The effect of tumour necrosis factor-α (TNF-α) muteins on human neutrophils in vitro

Tumour necrosis factor-α (TNF-α) has been implicated as an important inflammatory mediator. In vitro, TNF-α is reported to activate human polymorphonuclear neutrophils (PMN), inducing responses such as phagocytic activity, degranulation and oxidative metabolism. Biological responses to TNF-α are initiated by its binding to specific cell surface receptors, and various studies have shown that the major TNF receptor species on PMN is the 75 kDa receptor. To verify the suggestion that the receptor binding domain includes the region close to the N-terminus of the TNF-α molecule, four TNF-α derivatives termed muteins were constructed, using a synthetic cDNA fragment substituting the N-terminal 3–7 selected hydrophilic or hydrophobic amino acids in the original TNF-α genomic DNA. Binding of muteins to PMN was assessed using monoclonal antibodies recognizing either the 55 kDa (p55) or the 75 kDa (p75) TNF receptor subtypes. Blocking by muteins of anti-p75 antibody binding to PMN was as expected from their N-terminal amino acid composition and hydrophilic properties. Hydrophilic muteins competed well with anti-TNF receptor antibodies for binding to the p75 receptor. In contrast, hydrophobic muteins were unable to block anti-p75 binding. Similarly, degranulation, chemiluminescence or enhancement of the PMN response to specific stimuli by the muteins correlated with the hydrophilic properties of the muteins. The significance of these observations in relation to the molecular structure of TNF-α is discussed

Charged and Hydrophobic Surfaces on the A Chain of Shiga-Like Toxin 1 Recognize the C-Terminal Domain of Ribosomal Stalk Proteins

Shiga-like toxins are ribosome-inactivating proteins (RIP) produced by pathogenic E. coli strains that are responsible for hemorrhagic colitis and hemolytic uremic syndrome. The catalytic A1 chain of Shiga-like toxin 1 (SLT-1), a representative RIP, first docks onto a conserved peptide SD[D/E]DMGFGLFD located at the C-terminus of all three eukaryotic ribosomal stalk proteins and halts protein synthesis through the depurination of an adenine base in the sarcin-ricin loop of 28S rRNA. Here, we report that the A1 chain of SLT-1 rapidly binds to and dissociates from the C-terminal peptide with a monomeric dissociation constant of 13 µM. An alanine scan performed on the conserved peptide revealed that the SLT-1 A1 chain interacts with the anionic tripeptide DDD and the hydrophobic tetrapeptide motif FGLF within its sequence. Based on these 2 peptide motifs, SLT-1 A1 variants were generated that displayed decreased affinities for the stalk protein C-terminus and also correlated with reduced ribosome-inactivating activities in relation to the wild-type A1 chain. The toxin-peptide interaction and subsequent toxicity were shown to be mediated by cationic and hydrophobic docking surfaces on the SLT-1 catalytic domain. These docking surfaces are located on the opposite face of the catalytic cleft and suggest that the docking of the A1 chain to SDDDMGFGLFD may reorient its catalytic domain to face its RNA substrate. More importantly, both the delineated A1 chain ribosomal docking surfaces and the ribosomal peptide itself represent a target and a scaffold, respectively, for the design of generic inhibitors to block the action of RIPs

Natural humoral immune response to ribosomal P0 protein in colorectal cancer patients

Tumor associated antigens are useful in colorectal cancer (CRC) management. The ribosomal P proteins (P0, P1, P2) play an important role in protein synthesis and tumor formation. The immunogenicity of the ribosomal P0 protein in head and neck, in breast and prostate cancer patients and the overexpression of the carboxyl-terminal P0 epitope (C-22 P0) in some tumors were reported

The Worker Honeybee Fat Body Proteome Is Extensively Remodeled Preceding a Major Life-History Transition

