EGFR regulation of colon cancer stem-like cells during aging and in response to the colonic carcinogen dimethylhydrazine

One of the most consistent pathological conditions in the gastrointestinal tract with advancing age is malignancy, particularly gastrointestinal cancers, the incidence of which increases sharply with aging. Although the reasons for the age-related rise in colorectal cancer are not fully understood, we hypothesize that aging increases susceptibility of the colon to carcinogen(s)/toxicant(s), leading to an increase in cancer stem-like cells (CSLCs) that express cancer stem cell markers, in the colonic mucosa. The current study demonstrates that aging is associated with increased expression of several colon CSLC markers [CD44, CD166, and aldehyde dehydrogenase 1 (ALDH-1)] and a higher proportion of cells expressing these markers. Aging is also accompanied by increased expression of miR-21 in colon. These increases are further increased in response to the colonic carcinogen dimethylhydrazine (DMH). Aging is also associated with increased tyrosine-phosphorylated epidermal growth factor receptor (EGFR). Inhibition of EGFR using the EGFR inhibitor cetuximab abrogated the age-related increase in CD166 and ALDH-1 as well as miRNA (miR)-21. Our results provide new evidence that aging and DMH are associated with increases in CSLC biomarkers and miR21, each of which have been linked to colorectal cancer. EGFR inhibition attenuates these changes, indicating a role for EGFR in age- and mutagen-associated changes in CSLCs.     

Central administration of l- and d-aspartate attenuates stress behaviors by social isolation and CRF in neonatal chicks

Intracerebroventricular (i.c.v.) administration of l-aspartate (l-Asp) attenuates stress responses in neonatal chicks, but the mechanism has not been clarified. In the present study, three behavioral experiments were carried out under socially isolated stressful conditions exacerbated by the use of corticotrophin-releasing factor (CRF). In Experiment 1, i.c.v. injection of l-Asp attenuated behavioral stress responses (distress vocalization and active wakefulness) in a dose-dependent manner. Furthermore, l-Asp increased time spent standing/sitting motionless with eyes open and sitting motionless with head dropped (sleeping posture) in comparison with the group receiving CRF alone. In Experiment 2, i.c.v. injection of d-Asp dose-dependently decreased the number of distress vocalizations and the amount of time spent in active wakefulness. d-Asp increased the time spent standing/sitting motionless with eyes open compared with the group receiving CRF alone. In Experiment 3, we directly compared the effect of l-Asp with that of d-Asp. Both l- and d-Asp induced sedative effects under an acutely stressful condition. However, l-Asp, but not d-Asp, increased the time spent in a sleeping posture. These results indicate that both l- and d-Asp, when present in the brain, could induce a sedative effect, while the mechanism for hypnosis in neonatal chicks may be different for l-Asp in comparison with d-Asp.     

Effect of labelling with the radioisotope 65Zn on the performance of the eulophid, Colpoclypeus florus, a parasite of Tortricidae

Effects of the radioisotope ⁶⁵Zn used to label Colpoclypeus florus Wlk. were studied. Labelled parents did not show any decrease in parasitizing ability.Radioactive labelling shortened the adult life span to a certain degree depending on rearing conditions. It also increased embryonic and larval mortality of the progeny to some extent.

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Résumé L'effet du radioisotope ⁶⁵Zn, utilisé comme traceur, a été mis à l'épreuve sur Colpoclypeus florus, ectoparasite grégaire s'attaquant fréquemment aux Tortricidae dans les vergers de pommiers aux Pays-Bas. Les C. florus adultes, marqués avec ⁶⁵Zn en leurs donnant accès pendant 2 jours à du miel contenant cet isotope, présentent par comparaison avec des parasites alimentés de miel pur, une réduction d'environ 50% de la longévité dans des conditions permettant, ordinairement, une survie prolongée. L'aptitude à coloniser les larves-hôtes et le nombre d'oeufs déposés par larve-hôte ne sont pas modifiés, mais le taux d'éclosion et la survie des larves du parasite sont diminués de 5% à 10% par le traceur.Ainsi, avant toute étude de la dispersion du parasite après libération en verger, d'autres traceurs radioactifs doivent être testés.

