In situ generation of hydrogen peroxide by carbohydrate oxidase and cellobiose dehydrogenase for bleaching purposes

The carbohydrate oxidase from Microdochium nivale (CAOX), heterologously expressed in Aspergillus oryzae, and cellobiose dehydrogenase from Myriococcum thermophilum (MtCDH), were assessed for their ability to generate bleaching species at a pH suitable for liquid detergents. The substrate specificities of CAOX and MtCDH were analyzed on a large variety of soluble and insoluble substrates, using oxygen as an electron receptor. Even insoluble substrates like cellulose were oxidized from both CAOX and MtCDH, but only MtCDH produced H₂O₂ on cotton as the sole substrate. To enhance the amount of cello-oligosaccharides formed from cotton as substrates for CAOX and MtCDH, various cellulases were used in combination with MtCDH or CAOX, leading to a 10-fold increase in H₂O₂. As model substrates for colored stains, the degradation of pure anthocyanins and stain removal of blueberry stains by CAOX and MtCDH was examined in the absence and presence of a horseradish peroxidase. Both enzymes were able to produce an amount of H₂O₂ sufficient to decolorize the pure anthocyanins within 2 h and showed significant cleaning benefits on the stains.     

Machupo virus glycoprotein determinants for human transferrin receptor 1 binding and cell entry

Machupo virus (MACV) is a highly pathogenic New World arenavirus that causes hemorrhagic fever in humans. MACV, as well as other pathogenic New World arenaviruses, enter cells after their GP1 attachment glycoprotein binds to their cellular receptor, transferrin receptor 1 (TfR1). TfR1 residues essential for this interaction have been described, and a co-crystal of MACV GP1 bound to TfR1 suggests GP1 residues important for this association. We created MACV GP1 variants and tested their effect on TfR1 binding and virus entry to evaluate the functional significance of some of these and additional residues in human and simian cells. We found residues R111, D123, Y122, and F226 to be essential, D155, and P160 important, and D114, S116, D140, and K169 expendable for the GP1-TfR1 interaction and MACV entry. Several MACV GP1 residues that are critical for the interaction with TfR1 are conserved among other New World arenaviruses, indicating a common basis of receptor interaction. Our findings also open avenues for the rational development of viral entry inhibitors.     

Association of ebola virus matrix protein VP40 with microtubules

Viruses exploit a variety of cellular components to complete their life cycles, and it has become increasingly clear that use of host cell microtubules is a vital part of the infection process for many viruses. A variety of viral proteins have been identified that interact with microtubules, either directly or via a microtubule-associated motor protein. Here, we report that Ebola virus associates with microtubules via the matrix protein VP40. When transfected into mammalian cells, a fraction of VP40 colocalized with microtubule bundles and VP40 coimmunoprecipitated with tubulin. The degree of colocalization and microtubule bundling in cells was markedly intensified by truncation of the C terminus to a length of 317 amino acids. Further truncation to 308 or fewer amino acids abolished the association with microtubules. Both the full-length and the 317-amino-acid truncation mutant stabilized microtubules against depolymerization with nocodazole. Direct physical interaction between purified VP40 and tubulin proteins was demonstrated in vitro. A region of moderate homology to the tubulin binding motif of the microtubule-associated protein MAP2 was identified in VP40. Deleting this region resulted in loss of microtubule stabilization against drug-induced depolymerization. The presence of VP40-associated microtubules in cells continuously treated with nocodazole suggested that VP40 promotes tubulin polymerization. Using an in vitro polymerization assay, we demonstrated that VP40 directly enhances tubulin polymerization without any cellular mediators. These results suggest that microtubules may play an important role in the Ebola virus life cycle and potentially provide a novel target for therapeutic intervention against this highly pathogenic virus.     

Understanding the molecular basis of Apert syndrome

Apert syndrome, first described in 1906, is one of the most severe of the craniosynostosis syndromes and is further characterized by midface hypoplasia, syndactyly, and other visceral abnormalities. Affected individuals generally require lifelong management by a multidisciplinary team of health care specialists. Apert syndrome results almost exclusively from one or the other of two point mutations in fibroblast growth factor receptor 2. Tremendous scientific advances have been made recently in understanding the molecular basis for Apert syndrome through clinical genetic, biochemical, and structural approaches. In this review, the authors provide the clinician with a basic overview of these findings and their therapeutic implications.     

