Unfolding and refolding of the glutamine-binding protein from Escherichia coli and its complex with glutamine induced by guanidine hydrochloride

The aim of this work was to study the conformational changes of the Escherichia coli glutamine-binding protein (GlnBP) induced by GdnHCl and the effect of the binding of glutamine (Gln) on these processes. To this end, GdnHCl-induced unfolding of GlnBP alone and its GlnBP-Gln complex was studied by protein intrinsic fluorescence, ANS emission fluorescence, and far- and near-UV circular dichroism spectroscopy. The obtained spectroscopic data were interpreted taking into the account the peculiarities of protein three-dimensional structure. In particular, the fact that formation of a complex of GlnBP and Gln, which essentially changes the global structure of protein, affects only insignificantly the microenvironments of tryptophan residues elucidates the similarity of the emission spectra of GlnBP and the GlnBP-Gln complex, and the existence of quenching groups near tyrosine residues and an effective nonradiative Tyr --> Trp and/or Tyr --> Tyr --> Trp energy transfer provide an explanation for the negligibly small contribution of tyrosine to the bulk fluorescence of the native protein and for its increase in protein unfolding. The use of the parametric presentation of fluorescence data showed that both GlnBP unfolding and GlnBP-Gln unfolding are three-step processes (N --> I(1) --> I(2) --> U), though in the case of the GlnBP-Gln complex these stages essentially overlap. Despite the complex character, GlnBP unfolding is completely reversible. The dramatic shift of the N --> I(1) process to higher GdnHCl concentrations for the GlnBP-Gln complex in comparison with GlnBP was shown.     

Structure of minor oligosaccharides from the lipopolysaccharide fraction from Pseudomonas stutzeri OX1

A minor oligosaccharide fraction was isolated after complete de-acylation of the lipooligosaccharide extracted from Pseudomonas stutzeri OX1. The full structure of this oligosaccharide was obtained by chemical degradation, NMR spectroscopy and MALDI-TOF MS spectrometry. These experiments showed the presence of two novel oligosaccharides (OS1 and OS2): [structure: see text] where R=(S)-Pyr(-->4,6) in OS1 and alpha-Rha-(1-->3) in OS2. All sugars are D-pyranoses, except Rha, which is L-pyranose. Hep is L-glycero-D-manno-heptose, Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid, Pyr is pyruvic acid, P is phosphate.     

In vitro assembly into virus-like particles is an intrinsic quality of Pichia pastoris derived HCV core protein

Different variants of hepatitis C virus core protein (HCcAg) have proved to self-assemble in vitro into virus-like particles (VLPs). However, difficulties in obtaining purified mature HCcAg have limited these studies. In this study, a high degree of monomeric HCcAg purification was accomplished using chromatographic procedures under denaturing conditions. Size exclusion chromatography and sucrose density gradient centrifugation of renatured HCcAg (in the absence of structured RNA) under reducing conditions suggested that it assembled into empty capsids. The electron microscopy analysis of renatured HCcAg showed the presence of spherical VLPs with irregular shapes and an average diameter of 35nm. Data indicated that HCcAg monomers assembled in vitro into VLPs in the absence of structured RNA, suggesting that recombinant HCcAg used in this work contains all the information necessary for the assembly process. However, they also suggest that some cellular factors might be required for the proper in vitro assembly of capsids.     

Loss of negative regulation by Numb over Notch is relevant to human breast carcinogenesis

The biological antagonism between Notch and Numb controls the proliferative/differentiative balance in development and homeostasis. Although altered Notch signaling has been linked to human diseases, including cancer, evidence for a substantial involvement of Notch in human tumors has remained elusive. Here, we show that Numb-mediated control on Notch signaling is lost in approximately 50% of human mammary carcinomas, due to specific Numb ubiquitination and proteasomal degradation. Mechanistically, Numb operates as an oncosuppressor, as its ectopic expression in Numb-negative, but not in Numb-positive, tumor cells inhibits proliferation. Increased Notch signaling is observed in Numb-negative tumors, but reverts to basal levels after enforced expression of Numb. Conversely, Numb silencing increases Notch signaling in normal breast cells and in Numb-positive breast tumors. Finally, growth suppression of Numb-negative, but not Numb-positive, breast tumors can be achieved by pharmacological inhibition of Notch. Thus, the Numb/Notch biological antagonism is relevant to the homeostasis of the normal mammary parenchyma and its subversion contributes to human mammary carcinogenesis.     

