Mass spectrometric identification of the trypsin cleavage pathway in lysyl-proline containing oligotuftsin peptides.

Trypsin cleaves specifically peptide bonds at the C-terminal side of lysine and arginine residues, except for -Arg-Pro- and -Lys-Pro- bonds which are normally resistant to proteolysis. Here we report evidence for a -Lys-Pro- tryptic cleavage in modified oligotuftsin derivatives, Ac-[TKPKG]4-NH2) (1), using high-resolution mass spectrometry and HPLC as primary methods for analysis of proteolytic reactions. The proteolytic susceptibility of -Lys-Pro- bonds was strongly dependent on flanking residues, and the flexibility of the peptide backbone might be a prerequisite for this unusual cleavage. While -Lys-Gly- bonds in 1 were rapidly cleaved, the modification of these Lys residues by the attachment of a ss-amyloid(4-10) epitope to yield -Lys(X)-Gly derivatives prevented cleavage of this bond, and provided trypsin cleavage of -Lys-Pro- bonds, the pathway of this degradation being independent on the type of Lys-N(epsilon)-side chains (acetyl group, amino acid, peptide). Substitution of the Lys residues by Ala at the P'2 positions decreased the tryptic cleavage, while replacement of the bulky side chain of Thr at the P2 positions strongly increased the cleavage of -Lys-Pro- bonds. Circular dichroism (CD) data of the modified oligotuftsin derivatives are in accord with enhanced flexibility of the peptide backbone, as a prerequisite for increased susceptibility to cleavage of -Lys-Pro- bonds. These results obtained of oligotuftsin derivatives might have implications for the proteolytic degradation of target peptides that require specific conformational preconditions.     

The Depolarizing Action of GABA Controls Early Network Activity in the Developing Hippocampus

Early in postnatal life γ-aminobutyric acid (GABA), the primary inhibitory transmitter in adults, excites targeted neurons by an outwardly directed flux of chloride which results from the unbalance between the cation–chloride cotransporters NKCC1 and KCC2, involved in chloride uptake and extrusion, respectively. This effect contributes to generate synchronized network activity or giant depolarizing potentials (GDPs) in the developing hippocampus. Here, we review some recent data concerning the mechanisms by which GDPs are generated and their functional role in enhancing synaptic efficacy at poorly developed GABAergic and glutamatergic synapses. In adulthood, reshaping neuronal circuits due to changes in chloride homeostasis and to the shift of GABA from hyperpolarizing to depolarizing, has been implicated in several neurological disorders, including epilepsy. Evidence has been recently provided that in chronically nerve growth factor-deprived mice expressing a progressive age-dependent neurodegenerative pathology resembling that observed in patients with Alzheimer’s disease, the reduced expression of mRNA encoding for the Kcc2 gene and the depolarizing action of GABA lead to the reorganization of the neuronal hippocampal network. This may represent a novel mechanism by which GABAergic signaling counterbalances the loss of synaptic activity in neurodegenerative diseases.     

A Population-based study of dementia in the oldest old: the Monzino 80-plus Study

Background

Despite being the fastest growing and the most cognitively impaired age group, the oldest olds are under-represented in clinical research. The purpose of this study was to describe the design, methods, and baseline characteristics of the survey population and investigate possible differences in demographic, cognitive, functional, and behavioral characteristics between oldest old with and without any performance on cognitive tests and between oldest old alive and those deceased prior to the interview.

Methods

The Monzino 80-plus Study is a prospective door-to-door population-based survey among 80 years or older residents in the municipalities in the province of Varese, Italy. Dementia cases were identified with a one-phase design. Trained psychologists interviewed both the subject and a proxy informant. The interview included a comprehensive standardized questionnaire together with an array of rating scales and a multidomain cognitive battery to assess cognitive and functional ability, behavioral disturbances and mood.

Results

Information was available for 2,139 of the 2,428 registered individuals aged 80 years or older. Main baseline characteristics of the population are reported and discussed. In comparison with those living, elderly persons who had died before the first visit were older, had twice the rate of institutionalization, poorer cognitive performance and competence, and significantly greater instrumental and basic functional disability. The percentage of elderly persons, alive at baseline, without Mini-Mental State Examination rose rather evenly with age. Moreover, they had significantly worse cognitive competence and functional ability, and reported higher prevalences of depressive symptoms and problem behaviors than those with Mini-Mental State Examination.

Conclusions

Prospective investigation of a large population of oldest old can contribute significantly to understanding the relations between age, cognitive decline, and dementia occurrence. Use of informant-based instruments in surveys in the oldest old is crucial in assessing everyday functioning and changes, especially in participants with no cognitive test performance available. Failure to include information on deceased elderly would underestimate, increasingly with age, the prevalence of cognitive and functional disability in the elderly population.     