The new higher level classification of eukaryotes with emphasis on the taxonomy of protists

This revision of the classification of unicellular eukaryotes updates that of Levine et al. (1980) for the protozoa and expands it to include other protists. Whereas the previous revision was primarily to incorporate the results of ultrastructural studies, this revision incorporates results from both ultrastructural research since 1980 and molecular phylogenetic studies. We propose a scheme that is based on nameless ranked systematics. The vocabulary of the taxonomy is updated, particularly to clarify the naming of groups that have been repositioned. We recognize six clusters of eukaryotes that may represent the basic groupings similar to traditional "kingdoms." The multicellular lineages emerged from within monophyletic protist lineages: animals and fungi from Opisthokonta, plants from Archaeplastida, and brown algae from Stramenopiles.     

Transhiatal and transthoracic resection in adenocarcinoma of the esophagus: Does the operative approach have an influence on the long-term prognosis?

Background

Goblet cell carcinoid (GCC) of the appendix is a rare neoplasm that share histological features of both adenocarcinoma and carcinoid tumor. While its malignant potential remains unclear, GCC's are more aggressive than conventional carcinoid. The clinical presentations of this neoplasm are also varied. This review summarizes the published literature on GCC of the appendix. The focus is on its diagnosis, histopathological aspects, clinical manifestations, and management.

Methods

Published studies in the English language between 1966 to 2004 were identified through Medline keyword search utilizing terms "goblet cell carcinoid," "adenocarcinoid", "mucinous carcinoid" and "crypt cell carcinoma" of the appendix.

Results

Based on the review of 57 published papers encompassing nearly 600 diagnosed patients, the mean age of presentation for GCC of the appendix was 58.89 years with equal representation in both males and females. Accurate diagnosis of this neoplasm requires astute observations within an acutely inflamed appendix as this neoplasm has a prominent pattern of submucosal growth and usually lacks the formation of a well-defined tumor mass. The mesoappendix was involved in 21.64% followed by perineural involvement in 2.06%. The most common clinical presentations in order of frequency were acute appendicitis in 22.5%; asymptomatic in 5.4%; non-localized abdominal pain in 5.15% and an appendicular mass in 3.09%. The most common surgical treatment of choice was appendectomy with right hemicolectomy in 34.70% followed by simple appendectomy in 24.57%. Concomitant distant metastasis at diagnosis was present in 11.16% of patients with the ovaries being the most common site in 3.60% followed by disseminated abdominal carcinomatosis in 1.03%. Local lymph node involvement was seen in 8.76% of patients at the time of diagnosis. The reported 5-year survival ranges from 60 % to 84%. GCC's of the appendix remains a neoplasm of unpredictable biological behavior and thus warrants lifelong surveillance for recurrence of the disease upon diagnosis and successful surgical extirpation.

Conclusion

GCC of the appendix is a rare neoplasm. Due to its wide range of presentation, this tumor should be considered as a possible diagnosis in many varied situations leading to abdominal surgery. Histopathological features such as increased number of Paneth cells, increased amount of mucin secretion and presence of pancreatic polypeptide may predict a more aggressive behavior. The advocated plan of management recommended for patients with tumors that involve the adjacent caecum or with high-grade tumors with histological features such as an increased mitotic rate involve initial appendectomy with completion right hemicolectomy due to the high possibility of local recurrence with intraperitoneal seeding prior to lymph node involvement and a 20% risk of metastatic behavior. In female patients with GCC of the appendix regardless of age, bilateral salpingo-oophorectomy is advocated. In cases with obvious spread of the disease chemotherapy, mostly with 5-FU and leucovorin is advised. Cytoreductive surgery with adjuvant intraperitoneal chemotherapy can offer improved survival in cases with advanced peritoneal dissemination.     

High-performance liquid chromatographic determination of lamivudine in human serum using liquid-liquid extraction; application to pharmacokinetic studies

