Evaluation of three E-rosette assays in melanoma patients

Summary The percentage of E-rosette-forming cells (E-RFC) was determined repeatedly in the peripheral blood of 43 patients with malignant melanoma. Three methods of E-rosette assaying were used: total E-rosette test, active E-rosette test, and 29° C E-rosette assay.Depressed E-RFC values were found in fewer assays than normal values. The mean values of E-RFC and the proportion of depressed E-RFC in patients in stage I did not differ from the values in healthy control persons whatever method of assay was used.The frequency of depressed E-RFC values was higher in advanced stages. Significant differences were demonstrated between stage I and stages II and III combined in the values for total and for active E-rosettes (P<0.01).The active E-rosette test and the 29° C E-rosette assay gave no more positive results (i.e., depressed E-RFC values) than the total E-rosette test in melanoma patients.     

Adjacent repeating units of xenopus laevis 5S DNA can be heterogeneous in length

The distribution of length heterogeneity in adjacent repeating units of X. laevis 5S DNA has been examined by “cloning” 5S DNA in bacteria. Fragments of 5S DNA produced by partial digestion with Hind III and containing 1, 4, and 5 repeating units have been inserted at the single Hind III site of the tetracycline-resistance plasmid, pSC101, and the hybrid plasmids cloned in E. coli. Adjacent 5S DNA repeats in the cloned multi-repeat fragments can differ in length. This finding rules out some mechanisms which have been proposed to account for the parallel evolution of tandem repeated DNAs. The results are consistent with an unequal crossing-over mechanism and place some constraints on the molecular processes in this recombinatory event.     

Permeability of sarcoplasmic reticulum membrane. The effect of changed ionic environments on Ca2+ release

Summary Permeability properties and the effects of a changed membrane potential on Ca²⁺ release of sarcoplasmic reticulum vesicles of rabbit skeletal muscle were investigated by Millipore filtration. The relative permeability of sarcoplasmic reticulum to solutes determined under conditions of isotope exchange at equilibrium and/or under conditions of net flow of solute and water into the vesicles was as follows: sucrose, Ca²⁺, Mn²⁺–, choline⁺, Tris⁺–+, Na⁺, Li⁺, Cl^–. Transient membrane potentials were induced by rapidly changing the ionic environment of the vesicles. Knowledge of the relative permeation rates of the above ions allowed prediction of the direction and extent of membrane polarization. Osmotic effects in the polarization measurements due to the rapid influx of solute and water into the vesicles were minimized by using media containing a fast (K⁺ or Cl^–) and a relatively slow (gluconate^– or choline⁺) penetrating ion.⁴⁵Ca²⁺ efflux from vesicles derived from different parts of the sarcoplasmic reticulum structure was not appreciably changed when vesicles were made more positive inside (choline chloride potassium gluconate) or more negative inside (potassium gluconate choline chloride). These studies suggest that part or all of the ion-induced changes in sarcoplasmic reticulum membrane permeability, previously interpreted to indicate depolarization-induced Ca²⁺ release, may be due to osmotic effects.     

Water in normal muscle and muscle with a tumor

The total water content, the amount of non-freezable water, and the Na⁺ and K⁺ contents in the gastrocnemius muscle of albino mice with and without a solid tumor were determined. The spin-lattice relaxation time (T₁) for the water protons in the two kinds of muscle were measured at six resonance frequencies ranging from 4.5 to 60 MHz over the temperature range +37 to −65°C. Quantitatively calculated T₁ values are given. The difference in T₁ for the two types of muscle at temperatures above −5°C is attributed to the difference in the distribution ratio of water between hydration and free states, and bears no direct relation to the concentration of Na⁺.     

The Treatment with Interleukin 17 Inhibitors and Immune-Mediated Inflammatory Diseases

IL-17 inhibitors (IL-17i) are medicines used to treat dermatological and rheumatic diseases They belong to a class of medicines called biological disease-modifying anti-rheumatic drugs (bDMARDs). This class of drugs has had a major impact on the therapy of autoimmune diseases, being much safer and more effective than treatment with small molecules. At the same time, they have highly beneficial effects on skin and joint changes, and their efficacy has been extensively monitored and demonstrated in numerous clinical trials. More and more such drugs are still being discovered today to ensure the best possible treatment of these patients, but more frequently and relatively constantly three agents are used. Two of them (Secukinumab and Ixekizumab) inhibit IL-17A directly, and the third, Brodamulab, inhibits the IL-17A receptor. Although they are extremely effective in the treatment of these diseases, sometimes their administration has been associated with paradoxical effects, i.e., there is an exacerbation of the inflammatory process. Tough, clinical trials of IL-17i have described cases of exacerbation or even onset of inflammatory bowel disease (IBD), such as Crohn’s disease and ulcerative colitis, after administration of these drugs in patients previously diagnosed with psoriasis (PS), psoriatic arthritis (PsA), or ankylosing spondylitis (AS). The pathophysiological mechanism of action is not well understood at present. One explanation would be that this hyperreactive inflammatory process would be triggered by Interferon 1 derived from dendritic plasma cells. Even though there are many reports in the recent literature about the role of IL17i in the onset of IBD, conclusions of studies do not converge. Some of them show an increased incidence of IBD in patients treated with IL17i, while some others affirm their safety of them. In the near future we will surely have more data emerging from ongoing meta-analyses regarding safety of use IL17i in patients who are at risk of developing IBD. Clinical and paraclinical evaluation (inflammatory intestinal markers) are carefully advised before recommending treatment with IL-17i and after initiation of treatment, and prospective surveillance by clinical and biomarkers of patients treated with IL-17i is absolutely essential to capture the onset of IBD.     

