Establishment of polarized endocytosis in differentiable intestinal HT29-18 subclones

Subclones of the HT29-18 clone, derived from a human adenocarcinoma, are able to acquire an enterocyte-like phenotype depending on the culture conditions. To investigate fluid-phase and receptor-mediated endocytosis in the polarized subclone HT29-18-C1, we established culture conditions that allowed cell growth on permeable supports. HT29-18-C1 monolayers had an electrical resistance of 43 ohms.cm2 and developed a transepithelial potential of about 2 mV. Transferrin receptors were uniformly distributed on the entire cell surface of undifferentiated HT29-18 cells but were located on the basolateral membrane of differentiated cells. Transferrin had a high affinity (Kd = 2.5 x 10(-9) M) for its receptor independent of the state of differentiation. The number of transferrin receptors and the mRNA amounts encoding them were comparable in the undifferentiated and differentiated HT29-18 cells. Transferrin was quickly internalized and recycled back to the cell surface of undifferentiated HT29-18 cells. The same phenomenon also occurred in differentiated HT29-18 cells, but the receptors were limited to the basolateral membrane. In the presence of ammonium chloride, the process was slower but remained polarized. Fluid-phase uptake was also investigated with horseradish peroxidase (HRP) in differentiated HT29-18 C1 cells. HRP that was internalized in 1 hour from a given membrane domain preferentially recycled back to the same membrane domain. No significant accumulation of the enzyme in the late endosomes and lysosomes of the differentiated HT29-18-C1 cells was observed.     

Changes in total and free tryptophan levels in serum following acute morphine administration in arthritic rats

In ‘arthritic’ rats a decrease in total tryptophan and an increase in free tryptophan levels was observed in serum after morphine administration (10 mg kg, s.c.). These changes were maximum within 15 and 30 min after injection.A decrease in total and an increase in free tryptophan levels in serum were observed 30 min after naloxone administration (1 mg/kg, i.m.).An increase in tryptophan and 5-hydroxyindoleacetic acid levels was also observed in the brain after morphine and naloxone.These observations suggest that the rise in 5-hydroxytryptamine synthesis provoked by morphine may be partly related to an increase in the availability of tryptophan from blood. However, the analgesia induced by the opiate appears unlikely to be directly related to this effect.     

The Association of Myosin IB with Actin Waves in Dictyostelium Requires Both the Plasma Membrane-Binding Site and Actin-Binding Region in the Myosin Tail

F-actin structures and their distribution are important determinants of the dynamic shapes and functions of eukaryotic cells. Actin waves are F-actin formations that move along the ventral cell membrane driven by actin polymerization. Dictyostelium myosin IB is associated with actin waves but its role in the wave is unknown. Myosin IB is a monomeric, non-filamentous myosin with a globular head that binds to F-actin and has motor activity, and a non-helical tail comprising a basic region, a glycine-proline-glutamine-rich region and an SH3-domain. The basic region binds to acidic phospholipids in the plasma membrane through a short basic-hydrophobic site and the Gly-Pro-Gln region binds F-actin. In the current work we found that both the basic-hydrophobic site in the basic region and the Gly-Pro-Gln region of the tail are required for the association of myosin IB with actin waves. This is the first evidence that the Gly-Pro-Gln region is required for localization of myosin IB to a specific actin structure in situ. The head is not required for myosin IB association with actin waves but binding of the head to F-actin strengthens the association of myosin IB with waves and stabilizes waves. Neither the SH3-domain nor motor activity is required for association of myosin IB with actin waves. We conclude that myosin IB contributes to anchoring actin waves to the plasma membranes by binding of the basic-hydrophobic site to acidic phospholipids in the plasma membrane and binding of the Gly-Pro-Gln region to F-actin in the wave.     

