Delayed allograft rejection in mice transgenic for a soluble form of the IL-4 receptor.

Accumulating evidence suggests that in serum and other biological fluids, cytokine binding is a property associated with soluble proteins, including a high-affinity soluble version of the IL-4 receptor (sIL-4R). While it is tempting to speculate that sIL-4R might act as a serum carrier protein or serve to inhibit or modulate IL-4 action, specific biological roles for sIL-4R remain to be established. To further assess the immunoregulatory and therapeutic potential of sIL-4R and other soluble receptors, we have created transgenic mice which constitutively express elevated levels of biologically active sIL-4R. Phenotypic characterization of lymphoid organs in sIL-4R transgenic mice revealed normal numbers of B and T cells and normal surface marker expression. Splenic lymphocytes displayed normal in vitro activities as measured by the PFC response and generation of cytotoxic T cells. In addition, antigen-specific IgE and IgG1 in vivo responses were similar in control and transgenic mice. Despite the apparent developmental normality of the sIL-4R transgenic mice, these animals were markedly deficient in the ability to reject cardiac allografts, suggesting that IL-4 is critical for the generation of alloreactivity. The results further suggest that the ability of sIL-4R to regulate IL-4 activities may be under the control of complex interactions that remain to be elucidated.     

Regulation of RhoA Signaling by the cAMP-dependent Phosphorylation of RhoGDIα

RhoA plays a pivotal role in regulating cell shape and movement. Protein kinase A (PKA) inhibits RhoA signaling and thereby induces a characteristic morphological change, cell rounding. This has been considered to result from cAMP-induced phosphorylation of RhoA at Ser-188, which induces a stable RhoA-GTP-RhoGDIα complex and sequesters RhoA to the cytosol. However, few groups have shown RhoA phosphorylation in intact cells. Here we show that phosphorylation of RhoGDIα but not RhoA plays an essential role in the PKA-induced inhibition of RhoA signaling and in the morphological changes using cardiac fibroblasts. The knockdown of RhoGDIα by siRNA blocks cAMP-induced cell rounding, which is recovered by RhoGDIα-WT expression but not when a RhoGDIα-S174A mutant is expressed. PKA phosphorylates RhoGDIα at Ser-174 and the phosphorylation of RhoGDIα is likely to induce the formation of a active RhoA-RhoGDIα complex. Our present results thus reveal a principal molecular mechanism underlying G_s/cAMP-induced cross-talk with G_q/G₁₃/RhoA signaling.     

His499 Regulates Dimerization and Prevents Oncogenic Activation by Asparagine Mutations of the Human Thrombopoietin Receptor

Ligand binding to the extracellular domain of the thrombopoietin receptor (TpoR) imparts a specific orientation on the transmembrane (TM) and intracellular domains of the receptors that is required for physiologic activation via receptor dimerization. To map the inactive and active dimeric orientations of the TM helices, we performed asparagine (Asn)-scanning mutagenesis of the TM domains of the murine and human TpoR. Substitution of Asn at only one position (S505N) activated the human receptor, whereas Asn substitutions at several positions activated the murine receptor. Second site mutational studies indicate that His⁴⁹⁹ near the N terminus of the TM domain is responsible for protecting the human receptor from activation by Asn mutations. Structural studies reveal that the sequence preceding His⁴⁹⁹ is helical in the murine receptor but non-helical in peptides corresponding to the TM domain of the inactive human receptor. The activating S505N mutation and the small molecule agonist eltrombopag both induce helix in this region of the TM domain and are associated with dimerization and activation of the human receptor. Thus, His⁴⁹⁹ regulates the activation of human TpoR and provides additional protection against activating mutations, such as oncogenic Asn mutations in the TM domain.     

Microquantification of proteins with low chromophore content

The advantages of the intrinsic fluorescence of the tryptophan and the absorbance of the methionine residues of the 18 kDa-hsp - a recombinant protein from Mycobacterium leprae - was exploited here to develop a sensitive and low costs method for protein assaying. They presented linearity between 3 and 1000 g of protein. The correlations between intrinsic fluorescence or absorbance at 230 nm and protein contents were both superiors to 0.99. These methods can be extended to others proteins with low aromatic residue contents.     