     

Behavioral and olfactory antennal responses of Solenopsis geminata (Fabricius) (Hymenoptera: Formicidae) workers to their dufour gland secretion

Behavioral and electrophysiological tests were performed to evaluate the responses of workers of the ant Solenopsis geminata (Fabricius) from different size categories to Dufour gland extracts. Morphometric measures based in head widths across eyes were used to determine worker sizes. Trail following response of different worker sizes to Dufour gland extract from workers of different sizes was assessed. For each worker size category olfactory responses to Dufour gland extracts were determined using electroantennography (EAG). Gas chromatography and mass spectrometry (GC-MS) were used to determine the chromatographic profile of Dufour gland secretion for each worker size. Morphometric measures permitted to classify the workers of S. geminata as large, medium and small workers. Medium S. geminata workers displayed a significantly higher behavioral response to Dufour gland extracts produced by medium size workers. Similarly, medium workers showed a significantly higher EAG response to Dufour gland extracts produced by medium sized workers. Chromatographic profile of Dufour gland secretions produced by workers showed that each size category exhibited a characteristic profile of the three main components considered as potential trail pheromone constituents. This work showed that medium workers of S. geminata exhibited a high trail-following behavior as well as a high antennal response to Dufour gland secretion. This and their relative abundance in field foraging areas, suggest that medium-sized workers are specialized in foraging activities.     

Efficient biosynthetic incorporation of tryptophan and indole analogs in an integral membrane protein

Biosynthetic incorporation of tryptophan (Trp) analogs such as 7-azatryptophan, 5-hydroxytryptophan, and fluorotryptophan into a protein can facilitate its structural analysis by spectroscopic techniques such as fluorescence, phosphorescence, nuclear magnetic resonance, and Fourier transform infrared. Until now, the approach has dealt primarily with soluble proteins. In this article, we demonstrate that four different Trp analogs can be very efficiently incorporated into a membrane protein as demonstrated for the mannitol transporter of Escherichia coli (EII(mtl)). EII(mtl) overexpression was under control of the lambdaP(R) promoter, and the E. coli Trp auxotroph M5219 was used as host. This strain constitutively expresses the heat labile repressor protein of the lambdaP(R) promoter. Together with the presence of the repressor gene on the EII(mtl) plasmid, this resulted in a tightly controlled promoter system, a prerequisite for high Trp analog incorporation. A new method for determining the analog incorporation efficiency is presented that is suitable for membrane proteins. The procedure involves fitting of the phosphorescence spectrum as a linear combination of the Trp and Trp analog contributions, taking into account the influence of the protein environment on the Trp analog spectrum. The data show that the analog content of EII(mtl) samples is very high (>95%). In addition, we report here that biosynthetic incorporation of Trp analogs can also be effected with less expensive indole analogs, which in vivo are converted to L-Trp analogs.     

Prevalence and evolutionary origins of the del(GJB6-D13S1830) mutation in the DFNB1 locus in hearing-impaired subjects: a multicenter study

Mutations in GJB2, the gene encoding connexin-26 at the DFNB1 locus on 13q12, are found in as many as 50% of subjects with autosomal recessive, nonsyndromic prelingual hearing impairment. However, genetic diagnosis is complicated by the fact that 10%-50% of affected subjects with GJB2 mutations carry only one mutant allele. Recently, a deletion truncating the GJB6 gene (encoding connexin-30), near GJB2 on 13q12, was shown to be the accompanying mutation in approximately 50% of these deaf GJB2 heterozygotes in a cohort of Spanish patients, thus becoming second only to 35delG at GJB2 as the most frequent mutation causing prelingual hearing impairment in Spain. Here, we present data from a multicenter study in nine countries that shows that the deletion is present in most of the screened populations, with higher frequencies in France, Spain, and Israel, where the percentages of unexplained GJB2 heterozygotes fell to 16.0%-20.9% after screening for the del(GJB6-D13S1830) mutation. Our results also suggest that additional mutations remain to be identified, either in DFNB1 or in other unlinked genes involved in epistatic interactions with GJB2. Analysis of haplotypes associated with the deletion revealed a founder effect in Ashkenazi Jews and also suggested a common founder for countries in Western Europe. These results have important implications for the diagnosis and counseling of families with DFNB1 deafness.     

