Separation of metabolically radiolabeled O-methylmannitols and O-methylfucitol by reverse-phase high-performance liquid chromatography: applications to animal cell oligosaccharide structural analysis

Reverse-phase high-performance liquid chromatography was utilized to separate efficiently and rapidly a standard mixture of various radiolabeled O-methylated mannitols and O-methylfucitol commonly encountered when vertebrate asparagine-linked oligosaccharides are subjected to permethylation, hydrolysis, and reduction with NaBH4. The following reduced, radioactive O-methylhexitols were resolved: 2,4-, 3,4-, and 3,6-di-O-methylmannitols; 3,4,6-tri-O-methylmannitol, 2,3,4-tri-O-methylfucitol, and 2,3,4,6-tetra-O-methylmannitol. To demonstrate the utility of this separation method in the analysis of metabolically radiolabeled asparagine-linked oligosaccharides, mouse lymphoma BW 5147 cells were metabolically radiolabeled with [2-3H]mannose and their glycopeptides prepared by Pronase digestion and fractionated by serial chromatography on immobilized lectins. Each fraction was subjected to methylation and hydrolysis, the released monosaccharides were reduced, and the radioactive O-methylhexitols were separated by reverse-phase HPLC. The relative amounts of the O-methylhexitols in each glycopeptide fraction analyzed were similar to those values determined by a combination of other separation systems.     

T-cell dysfunction and hyperimmunoglobulinemia E in paracoccidioidomycosis

Various aspects of T and B cell mediated immunity were investigated in 20 well documented cases of active (10) or inactive (10) paracoccidioidomycosis (Pcm), as well as in 8 healthy individuals living in the endemic area of the disease. The results confirm previous reports that active Pcm produces diverse grades of depression of T cell mediated immunity. Such T cell dysfunction is not associated with a reduction in the number of peripheral E rosette-forming cells, and the immunodepression is reversed by chemotherapy. Sera from Pcm (active or inactive) patients have significantly increased levels of total IgE, but the actual proportion of IgE antibodies against P. brasiliensis was very low (0.4–0.6%). The highest levels of total IgE were found in active patients with disease-related immune depression, suggesting that T cell dysfunction might contribute to the excessive IgE production.     

Rous sarcoma virus-transformed baby hamster kidney cells express higher levels of asparagine-linked tri- and tetraantennary glycopeptides containing [GlcNAc-beta (1,6)Man-alpha (1,6)Man] and poly-N-acetyllactosamine sequences than baby hamster kidney cells

The alterations in complex-type N-linked oligosaccharides that can occur when an animal cell line is transformed by two dissimilar viruses were examined by comparing the N-linked oligosaccharides of baby hamster kidney (BHK) cells, metabolically radiolabeled with [2-3H]mannose, to the same class of oligosaccharides from BHK cells separately transformed by Rous sarcoma virus (RS-BHK), an RNA retrovirus, and polyoma virus (PY-BHK), a DNA papovavirus. Based on experiments that utilized serial lectin affinity chromatography, glycosidase digestions, and methylation analyses, both RS-BHK and PY-BHK cells demonstrated a significant increase in the relative amounts of tri- and tetraantennary complex-type N-linked oligosaccharides containing the branching sequence, [GlcNAc-beta(1,6)Man-alpha(1,6)Man], compared to the nontransformed BHK cells. In addition, almost all of the poly-N-acetyllactosamine sequence, [GlcNAc-beta(1,3)-Gal-beta(1,4)], was expressed on the tri- and tetraantennary N-linked oligosaccharides from BHK and RS-BHK cells that contain the sequence, [GlcNAc-beta(1,6)Man-beta(1,6)Man]. The increase in the relative amounts of this latter sequence in the transformed cells, therefore, most likely results in an increase in the amount of poly-N-acetyllactosamine sequence on the N-linked glycopeptides of these cells. The analysis of the degree of sialylation of the complex-type N-linked oligosaccharides from BHK and RS-BHK cells by ion exchange chromatography revealed no apparent differences, and in both of these cell types approximately 3% of the glycopeptide fraction radiolabeled with mannose was recovered in a highly negatively charged fraction that was identified by keratanase digestion to be keratan sulfate.     

