CSF3R,SETBP1 and CALR mutations in chronic neutrophilic leukemia

The WHO 2008 definition of chronic neutrophilic leukemia (CNL) is based on clinical and laboratory parameters but not on molecular abnormalities. Mutations in CSF3R, SETBP1 and CALR are reported in patients with chronic neutrophilic leukemia (CNL). However, because CNL is rare, there are few large studies of this issue. We sequenced these genes in 14 patients who met the WHO-criteria of CNL. 8 subjects had CSF3R ^T618I , 6 SETBP1 mutations and 1 a CALR mutation. Our data suggest mutation analysis of CSF3R, SETBP1 and CALR should be included in the diagnostic criteria for CNL. These data may also have therapy implications.     

Cranial nerve lesion in diabetic patients

The retrospective investigation was done about relationships between diabetes and cranial nerve lesions (CNL) on the sample of hospitalized neurological patients in Clinical Hospital Dubrava (CHD) in 6 yrs. period (2001-2006). The goal was to expand the cognition about CNL as a complication of diabetes, to investigate possibility of better therapy models as well as to investigate the prevention possibilities. The results show that CNL are significantly more present by the diabetic patients vrs. the other hospitalized neurological patients. The main risk factors for CNL development are the duration of diabetes, patient's age and diabetes per se. No significant differences between masculine and feminine patients were registered nor by diabetics neither by other patients. For CNL are also not from significant importance the successfully treatment of diabetes, as well as type of antidiabetic and other medication. This investigation can not confirm the suspicion that some of antidiabetic medicaments are responsible for CNL due to their neurotoxic side effects.     

Lysosomal Degradation of CD44 Mediates Ceramide Nanoliposome-induced Anoikis and Diminished Extravasation in Metastatic Carcinoma Cells

The ceramide nanoliposome (CNL) has shown promise in being able to treat a variety of primary tumors. However, its potential for treating metastatic cancer remains unknown. In this study, we demonstrate that CNL increases anoikis while preventing cancer cell extravasation under both static and physiological fluid flow conditions. Mechanistically, CNL limits metastases by decreasing CD44 protein levels in human breast and pancreatic cancer cells via lysosomal degradation of CD44, independent of palmitoylation or proteasome targeting. siRNA down-regulation of CD44 mimics CNL-induced anoikis and diminished extravasation of cancer cells. Taken together, our data indicate that ceramide limits CD44-dependent cancer cell migration, suggesting that CNL could be used to prevent and treat solid tumor metastasis.     

Differential photoperiodic responses in genetically identical winged and wingless pea aphids, Acyrthosiphon pisum, and the effect of day length on wing development

Abstract. Winged and wingless individuals of a pink clone of the pea aphid, Acyrthosiphon pisum (Harris), showed differences in the response curves for photoperiodic induction of both males and sexual females (oviparae). The critical night length (CNL) for ovipara induction in winged aphids was 0.75 h shorter than in wingless aphids, whereas the CNL for male induction in winged aphids was 1.0h longer than in wingless aphids. This means that in winged aphids the CNL for male induction in winged aphids was 0.5 h longer than that for ovipara induction, while in wingless aphids the CNL for male induction was 1.0–1.5 h shorter than that for ovipara induction, and also the shapes of the curves differed.
Winged aphids were produced by wingless mothers which were crowded as young adults. However, when young adults were crowded in long nights, winged aphids were not produced, and the CNL for wing inhibition was between 9.5 and 10h. This effect of photoperiod on wing induction was maternal.     

Bivalent carbohydrate binding is required for biological activity of Clitocybe nebularis lectin (CNL), the N,N'-diacetyllactosediamine (GalNAcβ1-4GlcNAc, LacdiNAc)-specific lectin from basidiomycete C. nebularis