Co-localization of susceptibility loci for psoriasis (PSORS4) and atopic dermatitis (ATOD2) on human chromosome 1q21

Psoriasis (PS) is a chronic inflammatory skin disorder characterized by keratinocyte hyperproliferation and altered differentiation. Atopic dermatitis (ATOD) is a chronic inflammatory, pruritic and eczematous disease frequently associated with respiratory atopy. These diseases are associated with distinct immunologic abnormalities and represent typical examples of complex diseases triggered by both genetic and environmental factors, as demonstrated by independent twin studies. Genome wide linkage studies have mapped susceptibility loci on several chromosomes (PSORS1-9; ATOD1-5). Four of them overlap on chromosomes 1q21, 3q21, 17q25 and 20p although ATOD is quite distinct from PS and these two diseases rarely occur together in the same patient. An association fine-mapping study has been performed to refine PSORS4 and ATOD2 susceptibility loci on chromosome 1q21 analyzing two independently collected cohorts of 128 PS and 120 ATOD trios. Genotype and haplotype analysis of PSORS4 and ATOD2 led us to detect significant p value for haplotypes defined by MIDDLE and ENDAL16 markers in both PS (p = 0.0000036) and ATOD (p = 0.0276), suggesting a strict co-localization within an interval of 42 kb. This genomic interval contains a single gene, LOR, encoding for loricrin. Polymorphic markers mapping in regulatory and coding regions did not show evidence of association in neither of the two diseases. However, expression profiles of LOR in skin biopsies have shown reduced levels in PS and increased levels in ATOD, suggesting the existence of a specific misregulation in LOR mRNA production.     

Immunohistochemical targeting of sea anemone cytolysins on tentacles, mesenteric filaments and isolated nematocysts of Stichodactyla helianthus

The toxicity of biomolecules obtained from sea anemones in vitro does not necessarily justify their function as toxins in the physiology of the anemone. That is why anatomical and physiological considerations must be taken into account in order to define their physiological role in the organism. In this work, antibodies generated to Sticholysin II, a cytolysin produced by the Caribbean Sea anemone Stichodactyla helianthus, are used as specific markers to explore the sites of production and storage of the cytolysin in the sea anemone. The immunoperoxidase staining developed gave specific dark-brown staining in tentacles and mesenteric filaments as well as in basitrichous nematocysts isolated from tentacles of S. helianthus. These results support the role of these proteins as toxins in the physiology of the anemone, especially in functions such as in predation, defense and digestion.     

Geographic patterns of morphological variation in Turnera sidoides subsp. pinnatifida (Turneraceae)

With the objective of contributing to understanding the patterns of variation within the Turnera sidoides complex, a detailed evaluation of morphological variation along the range of T. sidoides subsp. pinnatifida was performed. A multivariate analysis based on leaf traits and flower colour data enabled differentiation of five morphotypes. Common-garden experiments demonstrated that the morphological variants have a strong genetic basis. It was also found that the morphotypes are geographically structured along the subspecies range, display different habitat preferences, and occur in regions with different climatic regimes. Although these results are suggestive of adaptive differentiation of T. sidoides subsp. pinnatifida, comparisons between morphological and bioclimatic ordinations showed that the patterns observed cannot be fully explained by current climatic conditions. It is proposed that Miocene–Pleistocene events may explain the origin of the five morphotypes and that current climatic and ecological factors may be contributing to the maintenance of the extent and patterns of morphological differentiation in T. sidoides subsp. pinnatifida.     