Utilisation illégale d’androgènes

Drug abuse for synthetic anabolic androgenic steroids in order to ameliorate sports results is illegal since the law of june 89 in France. No exception whatsoever, therapeutical purpose(s) included, is accepted. Means for controlling such abuse are reviewed briefly here together with data from our research on epitestosterone modifications following physical exercise and testosterone undecanoate controled administration in 15 nor In France the national body responsible for doping analysis in sports is the «Laboratoire National de Dépistage du Dopage» (LNDD). In fact, the control of drug abuse in sports requires laboratory means enabling the detection of banned substances with unlimited certainty. Briefly, untimed urine samples are analyzed for such purpose by gas (liquid or high pressure) chromatography coupled to mass spectrometry (GC/MS) which is the only technique accepted by the medical commission of I.O. C. and relevant bodies world-wide. However, since testosterone itself can now be used as a mean of steroid abuse in man, detection has to solve new problems arising from such manipulation. After considering various approches in order to prove the offense, such as the isotopic ratio for testosterone and different urinary metabolites, and indirect technique, based on the ratio of testosterone to epitestosterone glucuronides, has proved valuable but is now questionned. Epitestosterone is the 17α epimere of testosterone. It is not readily known to clinicians as it has no androgenic potency [5] and does not bind to the specific plasma protein TeBG (26) or androgen receptor [5, 26]. Epitestosterone was first isolated from human urines, simultaneously, by Brooks [6] and Korenman [18]. Wilson and Lipsett [30] among others demonstrated, in man, that this steroid originated both from adrenals and testis, results confirmed recently by Dehennin [8]. Dray et al. and studied its production and circadian rythm in man pointing out that epitestosterone is found in the sulphate fraction of plasma steroids and in the glucuronide fraction of urinary androgen metabolites [12, 20]. Epitestosterone is recovered as such in urines. It is not metabolized [12, 28]. However the pathway for epitestosterone biosynthesis is still uncertain today. The best (for it’s the only one!) hypothesis at present being that of Weustein et al. [30] who reported that epitestosterone could be synthesyzed in man from Δ5 androstène- 3 β, 17α-diol through a possible non enzymic modification of Δ5 androstène-3β, 17β- diol. Donike et al. [10] showed, in man, that the mean GT/GEPIT ratio in urines was 1,5±0,9 (±SD) using epitestosterone as a marker for endogenous androgens. This added index has being implemented, as an official test for androgen abuse’s detection in sports, since the Los Angeles Olympic Games in 1982. All other parameters being normal the definition of a positive androgen doped case is based on GT/GEPIT values over 6. This value of 6 was obtained by adding 6 SD to the mean obtained in man. However, is the use of this parameter justified and 100% safe? Some have questioned its used arguing that epitestosterone, not a well known substance, can not be reliable and could lead to false positive results. We summarize here the results of the French Research Network to which we participated. Mathian et al. [22] showed that epitestosterone production follows testosterone production whatever the age of the subject. The ratio GT/GEPIT doesn’t vary according to age, even over puberty, it remains at 1,40±0,86 from Tanner Stade II to Tanner Stade V. It doesn’t vary significantly after exercise or with fatigue. We also report our study of 15 young men (18–45 year old) over a year. Extensive blood (T, Δ₄, DHT, DHA, SDHA, E2, TeBG, FSH, LH) and urinary parameters (GT, DHT, GEPIT, ADIOL, BDIOL) were measured before, during and after a 21 days course of testosterone undecanoate (TU). Whatever the technique used (GC/MS or RIA) results are identical. We confirmed that GT/GEPIT was very stable for each individual and could be considered as a personnal marker. After TU, at the dosage of 40 to 80 mg/day, GT/GEPIT increased significantly in all instances (athletes and sedentary subjects alike), but not permanently. This change resulted from an increase in testosterone excretion wheras epitestosterone remained non statistically changed. However at the dosage used no permanent modification was found and most of the time GT/GEPIT returned to basal values rapidly. The analysis of the results of our study, according to the limit set by the I.O.C. at 6 for GT/GEPIT, pointed out a lot of false negative (over 50%). Values for GT excretion rate corrected with the creatinine content in the same urinary sample (GT/mg creatinine) have therefore been considered together with GT/GEPIT values. In our opinion, a more suitable and reliable index is thus obtained. The setting of a new limit at 3 for GT/GEPIT (Mean ± 3 SD) together with values under 75 ng/mg creatinine for GT is analyzed. It is also stressed that only a medical commission (aware of the significance of epitestosterone) can interpret the results obtained by analytical chemistry. This is the case in France.     

Rapamycin-mediated enrichment of T cells with regulatory activity in stimulated CD4+ T cell cultures is not due to the selective expansion of naturally occurring regulatory T cells but to the induction of regulatory functions in conventional CD4+ T cells

Rapamycin is an immunosuppressive drug currently used in different clinical settings. Although the capacity of rapamycin to inhibit the mammalian target of rapamycin serine/threonine protein kinase and therefore T cell cycle progression is well known, its effects are complex and not completely understood. It has been reported recently that TCR-mediated stimulation of murine CD4+ T cells in the presence of rapamycin results in increased proportions of CD4+ T cells with suppressive functions, suggesting that the drug may also exert its immunosuppressive activity by promoting the selective expansion of naturally occurring CD4+ regulatory T cells (Treg). In this study, we show that stimulation of human circulating CD4+ T cells in the presence of rapamycin results indeed in highly increased suppressor activity. By assessing the effect of rapamycin on the growth of nonregulatory and Treg populations of defined differentiation stages purified ex vivo from circulating CD4+ T cells, we could demonstrate that this phenomenon is not due to a selective expansion of naturally occurring Tregs, but to the capacity of rapamycin to induce, upon TCR-mediated stimulation, suppressor functions in conventional CD4+ T cells. This condition, however, is temporary and reversible as it is dependent upon the continuous presence of rapamycin.     