Lectins are carbohydrate-binding proteins that exert their biological activity by binding to specific cell glycoreceptors. We have expressed CNL, a ricin B-like lectin from the basidiomycete Clitocybe nebularis in Escherichia coli. The recombinant lectin, rCNL, agglutinates human blood group A erythrocytes and is specific for the unique glycan N,N'-diacetyllactosediamine (GalNAcβ1-4GlcNAc, LacdiNAc) as demonstrated by glycan microarray analysis. We here describe the crystal structures of rCNL in complex with lactose and LacdiNAc, defining its interactions with the sugars. CNL is a homodimeric lectin, each of whose monomers consist of a single ricin B lectin domain with its β-trefoil fold and one carbohydrate-binding site. To study the mode of CNL action, a nonsugar-binding mutant and nondimerizing monovalent CNL mutants that retain carbohydrate-binding activity were prepared. rCNL and the mutants were examined for their biological activities against Jurkat human leukemic T cells and the hypersensitive nematode Caenorhabditis elegans mutant strain pmk-1. rCNL was toxic against both, although the mutants were inactive. Thus, the bivalent carbohydrate-binding property of homodimeric CNL is essential for its activity, providing one of the rare pieces of evidence that certain activities of lectins are associated with their multivalency.     

Production and properties of two collagenases from bacteria and their application for collagen extraction

Two collagenolytic protease (collagenase) producing bacteria, a Gram positive Bacillus cereus CNA1 and a Gram negative Klebsiella pneumoniae CNL3, were isolated under alkaline and acidic conditions, respectively. The production of collagenase by these two bacteria was optimized. Glycerol was the suitable carbon source for collagenase production by both strains. The optimal initial pH values for collagenase production by CNA1 and CNL3 were 7.5 and 6.0, respectively, and the optimal temperature was 37°C for both strains. The maximum activity of the partially purified collagenase from CNA1 was at pH 7.0 and 45°C. Its pH and thermal stability were in the range of 6-8 and below 40°C, respectively. The maximum activity of the partially purified collagenase from CNL3 was at pH 6.0 and 40°C. Its pH and thermal stability were in the range of 5-7 and below 37°C, respectively. The collagenase from CNL3 was more stable at a low pH compared with that from CNA1. Collagenases from both strains were used to extract collagen from salmon fish skin. The use of collagenases from CNA1 and CNL3 combined with acid treatment yielded a high collagen extraction of 54.6% and 53.0%, of the fish skin dry weight, respectively.     

Genome-wide analysis of NBS-LRR-encoding genes in Arabidopsis

The Arabidopsis genome contains approximately 200 genes that encode proteins with similarity to the nucleotide binding site and other domains characteristic of plant resistance proteins. Through a reiterative process of sequence analysis and reannotation, we identified 149 NBS-LRR-encoding genes in the Arabidopsis (ecotype Columbia) genomic sequence. Fifty-six of these genes were corrected from earlier annotations. At least 12 are predicted to be pseudogenes. As described previously, two distinct groups of sequences were identified: those that encoded an N-terminal domain with Toll/Interleukin-1 Receptor homology (TIR-NBS-LRR, or TNL), and those that encoded an N-terminal coiled-coil motif (CC-NBS-LRR, or CNL). The encoded proteins are distinct from the 58 predicted adapter proteins in the previously described TIR-X, TIR-NBS, and CC-NBS groups. Classification based on protein domains, intron positions, sequence conservation, and genome distribution defined four subgroups of CNL proteins, eight subgroups of TNL proteins, and a pair of divergent NL proteins that lack a defined N-terminal motif. CNL proteins generally were encoded in single exons, although two subclasses were identified that contained introns in unique positions. TNL proteins were encoded in modular exons, with conserved intron positions separating distinct protein domains. Conserved motifs were identified in the LRRs of both CNL and TNL proteins. In contrast to CNL proteins, TNL proteins contained large and variable C-terminal domains. The extant distribution and diversity of the NBS-LRR sequences has been generated by extensive duplication and ectopic rearrangements that involved segmental duplications as well as microscale events. The observed diversity of these NBS-LRR proteins indicates the variety of recognition molecules available in an individual genotype to detect diverse biotic challenges.     

Different cellular fatty acid pattern behaviours of two strains of Listeria monocytogenes Scott A and CNL 895807 under different temperature and salinity conditions

Cells of two strains of Listeria monocytogenes CNL 895807 and Scott A were grown to late exponential phase at different growth temperatures (37, 20 and 4 degrees C) with or without NaCl (7%), and their fatty acid compositions were analysed. The results showed that low thermal adaptation response of L. monocytogenes CNL was different than that of the Scott A strain, and it was based on both an increase of anteiso-branched-chain fatty acids and a significant decrease of straight-chain fatty acids. However, the main modifications observed in the Scott A strain when grown at a low temperature were a decrease of the proportion of ai17:0 and an increase of ai15:0. In hyperosmotic medium and over the entire temperature range (4 degrees C, 20 degrees C and 37 degrees C) the two L. monocytogenes strains showed a cellular fatty acid profile dominated by ai15:0. In addition, a decrease of the two major straight-chain fatty acids (14:0 and 16:0) was observed in the CNL strain. These results demonstrated that the CNL strain showed different behaviours of low thermal and salt adaptation to maintain membrane fluidity, which are based both on an increase of anteiso-branched-chain fatty acids, and a significant decrease of straight-chain fatty acids.     

A genome-wide genetic map of NB-LRR disease resistance loci in potato

Like all plants, potato has evolved a surveillance system consisting of a large array of genes encoding for immune receptors that confer resistance to pathogens and pests. The majority of these so-called resistance or R proteins belong to the super-family that harbour a nucleotide binding and a leucine-rich-repeat domain (NB-LRR). Here, sequence information of the conserved NB domain was used to investigate the genome-wide genetic distribution of the NB-LRR resistance gene loci in potato. We analysed the sequences of 288 unique BAC clones selected using filter hybridisation screening of a BAC library of the diploid potato clone RH89-039-16 (S. tuberosum ssp. tuberosum) and a physical map of this BAC library. This resulted in the identification of 738 partial and full-length NB-LRR sequences. Based on homology of these sequences with known resistance genes, 280 and 448 sequences were classified as TIR-NB-LRR (TNL) and CC-NB-LRR (CNL) sequences, respectively. Genetic mapping revealed the presence of 15 TNL and 32 CNL loci. Thirty-six are novel, while three TNL loci and eight CNL loci are syntenic with previously identified functional resistance genes. The genetic map was complemented with 68 universal CAPS markers and 82 disease resistance trait loci described in literature, providing an excellent template for genetic studies and applied research in potato.     

The enzyme cytochemistry and composition of elasmobranch granulocytes

Peroxidase, alkaline phosphatase, acid phosphatase, β-glucuronidase, α-naphthyl acetate esterase (ANAE), α-naphthyl butyrate esterase, naphthol AS-D chloroacetate esterase, acetyl-L-tyrosine-α-naphthyl esterase (ATNE), tosyl-L-lysine-α-naphthyl esterase (TLNE) and periodic acid-Schiff (PAS) were studied in 17 species of elasmobranchs in which granulocytes had previously been identified at the ultrastructural level.
Eosinophils, eosinophilic and neutrophilic granulocytes contained variable acid phosphatase, esterases and PAS, but they were strongest in neutrophilic granulocytes; particularly ANAE. Esterases were released into surrounding plasma and therefore probably function as ectoenzymes. In eosinophils and some neutrophilic granulocytes there were indications of weak peroxidase, but this could not be conclusively demonstrated. Alkaline phosphatase was diffuse between granules in some eosinophils of Pavoraja , and (β-glucuronidase was diffuse in neutrophilic granulocytes of Etmopterus baxteri , otherwise granulocytes lacked these enzymes. Neutrophilic granulocytes stained moderately to strongly for ATNE and weakly and inconsistently for TLNE in Squalus acanthias and Dalatias licha . with a similar reaction in granular lymphocytoid and thrombocytoid cells of Galeorhinus ausiralis and Raja nasuta . The enzyme composition of these granulocytes is discussed.     

Six amino acid changes confined to the leucine-rich repeat beta-strand/beta-turn motif determine the difference between the P and P2 rust resistance specificities in flax

At least six rust resistance specificities (P and P1 to P5) map to the complex P locus in flax. The P2 resistance gene was identified by transposon tagging and transgenic expression. P2 is a member of a small multigene family and encodes a protein with nucleotide binding site (NBS) and leucine-rich repeat (LRR) domains and an N-terminal Toll/interleukin-1 receptor (TIR) homology domain, as well as a C-terminal non-LRR (CNL) domain of approximately 150 amino acids. A related CNL domain was detected in almost half of the predicted Arabidopsis TIR-NBS-LRR sequences, including the RPS4 and RPP1 resistance proteins, and in the tobacco N protein, but not in the flax L and M proteins. Presence or absence of this domain defines two subclasses of TIR-NBS-LRR resistance genes. Truncations of the P2 CNL domain cause loss of function, and evidence for diversifying selection was detected in this domain, suggesting a possible role in specificity determination. A spontaneous rust-susceptible mutant of P2 contained a G-->E amino acid substitution in the GLPL motif, which is conserved in the NBS domains of plant resistance proteins and the animal cell death control proteins APAF-1 and CED4, providing direct evidence for the importance of this motif in resistance gene function. A P2 homologous gene isolated from a flax line expressing the P resistance specificity encodes a protein with only 10 amino acid differences from the P2 protein. Chimeric gene constructs indicate that just six of these amino acid changes, all located within the predicted beta-strand/beta-turn motif of four LRR units, are sufficient to alter P2 to the P specificity.     

Polyclonal Antibody Development Against Purified CC-NBS-LRR like Protein Fragment from Mature <Emphasis Type="Italic">Lageneria siceraria</Emphasis> Seeds and Immunolocalization

CC-NBS-LRR (CNL) plant proteins are related with highly conserved family of disease resistance protein distinguished by a coiled-coil domain, which plays an important role in innate immunity. The present study reports the purification and identification of CNL like protein fragment (CNL-LPF) by two step chromatography and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF/MS), respectively. Furthermore, current study also illustrated the development of polyclonal antibody against purified CNL-LPF, which was used for immunolocalization of CNL-LPF in cytoplasm of cotyledon, using Fluorescence microscopy and Transmission electron microscopy. Lastly, present study also demonstrates in vitro oligomerization of purified CNL-LPF with multiple bands on 4–10 % gradient native-PAGE; each band representing a small fraction of each oligomer population as evident by immunoblots. In conclusion, the current study deals with the purification and polyclonal antibody development against CNL-LPF.     

Photoperiodic induction of winged females in the black bean aphid. Aphis fabae

Abstract. The Photoperiodic of winged females (alatae) in the black bean aphid, Aphis fabae Scop. (Homopetera: Aphididae), is investigated in detail with emphasis on the interaction of the maternal and embryonic/young larval photoperiodic clocks. Previous work had shown that in uncrowded conditions the induction of gynoparae (winged females that produce sexual females) requires both prenatal and postnatal exposure to long-night (12 h) Photoperidic cycles: present results show that sole postantal exposure to long nights of any lenght does not induce wing formation in early-born aphids.
When aphids were exposed to experimental light-dark cycles postanatally only, their daughters developed as alate in long nights and as apterae in short nights: the critical night lenght (CNL) was 11:1 h. Additional prenatal exposure to experimental regimes resulted in a significantly shorter CNL (10.6 h). This difference could be accounted for by the fact that more experimental light-dark cycles were experienced in the latter case.
Apterous aphids transferred from LD 16:8 h to LD 12:12 h as either third-or fourth-stadium larvae, or young adults, switched for aptera-production to alata-production. The transition form aptera- to alata-production was rather abrupt in third-stadium transfers but more gradual when transfers occurred as fourth-stadium larvae and adults. Moreover, s the number of days required for 50% of the aphids to become alata-producers increased from 7–8 in third-stadium transfers, to 9–10 and 11–12 in the later transfers.     

Molecular cloning, nucleotide sequence, and expression of the structural gene for alkaline serine protease from alkaliphilic Bacillus sp. 221.

The gene encoding an alkaline serine protease from alkaliphilic Bacillus sp. 221 was cloned in Escherichia coli and expressed in Bacillus subtilis. An open reading frame of 1,140 bases, identified as the protease gene was preceded by a putative Shine-Dalgarno sequence (AGGAGG) with a spacing of 7 bases. The deduced amino acid sequence had a pre-pro-peptide of 111 residues followed by the mature protease comprising 269 residues. The alkaline protease from alkaliphilic Bacillus sp. 221 had higher homology to the protease from alkaliphilic bacilli (82.1% and 99.6%) than to those from neutrophilic bacilli (60.6-61.7%). Also Bacillus sp. 221 protease and other protease from alkaliphilic bacilli shared common amino acid changes and 4 amino acid deletions that seemed to be related to characteristics of the enzyme of alkaliphilic bacilli when compared to the proteases from neutrophilic bacilli.     

Thioredoxin-1 Protects against Neutrophilic Inflammation and Emphysema Progression in a Mouse Model of Chronic Obstructive Pulmonary Disease Exacerbation

Background

Exacerbations of chronic obstructive pulmonary disease (COPD) are characterized by acute enhancement of airway neutrophilic inflammation under oxidative stress and can be involved in emphysema progression. However, pharmacotherapy against the neutrophilic inflammation and emphysema progression associated with exacerbation has not been established. Thioredoxin-1 has anti-oxidative and anti-inflammatory properties and it can ameliorate neutrophilic inflammation through anti-chemotactic effects and prevent cigarette smoke (CS)-induced emphysema. We aimed to determine whether thioredoxin-1 can suppress neutrophilic inflammation and emphysema progression in a mouse model of COPD exacerbation and if so, to reveal the underlying mechanisms.

Results

Mice were exposed to CS and then challenged with polyinosine-polycytidylic acid [poly(I:C)], an agonist for virus-induced innate immunity. Airway neutrophilic inflammation, oxidative stress and lung apoptosis were enhanced in smoke-sensitive C57Bl/6, but not in smoke-resistant NZW mice. Exposure to CS and poly(I:C) challenge accelerated emphysema progression in C57Bl/6 mice. Thioredoxin-1 suppressed neutrophilic inflammation and emphysema progression. Poly(I:C) caused early neutrophilic inflammation through keratinocyte-derived chemokine and granulocyte-macrophage colony-stimulating factor (GM-CSF) release in the lung exposed to CS. Late neutrophilic inflammation was caused by persistent GM-CSF release, which thioredoxin-1 ameliorated. Thioredoxin-1 enhanced pulmonary mRNA expression of MAP kinase phosphatase 1 (MKP-1), and the suppressive effects of thioredoxin-1 on prolonged GM-CSF release and late neutrophilic inflammation disappeared by inhibiting MKP-1.

Conclusion

Using a mouse model of COPD exacerbation, we demonstrated that thioredoxin-1 ameliorated neutrophilic inflammation by suppressing GM-CSF release, which prevented emphysema progression. Our findings deepen understanding of the mechanisms underlying the regulation of neutrophilic inflammation by thioredoxin-1 and indicate that thioredoxin-1 could have potential as a drug to counteract COPD exacerbation.     

A primary survey on bryophyte species reveals two novel classes of nucleotide-binding site (NBS) genes

Due to their potential roles in pathogen defense, genes encoding nucleotide-binding site (NBS) domain have been particularly surveyed in many angiosperm genomes. Two typical classes were found: one is the TIR-NBS-LRR (TNL) class and the other is the CC-NBS-LRR (CNL) class. It is seldom known, however, what kind of NBS-encoding genes are mainly present in other plant groups, especially the most ancient groups of land plants, that is, bryophytes. To fill this gap of knowledge, in this study, we mainly focused on two bryophyte species: the moss Physcomitrella patens and the liverwort Marchantia polymorpha, to survey their NBS-encoding genes. Surprisingly, two novel classes of NBS-encoding genes were discovered. The first novel class is identified from the P. patens genome and a typical member of this class has a protein kinase (PK) domain at the N-terminus and a LRR domain at the C-terminus, forming a complete structure of PK-NBS-LRR (PNL), reminiscent of TNL and CNL classes in angiosperms. The second class is found from the liverwort genome and a typical member of this class possesses an α/β-hydrolase domain at the N-terminus and also a LRR domain at the C-terminus (Hydrolase-NBS-LRR, HNL). Analysis on intron positions and phases also confirmed the novelty of HNL and PNL classes, as reflected by their specific intron locations or phase characteristics. Phylogenetic analysis covering all four classes of NBS-encoding genes revealed a closer relationship among the HNL, PNL and TNL classes, suggesting the CNL class having a more divergent status from the others. The presence of specific introns highlights the chimerical structures of HNL, PNL and TNL genes, and implies their possible origin via exon-shuffling during the quick lineage separation processes of early land plants.     

Activation of the Na+/H+ exchanger by phorbol ester and osmotic shock is dependent on the degree of neutrophilic maturation

Activation of neutrophils leading to superoxide production is accompanied by cytoplasmic alkalinization, which results from stimulation of the Na+/H+ exchanger. Since the exchanger undergoes permanent alterations during neutrophilic maturation of HL-60 cells (Costa-Casnellie et al.: Journal of Biological Chemistry 263:11851-11855, 1988), we investigated whether its response to external stimuli such as phorbol esters or osmotic shock also was modified during cell maturation. Mature HL-60 cells produce superoxide in response to active phorbol esters, whereas immature HL-60 cells do not. Stimulation of the exchanger by active phorbol esters (phorbol 12-myristate 13-acetate or phorbol 12,13-dibutyrate) was observed in mature neutrophilic HL-60 cells but not in their immature counterparts. Inactive 4-alpha phorbol had no effect in either cell population. Compound H7 inhibited phorbol ester activation by 65%. In mature neutrophilic cells activation of the exchanger by phorbol esters caused two novel changes of its properties: 1) its apparent Km for Na+ transport increased 2-fold; 2) its Vmax increased 2.6-fold. Phorbol esters also caused a shift in pH dependence of activation similar to that induced in other cells. Osmotic shock, a different method known to activate the exchanger of other cells, induced activation in mature neutrophilic cells but not in immature cells. Thus, the response of the exchanger to external stimuli is affected by alterations occurring in association with cell maturation.     

Src homology 2 domain of overexpressed Lyn kinase is responsible for the acceleration of granulocyte colony-stimulating factor-induced neutrophilic nuclear lobulation

We have previously shown that the overexpression of a Src family kinase, Lyn, and its kinase-negative form, LynKN, in a granulocyte progenitor cell line, GM-I62M, accelerates neutrophilic nuclear lobulation when the cells are cultured in the presence of granulocyte colony-stimulating factor. In this study, we investigated the role of the Src homology 2 (SH2) and SH3 domains of Lyn in the accelerated induction of nuclear lobulation. In contrast to wild-type Lyn, the overexpression of its SH2 domain mutant did not induce the accelerated nuclear morphological changes, but the overexpressed SH3 domain mutant had the same effects as wild-type Lyn. Therefore, the SH2 domain of Lyn is responsible for the accelerated induction of neutrophilic nuclear lobulation upon G-CSF stimulation.     

Acid aspiration-induced lung inflammation and injury are exacerbated in NADPH oxidase-deficient mice

Increased reactive oxidant intermediates (ROIs) from primed leukocytes have been implicated in the pathogenesis of acid aspiration lung injury. To evaluate the specific role of the phagocyte NADPH oxidase-derived ROIs in acid lung injury, the p47phox-/- knockout mouse model of chronic granulomatous disease was used. p47phox-/- mice developed a significantly greater alveolar neutrophilic leukocytosis compared with wild-type mice at all time points after acid injury, with the difference between genotypes being most marked at 48 h. In contrast, the p47phox-/- mice had a decreased number of macrophages in bronchoalveolar lavage (BAL) compared with wild-type at 48 h after acid or saline aspiration. Albumin concentration in BAL reflecting capillary leak was also greater in p47phox-/- compared with wild-type mice. BAL concentrations of proinflammatory cytokines and chemokines were greater in p47phox-/- compared with wild-type mice. These findings suggest that NADPH oxidase, directly or indirectly, plays a role in attenuating the acute neutrophilic response after acid lung injury. We speculate that this downmodulating effect may be mediated by promoting the transition from production of cytokines and chemokines involved in neutrophilic infiltration to a less injurious, chronic inflammatory response.