Novel Approach to the Formulation of an Epstein-Barr Virus Antigen-Based Nasopharyngeal Carcinoma Vaccine

Epstein-Barr virus (EBV) is associated with several malignant diseases including nasopharyngeal carcinoma (NPC), a common neoplasm throughout southeast Asia. Radiotherapy and chemotherapy can achieve remission, but a reemergence of disease is not uncommon. Therefore, there is a need for specific therapies that target the tumor through the recognition of EBV antigens. In NPC, latent membrane protein 1 (LMP1) and LMP2 offer the best opportunity for specific targeting since they are typically expressed and T-cell determinants in each of these proteins have been defined. We have attempted to maximize the opportunity of incorporating every possible CD4 and CD8 determinant in a single formulation. We have achieved this by generating a scrambled protein incorporating random overlapping peptide sets from EBNA1, LMP1, and LMP2, which was then inserted into a replication-deficient strain of adenovirus (adenovirus scrambled antigen vaccine [Ad-SAVINE]). This report describes the construction of this Ad-SAVINE construct, its utility in generating LMP1 and LMP2 responses in healthy individuals as well as NPC patients, and its capacity to define new epitopes. This formulation could have a role in NPC immunotherapy for all ethnic groups since it has the potential to activate all possible CD4 and CD8 responses within EBNA1 and LMPs.Epstein-Barr virus (EBV) is a member of the herpesvirus family and is one of the most common human viruses. It occurs worldwide, and most people become infected with the virus sometime during their lives. EBV is associated with a range of neoplasms. These include various B- and T-cell non-Hodgkin''s lymphomas such as posttransplant lymphoproliferative disease (PTLD), Hodgkin''s lymphoma (HL), and several lymphoepithelioma-like carcinomas, of which nasopharyngeal carcinoma (NPC) is the archetype (1). The association of the virus with these malignancies and its oncogenic potential have been well established (19).Worldwide, NPC is characterized epidemiologically by foci of relatively high endemicity in certain geographic regions including southern China, Hong Kong, Taiwan, the Philippines, Singapore, Vietnam, Kenya, Tunisia, Sudan, and Uganda. The reason for the focal distribution of NPC is uncertain, although genetics and environmental factors have been suggested to be causes (14, 49).Currently, the mainstay for the treatment of NPC is radiation and chemotherapy. Indeed, this treatment is frequently successful when the extent of the tumor is small and confined. However, when disease is advanced at diagnosis and where metastatic spread has become apparent, more radical treatments may need to be adopted, including surgery. In either case, all these treatments are associated with severe short- and long-term side effects including secondary malignancies (16). Hence, there is a need for specific therapies that target the tumor itself rather than therapies that are associated with the destruction of normal tissue.Virus-associated malignancies offer a distinct advantage in this regard since therapy can be directed specifically toward viral proteins expressed in the tumor, thus avoiding collateral damage to normal tissue. This has been dramatically demonstrated in the case of PTLD, where the adoptive transfer of EBV-specific cytotoxic T lymphocytes (CTLs) activated in vitro by using autologous lymphoblastoid cell lines (LCLs) has resulted in a resolution of disease with a very low frequency of side effects (9, 18, 40). In this case, it is likely that the effector cells infused into these patients are directed mainly toward the dominant EBV nuclear antigen 3A (EBNA3A), EBNA3B, and EBNA3C. The concept of immunological intervention as a treatment option for NPC is greatly enhanced by a range of previously reported studies that indicated the presence of transport-associated proteins (TAP1 and TAP2) and major histocompatibility complex class I and class II in NPC (23, 37, 42, 48), all of which are required for efficient CTL recognition. In NPC, EBNA1, latent membrane protein 1 (LMP1), and LMP2 offer the best opportunity for specific targeting since these are the only EBV proteins expressed in this malignancy. This is particularly so in the case of immunotherapy since defined CD4⁺ and