What is the impact of radical hysterectomy on endometrial cancer with cervical involvement?

BACKGROUND: When endometrial carcinoma invades the cervical stroma, overall survival and disease-free survival decrease. However, it is still controversial whether patients in suspected stage II should be treated with radical hysterectomy. The goal of this study is to describe the role of radical hysterectomy in patients with endometrial carcinoma and cervical involvement. METHODS: This was a retrospective cohort study were a total of 239 patients with endometrial carcinoma with cervical involvement from Mexico City's National Cancer Institute were divided according to the type of hysterectomy, and the outcomes were compared using statistical analysis. RESULTS: The 5-year overall survival was 75.76% for the simple hysterectomy group and 89.19% for the radical hysterectomy group, without achieving statistical significance. The 5-year disease-free survival was 72.95% for the simple hysterectomy group and 64.31% for the radical hysterectomy group, without achieving statistical significance. Radicality was associated with longer surgical times, intraoperative complications, and bleeding over 500 ml. CONCLUSIONS: In patients with endometrial carcinoma with cervical involvement, radical hysterectomy does not improve prognosis or alter adjuvant therapy.

Antimicrobial activity of broccoli (Brassica oleracea var. italica) cultivar Avenger against pathogenic bacteria, phytopathogenic filamentous fungi and yeast.

AIMS: The objective of this study was to show whether the edible part of broccoli has antibacterial and antifungal activity against micro-organism of importance in human health and vegetable spoilage, and to test if this effect was partially due to antimicrobial peptides (AMPs). METHODS AND RESULTS: Crude extracts were obtained from florets and stems of broccoli cultivar Avenger and the inhibitory effect was demonstrated against pathogenic bacteria (Bacillus cereus, Staphylococcus xylosus, Staphylococcus aureus, Shigella flexneri, Shigella sonnei, Proteus vulgaris), phytopathogenic fungi (Colletotrichum gloeosporioides, Asperigillus niger) and yeasts (Candida albicans and Rhodotorula sp.). It was shown that samples treated with proteolytic enzymes had a reduction of approximately 60% in antibacterial activity against Staph. xylosus, suggesting that proteinaceous compounds might play a role in the inhibitory effect. Antimicrobial components in crude extracts were thermoresistant and the highest activity was observed under acidic conditions. It was shown that antifungal activity of broccoli's crude extracts might not be attributed to chitinases. CONCLUSIONS: Organic broccoli cultivar Avenger has antimicrobial activity against pathogenic bacteria, yeast and phytophatogenic fungi. Data suggest that this effect is partially due to AMPs. SIGNIFICANCE AND IMPACT OF THE STUDY: Broccoli's crude extracts have activity not only against pathogenic bacteria but also against phytophatogenic fungi of importance in agriculture. We suggest for first time that the inhibitory effect is probably due to AMPs.

Role of the C-terminal and chitin insertion domains on enzymatic activity of endochitinase ChiA74 of Bacillus thuringiensis.

ChiA74 has modular structure that includes a secretion signal peptide (sp) sequence, and catalytic (CD), chitin insertion (CID), fibronectin type-III (FnIII) and chitin binding (CBD) domains. We described for the first time the existence of a putative CID in ChiA74. Mature ChiA74 lacking its sp sequence (rChiA74Δsp, ∼70kDa) and two truncated versions, rChiA74Δsp-60, rChiA74Δsp-50 lacking, respectively, CBD and CDB-FnIII were produced. rChiA74Δsp and rChiA74Δsp-60 are unstable and were processed to generate stable proteins of ∼50kDa. With colloidal chitin, rChiA74Δsp and rChiA74Δsp-50 had higher activity than rChiA74Δsp-60. rChiA74Δsp showed similar ability to bind chitin than rChiA74Δsp-50. The catalytic efficiencies (kcat/Km) of rChiA74Δsp and rChiA74Δsp-50 were higher, ∼ 21-fold than rChiA74Δsp-60, using chitin as the substrate. Optimal activity was detected at pH 7 and 40°C. Data suggest that the CBD in ChiA74 is important for binding to chitin, but not necessary as the presence of a CID together with the CD in a stable truncated version (i.e. ChiA74Δsp-50) has similar affinity and hydrolytic activity as the mature enzyme. The CID of ChiA74 showed identities of ∼ 55% with CIDs of other chitinases such as those from B. circulans and B. licheniformis, respectively, and conserved residues important for interacting with chitin.

