Effector CD4 T cells are biochemically distinct from the memory subset: evidence for long-term persistence of effectors in vivo.

Memory T cell responses are believed to be mediated by long-lived memory T cells that arise directly from a subset of short-lived, activated effector T cells that have reverted to the resting state. Although widely accepted, definitive proof that memory T cells arise from effectors is lacking because of the inability to reliably distinguish these subsets based on known phenotypic or functional parameters. We have used a biochemical approach to distinguish effector and memory CD4 T cell subsets and follow the differentiative fate of effector cells in vivo. When examined biochemically, effector and memory CD4 T cells are strikingly distinct and exhibit qualitative and quantitative differences in tyrosine phosphorylation. These effector-specific patterns were identical in effectors derived either from naive CD4 T cells (primary effectors) or memory CD4 T cells (memory effectors). To monitor the fate of effector cells in vivo, Ag-activated CD4+ TCR-transgenic T cells were transferred into irradiated BALB/c mice. These TCR-transgenic CD4 T cells persisted in adoptive hosts for several months, gave a recall response to Ag, yet exhibited effector-specific biochemical profiles. These results suggest that a subset of effector CD4 T cells can persist in vivo and contribute to long-term immunity by mediating secondary immune responses.     

Effect of clinical mastitis and other diseases on reproductive performance of Holstein cows

The objective of this study was to evaluate the effect of clinical mastitis and (or) other diseases on reproductive performance in lactating Holstein cows. Cows (n=967) from a commercial dairy farm were divided into four groups retrospectively: cows with clinical mastitis and other diseases (MD, n=54), clinical mastitis only (M, n=154), other diseases only (D, n=187), and cows with no record of clinical mastitis or other diseases (H, n=572). Days in milk at first service (DIMFS), services per conception (S/C), days not pregnant (DNP), the rate at which animals became pregnant over time and the proportion of cows that remained non-pregnant during 224 days of lactation were evaluated. Groups MD and M had greater (P<0.05) DNP compared with H (155+/-15 and 140+/-5 vs. 88+/-2, respectively). Moreover, MD and M had greater (P<0.05) S/C compared with H (3.0+/-0.4 and 2.1+/-0.1 vs. 1.6+/-0.1, respectively). The rate at which animals became pregnant over time was less (P<0.05) for MD and M and tended (P=0.1) to be less for D when compared with H. In addition, proportion of cows that remained non-pregnant by 224 days of lactation was greater (P<0.05) in MD, M, and D compared with H. Cows with mastitis were also divided into three groups according to the day of occurrence of the first case of clinical mastitis: (1) clinical mastitis occurred before 56 days postpartum (MP1); (2) clinical mastitis occurred between 56 and 105 days after parturition (MP2); and (3) clinical mastitis occurred after 105 days postpartum (MP3) Regardless of the time of occurrence, DNP was greater (P<0.05) for cows with mastitis compared with H. Time of mastitis occurrence affected S/C in that cows in MP2 and MP3 had a greater S/C compared with H cows (P<0.05). Reproductive efficiency was decreased by the presence of clinical mastitis alone because a greater proportion of cows with mastitis remained non-pregnant over time. Moreover, a greater proportion of cows with mastitis or diseases remained non-pregnant by 224 postpartum. Furthermore, the negative effects on reproduction were exacerbated when cows experienced both clinical mastitis and other diseases.     

Evaluation of fluorescent pseudomonads for plant growth promotion,antifungal activity against Rhizoctonia solani on common bean,and biocontrol potential

Fluorescent pseudomonads are ubiquitous bacteria that are common inhabitants of the rhizosphere and are the most studied group within the genus Pseudomonas. Bacterial isolates (n = 103) from the rhizosphere of wheat and common bean were assessed as potential biocontrol agents in this study. Fungal inhibition tests were performed by a plate assay in which each isolate was tested directly for the production of hydrogen cyanide, protease, siderophore and cellulase. Production of DAPG was verified by using an analytical high performance liquid chromatography assay (HPLC). Plant growth promotion was assessed in phytochamber trials and biocontrol activity was evaluated in greenhouse trials. In all, 52 bacterial isolates with antifungal activity against Rhizoctonia solani were found. Of the 52 isolates, 41 were selected according to their high efficiency in in vitro antagonism, which was shown as inhibition zones in the dual-culture assay. Six of the 41 rhizobacteria, including isolates UTPF7, UTPF13, UTPF18, UTPF22, UTPF27 and strain CHA0 produced HCN. Production of protease enzyme was detected for all isolates excluding UTPF30 isolate. Although some stains appeared not to produce any compound with affinity for ferric iron, other isolates produced prolific amounts, creating a large zone of orange (up to 160 mm², i.e., UTPF16). Seventeen of 41 isolates of fluorescent pseudomonads including strain CHAO produced different amounts of DAPG ranging from 0.6 to 11.4 ng/10⁸ cfu. A total of 39 isolates induced statistically significant effects on plant growth compared with the non-treated control for at least one parameter. The predominant influence observed was increased root length. No bacteria could completely protect the plant against R. solani, although all isolates significantly increased fresh weight as compared to the infested control in greenhouse trials. Pseudomonas fluorescens isolates UTPF16 and UTPF26 significantly (P < 0.05) decreased the number of seedlings with damping-off symptoms in the means of the experiments.     