CD8⁺ T-cell determinants in each of these proteins have been defined (12, 15, 20, 31). However, the CTL response in the case of the LMPs is relatively weak (particularly LMP1), and the glycine-alanine repeat sequence within EBNA1 may affect immunological processing (29), although this may not be the absolute barrier that was first hypothesized (46). Recent studies have provided some encouragement that immunotherapeutic intervention may be a realistic treatment option for NPC (4, 5, 7, 8, 10, 30, 43, 45). For example, Straathof et al. (43) and Comoli et al. (10) adoptively transferred effector cells expanded in vitro by using LCLs to activate CTLs in patients with advanced NPC, resulting in some cases in the resolution of disease, although in other cases, efficacy was limited and transient (3). Those studies, however, have provided a promising hint that the immunotherapeutic control of NPC might be feasible.Indeed, recent studies have shown that multiple human leukocyte antigen (HLA) A2-restricted LMP1 CTL epitopes, when used as a polyepitope vaccine in a poxvirus vector, efficiently induced a strong CTL response, and this response could reverse the outgrowth of LMP1-expressing tumors in HLA-A2/Kb mice (13). The poxvirus-based LMP1-polyepitope vaccine tested in these studies contained only HLA A2-restricted epitopes, and targeting just one HLA allele will not be suitable for all ethnic groups. If a CTL-based therapy for NPC is to be universally applicable, the target epitopes must bind to a range of HLA alleles preferably present at a high frequency in patient populations and include determinants irrespective of whether they have previously been defined.It is likely that the essential difference between the very successful treatment of patients with PTLD and the partial success in the case of NPC is that in the former case, immunodominant targets are available, while in the latter case, only relatively weak responses are seen even for healthy individuals. The present communication has arisen in an attempt to maximize the possibility of activating a response toward the three proteins present in NPC rather than skewing the effector population toward the immunodominant EBNA3A, -B, and -C proteins. We have achieved this by generating a “scrambled-antigen vaccine” (referred to as SAVINE) incorporating random overlapping peptide sets from EBNA1, LMP1, and LMP2. This SAVINE has been incorporated into a replication-deficient adenovirus (Ad5/F35) as a 6.9-kb insert (Ad-SAVINE). An important feature of the Ad-SAVINE strategy is that it provides a platform for the activation of all possible immunological determinants (including helper cells and CTLs) within EBNA1, LMP1, and LMP2 and should be applicable to all populations for which NPC is endemic. This report describes the construction of this Ad-SAVINE construct and its utility in generating LMP1 and LMP2 responses from peripheral blood mononuclear cells (PBMCs) from healthy individuals and NPC patients.     

Culture of bovine hepatocytes: a non-perfusion technique for cell isolation

In this work we have studied the isolation and culture of mature bovine hepatocytes on plastic dishes without exogenous matrix. The liver has been disaggregated in a collagenase solution instead of undergoing a perfusion step. After a few days in culture, the plates showed several clusters of different cell types. Although the average yield was 1.60±0.57×10⁸ viable liver cells per gram of tissue, these cultures were formed by non-parenchymal cells and only very few or none by parenchymal cells. In these cultures, actin structures used as a marker for Stellate (Ito) cells have been visualized by immunocytochemical techniques. In order to increase the proportion of parenchymal cells a centrifugation on Percoll, which separates cell sub-populations, has been introduced. Though the yield was lower than in the previous method, these pre-purified cultures were only composed of hepatocytes. It has been shown that these cells exhibited albumin synthesis, which is a specific hepatocytes function. In addition, these cultures were capable of producing metabolites of 7-ethoxycoumarin at a higher rate than non purified cell cultures. Therefore this simplified procedure for the isolation and culture of functional and viable hepatocytes may be applied for in vitro studies in bovine.