Bacillus thuringiensis subsp. israelensis producing endochitinase ChiA74Δsp inclusions and its improved activity against Aedes aegypti.

AIMS: The objective of this study was to produce stable inclusions of chitinase ChiA74Δsp in Bacillus thuringiensis subsp. israelensis (Bti) and to assay its insecticidal activity against Aedes aegypti larvae. METHODS AND RESULTS: Bti was transformed with chiA74Δsp regulated by its own promoter or by the strong chimeric cytAp/STAB-SD promoter system to generate two recombinant Bti strains. These recombinants produced their native parasporal bodies composed of Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa and ChiA74Δsp inclusions, and showed a approx. threefold increase in both endochitinase activity and viable spore count when compared with the parental strain. Both recombinants were approximately twofold more toxic (LC50s 8·02, 9·6 ng ml(-1) ) than parental Bti (19·8 ng ml(-1) ) against 4(th) instars of A. aegypti larvae. CONCLUSIONS: ChiA74Δsp inclusions, together with the insecticidal crystals and spores of Bti increased the toxicity against A. aegypti larvae by at least twofold. SIGNIFICANCE AND IMPACT OF THE STUDY: We report for the first time the engineering of Bti to produce spore-parasporal body-ChiA74∆sp inclusions in the same sporangium, which are released together following autolysis. Our work lays a foundation for engineering Bti to produce more efficacious combinations of Cry4Aa, Cry4Ba, Cry11Aa, Cyt1Aa and chitinase inclusions.

Detection of ß-exotoxin synthesis in Bacillus thuringiensis using an easy bioassay with the nematode Caenorhabditis elegans.

UNLABELLED: The insecticidal activity of Bacillus thuringiensis is owing to the action of Cry and Cyt proteins. In addition to the synthesis of insecticidal proteins, some strains are able to synthesize ß-exotoxin, which is highly toxic to humans. In this regard, it is very important to have a simple method to detect ß-exotoxin to avoid the commercial production of this type of strains. In this work, we developed a simple and fast method, using the nematode Caenorhabditis elegans to detect indirectly the synthesis of ß-exotoxin by B. thuringiensis strain. Using this assay, we detected that ~60% of Mexican native strains (i.e. LBIT-471, 491, 492, 497, 507, 511, 515, 536 and 537) were toxic to the nematode (44-97% mortalities) and their ß-exotoxin (ßEx(+) ) production, including a positive control (NRD-12), was confirmed by HPLC. In addition, the negative controls (ßEx(-) ) LBIT-436 (HD-1) and LBIT-438 and also the native strains LBIT-499, 500, 521, 522, 533 and 542, did not show a detrimental effect against nematodes larvae, neither the synthesis of ß-exotoxin as determined by HPLC. Finally, we did not find a correlation between B. thuringiensis strains with similar plasmid patterns and the ß-exotoxin production. SIGNIFICANCE AND IMPACT OF THE STUDY: In this work, we implemented a qualitative and fast bioassay using the nematode Caenorhabditis elegans to detect the production of ß-exotoxin in different strains of Bacillus thuringiensis. We show that this assay is useful to detect ß-exotoxin in B. thuringiensis with high reliability, helping to discriminate strains that could not be used as bioinsecticides because of their putative risk to humans. Data show that qualitative bioassay with nematodes is a potential alternative to fly larvae bioassays, and correlated with the determination of ß-exotoxin by HPLC.

Isolation of a new Mexican strain of Bacillus subtilis with antifungal and antibacterial activities.

