BBX32, an Arabidopsis B-Box protein, functions in light signaling by suppressing HY5-regulated gene expression and interacting with STH2/BBX21

A B-box zinc finger protein, B-BOX32 (BBX32), was identified as playing a role in determining hypocotyl length during a large-scale functional genomics study in Arabidopsis (Arabidopsis thaliana). Further analysis revealed that seedlings overexpressing BBX32 display elongated hypocotyls in red, far-red, and blue light, along with reduced cotyledon expansion in red light. Through comparative analysis of mutant and overexpression line phenotypes, including global expression profiling and growth curve studies, we demonstrate that BBX32 acts antagonistically to ELONGATED HYPOCOTYL5 (HY5). We further show that BBX32 interacts with SALT TOLERANCE HOMOLOG2/BBX21, another B-box protein previously shown to interact with HY5. Based on these data, we propose that BBX32 functions downstream of multiple photoreceptors as a modulator of light responses. As such, BBX32 potentially has a native role in mediating gene repression to maintain dark adaptation.     

Identical amino acid substitutions in the repression domain of auxin/indole-3-acetic acid proteins have contrasting effects on auxin signaling

Auxin/indole-3-acetic acid (Aux/IAA) proteins function as repressors of auxin response gene expression when auxin concentrations in a cell are low. At elevated auxin concentrations, these repressors are destroyed via the ubiquitin-proteasome pathway, resulting in derepression/activation of auxin response genes. Most Aux/IAA repressors contain four conserved domains, with one of these being an active, portable repression domain (domain I) and a second being an auxin-dependent instability domain (domain II). Here, we have analyzed the effects of amino acid substitutions in the repression domain of selected Aux/IAA proteins. We show that stabilized versions of Aux/IAA proteins with amino acid substitutions in domain I display contrasting phenotypes when expressed in transformed Arabidopsis (Arabidopsis thaliana) plants. An alanine-for-leucine substitution in the LxLxL (where L is leucine and x is another amino acid) repression domain of IAA3, IAA6, or IAA19 confers enhanced auxin response gene expression and "high-auxin" phenotypes when expressed from the 35S or IAA19 promoter (as tested with IAA19) in transformed Arabidopsis plants. In marked contrast, a single alanine-for-leucine substitution in domain I of IAA12 or IAA17 confers repression of auxin response genes and "low-auxin" phenotypes. These results point to intrinsic differences in the repression domain(s) of IAA proteins and suggest that some IAA proteins have stronger or more complex repression domains than others.     

Non-Smad transforming growth factor-β signaling regulated by focal adhesion kinase binding the p85 subunit of phosphatidylinositol 3-kinase

TGF-β modulates numerous diverse cellular phenotypes including growth arrest in epithelial cells and proliferation in fibroblasts. Although the Smad pathway is fundamental for the majority of these responses, recent evidence indicates that non-Smad pathways may also have a critical role. Here we report a novel mechanism whereby the nonreceptor tyrosine focal adhesion kinase (FAK) functions as an adaptor necessary for cell type-specific responses to TGF-β. We show that in contrast to Smad actions, non-Smad pathways, including c-Abl, PAK2, and Akt, display an obligate requirement for FAK. Interestingly, this occurs in Src null SYF cells and is independent of FAK tyrosine phosphorylation, kinase activity, and/or proline-rich sequences in the C-terminal FAT domain. FAK binds the phosphatidylinositol 3-kinase (PI3K) p85 regulatory subunit following TGF-β treatment in a subset of fibroblasts but not epithelial cells and has an obligate role in TGF-β-stimulated anchorage-independent growth and migration. Together, these results uncover a new scaffolding role for FAK as the most upstream component regulating the profibrogenic action of TGF-β and suggest that inhibiting this interaction may be useful in treating a number of fibrotic diseases.     

Subtilase from Beauveria sp.: conformational and functional investigation of unusual stability

Retention of total activity of the subtilisin-like serine protease from Beauveria sp. MTCC 5184 (Bprot) in the vicinity of (1) 3 M GdnHCl for 12 h, (2) 50 % methanol and dimethyl sulfoxide each for 24 h, and (3) proteolytic enzymes (trypsin, chymotrypsin, and proteinase K) for 48 h led to expect the enzyme to be a kinetically stable protein. Also, the structure of the protein was stable at pH 2.0. Biophysical characterization and conformational transitions were monitored using steady-state and time-resolved fluorescence, FTIR, and CD spectroscopy. Single tryptophan in the protein exists as two conformers, in hydrophobic and polar environment. The secondary structure of Bprot was stable in 3 M GdnHCl as seen in far-UV CD spectra. The active fraction of Bprot obtained from size-exclusion chromatography in the presence of GdnHCl (1.0–3.0 M) eluted at reduced retention time. The peak area of inactive or denatured protein with the same retention time as that of native protein increased with increasing concentration of denaturant (1.0–4.0 M GdnHCl). However, the kinetics of GdnHCl-induced unfolding as studied from intrinsic fluorescence revealed k _unf of native protein to be 5.407 × 10^?5 s^?1 and a half-life of 3.56 h. The enzyme is thermodynamically stable in spite of being resistant to the denaturant, which could be due to the effect of GdnHCl imparting rigidity to the active fraction and simultaneously unfolding the partially unfolded protein that exists in equilibrium with the folded active protein. Thermal and pH denaturation of Bprot exhibited interesting structural transitions.     

