Fyn Mediates Leptin Actions in the Thymus of Rodents

BACKGROUND:Several effects of leptin in the immune system rely on its capacity to modulate cytokine expression and apoptosis in the thymus. Surprisingly, some of these effects are dependent on signal transduction through the IRS1/PI3-kinase, but not on the activation of JAK2. Since all the well known effects of leptin in different cell types and tissues seem to be dependent on JAK2 activation, we hypothesized that, at least for the control of thymic function, another, unknown kinase could mediate the transduction of the leptin signal from the ObR towards the IRS1/PI3-kinase signaling cascade. METHODOLOGY/PRINCIPAL FINDINGS:Here, by employing immunoblot, real-time PCR and flow citometry we show that the tyrosine kinase, Fyn, is constitutively associated with the ObR in thymic cells. Following a leptin stimulus, Fyn undergoes an activating tyrosine phosphorylation and a transient association with IRS1. All these effects are independent of JAK2 activation and, upon Fyn inhibition, the signal transduction towards IRS1/PI3-kinase is abolished. In addition, the inhibition of Fyn significantly modifies the effects of leptin on thymic cytokine expression. CONCLUSION/SIGNIFICANCE:Therefore, in the thymus, Fyn acts as a tyrosine kinase that transduces the leptin signal independently of JAK2 activation, and mediates some of the immunomodulatory effects of leptin in this tissue

Evolution of Skin Temperature after the Application of Compressive Forces on Tendon, Muscle and Myofascial Trigger Point.

Some assessment and diagnosis methods require palpation or the application of certain forces on the skin, which affects the structures beneath, we highlight the importance of defining possible influences on skin temperature as a result of this physical contact. Thus, the aim of the present study is to determine the ideal time for performing thermographic examination after palpation based on the assessment of skin temperature evolution. Randomized and crossover study carried out with 15 computer-user volunteers of both genders, between 18 and 45 years of age, who were submitted to compressive forces of 0, 1, 2 and 3 kg/cm2 for 30 seconds with a washout period of 48 hours using a portable digital dynamometer. Compressive forces were applied on the following spots on the dominant upper limb: myofascial trigger point in the levator scapulae, biceps brachii muscle and palmaris longus tendon. Volunteers were examined by means of infrared thermography before and after the application of compressive forces (15, 30, 45 and 60 minutes). In most comparisons made over time, a significant decrease was observed 30, 45 and 60 minutes after the application of compressive forces (p < 0.05) on the palmaris longus tendon and biceps brachii muscle. However, no difference was observed when comparing the different compressive forces (p > 0.05). In conclusion, infrared thermography can be used after assessment or diagnosis methods focused on the application of forces on tendons and muscles, provided the procedure is performed 15 minutes after contact with the skin. Regarding to the myofascial trigger point, the thermographic examination can be performed within 60 minutes after the contact with the skin

Expression of Src family members in the thymus.

<p>(A) The thymus from 21-d rats was homogenized and samples containing 0.2 mg total protein were separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted with anti-Scr, Fyn or Lck antibodies; the specific bands are indicated by arrows. (B) Rats were anesthetized and a single injection of leptin (100 µL, 10⁻⁶M) was performed through the cava vein; thymuses were obtained after the times depicted in the figure and homogenized; samples containing 0.5 mg total protein were used in immunoprecipitation assays with anti-Src, Fyn or Lck antibodies; immunocomplexes were separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted with anti-phosphotyrosine antibodies. (C) Rats were anesthetized and a single injection of leptin (100 µL, concentrations ranging from 10⁻¹² to 10⁻⁴M) was performed through the cava vein; thymuses were obtained after 10 min and homogenized; samples containing 0.5 mg total protein were used in immunoprecipitation assays with anti-Fyn antibodies; immunocomplexes were separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted with anti-phosphotyrosine antibodies. The depicted blots are representative of n = 5. MW, molecular mass; Thy, thymus.</p

Fyn expression and activation in the hypothalmus.

<p>(A–B) Some rats were used without previous treatment (A), or some rats were treated with Fyn inhitor PP2 (5 nmol in 100 µl buffer, ip) 30 min before leptin injection (B). Anesthetized rats were injected via intra cava vein either with 100 µl saline (C) or with an equal volume of leptin (10⁻⁶M) (Lep and PP2+Lep); the hypothalami were obtained, homogenized and samples containing 0.5 mg total protein were used in immunoprecipitation assays with anti-Fyn or anti-JAK2 antibodies; immunocomplexes were separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted with anti-phosphotyrosine antibody; or 0.2 mg protein was separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted with anti-Fyn or anti-JAK2 antibodies. The depicted blots are representative of n = 5. In all experiments, n = 5; *p<0.05 vs. C.</p

Effects of Fyn inhibition on apoptosis and cytokine expression.