Honeybee workers are essentially sterile female helpers that make up the majority of individuals in a colony. Workers display a marked change in physiology when they transition from in-nest tasks to foraging. Recent technological advances have made it possible to unravel the metabolic modifications associated with this transition. Previous studies have revealed extensive remodeling of brain, thorax, and hypopharyngeal gland biochemistry. However, data on changes in the abdomen is scarce. To narrow this gap we investigated the proteomic composition of abdominal tissue in the days typically preceding the onset of foraging in honeybee workers

Starość jako wartość i fenomen pedagogiczny

Starość ma niejedno oblicze. Różne dyscypliny naukowe definiują ją na różne sposoby. Niemniej jest ona powiązana z określonym etapem życia i osobistym doświadczaniem jej przez konkretnego człowieka. Żadna z form starzenia się nie może wykluczać człowieka ze społeczności. Każdy stary człowiek ma pełne prawo do zachowania godności i okazywania mu szacunku, a jeśli zachodzi konieczność, do wsparcia i opieki. Wraz z kolejnymi latami życia przestrzeń życiowa i społeczna seniorów zawęża się. Narasta w nich poczucie samotności i osamotnienia. Okres starości nie wyklucza automatycznie ze świadomości seniorów poczucia własnej podmiotowości i tożsamości. Przeprowadzanie„bilansu życia” w okresie starości pozwala, chociaż niekoniecznie prowadzić musi do wnikliwej oceny moralnej swojej osoby. Rzetelny bilans życia polega na indywidualnym odniesieniu się do pytania stawianego samemu sobie o jego ocenę. Upowszechnianie się typu rodziny nuklearnej, w skład której wchodzą jedynie rodzice i dzieci, narastające zjawisko dysfunkcjonalności rodzin, spowodowało zmianę pozycji i roli człowieka starego w rodzinie. Pogarszające się sukcesywnie stan zdrowia i kondycja psychiczna człowieka starego powodują, że jest on marginalizowany przez najbliższe otoczenie, często pozostawiony sam sobie. Nie zawsze rodziny są przygotowane i dojrzałe do współbycia z osobami starszymi. Wejście w wiek senioralny każdego człowieka w różny sposób wieńczy przebytą drogę, która wiodła ku wszelkim potencjalnym możliwościom i prowadziła go na szczyt indywidualnych osiągnięć życiowych. Nie jest i nie musi być ona jego kresem. Ważne jest, aby senior zdawał sobie sprawę z tego, że może być nadal potrzebny innym, powinien wykazywać otwartość i gotowość zdobywania wiedzy i pracy nad samym sobą oraz w miarę sił i możliwości świadczyć dobro na rzecz wspólnoty, do której przynależy. Gerontologia pedagogiczna staje przed zadaniem upowszechniania pewnych norm etosu starości. Do nich zapewne można zaliczyć te, które przyczyniają się do uznania wieku senioralnego jako wartościowego czasu życia..

An Ann-Based Scalable Hashing Algorithm for Computational Clouds with Schedulers

The significant benefits of cloud computing (CC) resulted in an explosion of their usage in the last several years. From the security perspective, CC systems have to offer solutions that fulfil international standards and regulations. In this paper, we propose a model for a hash function having a scalable output. The model is based on an artificial neural network trained to mimic the chaotic behaviour of the Mackey-Glass time series. This hashing method can be used for data integrity checking and digital signature generation. It enables constructing cryptographic services according to the user requirements and time constraints due to scalable output. Extensive simulation experiments are conduced to prove its cryptographic strength, including three tests: a bit prediction test, a series test, and a Hamming distance test. Additionally, flexible hashing function performance tests are run using the CloudSim simulator mimicking a cloud with a global scheduler to investigate the possibility of idle time consumption of virtual machines that may be spent on the scalable hashing protocol. The results obtained show that the proposed hashing method can be used for building light cryptographic protocols. It also enables incorporating the integrity checking algorithm that lowers the idle time of virtual machines during batch task processing

Elevated copy number of L-A virus in yeast mutant strains defective in ribosomal stalk.