Contribution of physical fitness component to health status in middle-aged and elderly females

This study determined the physical fitness component that contributes to improving and maintaining health status for each age group as well as quantifying the degree of the relationship between health status and physical fitness in middle-aged and elderly females. The participants were 2,371 females aged 30 to 69 years. Ten physical fitness tests and medical checkups were performed. The participants were divided into a healthy group and an unhealthy group according to health status. Multiple discriminant analysis was applied to the multivariate data. Correct discriminant probabilities of the multiple discriminant function to discriminate the healthy and unhealthy groups for females ranged from 63.0% to 77.5%. These results suggest that there is a relatively high relationship between health status and physical fitness level for middle-aged and elderly females. With each individual's discriminant score calculated by the obtained multiple discriminant function as the index of the degree of health, the Pearson's correlation coefficient of the discriminant score and the performance in each physical fitness test were calculated. The aging change from 30 to 69 years old was classified into four patterns according to the contribution. The result of this study is considered to be useful as objective data to prepare an exercise program considering the contribution of the physical fitness component of health status.     

Calcium-independent induction of cytocidal activity of polymorphonuclear leukocytes by phorbol myristate acetate-like tumor promoters

Tumor promoters were tested for the ability to induce cytocidal activity of polymorphonuclear leukocytes (PMNs), and the extracellular calcium-dependency of their PMN cytotoxicities were examined in comparison with that by some immunomodulators. Immunomodulators such as linear beta-1, 3-D-glucan (TAK) induced potent cytocidal activity of PMNs. The induction was dependent on extracellular Ca2+. Tumor promoters such as phorbol 12-myristate 13-acetate (PMA) and its derivatives, teleocidin which is structurally unrelated to PMA, and croton oil as an example of mixture also induced potent PMN cytotoxicities. In the latter cases, however, the induction was not dependent on extracellular Ca2+. The ability of these tumor promoters to induce PMN cytotoxicity correlated well with their skin-tumor promoting activities. These results indicate that inductions by PMA-like tumor promoters are distinguishable from those by TAK-like immunomodulators in not being Ca2+-dependent. The application of Ca2+-independent PMN cytotoxicity to detect PMA-like tumor promoters is discussed.     

Spread of X-chromosome inactivation into chromosome 15 is associated with Prader–Willi syndrome phenotype in a boy with a t(X;15)(p21.1;q11.2) translocation

X-chromosome inactivation (XCI) is an essential mechanism in females that compensates for the genome imbalance between females and males. It is known that XCI can spread into an autosome of patients with X;autosome translocations. The subject was a 5-year-old boy with Prader?CWilli syndrome (PWS)-like features including hypotonia, hypo-genitalism, hypo-pigmentation, and developmental delay. G-banding, fluorescent in situ hybridization, BrdU-incorporated replication, human androgen receptor gene locus assay, SNP microarrays, ChIP-on-chip assay, bisulfite sequencing, and real-time RT-PCR were performed. Cytogenetic analyses revealed that the karyotype was 46,XY,der(X)t(X;15)(p21.1;q11.2),?15. In the derivative chromosome, the X and half of the chromosome 15 segments showed late replication. The X segment was maternal, and the chromosome 15 region was paternal, indicating its post-zygotic origin. The two chromosome 15s had a biparental origin. The DNA methylation level was relatively high in the region proximal from the breakpoint, and the level decreased toward the middle of the chromosome 15 region; however, scattered areas of hypermethylation were found in the distal region. The promoter regions of the imprinted SNRPN and the non-imprinted OCA2 genes were completely and half methylated, respectively. However, no methylation was found in the adjacent imprinted gene UBE3A, which contained a lower density of LINE1 repeats. Our findings suggest that XCI spread into the paternal chromosome 15 led to the aberrant hypermethylation of SNRPN and OCA2 and their decreased expression, which contributes to the PWS-like features and hypo-pigmentation of the patient. To our knowledge, this is the first chromosome-wide methylation study in which the DNA methylation level is demonstrated in an autosome subject to XCI.