Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Non-peptide oxytocin agonists

A library of compounds targeted to the vasopressin/oxytocin family of receptors was screened for activity at a cloned human oxytocin receptor using a reporter gene assay. Potency and selectivity were optimised to afford compound 39, EC50 = 33 nM. This series of compounds represents the first disclosed, non-peptide, low molecular weight agonists of the hormone oxytocin (OT).     

The effect of erectile dysfunction on quality of life following treatment for localized prostate cancer

All forms of prostate cancer therapy carry significant risk of erectile dysfunction, but patients value sexual function so highly that they are often willing to choose a therapy that offers a shorter life expectancy but better potency following treatment. Advances in research methodology now allow reliable collection of meaningful data regarding patients' health-related quality of life, including both objective evaluation of patients' functional status and their perceptions of their own health and its impact on their existence. In the past decade, several validated and reliable questionnaires have been developed that are specifically designed to measure HRQOL in men with prostate cancer. Studies using these instruments have found that function and perceived bother may not be correlated; patients may express satisfaction with their therapy despite loss of sexual function. Erectile aids, including sildenafil, can be helpful for patients following treatment for localized prostate cancer.     

Overexpression of IL-6 but not IL-8 increases paclitaxel resistance of U-2OS human osteosarcoma cells

The cytokines IL-6, initially recognized as a regulator of immune and inflammatory response and IL-8, a potential regulator of angiogenesis, also regulate the growth of many tumor cells. Human cancer cells selected for multidrug resistance to common chemotherapeutic agents demonstrate increased expression of IL-6 and IL-8. To determine whether IL-6 or IL-8 overexpression contributes directly to the drug resistant phenotype, IL-6 or IL-8 cDNA were introduced into the paclitaxel sensitive human osteosarcoma cell line U-2OS using the pIRESneo bicistronic expression vector. Interleukin-6 and IL-8 transfectants were selected for either high IL-6 or IL-8 secretion and evaluated in drug resistance assays. Two IL-6 and two IL-8 secreting clones express IL-6 or IL-8 levels of 10 ng/ml and 1 ng/ml in culture, while parental U-2OS and pIRESneo vector transfected control cells express IL-6 and IL-8 levels of 0.005 ng/ml and 0.1 ng/ml, respectively. MTT cytotoxicity with IL-6 transfected cells demonstrates a five-fold increase in resistance to paclitaxel and a four-fold increase in resistance to doxorubicin as compared to U-2OS. There are no changes in mitoxantrone or topotecan resistance in the IL-6 transfectants as compared to parental U-2OS. Northern analysis of IL-6 transfectants demonstrates that the resistant phenotype is not related to increased levels of MDR-1, MRP-1, or LRP. Western analysis also confirms that P-glycoprotein levels are not altered in IL-6 transfectants. Further supporting an MDR-1 independent mechanism of drug resistance, verapamil cannot reverse paclitaxel resistance in transfected cells, findings further supported by rhodamine 123 exclusion data. Treatment of IL-6 transfected cells with paclitaxel, compared with drug-sensitive parental U-2OS, shows U-2OS(IL-6) are significantly more resistant to apoptosis induced by paclitaxel and exhibit decreased proteolytic activation of caspase-3. In contrast U-2OS(IL-8) transfectants demonstrate no appreciable increase in paclitaxel resistance when compared with parental cells. In summary, while both IL-6 and IL-8 are overexpressed in paclitaxel resistant cell lines, only IL-6 has the potential to contribute directly to paclitaxel and doxorubicin resistance in U-2OS. This resistance is through a non-MDR-1 pathway.     

Tyrosine phosphorylation of Munc18‐1 inhibits synaptic transmission by preventing SNARE assembly

Tyrosine kinases are important regulators of synaptic strength. Here, we describe a key component of the synaptic vesicle release machinery, Munc18‐1, as a phosphorylation target for neuronal Src family kinases (SFKs). Phosphomimetic Y473D mutation of a SFK phosphorylation site previously identified by brain phospho‐proteomics abolished the stimulatory effect of Munc18‐1 on SNARE complex formation (“SNARE‐templating”) and membrane fusion in vitro. Furthermore, priming but not docking of synaptic vesicles was disrupted in hippocampal munc18‐1‐null neurons expressing Munc18‐1_Y473D. Synaptic transmission was temporarily restored by high‐frequency stimulation, as well as by a Munc18‐1 mutation that results in helix 12 extension, a critical conformational step in vesicle priming. On the other hand, expression of non‐phosphorylatable Munc18‐1 supported normal synaptic transmission. We propose that SFK‐dependent Munc18‐1 phosphorylation may constitute a potent, previously unknown mechanism to shut down synaptic transmission, via direct occlusion of a Synaptobrevin/VAMP2 binding groove and subsequent hindrance of conformational changes in domain 3a responsible for vesicle priming. This would strongly interfere with the essential post‐docking SNARE‐templating role of Munc18‐1, resulting in a largely abolished pool of releasable synaptic vesicles.     

The cost of incubation in relation to clutch-size in the Collared Flycatcher Ficedula albicollis

This study examines the importance of avian incubation costs as determinants of clutch-size variation by performing clutch-size and brood-size manipulations in the same population of Collared Flycatchers Ficedula albicollis during the same breeding season. In 2 5 cases when three or more clutches of the same size were completed on the same day, we moved two eggs on the day after the last egg had been laid from one randomly selected clutch (C) to another (C) and moved two other eggs from this to a third clutch (C⁺). In 20 other cases of simultaneously completed clutches of the same size, we moved two randomly selected young from one brood to a second and from that moved two other young to a third (B, B and B⁺groups). Most females were weighed the day after completion of the clutch and 1–4 days before hatching of the young, and some of them also 10–14 days after hatching of the young. We measured the daily energy expenditure of females incubating manipulated clutches of 4, 6 and 8 eggs by means of the doubly-labelled water (D₂¹⁸O) technique and also recorded their nest attendance. Hatching success of fertilized eggs was reduced in the enlarged clutches compared with control and reduced clutches. Females expired on average 3142.6 ml CO₂ and expended 78.6 kJ per day while incubating, which corresponds to a metabolic intensity of 3.3 times BMR. Daily energy expenditure increased with clutch-size due to higher costs while incubating, and not because of changed activity patterns. There were no significant differences in length of incubation, female mass or mass changes between phases for the C, C and C⁺groups. In both the C and B groups, enlarged broods produced significantly more fledged young than control broods, and those significantly more than reduced broods. Fledgling tarsus-length and mass did not differ significantly between treatments in either the C or B groups. There was no significant difference in breeding success between clutch and brood manipulations. In this season, incubation costs did not entail significant fitness losses, expressed either as fledgling production or female condition. Also, control females could have raised more young to fledging age than they did with no apparent costs.