Influence of the Surface Topography of Stainless Steel on Bacterial Adhesion

Bacterial adhesion on stainless steel may cause problems such as microbially induced corrosion or represent a chronic source of microbial contamination. The investigation focussed on how the extent and patterns of four bacterial species comprising three different phyla and a broad variety of physicochemical characteristics was influenced by the surface topography of AISI 304 stainless steel. Five types of surface finish corresponding to roughness values R a between 0.03 and 0.89 w m were produced. Adhesion of all four bacteria was minimal at R a =0.16 w m, whereas smoother and rougher surfaces gave rise to more adhesion. This surface exhibited parallel scratches of 0.7 w m, in which a high proportion of bacteria of three of the strains aligned. Reduced overall adhesion was attributed to unfavorable interactions between this surface and bacteria oriented other than parallel to the scratches. Interaction energy calculations and considerations of micro-geometry confirmed this mechanism. Rougher surfaces exhibiting wider scratches allowed a higher fraction of bacteria to adhere in other orientations, whereas the orientation of cells adhered to the smoothest surface was completely random.     

A Novel Whole-Cell Mechanism for Long-Term Memory Enhancement

Olfactory-discrimination learning was shown to induce a profound long-lasting enhancement in the strength of excitatory and inhibitory synapses of pyramidal neurons in the piriform cortex. Notably, such enhancement was mostly pronounced in a sub-group of neurons, entailing about a quarter of the cell population. Here we first show that the prominent enhancement in the subset of cells is due to a process in which all excitatory synapses doubled their strength and that this increase was mediated by a single process in which the AMPA channel conductance was doubled. Moreover, using a neuronal-network model, we show how such a multiplicative whole-cell synaptic strengthening in a sub-group of cells that form a memory pattern, sub-serves a profound selective enhancement of this memory. Network modeling further predicts that synaptic inhibition should be modified by complex learning in a manner that much resembles synaptic excitation. Indeed, in a subset of neurons all GABA_A-receptors mediated inhibitory synapses also doubled their strength after learning. Like synaptic excitation, Synaptic inhibition is also enhanced by two-fold increase of the single channel conductance. These findings suggest that crucial learning induces a multiplicative increase in strength of all excitatory and inhibitory synapses in a subset of cells, and that such an increase can serve as a long-term whole-cell mechanism to profoundly enhance an existing Hebbian-type memory. This mechanism does not act as synaptic plasticity mechanism that underlies memory formation but rather enhances the response of already existing memory. This mechanism is cell-specific rather than synapse-specific; it modifies the channel conductance rather than the number of channels and thus has the potential to be readily induced and un-induced by whole-cell transduction mechanisms.     

Cell cycle and apoptosis regulatory protein-1: a novel regulator of apoptosis in the colonic mucosa during aging

Although the regulatory mechanisms for the age-related rise in proliferation and reduction in apoptosis in the colonic mucosa are yet to be fully delineated, we have demonstrated that these events are associated with increased expression and activation of epithelial growth factor receptor (EGFR)/ErbB-1 and some of its receptor family members (EGFRs), indicating their involvement in these processes. However, the downstream signaling events of EGFR and/or its family members regulating age-related changes in mucosal proliferation and apoptosis remain to be delineated. Cell cycle and apoptosis regulatory protein-1 (CARP-1), a novel growth signaling regulator that we isolated, participates in EGFR-dependent signaling. In the current investigation, we examined the involvement of CARP-1 in colonic mucosal growth-related processes during aging. We report that the age-related reduction in apoptosis in the colonic mucosa is associated with increased expression and tyrosine phosphorylation of not only EGFR but also ErbB-2 and ErbB-3. In contrast, protein and mRNA levels of CARP-1 as well as tyrosine phosphorylation of CARP-1 are decreased. Additionally, we have observed that administration of wortmannin, an inhibitor of phosphatidylinositol 3-kinase activity that accelerates apoptosis in the colonic mucosa of aged rats, causes a marked increase in expression and tyrosine phosphorylation of CARP-1. The age-related decline in CARP-1 expression could partly be attributed to increased methylation of the CARP-1 promoter. Taken together, our data suggest that not only EGFR but also its other members are involved in regulating colonic mucosal growth during aging and that CARP-1 may play a crucial role in transducing EGFRs signals.