Dynamics of methyl groups in proteins as studied by proton-detected 13C NMR spectroscopy. Application to the leucine residues of staphylococcal nuclease.

This paper describes the application of recently developed nuclear magnetic resonance (NMR) pulse sequences to obtain information about the internal dynamics of isotopically enriched hydrophobic side chains in proteins. The two-dimensional spectra provided by the pulse sequences enable one to make accurate measurements of nuclear Overhauser effects (NOE) and longitudinal (T1) and transverse (T2) relaxation times of enriched methyl carbons in proteins. Herein, these techniques are used to investigate the internal dynamics of the 11 leucine side chains of staphylococcal nuclease (SNase), a small enzyme having Mr = 16.8K, in the absence and presence of ligands thymidine 3',5'-bisphosphate (pdTp) and Ca2+. We report the synthesis of [5,5'-13C2]leucine, the preparation of SNase containing the labeled leucine, the sequential assignment of the leucine methyl carbons and protons in the liganded and unliganded proteins, and the measurement of the 13C T1, T2, and NOE values for the SNase leucine methyl carbons. Analysis of the relaxation parameters using the formalism of Lipari and Szabo shows that the internal motions of the leucine methyl carbons are characterized by effective correlation times tau f (5-80 ps) and tau s (less than 2 ns). The fast motion is identified with the rapid rotation of the methyl group about the C gamma-C delta bond axis, while the slow motion is associated with reorientation of the C gamma-C delta bond axis itself. The mean squared order parameters associated with the latter motion, Ss2, lie in the range 0.34-0.92. The values of Ss2 correlate reasonably well with the temperature factors of the leucine methyl carbons obtained from the crystal structures, but some are smaller than anticipated on the basis of the fact that nearly all leucine methyl carbons are buried and have temperature factors no larger than that of the leucine backbone atoms. Five leucine residues in liganded SNase and eight in unliganded SNase have values of Ss2 less than 0.71. These order parameters correspond to large amplitude motions (angular excursions of 27-67 degrees) of the C gamma-C delta bond axis. These results indicate that, in solution, the internal motions of the leucine side chains of SNase are significantly larger than suggested by the X-ray structures or by qualitative analysis of NOESY spectra. Comparison of Ss2 values obtained from liganded and unliganded SNase reveals a strong correlation between delta Ss2 and distance between the leucine methyl carbon and the ligands.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Cultivo biotecnológico de macrohongos comestibles: una alternativa en la obtención de nutracéuticos

Macromycetes have been part of the human culture for thousand years, and have been reported as food in the most important civilizations in history. Many nutraceutical properties of macromycetes have been described, such as anti-cancer, anti-tumour, cholesterol lowering, antiviral, antibacterial, or immunomodulatory, among others. Given that production of mushrooms by traditional cultivation and extraction of bioactive metabolites is very difficult in some cases, biotechnology is essential for the development of profitable and productive techniques for obtaining these metabolites. It is the development of this technology, and the ease in which it enables the use of its variables that has allowed mycelium to be cultivated in liquid medium of macrofungi, with a significant reduction in time and an increased production of metabolites. This increased production has led to the study of compounds that have medicinal, nutriceutical and quasi-farmaceutical potential, in the exhausted media and the mycelium. The aim of this review is to provide an overview of the use of liquid-state fermentation as a technological tool for obtaining edible fungi, and the study of these and their metabolites, by describing the different cultivation conditions used in recent years, as well as the results obtained. The relevance of Agaricus, Flammulina, Grifola, Pleurotus and Lentinula genera, will also be discussed, with emphasis on the last one, since Shiitake has been always considered as the ultimate medicinal mushroom.     

QTL mapping of egg albumen quality in egg layers

Background

A fresh, good quality egg has a firm and gelatinous albumen that anchors the yolk and restricts growth of microbiological pathogens. As the egg ages, the gel-like structure collapses, resulting in thin and runny albumen. Occasionally thin albumen is found in a fresh egg, giving the impression of a low quality product. A mapping population consisting of 1599 F₂ hens from a cross between White Rock and Rhode Island Red lines was set up, to identify loci controlling albumen quality. The phenotype for albumen quality was evaluated by albumen height and in Haugh units (HU) measured on three consecutive eggs from each F₂ hen at the age of 40 weeks. For the fine-mapping analysis, albumen height and HU were used simultaneously to eliminate contribution of the egg size to the phenotype.

