, the cleavage sites in supercoiled pGEM-4Z DNA are random.

The three-dimensional structure and X-ray sequence reveal that trichomaglin is a novel S-like ribonuclease.

Trichomaglin is a protein isolated from root tuber of the plant Maganlin (Trichosanthes Lepiniate, Cucurbitaceae). The crystal structure of trichomaglin has been determined by multiple-isomorphous replacement and refined at 2.2 A resolution. The X-ray sequence was established, based on electron density combined with the experimentally determined N-terminal sequence, and the sequence information derived from mass spectroscopic analysis. X-ray sequence-based homolog search and the three-dimensional structure reveal that trichomaglin is a novel S-like RNase, which was confirmed by biological assay. Trichomaglin molecule contains an additional beta sheet in the HV(b) region, compared with the known plant RNase structures. Fourteen cystein residues form seven disulfide bridges, more than those in the other known structures of S- and S-like RNases. His43 and His105 are expected to be the catalytic acid and base, respectively. Four hydrosulfate ions are bound in the active site pocket, three of them mimicking the substrate binding sites.

A novel ribotoxin with ribonuclease activity that specifically cleaves a single phosphodiester bond in rat 28S ribosomal RNA and inactivates ribosome.

A unique ribonuclease named Biota orientalis ribonuclease (Biota orientalis RNase) is purified to homogeneity from mature seeds of oriental arborvitae (Biota orientalis). The molecular mass of Biota orientalis RNase is about 13 kDa. When the concentration of Mg(2+) is 25 mM in the incubation buffer, the ribonuclease specifically cleaves the phosphodiester bond between C4453 and A4454 in region K (a region in domain VII) of 28S RNA in rat ribosome, resulting in inactivation of ribosome. Thus, it is a ribotoxin similar to alpha-sarcin. The region around C4453-A4454 in rat 28S rRNA is named "Biota orientalis RNase region." Rat ribosome treated by Biota orientalis RNase produces a small RNA fragment (S-fragment) that contains 333 nucleotides from the 3'-terminus of rat 28S rRNA. The distance between the cleavage-sites of alpha-sarcin (G4325) and Biota orientalis RNase (C4453) is 128 nucleotides. Under restricted conditions (25 mM Mg(2+)), the substrate specificity of Biota orientalis RNase is extremely high: it acts only on the "Biota orientalis RNase region" of the largest RNA in ribosomes from certain eukaryotes. The ribosome specifically damaged by Biota orientalis RNase is unable to EF-1alpha-dependently bind aminoacyl-tRNA, whereas the formation of the EF-2/GDP/ribosome complex is not affected. It is proposed that Biota orientalis RNase inactivates ribosome at least partially by interfering with the EF-1alpha-dependent binding of aminoacyl-tRNA to ribosome. Biota orientalis RNase might be a useful tool in studying the structure/function of ribosome.

Cinnamomin--a versatile type II ribosome-inactivating protein.

Ribosome-inactivating proteins (RIPs) are a group of toxic proteins that can specifically act on the universally conserved sarcin/ricin domain (S/R domain) of the largest RNA in ribosome and thus irreversibly inactivate ribosome for protein synthesis. Cinnamomin is a multifunctional type II RIP isolated in our laboratory from the mature seeds of the camphor tree. This protein has been extensively studied with regard to its purification, characteristics, structure and function, genetic expression, enzymatic mechanism, physiological role in seed cell and toxicity to cancer cells and insect larvae. The research results of cinnamomin obtained in our laboratory are summarized in this review. Understanding of cinnamomin and the relative new proteins will help expand our knowledge of RIPs and may accelerate theoretical study and the development of their potential applications.