Detection of the TNFSF members BAFF, APRIL, TWEAK and their receptors in normal kidney and renal cell carcinomas

In advanced renal cell carcinoma (RCC), surgery combined with systemic chemotherapy and immunotherapy have had limited effectiveness. Therapeutic modalities targeting VEGF, PDGF, and c-kit using tyrosine kinase inhibitors and m-TOR using specific biologic factors are in development. Therapeutic approaches targeting TNF-alpha have shown limited efficacy, while anti-TRAIL (TNFSF10) antibodies have shown enhanced activity. The presence and potential significance of other members of the TNFSF has not been investigated. Here, we assayed the TNFSF members APRIL, BAFF, TWEAK and their receptors (BCMA, TACI, BAFFR, Fn14) in 86 conventional type clear cell RCC, using immunohistochemistry and correlated our findings with histological data and, in a limited series, follow-up of patients. We observed a differential expression of these TNFSF ligands and receptors in cancerous and non-cancerous structures. BAFF was found in all RCC; APRIL expression is associated with an aggressive phenotype, correlating negatively with patients' disease-free survival, while TWEAK and its receptor Fn14 are heterogeneously expressed, correlating negatively with the grade and survival of RCC patients. This is the first study, presenting together the TNFSF members APRIL, BAFF, TWEAK and their receptors in different areas of normal renal tissue and RCC, suggesting a potential role of these TNFSF members in renal tumor biology.     

3D tumour models: novel in vitro approaches to cancer studies

3D in vitro models have been used in cancer research as a compromise between 2-dimensional cultures of isolated cancer cells and the manufactured complexity of xenografts of human cancers in immunocompromised animal hosts. 3D models can be tailored to be biomimetic and accurately recapitulate the native in vivo scenario in which they are found. These 3D in vitro models provide an important alternative to both complex in vivo whole organism approaches, and 2D culture with its spatial limitations. Approaches to create more biomimetic 3D models of cancer include, but are not limited to, (i) providing the appropriate matrix components in a 3D configuration found in vivo, (ii) co-culturing cancer cells, endothelial cells and other associated cells in a spatially relevant manner, (iii) monitoring and controlling hypoxia- to mimic levels found in native tumours and (iv) monitoring the release of angiogenic factors by cancer cells in response to hypoxia. This article aims to overview current 3D in vitro models of cancer and review strategies employed by researchers to tackle these aspects with special reference to recent promising developments, as well as the current limitations of 2D cultures and in vivo models. 3D in vitro models provide an important alternative to both complex in vivo whole organism approaches, and 2D culture with its spatial limitations. Here we review current strategies in the field of modelling cancer, with special reference to advances in complex 3D in vitro models.     

FLO11-based model for air-liquid interfacial biofilm formation by Saccharomyces cerevisiae

Sardinian wine strains of Saccharomyces cerevisiae used to make sherry-like wines form a biofilm at the air-liquid interface at the end of ethanolic fermentation, when grape sugar is depleted and further growth becomes dependent on access to oxygen. Here, we show that FLO11, which encodes a hydrophobic cell wall glycoprotein, is required for the air-liquid interfacial biofilm and that biofilm cells have a buoyant density greater than the suspending medium. We propose a model for biofilm formation based on an increase in cell surface hydrophobicity occurring at the diauxic shift. This increase leads to formation of multicellular aggregates that effectively entrap carbon dioxide, providing buoyancy. A visible biofilm appears when a sufficient number of hydrophobic cell aggregates are carried to and grow on the liquid surface.     

CD8+ T lymphocytes in lung tissue from patients with idiopathic pulmonary fibrosis

Objective

To study the prevalence of respiratory and atopic symptoms in (young) adults born prematurely, differences between those who did and did not develop Bronchopulmonary Disease (BPD) at neonatal age and differences in respiratory health between males and females.

Methods

Design: Prospective cohort study. Setting: Nation wide follow-up study, the Netherlands. Participants: 690 adults (19 year old) born with a gestational age below 32 completed weeks and/or with a birth weight less than 1500 g. Controls were Dutch participants of the European Community Respiratory Health Survey (ECRHS). Main outcome measures: Presence of wheeze, shortness of breath, asthma, hay fever and eczema using the ECRHS-questionnaire

Results

The prevalence of doctor-diagnosed asthma was significantly higher in the ex-preterms than in the general population, whereas eczema and hay fever were significant lower. Women reported more symptoms than men. Preterm women vs controls: asthma 13% vs 5% (p < 0.001); hay fever 8% vs 20% (p < 0.001); eczema 10% vs 42% (p < 0.001). Preterm men vs controls: asthma 9% vs 4% (p = 0.007); hay fever 8% vs 17% (p = 0.005); eczema 9% vs 31% (p < 0.001) Preterm women reported more wheeze and shortness of breath during exercise (sob) than controls: wheeze 30% vs 22% (p = 0.009); sob 27% vs 16% (p < 0.001); 19-year-old women with BPD reported a higher prevalence of doctor diagnosed asthma compared to controls (24% vs 5% p < 0.001) and shortness of breath during exercise (43% vs 16% p = 0.008). The prevalence of reported symptoms by men with BPD were comparable with the controls.

Conclusion

Our large follow-up study shows a higher prevalence of asthma, wheeze and shortness of breath in the prematurely born young adults. 19-year-old women reported more respiratory symptoms than men. Compared to the general population atopic diseases as hay fever and eczema were reported less often.