Results

Linkage analysis in a small population of seven half-sib families (668 F₂) with 162 microsatellite markers spread across 27 chromosomes revealed two genome-wide significant regions with additive effects for HU on chromosomes 7 and Z. In addition, two putative genome-wide quantitative trait loci (QTL) regions were identified on chromosomes 4 and 26. The QTL effects ranged from 2 to 4% of the phenotypic variance. The genome-wide significant QTL regions on chromosomes 7 and Z were selected for fine-mapping in the full set composed of 16 half-sib families. In addition, their existence was confirmed by an association analysis in an independent commercial Hy-Line pure line.

Conclusions

We identified four chicken genomic regions that affect albumen quality. Our results also suggest that genes that affect albumen quality act both directly and indirectly through several different mechanisms. For instance, the QTL regions on both fine-mapped chromosomes 7 and Z overlapped with a previously reported QTL for eggshell quality, indicating that eggshell membranes may play a role in albumen quality.     

Disorders related to sexuality and gender identity in the ICD‐11: revising the ICD‐10 classification based on current scientific evidence,best clinical practices,and human rights considerations

In the World Health Organization's forthcoming eleventh revision of the International Classification of Diseases and Related Health Problems (ICD‐11), substantial changes have been proposed to the ICD‐10 classification of mental and behavioural disorders related to sexuality and gender identity. These concern the following ICD‐10 disorder groupings: F52 Sexual dysfunctions, not caused by organic disorder or disease; F64 Gender identity disorders; F65 Disorders of sexual preference; and F66 Psychological and behavioural disorders associated with sexual development and orientation. Changes have been proposed based on advances in research and clinical practice, and major shifts in social attitudes and in relevant policies, laws, and human rights standards. This paper describes the main recommended changes, the rationale and evidence considered, and important differences from the DSM‐5. An integrated classification of sexual dysfunctions has been proposed for a new chapter on Conditions Related to Sexual Health, overcoming the mind/body separation that is inherent in ICD‐10. Gender identity disorders in ICD‐10 have been reconceptualized as Gender incongruence, and also proposed to be moved to the new chapter on sexual health. The proposed classification of Paraphilic disorders distinguishes between conditions that are relevant to public health and clinical psychopathology and those that merely reflect private behaviour. ICD‐10 categories related to sexual orientation have been recommended for deletion from the ICD‐11.     

Focal adhesion kinase: predictor of tumour response and risk factor for recurrence after neoadjuvant chemoradiation in rectal cancer

Rectal cancer represents about 30% of colorectal cancers, being around 50% locally advanced at presentation. Chemoradiation (CRT) followed by total mesorectal excision is the standard of care for these locally advanced stages. However, it is not free of adverse effects and toxicity and the complete pathologic response rate is between 10% and 30%. This makes it extremely important to define factors that can predict response to this therapy. Focal adhesion kinase (FAK) expression has been correlated with worse prognosis in several tumours and its possible involvement in cancer radio‐ and chemosensitivity has been suggested; however, its role in rectal cancer has not been analysed yet. To analyse the association of FAK expression with tumour response to CRT in locally advanced rectal cancer. This study includes 73 patients with locally advanced rectal cancer receiving standard neoadjuvant CRT followed by total mesorectal excision. Focal adhesion kinase protein levels were immunohistochemically analysed in the pre‐treatment biopsies of these patients and correlated with tumour response to CRT and patients survival. Low FAK expression was significantly correlated with local and distant recurrence (P = 0.013). Low FAK expression was found to be a predictive marker of tumour response to neoadjuvant therapy (P = 0.007) and patients whose tumours did not express FAK showed a strong association with lower disease‐free survival (P = 0.01). Focal adhesion kinase expression predicts neoadjuvant CRT response in rectal cancer patients and it is a clinically relevant risk factor for local and distant recurrence.