A multiple-capillary electrophoresis system for small-scale DNA sequencing and analysis

A five-capillary system has been developed for DNA sequencing and analysis. The post-column fluorescence detector is based on a sheath-flow cuvette. The instrument provides uniform and continuous illumination of the samples. The cuvette virtually eliminates cross-talk in the fluorescence signal between capillaries. Discrete single-photon counting avalanche photodiodes provide high efficiency light detection. The instrument has detection limits (3sigma) of 130 +/- 30 fluorescein molecules injected onto each capillary. Over 650 bases of sequence at 98.8% accuracy were generated in 100 min at 50 degrees C from M13mp18. Separation and detection of short tandem repeats proved efficient and accurate with the use of internal standards for direct comparison of migration times between capillaries.     

Heterogeneity of protein labeling with a fluorogenic reagent, 3-(2-furoyl)quinoline-2-carboxaldehyde

Fluorogenic reagents are used for protein labeling when high-sensitivity fluorescence detection is required. Similar to traditional labeling with activated fluorescent dyes, such as fluorescein isothiocyanate, a fluorogenic reaction is expected to change the physical-chemical properties of proteins. Knowledge of these changes may be essential for efficient separation and identification of labeled proteins. Here we studied the effect of labeling of myoglobin with a fluorogenic reagent on the acid-base properties of the protein. The fluorogenic reagent used was 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ). In slab-gel isoelectric focusing, we found that the labeling reaction generated at least six species with pI values lower than that of non-labeled myoglobin. These species can be identified as products of progressive labeling of myoglobin with one to six FQ molecules. The same series of FQ-labeled species were observed when the reaction products were analyzed by capillary zone electrophoresis. The comparison of experimental and theoretical pI values allowed us to elucidate the labeling pattern--the number of FQ molecules corresponding to each labeled product detected by isoelectric focusing.     

Cloning,Expression and One-Step Purification of a Novel IP-10-(anti-HER2 scFv) Fusion Protein in Escherichia coli

International Journal of Peptide Research and Therapeutics - Breast cancer is the most common cancer in women, worldwide. The correlation between breast cancer malignancy and human epidermal growth...     

Determining the contribution of glycosaminoglycans to tendon mechanical properties with a modified shear-lag model

Tendon has a complex hierarchical structure composed of both a collagenous and a non-collagenous matrix. Despite several studies that have aimed to elucidate the mechanism of load transfer between matrix components, the roles of glycosaminoglycans (GAGs) remain controversial. Thus, this study investigated the elastic properties of tendon using a modified shear-lag model that accounts for the structure and non-linear mechanical response of the GAGs. Unlike prior shear-lag models that are solved either in two dimensions or in axially symmetric geometries, we present a closed-form analytical model for three-dimensional periodic lattices of fibrils linked by GAGs. Using this approach, we show that the non-linear mechanical response of the GAGs leads to a distinct toe region in the stress–strain response of the tendon. The critical strain of the toe region is shown to decrease inversely with fibril length. Furthermore, we identify a characteristic length scale, related to microstructural parameters (e.g. GAG spacing, stiffness, and geometry) over which load is transferred from the GAGs to the fibrils. We show that when the fibril lengths are significantly larger than this length scale, the mechanical properties of the tendon are relatively insensitive to deletion of GAGs. Our results provide a physical explanation for the insensitivity for the mechanical response of tendon to the deletion of GAGs in mature tendons, underscore the importance of fibril length in determining the elastic properties of the tendon, and are in excellent agreement with computationally intensive simulations.     

Multiple dispersal out of Anatolia: biogeography and evolution of oriental green lizards

The oriental green lizards of the Lacerta trilineata group are widely distributed in Greece, Anatolia, the eastern Mediterranean, the southern Caucasus, and the Zagros mountains in Iran. We studied their phylogeography using three mitochondrial markers with comprehensive sampling from most representatives of the group. Their phylogeny and divergence times (implementing fossil‐based molecular clock calibrations) were inferred using Bayesian methods, and haplotype networks were reconstructed to assess how genetic diversity and current distributional patterns were shaped. According to our phylogenetic analyses, the group constitutes a well‐supported monophylum containing several distinct evolutionary lineages with high haplotype diversity. Vicariance might explain the divergences within most lineages that have accumulated by range restriction and expansion of populations as a result of Quaternary climate oscillations and glacial refugia. However, niche divergence appears to be a major force promoting speciation, and large scale distributional patterns between lineages were shaped earlier by multiple, independent dispersals out of Anatolia during the Pliocene and early Pleistocene. The results of the present study also suggest that the group is in need of a taxonomical revision because the identified lineages and genetic diversity are not congruent with the currently recognized subspecies. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 398–408.