Expression and Activation of Caspase-6 in Human Fetal and Adult Tissues

Caspase-6 is an effector caspase that has not been investigated thoroughly despite the fact that Caspase-6 is strongly activated in Alzheimer disease brains. To understand the full physiological impact of Caspase-6 in humans, we investigated Caspase-6 expression. We performed western blot analyses to detect the pro-Caspase-6 and its active p20 subunit in fetal and adult lung, kidney, brain, spleen, muscle, stomach, colon, heart, liver, skin, and adrenals tissues. The levels were semi-quantitated by densitometry. The results show a ubiquitous expression of Caspase-6 in most fetal tissues with the lowest levels in the brain and the highest levels in the gastrointestinal system. Caspase-6 active p20 subunits were only detected in fetal stomach. Immunohistochemical analysis of a human fetal embryo showed active Caspase-6 positive apoptotic cells in the dorsal root ganglion, liver, lung, kidney, ovary, skeletal muscle and the intestine. In the adult tissues, the levels of Caspase-6 were lower than in fetal tissues but remained high in the colon, stomach, lung, kidney and liver. Immunohistological analyses revealed that active Caspase-6 was abundant in goblet cells and epithelial cells sloughing off the intestinal lining of the adult colon. These results suggest that Caspase-6 is likely important in most tissues during early development but is less involved in adult tissues. The low levels of Caspase-6 in fetal and adult brain indicate that increased expression as observed in Alzheimer Disease is a pathological condition. Lastly, the high levels of Caspase-6 in the gastrointestinal system indicate a potential specific function of Caspase-6 in these tissues.     

Assessment of CD4<Superscript>+</Superscript> T cells specific for the tumor antigen SSX-1 in cancer-free individuals

Proteins encoded by genes of the SSX family are specifically expressed in tumors and are therefore relevant targets for cancer immunotherapy. One of the first identified family members, SSX-1, is expressed in a large fraction of synovial sarcomas as a fusion protein together with the product of the SYT gene. In addition, the full-length SSX-1 antigen is frequently expressed in tumors of several other histological types such as sarcoma, melanoma, hepatocellular carcinoma, ovarian cancer and myeloma. To date, however, SSX-1 specific T cell responses have not been investigated and no SSX-1 derived T cell epitopes have been described. Here, we have assessed the presence of CD4(+) T cells directed against the SSX-1 antigen in circulating lymphocytes of cancer-free individuals. After a single in vitro stimulation with a pool of peptides spanning the entire SSX-1 protein we could detect and isolate SSX-1-specific CD4(+) T cells from 5/5 donors analyzed. SSX-1-specific polyclonal populations isolated from these cultures recognized peptides located in three distinct regions of the protein containing clusters of sequences with significant predicted binding to frequently expressed MHC class II alleles. Characterization of specific clonal CD4(+) T cell populations derived from one donor allowed the identification of several naturally processed epitopes recognized in association with HLA-DR. These data document the existence of a significant repertoire of CD4(+) T cells specific for SSX-1 derived sequences in circulating lymphocytes of any individual that can be exploited for the development of both passive and active immunotherapeutic approaches to control disease evolution in cancer patients.     

Structure and DNA-binding properties of the Bacillus subtilis SpoIIIE DNA translocase revealed by single-molecule and electron microscopies

SpoIIIE/FtsK are a family of ring-shaped, membrane-anchored, ATP-fuelled motors required to segregate DNA across bacterial membranes. This process is directional and requires that SpoIIIE/FtsK recognize highly skewed octameric sequences (SRS/KOPS for SpoIIIE/FtsK) distributed along the chromosome. Two models have been proposed to explain the mechanism by which SpoIIIE/FtsK interact with DNA. The loading model proposes that SpoIIIE/FtsK oligomerize exclusively on SpoIIIE recognition sequence/orienting polar sequences (SRS/KOPS) to accomplish directional DNA translocation, whereas the target search and activation mechanism proposes that pre-assembled SpoIIIE/FtsK hexamers bind to non-specific DNA, reach SRS/KOPS by diffusion/3d hopping and activate at SRS/KOPS. Here, we employ single-molecule total internal reflection imaging, atomic force and electron microscopies and ensemble biochemical methods to test these predictions and obtain further insight into the SpoIIIE–DNA mechanism of interaction. First, we find that SpoIIIE binds DNA as a homo-hexamer with neither ATP binding nor hydrolysis affecting the binding mechanism or affinity. Second, we show that hexameric SpoIIIE directly binds to double-stranded DNA without requiring the presence of SRS or free DNA ends. Finally, we find that SpoIIIE hexamers can show open and closed conformations in solution, with open-ring conformations most likely resembling a state poised to load to non-specific, double-stranded DNA. These results suggest how SpoIIIE and related ring-shaped motors may be split open to bind topologically closed DNA.