<p>(A) Rats were treated ip for three days with Fyn antisense phosphothioate modified oligonucleotide (FynAS) (400 µl, 2 nmol). On the fourth day, the rats were injected via intra cava vein either with 100 µl saline (C and FynAS) or with an equal volume of leptin (10⁻⁶M) (Lep and FynAS+Lep); the thymuses were obtained, homogenized and 0.2 mg protein was separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted with anti-Bax or anti-Bcl-2 antibodies. (B) Isolated thymocytes were treated with leptin (10⁻⁸M)(Lep) or PP2 (10⁻⁸M) or both together and apoptosis was determined by the annexin method after 24h. (C–D) Rats were treated ip with a single dose of 100 µL of saline solution (C); 100 µL of lipopolysaccharide (LPS) 1 mg/mL; 150 µL of PP2 5 nM; 100 µL of leptin 31.2 µM (Lep) or with different combinations of these treatments; the sequence of treatment was, PP2, followed by leptin after 30 min and LPS after 30 min. Thymus was obtained after 2 h and RNA was prepared for determination of IL-1b (C) and TNF-α expression by real-time PCR. In all experiments n = 5. *p<0.05 <i>vs.</i> C; #p,0.05 <i>vs.</i> lep; §p<0.05 <i>vs.</i> Lep+LPS.</p

Photograph and infrared image of the palmaris longus tendon (A and D), biceps brachii muscle (B and E) and active myofascial trigger point in the insertion of the levator scapulae (C and F).

<p>Photograph and infrared image of the palmaris longus tendon (A and D), biceps brachii muscle (B and E) and active myofascial trigger point in the insertion of the levator scapulae (C and F).</p

Comparison of the skin temperature (degrees Celsius) on biceps the brachii muscle over time and at different compressive forces.

<p>Values shown in mean (95% confidence interval). All comparisons were made by means of two-way repeated measures analysis of variance post hoc Bonferroni. P0: Before the application of compressive force; P15: Fifteen minutes after; P30: Thirty minutes after; P45: Forty-five minutes after; P60: Sixty minutes after.</p><p>^aDiffers significantly from P60 (p < 0.05)</p><p>^bDiffers significantly from P45 (p < 0.05)</p><p>^cDiffers significantly from P30 (p < 0.05).</p><p>Comparison of the skin temperature (degrees Celsius) on biceps the brachii muscle over time and at different compressive forces.</p

Comparison of the skin temperature (in degrees Celsius) on myofascial trigger point over time and at different compressive forces.

<p>Values shown in mean (95% confidence interval). All comparisons were made by means of two-way repeated measures analysis of variance post hoc Bonferroni. P0: Before the application of compressive force; P15: Fifteen minutes after; P30: Thirty minutes after; P45: Forty-five minutes after; P60: Sixty minutes after.</p><p>No significant differences in comparisons over time or between different compressive forces (p > 0.05).</p><p>Comparison of the skin temperature (in degrees Celsius) on myofascial trigger point over time and at different compressive forces.</p

Inhibiting Fyn.

<p>(A) Rats were treated once a day for three days with a single 400 µl ip dose of buffer containing 0–8 nmol Fyn antisense (FynAS) or scrambled (FynSCR) phosphorthioate modified oligonucleotides; at the end of the experimental period the thymuses were obtained, homogenized and samples containing 0.2 mg total protein were separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted with anti-Fyn or anti-IRS1 antibodies. (B) Rats were pre-treated with a single dose of PP2 (5 nmol in 100 µl buffer, ip) (+) or saline (−), 30 min prior to leptin treatment. A single dose of leptin (100 µl 10⁻⁶M, via cava vein) (+), or similar volume of saline (−) was then injected; the thymuses were obtained for homogenization; samples containing 0.5 mg total protein were used in immunoprecipitation assays with anti-Fyn or anti-JAK2 antibodies; immunocomplexes were separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted (IB) with anti-phosphotyrosine antibody; or 0.2 mg protein was separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted with anti-phospho ERK antibody. The depicted blots are representative of n = 5.</p

Identification and Genomic Characterization of a New Virus (Tymoviridae Family) Associated with Citrus Sudden Death Disease

Citrus sudden death (CSD) is a new disease that has killed approximately 1 million orange trees in Brazil. Here we report the identification of a new virus associated with the disease. RNAs isolated from CSD-affected and nonaffected trees were used to construct cDNA libraries. A set of viral sequences present exclusively in libraries of CSD-affected trees was used to obtain the complete genome sequence of the new virus. Phylogenetic analysis revealed that this virus is a new member of the genus Marafivirus. Antibodies raised against the putative viral coat proteins allowed detection of viral antigens of expected sizes in affected plants. Electron microscopy of purified virus confirmed the presence of typical isometric Marafivirus particles. The screening of 773 affected and nonaffected citrus trees for the presence of the virus showed a 99.7% correlation between disease symptoms and the presence of the virus. We also detected the virus in aphids feeding on affected trees. These results suggest that this virus is likely to be the causative agent of CSD. The virus was named Citrus sudden death-associated virus