A simple, fast, and sensitive high performance liquid chromatographic (HPLC) assay was developed for quantitation of lamivudine in human serum. Lamivudine is polar compound and its extraction from the human serum in previously published HPLC methods involved either protein precipitation or solid phase extraction techniques. However, existence of endogenous peaks which interfere with the drug or appeared as late eluting peaks and lead to long run time of analysis has been reported. Application of either an ion pairing agent in the mobile phase or time consuming column purge has been used in the published methods. Present paper describes liquid - liquid extraction of lamivudine and internal standard (famotidine) using dichloromethane-isopropyl alcohol (1:1, v/v) as an extracting solvent and salting out approach. The mobile phase was a mixture of phosphate buffer (0.05 M) containing triethylamine (1 mL/L, v/v; pH 3.5) and methanol (91:9, v/v) at a flow rate of 2.2 mL/min. The analysis was performed on a column (150 mm x 6 mm i.d.) which was packed with 5 microm particles of ODS packing material. Under these conditions no interference in the assay from any endogenous substance was observed. The limit of quantification was evaluated to be 5 ng/mL. Accuracy and precision of the method were also studied and the technique was shown to be selective and linear into the concentration range of 5-2500 ng/mL. This method has been used in two randomized crossover bioequivalence studies of 100 and 150 mg lamivudine preparations in 12 and 24 healthy volunteers, respectively.     

Induction of humoral and CD8+ T cell responses are required for protection against lethal Ebola virus infection

Ebola virus (EBOV)-like particles (eVLP), composed of the EBOV glycoprotein and matrix viral protein (VP)40 with a lipid membrane, are a highly efficacious method of immunization against EBOV infection. The exact requirements for immunity against EBOV infection are poorly defined at this time. The goal of this work was to determine the requirements for EBOV immunity following eVLP vaccination. Vaccination of BALB/c or C57BL/6 mice with eVLPs in conjunction with QS-21 adjuvant resulted in mixed IgG subclass responses, a Th1-like memory cytokine response, and protection from lethal EBOV challenge. Further, this vaccination schedule led to the generation of both CD4(+) and CD8(+) IFN-gamma(+) T cells recognizing specific peptides within glycoprotein and VP40. The transfer of both serum and splenocytes, but not serum or splenocytes alone, from eVLP-vaccinated mice conferred protection against lethal EBOV infection in these studies. B cells were required for eVLP-mediated immunity to EBOV because B cell-deficient mice vaccinated with eVLPs were not protected from lethal EBOV challenge. We also found that CD8(+), but not CD4(+), T cells are absolutely required for eVLP-mediated protection against EBOV infection. Further, eVLP-induced protective mechanisms were perforin-independent, but IFN-gamma-dependent. Taken together, both EBOV-specific humoral and cytotoxic CD8(+) T cell responses are critical to mediate protection against filoviruses following eVLP vaccination.     

Revisiting the regiospecificity of Burkholderia xenovorans LB400 biphenyl dioxygenase toward 2,2'-dichlorobiphenyl and 2,3,2',3'-tetrachlorobiphenyl

2,2'-Dichlorobiphenyl (CB) is transformed by the biphenyl dioxygenase of Burkholderia xenovorans LB400 (LB400 BPDO) into two metabolites (1 and 2). The most abundant metabolite, 1, was previously identified as 2,3-dihydroxy-2'-chlorobiphenyl and was presumed to originate from the initial attack by the oxygenase on the chlorine-bearing ortho carbon and on its adjacent meta carbon of one phenyl ring. 2,3,2',3'-Tetrachlorobiphenyl is transformed by LB400 BPDO into two metabolites that had never been fully characterized structurally. We determined the precise identity of the metabolites produced by LB400 BPDO from 2,2'-CB and 2,3,2',3'-CB, thus providing new insights on the mechanism by which 2,2'-CB is dehalogenated to generate 2,3-dihydroxy-2'-chlorobiphenyl. We reacted 2,2'-CB with the BPDO variant p4, which produces a larger proportion of metabolite 2. The structure of this compound was determined as cis-3,4-dihydro-3,4-dihydroxy-2,2'-dichlorobiphenyl by NMR. Metabolite 1 obtained from 2,2'-CB-d(8) was determined to be a dihydroxychlorobiphenyl-d(7) by gas chromatographic-mass spectrometric analysis, and the observed loss of only one deuterium clearly shows that the oxygenase attack occurs on carbons 2 and 3. An alternative attack at the 5 and 6 carbons followed by a rearrangement leading to the loss of the ortho chlorine would have caused the loss of more than one deuterium. The major metabolite produced from catalytic oxygenation of 2,3,2',3'-CB by LB400 BPDO was identified by NMR as cis-4,5-dihydro-4,5-dihydroxy-2,3,2',3'-tetrachlorobiphenyl. These findings show that LB400 BPDO oxygenates 2,2'-CB principally on carbons 2 and 3 and that BPDO regiospecificity toward 2,2'-CB and 2,3,2,',3'-CB disfavors the dioxygenation of the chlorine-free ortho-meta carbons 5 and 6 for both congeners.