Asymmetrical intraguild interactions with coyotes,red foxes,and domestic dogs may contribute to competitive exclusion of declining gray foxes

Species coexistence is governed by availability of resources and intraguild interactions including strategies to reduce ecological overlap. Gray foxes are dietary generalist mesopredators expected to benefit from anthropogenic disturbance, but populations have declined across the midwestern USA, including severe local extirpation rates coinciding with high coyote and domestic dog occurrence and low red fox occurrence. We used data from a large‐scale camera trap survey in southern Illinois, USA to quantify intraguild spatial and temporal interactions among the canid guild including domestic dogs. We used a two‐species co‐occurrence model to make pairwise assessments of conditional occupancy and detection rates. We also estimated temporal activity overlap among species and fit a fixed‐effects hierarchical community occupancy model with the four canid species. We partitioned the posterior distributions to compare gray fox occupancy probabilities conditional on estimated state of combinations of other species to assess support for hypothesized interactions. We found no evidence of broadscale avoidance among native canids and conclude that spatial and temporal segregation were limited by ubiquitous human disturbance. Mean guild richness was two canid species at a site and gray fox occupancy was greater when any combination of sympatric canids was also present, setting the stage for competitive exclusion over time. Domestic dogs may amplify competitive interactions by increasing canid guild size to the detriment of gray foxes. Our results suggest that while human activities can benefit some mesopredators, other species such as gray foxes may serve as bellwethers for habitat degradation with trophic downgrading and continued anthropogenic homogenization.     

Replication study of 34 common SNPs associated with prostate cancer in the Romanian population

Prostate cancer is the third‐most common form of cancer in men in Romania. The Romanian unscreened population represents a good sample to study common genetic risk variants. However, a comprehensive analysis has not been conducted yet. Here, we report our replication efforts in a Romanian population of 979 cases and 1027 controls, for potential association of 34 literature‐reported single nucleotide polymorphisms (SNPs) with prostate cancer. We also examined whether any SNP was differentially associated with tumour grade or stage at diagnosis, with disease aggressiveness, and with the levels of PSA (prostate specific antigen). In the allelic analysis, we replicated the previously reported risk for 19 loci on 4q24, 6q25.3, 7p15.2, 8q24.21, 10q11.23, 10q26.13, 11p15.5, 11q13.2, 11q13.3. Statistically significant associations were replicated for other six SNPs only with a particular disease phenotype: low‐grade tumour and low PSA levels (rs1512268), high PSA levels (rs401681 and rs11649743), less aggressive cancers (rs1465618, rs721048, rs17021918). The strongest association of our tested SNP's with PSA in controls was for rs2735839, with 29% increase for each copy of the major allele G, consistent with previous results. Our results suggest that rs4962416, previously associated only with prostate cancer, is also associated with PSA levels, with 12% increase for each copy of the minor allele C. The study enabled the replication of the effect for the majority of previously reported genetic variants in a set of clinically relevant prostate cancers. This is the first replication study on these loci, known to associate with prostate cancer, in a Romanian population.     

Characterization of the Phosphorylation Site of PP59, a Substrate for Cyclic AMP-Dependent Protein Kinase That Is Enriched in the Synaptic Membrane Fraction of Rat Cerebellum

Abstract: We have identified previously a synaptic membrane-associated protein, PP59, that serves as a substrate for cyclic AMP-dependent protein kinase and is enriched in rat cerebellum. We show here that PP59 can be extracted from synaptic plasma membranes with a combination of 2% Triton X-100 plus 1 M KCl. A 290-fold purification of PP59 was achieved by selective solubilization, followed by continuous-elution preparative gel electrophoresis. To determine the amino acid sequence surrounding the cyclic AMP-dependent protein kinase phosphorylation site within PP59, the partially purified ³²P-phosphorylated protein was digested with chymotrypsin, and radiolabeled peptides were purified by sequential reversed-phase HPLC in two different solvent systems. Automated Edman degradation revealed a single phosphorylation site contained within the sequence Ala-Arg-Glu-Arg-Ser-Asp-Ser(P)-Thr-Gly-Ser-Ser-Ser-Val-Tyr. No strong sequence homology to this peptide fragment with other known peptides or proteins in the SwissProt, PIR, or GenPept databases could be found. A synthetic peptide containing this unique 14-amino acid sequence was used to develop polyclonal anti-peptide antibodies that were affinity-purified and shown to recognize intact PP59 as determined by western blotting. These antibodies specifically inhibited the phosphorylation of PP59 by cyclic AMP-dependent protein kinase in an in vitro phosphorylation assay containing synaptic plasma membranes.