(d)-Amino acid analogues of DT-2 as highly selective and superior inhibitors of cGMP-dependent protein kinase Iα

The cGMP-dependent protein kinase type I (PKG I) is an essential regulator of cellular function in blood vessels throughout the body. DT-2, a peptidic inhibitor of PKG, has played a central role in determining the molecular mechanisms of vascular control involving PKG and its signaling partners. Here, we report the development of (d)-amino acid DT-2 derivatives, namely the retro-inverso ri-(d)-DT-2 and the all (d)-amino acid analog, (d)-DT-2. Both peptide analogs were potent PKG Iα inhibitors with K_i values of 5.5 nM (ri-(d)-DT-2) and 0.8 nM ((d)-DT-2) as determined using a hyperbolic mixed-type inhibition model. Also, both analogs were proteolytically stable in vivo, showed elevated selectivity, and displayed enhanced membrane translocation properties. Studies on isolated arteries from the resistance vasculature demonstrated that intraluminally perfused (d)-DT-2 significantly inhibited vasodilation induced by 8-Br-cGMP. Furthermore, in vivo application of (d)-DT-2 established a uniform translocation pattern in the resistance vasculature, with exception of the brain. Thus, (d)-DT-2 caused significant increases in mean arterial blood pressure in unrestrained, awake mice. Further, mesenteric arteries isolated from (d)-DT-2 treated animals showed a markedly reduced dilator response to 8-Br-cGMP in vitro. Our results clearly demonstrate that (d)-DT-2 is a superior inhibitor of PKG Iα and its application in vivo leads to sustained inhibition of PKG in vascular smooth muscle cells. The discovery of (d)-DT-2 may help our understanding of how blood vessels constrict and dilate and may also aid the development of new strategies and therapeutic agents targeted to the prevention and treatment of vascular disorders such as hypertension, stroke and coronary artery disease.     

Melatonin Modulates Thyroid Hormones and Splenocytes Proliferation Through Mediation of Its MT1 and MT2 Receptors in Hyperthyroid Mice

Hyperthyroidism is characterized by an increased metabolic rate with the alteration of immune activity. The pineal hormone melatonin regulates various physiological activities through sensitization of MT1 and MT2 membrane receptors in mammals. In the present study we have evaluated the involvement of MT1 and MT2 receptors in melatonin mediated modulation of thyroid hormones and splenocyte proliferation in experimentally induced hyperthyroidic mice. The l-thyroxine treatment induced the hyperthyroidism in mice evidenced with hypersecretion of T3 and T4 hormones from thyroid gland. Hyperthyroidic state increased the TSH hormone level which might be inducing hyper activity in thyroid gland. Exogenous melatonin suppressed the thyroid hormones level as well as TSH level in circulation. The l-thyroxine treatment increased the splenocyte proliferation and showed synergic effects along with melatonin. l-thyroxine treated mice alone or along with melatonin treatment showed differential expression pattern of MT1 and MT2 receptors protein in thyroid and spleen tissues. It seems that melatonin regulates thyroid hormones and splenocyte proliferation through activation of MT1 and MT2 receptors.     

Prostaglandin production contributes to exercise-induced vasodilation in heart failure

Lang, Chim C., Don B. Chomsky, Javed Butler, Shiv Kapoor,and John R. Wilson. Prostaglandin production contributes toexercise-induced vasodilation in heart failure. J. Appl. Physiol. 83(6): 1933-1940, 1997.Endothelial release of prostaglandins may contribute toexercise-induced skeletal muscle arteriolar vasodilation in patientswith heart failure. To test this hypothesis, we examined the effect ofindomethacin on leg circulation and metabolism in eight chronic heartfailure patients, aged 55 ± 4 yr. Central hemodynamics and legblood flow, determined by thermodilution, and leg metabolic parameterswere measured during maximum treadmill exercise before and 2 h afteroral administration of indomethacin (75 mg). Leg release of6-ketoprostaglandin F₁ was alsomeasured. During control exercise, leg blood flow increased from 0.34 ± 0.03 to 1.99 ± 0.19 l/min(P < 0.001), legO₂ consumption from 13.6 ± 1.8 to 164.5 ± 16.2 ml/min (P < 0.001), and leg prostanoid release from 54.1 ± 8.5 to267.4 ± 35.8 pg/min (P < 0.001).Indomethacin suppressed release of prostaglandinF₁(P < 0.001) throughout exercise anddecreased leg blood flow during exercise(P < 0.05). This was associated witha corresponding decrease in leg O₂ consumption (P < 0.05) and a higher level offemoral venous lactate at peak exercise(P < 0.01). These data suggest thatrelease of vasodilatory prostaglandins contributes to skeletal musclearteriolar vasodilation in patients with heart failure.

     

Plants used by the bhat community for regulating fertility

A survey was conducted among medicinal plant sellers, healers, priests and local people in the rural population of Bhat, India. In this rural community medicinal plants are used for regulating fertility. Due to the fact that the majority of the Bhat community is illiterate, oral interviews were held in the villages. The information was then recorded. Plant samples were collected and preserved for future reference. Information on 28 plants and 11 recipes for doses from 2 or more vegetable species are presented, and popular names, empirical properties, and manner of using recipes are described. More than 90% of the information gathered was consistent among the informants, but some of the informants differed in their reports on the way to use the same plants. There is no certainty as to which of the plants are effective. Since the Bhat community has frequently observed the positive effects of their preparations, they have much faith in their recipes. The acceptability of these preparations is quite high among 95% of the Bhat population.