The eukaryotic ribosomal stalk, composed of the P-proteins, is a part of the GTPase-associated-center which is directly responsible for stimulation of translation-factor-dependent GTP hydrolysis. Here we report that yeast mutant strains lacking P1/P2-proteins show high propagation of the yeast L-A virus. Affinity-capture-MS analysis of a protein complex isolated from a yeast mutant strain lacking the P1A/P2B proteins using anti-P0 antibodies showed that the Gag protein, the major coat protein of the L-A capsid, is associated with the ribosomal stalk. Proteomic analysis revealed that the elongation factor eEF1A was also present in the isolated complex. Additionally, yeast strains lacking the P1/P2-proteins are hypersensitive to paromomycin and hygromycin B, underscoring the fact that structural perturbations in the stalk strongly influence the ribosome function, especially at the level of elongation

The effects of tumor necrosis factor (TNF) derivatives on TNF receptors.

The pleiotropic cytokine TNF has been implicated in the regulation of many immune and inflammatory responses in vivo, and in addition exerts a wide range of effects on target cells in vitro. However, although two cell surface receptors for TNF have been identified, and their cDNAs cloned, the amino acid residues necessary for the biological activity of TNF have not been characterized. We have therefore constructed derivatives of TNF termed 'muteins', in which the first 3 to 7 amino acids of native TNF-alpha have been replaced, using synthetic cDNA expressed in E. coli. In the present study we compare the effects of native TNF-alpha and muteins III, IV, V and VI in several different in vitro systems and in one in vivo model. We observed binding to the p75 TNF receptor on Jijoye Burkitt lymphoma cells with native TNF-alpha and mutein III alone, whereas the p55 TNF receptor on the human epithelioid carcinoma cell line HeLa bound TNF-alpha, mutein III and mutein V. Muteins IV and VI failed to recognize either TNF receptor. WEHI 164 fibrosarcoma cells were killed by muteins III, V and VI. Human umbilical vein endothelial cells responded to native TNF-alpha and to muteins III, IV and V, but not to mutein VI, by increasing the surface expression of ICAM-1 antigen and secretion of the cytokines GM-CSF and IL-6. All four compounds were pro-inflammatory in a mouse in vivo model. The results presented in this report confirm that N-terminal amino acids are critical for both receptor binding and biological activity of TNF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS

The effects of tumor necrosis factor (TNF) derivatives on TNF receptors.

The pleiotropic cytokine TNF has been implicated in the regulation of many immune and inflammatory responses in vivo, and in addition exerts a wide range of effects on target cells in vitro. However, although two cell surface receptors for TNF have been identified, and their cDNAs cloned, the amino acid residues necessary for the biological activity of TNF have not been characterized. We have therefore constructed derivatives of TNF termed 'muteins', in which the first 3 to 7 amino acids of native TNF-alpha have been replaced, using synthetic cDNA expressed in E. coli. In the present study we compare the effects of native TNF-alpha and muteins III, IV, V and VI in several different in vitro systems and in one in vivo model. We observed binding to the p75 TNF receptor on Jijoye Burkitt lymphoma cells with native TNF-alpha and mutein III alone, whereas the p55 TNF receptor on the human epithelioid carcinoma cell line HeLa bound TNF-alpha, mutein III and mutein V. Muteins IV and VI failed to recognize either TNF receptor. WEHI 164 fibrosarcoma cells were killed by muteins III, V and VI. Human umbilical vein endothelial cells responded to native TNF-alpha and to muteins III, IV and V, but not to mutein VI, by increasing the surface expression of ICAM-1 antigen and secretion of the cytokines GM-CSF and IL-6. All four compounds were pro-inflammatory in a mouse in vivo model. The results presented in this report confirm that N-terminal amino acids are critical for both receptor binding and biological activity of TNF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS