immunocytochemical localization of urokinase-type plasminogen activator in lewis lung carcinoma

The invasively growing and metasizing Lewis lung carcinoma consistently contained urokinase-type plasminogen activator (u-PA) enzyme activity. When investigated immunocytochemically with antibodies against u-PA, different parts of individual tumors showed a pronounced heterogeneity in staining intensity. Strong staining was found in areas with invasive growth and degradation of surrounding normal tissue, while other areas were completely devoid of staining. Immunoreactivity occurred both with a perinuclear cytoplasmic localization in tumor cells and associated with apparently extracellular material. SDS PAGE of tumor extracts, under both reducing and nonreducing conditions, followed by immunoblotting, showed only one immunocytochemically stainable band with an electrophoretic mobility corresponding to that of purified proenzyme to u-PA, while no two-chain u-PA was detected. This indicates that the major part of the activator in Lewis lung carcinoma is present as one-chain pro-u-PA.     

HIBERNATION CHEZ TROIS ESPÈCES DE MÉTOPIINES: HYMENOPTERA,ICHNEUMONIDAE

Résumé Les conditions de l'hibernation sont étudiées chez les trois espèces de Métopiines Chorinaeus funebris Gravenhorst, Triclistus podagricus Gravenhorst et Triclistus pygmaeus Cresson. Ces espèces hibernent à des stades de développement compris entre l'éonymphe et l'adulte non émergé (Tableau I). La durée de l'incubation complémentaire après réactivation par le froid à 2^o varie entre 10 et 30 jours en moyenne (Tableaux II, IV et V). Chez T. podagricus et T. pygmaeus, on a observé également des émergences d'adultes avant l'hiver (Tableau III). I1 semble que T. podagricus ne parvienne au stade d'imago hibernant qu'après une baisse de la température d'élevage. Aucune souche dépourvue de diapause n'a pu être créée, mais pour les trois espèces, deux générations annuelles seraient possibles sous conditions contrôlées. Enfin, l'utilisation éventuelle de T. pygmaeus pour une lutte biologique contre Zeiraphera diniana Guénée est évoquée.

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Summary The conditions of hibernation have been investigated in the three metopiine species Chorinaeus funebris, Triclistus podagricus and Triclistus pygmaeus, all entomophagous on Zeiraphera diniana Guénée (Lepidoptera, Tortricidae) in the Engadine Valley (Switzerland). The hibernating stages are eonymph, pronymph or unemerged adult inside the host pupae (Table I). The length of the complementary incubation after reactivation at 2^o varies between 10–30 days (Tables II, IV and V). A small number of T. podagricus and of T. pygmaeus regularly appear as adults before winter, in rearings of both laboratory and field populations (Table III). It seems that T. podagricus larvae need a reduction in the initial rearing temperature before full development is achieved and the hibernating stage is reached (imago). No strain without diapause could be obtained for any of the three species, but the rearing of two annual generations under controlled conditions is possible in the laboratory. The eventual use of T. pygmaeus as a control agent against Z. diniana is discussed.

     

Biology of Ixodes (Pholeoixodes) hexagonus under laboratory conditions Part II. Effect of mating on feeding and fecundity of females

The effect of mating on the feeding and fecundity ofIxodes (Pholeoixodes) hexagonus females was studied under controlled laboratory conditions of 22–23°C and 98% relative humidity. The feeding period of mated females was 6–15 days and 11–13 days for unmated females. The mean weight of the engorged mated females was 114.84±45.89 mg, whereas, that of the engorged unmated females was significantly lower (80.61±28.84 mg). During the initial slow feeding period, the weight of mated females increased 6.6 times. At the end of the blood feeding, they had increased their initial weight 35.5 times. Unmated females never entered the rapid engorgement phase and up to 12 days of feeding period their mean weight did not increase more than 9.2 times. The pre-oviposition periods of mated and unmated females were 6–15 days and 4–12 days, respectively. The mean of the egg production efficiency was 40.26±12.47% for mated females and 35.68±12.2% for unmated females. The mean of the mass conversion efficiency was 73.6±13.7% for mated females and 66.48 ±16.55% for unmated females. Sixty per cent of the eggs deposited by mated females hatched whereas only 1% of the eggs deposited by unmated females hatched. These results indicate thatI. hexagonus females possess some predisposition for parthenogenesis and only fertility and not fecundity depends on mating.     

An association between the acute phase response and patterns of antigen induced T cell proliferation in juvenile idiopathic arthritis

The aim of this research was to determine whether all memory T cells have the same propensity to migrate to the joint in patients with juvenile idiopathic arthritis. Paired synovial fluid and peripheral blood mononuclear cell proliferative responses to a panel of antigens were measured and the results correlated with a detailed set of laboratory and clinical data from 39 patients with juvenile idiopathic arthritis. Two distinct patterns of proliferative response were found in the majority of patients: a diverse pattern, in which synovial fluid responses were greater than peripheral blood responses for all antigens tested; and a restricted pattern, in which peripheral blood responses to some antigens were more vigorous than those in the synovial fluid compartment. The diverse pattern was generally found in patients with a high acute phase response, whereas patients without elevated acute phase proteins were more likely to demonstrate a restricted pattern. We propose that an association between the synovial fluid T cell repertoire and the acute phase response suggests that proinflammatory cytokines may influence recruitment of memory T cells to an inflammatory site, independent of their antigen specificity. Additionally, increased responses to enteric bacteria and the presence of αEβ7 T cells in synovial fluid may reflect accumulation of gut associated T cells in the synovial compartment, even in the absence of an elevated acute phase response. This is the first report of an association between the acute phase response and the T cell population recruited to an inflammatory site.     

Transglutaminase 6: a protein associated with central nervous system development and motor function

Transglutaminases (TG) form a family of enzymes that catalyse various post-translational modifications of glutamine residues in proteins and peptides including intra- and intermolecular isopeptide bond formation, esterification and deamidation. We have characterized a novel member of the mammalian TG family, TG6, which is expressed in a human carcinoma cell line with neuronal characteristics and in mouse brain. Besides full-length protein, alternative splicing results in a short variant lacking the second β-barrel domain in man and a variant with truncated β-sandwich domain in mouse. Biochemical data show that TG6 is allosterically regulated by Ca²⁺ and guanine nucleotides. Molecular modelling indicates that TG6 could have Ca²⁺ and GDP-binding sites related to those of TG3 and TG2, respectively. Localization of mRNA and protein in the mouse identified abundant expression of TG6 in the central nervous system. Analysis of its temporal and spatial pattern of induction in mouse development indicates an association with neurogenesis. Neuronal expression of TG6 was confirmed by double-labelling of mouse forebrain cells with cell type-specific markers. Induction of differentiation in mouse Neuro 2a cells with NGF or dibutyryl cAMP is associated with an upregulation of TG6 expression. Familial ataxia has recently been linked to mutations in the TGM6 gene. Autoantibodies to TG6 were identified in immune-mediated ataxia in patients with gluten sensitivity. These findings suggest a critical role for TG6 in cortical and cerebellar neurons.     

Evolutionary history of the SARS-CoV-2 Gamma variant of concern (P.1): a perfect storm

Our goal was to describe in more detail the evolutionary history of Gamma and two derived lineages (P.1.1 and P.1.2), which are part of the arms race that SARS-CoV-2 wages with its host. A total of 4,977 sequences of the Gamma strain of SARS-CoV-2 from Brazil were analyzed. We detected 194 sites under positive selection in 12 genes/ORFs: Spike, N, M, E, ORF1a, ORF1b, ORF3, ORF6, ORF7a, ORF7b, ORF8, and ORF10. Some diagnostic sites for Gamma lacked a signature of positive selection in our study, but these were not fixed, apparently escaping the action of purifying selection. Our network analyses revealed branches leading to expanding haplotypes with sites under selection only detected when P.1.1 and P.1.2 were considered. The P.1.2 exclusive haplotype H_5 originated from a non-synonymous mutational step (H3509Y) in H_1 of ORF1a. The selected allele, 3509Y, represents an adaptive novelty involving ORF1a of P.1. Finally, we discuss how phenomena such as epistasis and antagonistic pleiotropy could limit the emergence of new alleles (and combinations thereof) in SARS-COV-2 lineages, maintaining infectivity in humans, while providing rapid response capabilities to face the arms race triggered by host immuneresponses.     

Leptin activation of mTOR pathway in intestinal epithelial cell triggers lipid droplet formation,cytokine production and increased cell proliferation

Accumulating evidence suggests that obesity and enhanced inflammatory reactions are predisposing conditions for developing colon cancer. Obesity is associated with high levels of circulating leptin. Leptin is an adipocytokine that is secreted by adipose tissue and modulates immune response and inflammation. Lipid droplets (LD) are organelles involved in lipid metabolism and production of inflammatory mediators, and increased numbers of LD were observed in human colon cancer. Leptin induces the formation of LD in macrophages in a PI3K/mTOR pathway-dependent manner. Moreover, the mTOR is a serine/threonine kinase that plays a key role in cellular growth and is frequently altered in tumors. We therefore investigated the role of leptin in the modulation of mTOR pathway and regulation of lipid metabolism and inflammatory phenotype in intestinal epithelial cells (IEC-6 cells). We show that leptin promotes a dose- and time-dependent enhancement of LD formation. The biogenesis of LD was accompanied by enhanced CXCL1/CINC-1, CCL2/MCP-1 and TGF-β production and increased COX-2 expression in these cells. We demonstrated that leptin-induced increased phosphorylation of STAT3 and AKT and a dose and time-dependent mTORC activation with enhanced phosphorilation of the downstream protein P70S6K protein. Pre-treatment with rapamycin significantly inhibited leptin effects in LD formation, COX-2 and TGF-β production in IEC-6 cells. Moreover, leptin was able to stimulate the proliferation of epithelial cells on a mTOR-dependent manner. We conclude that leptin regulates lipid metabolism, cytokine production and proliferation of intestinal cells through a mechanism largely dependent on activation of the mTOR pathway, thus suggesting that leptin-induced mTOR activation may contribute to the obesity-related enhanced susceptibility to colon carcinoma.     

A kinetic analysis of hybridoma growth and metabolism in continuous suspension culture on serum-free medium

The steady-state metabolic parameters for a murine hybridoma cell line have been determined in continuous suspension culture over a wide range of dilution rates. Long-term adaption occurred over seven months in culture and resulted in lower glucose consumption rates, reduced lactate production, higher cell viability, and, consequently, growth rates more nearly matching the dilution rate. Antibody production rates decreased over the first two months and then remained stable for at least 75 days. The antibody production rate was not found to be growth associated. Steadystate amino acid uptake rates are presented for a wide range of growth rates.     

Evolution of transglutaminase genes: identification of a transglutaminase gene cluster on human chromosome 15q15. Structure of the gene encoding transglutaminase X and a novel gene family member, transglutaminase Z

We isolated and characterized the gene encoding human transglutaminase (TG)(X) (TGM5) and mapped it to the 15q15.2 region of chromosome 15 by fluorescence in situ hybridization. The gene consists of 13 exons separated by 12 introns and spans about 35 kilobases. Further sequence analysis and mapping showed that this locus contained three transglutaminase genes arranged in tandem: EPB42 (band 4.2 protein), TGM5, and a novel gene (TGM7). A full-length cDNA for the novel transglutaminase (TG(Z)) was obtained by anchored polymerase chain reaction. The deduced amino acid sequence encoded a protein with 710 amino acids and a molecular mass of 80 kDa. Northern blotting showed that the three genes are differentially expressed in human tissues. Band 4.2 protein expression was associated with hematopoiesis, whereas TG(X) and TG(Z) showed widespread expression in different tissues. Interestingly, the chromosomal segment containing the human TGM5, TGM7, and EPB42 genes and the segment containing the genes encoding TG(C),TG(E), and another novel gene (TGM6) on chromosome 20q11 are in mouse all found on distal chromosome 2 as determined by radiation hybrid mapping. This finding suggests that in evolution these six genes arose from local duplication of a single gene and subsequent redistribution to two distinct chromosomes in the human genome.