Although several strains of B. subtilis with antifungal activity have been isolated worldwide, to date there are no published reports regarding the isolation of a native B. subtilis strain from strawberry plants in Mexico. A native bacterium (Bacillus subtilis 21) demonstrated in vitro antagonistic activity against different plant pathogenic fungi. Under greenhouse conditions, it was shown that plants infected with Rhizoctonia solani and Fusarium verticillioides and treated with B. subtilis 21 produced augment in the number of leaves per plant and an increment in the length of healthy leaves in comparison with untreated plants. In addition, B. subtilis 21 showed activity against pathogenic bacteria. Secreted proteins by B. subtilis 21 were studied, detecting the presence of proteases and bacteriocin-like inhibitor substances that could be implicated in its antagonistic activity. Chitinases and zwittermicin production could not be detected. Then, B. subtilis 21 could potentially be used to control phytopathogenic fungi that infect strawberry plants.

Colonic micropapillary carcinoma, a recently recognized subtype associated with histological adverse factors: clinicopathological analysis of 15 cases.

AIM: Micropapillary carcinoma (MPC) is regarded as an aggressive variant of adenocarcinoma in any location. The reported proportion of a micropapillary carcinoma component in an entire tumour ranges from 5 to 95% and only one case of pure MPC has been reported. To date, approximately 130 cases of MPC in the colorectum have been reported, but it is likely that this small number is to some extent due to under-reporting because this pattern is not well recognized by the general pathologist. All previous studies have combined colonic and rectal primary tumours and most have only analysed patients with clinical Stages I or II. METHOD: We analysed 15 cases of MPC of the colon alone, diagnosed in our institution, and compared them with 105 conventional carcinomas of the colon. RESULTS: An MPC component was present in 10% of all colonic carcinomas. These tumours presented at a median age of 56 years, and all were of American Joint Committee on Cancer Stages III and IV. Subserosal tissue invasion was present in every case, 60% had more than four positive lymph nodes, 60% were accompanied by poorly differentiated conventional carcinoma, 40% had had an incomplete resection and a third demonstrated lymphovascular invasion. Despite these adverse prognostic factors, tumours containing MPC showed the same survival, stage by stage, as conventional adenocarcinoma in multivariate analysis, although 3-year survival (81.7%vs 87.3%, P=0.035) was worse on univariate analysis. CONCLUSION: The histopathologist should be aware of the possibility of MPC. Three-year survival is worse than in patients with conventional colonic carcinomas in Stage III.

Generation of chitin-derived oligosaccharides toxic to pathogenic bacteria using ChiA74, an endochitinase native to Bacillus thuringiensis.

AIMS: To demonstrate that an endochitinase (ChiA74) native to Bacillus thuringiensis can be used to generate chitin-derived oligosaccharides (OGS) with antibacterial activity against a number of aetiological agents of disease, including bacteria that cause diarrhoeal and emetic syndromes in humans. METHODS AND RESULTS: The intact chiA74 with its cis elements was cloned into high and moderately high copy number Escherichia coli expression vectors. Functionally secreted ChiA74 was produced, and the endochitinase cleaved substrate colloidal chitin to produce OGS with 3, 5 and 6 degrees of polymerization. The enzyme was active for an extended period of incubation (24 h), but its activity showed a decrement of 73% and 87%, respectively, after 24 h of incubation at 37 and 55 degrees C. OGS showed inhibitory activity against Bacillus cereus, Listeria inoccua, E. coli, Staphylococcus xylosus, Salmonella species, Staphylococcus aureus, Pseudomona aeruginosa, Shigella flexneri, and Proteus vulgaris. CONCLUSIONS: Endochitinase ChiA74 is able to stably maintain hydrolytic activity during prolonged incubation in a mix reaction with chitin to produce bioactive OGS with inhibitory activity against important food-borne pathogenic bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study showing that an endochitinase (ChiA74) native of the most important bioinsecticide used worldwide (B. thuringiensis), but here produced in E. coli, is able to generate chitin-derived OGS with antibacterial activity against clinically significant food-borne pathogenic bacteria.

Enhanced synthesis and antimicrobial activities of bacteriocins produced by Mexican strains of Bacillus thuringiensis.

Recently, we reported the synthesis of five bacteriocin-like inhibitor substances (Bt-BLIS: morricin 269, kurstacin 287, kenyacin 404, entomocin 420, and tolworthcin 524) by Mexican strains of Bacillus thuringiensis. Here we show that, collectively, these Bt-BLIS have a moderate to broad spectrum of antibacterial activity, being toxic to clinically significant against Gram-positive and Gram-negative bacteria, including common etiological agents of human diseases, such as strep throat and scarlet fever, septicemia, pneumonia, urinary tract infection, and emetic and gastrointestinal syndromes. Although synthesis of the five Bt-BLIS was independent of the presence of a target inducing bacterium, we demonstrated for the first time that a proteinaceous component(s) secreted by, or liberated by proteolytic cleavage of Bacillus cereus 183 following treatment with proteinase K, enhanced Bt-BLIS synthesis.

Generation of antibacterial oligosaccharides derived from chitin using heterologous endochitinase synthesized in Escherichia coli.

AIMS: To synthesize two heterologous endochitinases in Escherichia coli and demonstrate their potential for applied use in generating antibacterial chitin-derived oligosaccharides (OGS). METHODS AND RESULTS: Heterologous endochitinase genes, chiA Nima and chiA74, were expressed in E. coli. Endochitinases were secreted by the E. coli export machinery and by approximately 20 h maximal chitinolytic activity was observed. The highest chitinolytic activity was observed with ChiA Nima, which produced antibacterial OGS with activities against Enterobacter cloacae, Escherichia coli, Staphylococcus aureus and S. xylosus. CONCLUSIONS: It was shown that the export machinery of E. coli is well suited for the secretion of bioactive ChiA74 and ChiA Nima endochitinases, and that the latter can generate antibacterial OGS. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study suggests that it is feasible to synthesize endochitinases ChiA Nima and ChiA74 codified by E. coli and mass-produce these enzymes in culture supernatants. As signal peptides in native ChiA Nima and ChiA74 were recognized by the protein export molecular apparatus in E. coli, these short peptides could be included as signal sequences for transport in E. coli of other proteins with applied value. This is the first report suggesting that ChiA Nima can be used to produce OGS to control food-borne pathogenic bacteria.

Chitinases from Serratia marcescens Nima.

Chitinolytic activity of Serratia marcescens Nima (130 U ml(-1)) was up to 43 times higher than those produced by other S. marcescens strains. This strain synthesized an endochitinase (Chi-60), an exochitinase (Chi-50) and a novel N-acetylglucosaminidase. This latter showed two putative isoforms (Chi-180.5 and Chi-180.8) with isoelectric points of 5 and 8.1, respectively.

Expression and one-step purification of a fully active polyhistidine-tagged cytochrome bc1 complex from Rhodobacter sphaeroides.

The fbcB and fbcC genes encoding cytochromes b and c1 of the bc1 complex were extended with a segment to encode a polyhistidine tag linked to their C-terminal sequence allowing a one-step affinity purification of the complex. Constructions were made in vitro in a pUC-derived background using PCR amplification. The modified fbc operons were transferred to a pRK derivative plasmid, and this was used to transform the fbc- strain of Rhodobacter sphaeroides, BC17. The transformants showed normal rates of growth. Chromatophores prepared from these cells showed kinetics of turnover of the bc1 complex on flash activation which were essentially the same as those from wild-type strains, and analysis of the cytochrome complement and spectral and thermodynamic properties by redox potentiometry showed no marked difference from the wild type. Chromatophores were solubilized and mixed with Ni-NTA-Sepharose resin. A modification of the standard elution protocol in which histidine replaced imidazole increased the activity 20-fold. Imidazole modified the redox properties of heme c1, suggesting ligand displacement and inactivation when this reagent is used at high concentration. The purified enzyme contained all four subunits in an active dimeric complex. This construction provides a facile method for preparation of wild-type or mutant bc1 complex, for spectroscopy and structural studies.

Matrix-assisted laser desorption ionization mass spectrometry of membrane proteins: demonstration of a simple method to determine subunit molecular weights of hydrophobic subunits.

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry has been used to obtain accurate molecular weight information for each subunit of several hydrophobic integral membrane proteins: cytochrome bo3 (4 subunits) and cytochrome bd (2 subunits) from E. coli, and the bc1 complex (3 subunits) and the cytochrome c oxidase (3 subunits) from Rhodobacter sphaeroides. The results demonstrate that the MALDI method is a convenient, quick, sensitive and reliable means for obtaining the molecular masses of the subunits of purified multisubunit membrane proteins.