New synthesis of L-FMAU from L-arabinose

A new synthesis of 2'-deoxy-2'-fluoro-5-methyl-beta-L-arabinofuranosyl uracil (13, L-FMAU) was achieved in 10 steps from L-arabinose.     

Identification of an apical Cl(-)/HCO3(-) exchanger in the small intestine

HCO3(-) secretion is the most important defense mechanism against acid injury in the duodenum. However, the identity of the transporter(s) mediating apical HCO3(-) secretion in the duodenum remains unknown. A family of anion exchangers, which include downregulated in adenoma (DRA or SLC26A3), pendrin (PDS or SLC26A4), and the putative anion transporter (PAT1 or SLC26A6) has recently been identified. DRA and pendrin mediate Cl(-)/base exchange; however, the functional identity and distribution of PAT1 (SLC26A6) is not known. In these studies, we investigated the functional identity, tissue distribution, and membrane localization of PAT1. Expression studies in Xenopus oocytes demonstrated that PAT1 functions in Cl(-)/HCO3(-) exchange mode. Tissue distribution studies indicated that the expression of PAT1 is highly abundant in the small intestine but is low in the colon, a pattern opposite that of DRA. PAT1 was also abundantly detected in stomach and heart. Immunoblot analysis studies identified PAT1 as a approximately 90 kDa protein in the duodenum. Immunohistochemical studies localized PAT1 to the brush border membranes of the villus cells of the duodenum. We propose that PAT1 is an apical Cl(-)/HCO3(-) exchanger in the small intestine.     

Signalling via type IA and type IB bone morphogenetic protein receptors (BMPR) regulates intramembranous bone formation, chondrogenesis and feather formation in the chicken embryo

Bone morphogenetic proteins (BMPs) signal via complexes of type I and type II receptors. In this study, we mapped the expression of type IA, type IB and type II receptors during craniofacial chondrogenesis and then perturbed receptor function in vivo with retroviruses expressing dominant-negative or constitutively active type I receptors. BmprIB was the only receptor expressed within all cartilages. BmprIA was initially expressed in cartilage condensations, but later decreased within cartilage elements. BmprII was expressed at low levels in the nasal septum and prenasal cartilage and at higher levels in other craniofacial cartilages. The maxillary prominence, which gives rise to several intramembranous bones, expressed both type I receptors. Misexpression of dnBMPRIB decreased the size of cartilages and bones on the treated side. In contrast, dnBMPRIA had no effect on the skeletal phenotype. The phenotypes of caBMPRIA and caBMPRIB were similar; both led to overgrowth of cartilage elements, thinner bones with fewer trabeculae and inhibition of feather development. Infection with constitutively active viruses resulted in ectopic expression of Msx1, Msx2 and Fgfr2 throughout the maxillary mesenchyme. These data suggest that the pattern of trabeculation in membranous bones derived from the maxillary prominence was related to the change in expression pattern and that Msx and Fgfr2 genes were downstream of both type I BMP receptors. We conclude that the requirement for the type IB is greater than for the type IA receptor but, when active, both receptors play similar roles in regulating bone, cartilage and feather formation in the skull.     

Therapeutic efficacy of melanoma-reactive TIL injected in stage III melanoma patients

Adoptive therapy for cancer using tumor-infiltrating lymphocytes (TIL) has mainly been investigated in cancer patients with advanced stage disease. The limited clinical success has not been encouraging, although this might be explained by poor TIL specificity and/or high tumor burden. To re-evaluate the effectiveness of adoptive therapy, we analyzed the capacity of tumor-reactive TIL injection in preventing the further development of disease in stage III melanoma patients after complete tumor resection. A phase II/III randomized trial was performed on 88 melanoma patients, who received autologous TIL plus interleukin-2 (IL-2) or IL-2 only. The duration of relapse-free survival was analyzed, taking into account the immunological specificity of injected TIL and the number of metastatic lymph nodes removed before treatment. Kaplan-Meyer analysis revealed that the injection of tumor-reactive TIL was statistically correlated with prolonged relapse-free survival in patients with only one metastatic lymph node. Therefore, improved clinical outcome could be obtained after adoptive therapy by selecting appropriate groups of patients and monitoring the specificity of the injected TIL populations.     

Light-induced hydrolysis and rebinding of nonisomerizable bacteriorhodopsin pigment

Bacteriorhodopsin (bR) is characterized by a retinal-protein protonated Schiff base covalent bond, which is stable for light absorption. We have revealed a light-induced protonated Schiff base hydrolysis reaction in a 13-cis locked bR pigment (bR5.13; lambda(max) = 550 nm) in which isomerization around the critical C13==C14 double bond is prevented by a rigid ring structure. The photohydrolysis reaction takes place without isomerization around any of the double bonds along the polyene chain and is indicative of protein conformational alterations probably due to light-induced polarization of the retinal chromophore. Two photointermediates are formed during the hydrolysis reaction, H450 (lambda(max) = 450 nm) and H430 (lambda(max) = 430 nm), which are characterized by a 13-cis configuration as analyzed by high-performance liquid chromatography. Upon blue light irradiation after the hydrolysis reaction, these intermediates rebind to the apomembrane to reform bR5.13. Irradiation of the H450 intermediate forms the original pigment, whereas irradiation of H430 at neutral pH results in a red shifted species (P580), which thermally decays back to bR5.13. Electron paramagnetic resonance (EPR) spectroscopy indicates that the cytoplasmic side of bR5.13 resembles the conformation of the N photointermediate of native bR. Furthermore, using osmotically active solutes, we have observed that the hydrolysis rate is dependent on water activity on the cytoplasmic side. Finally, we suggest that the hydrolysis reaction proceeds via the reversed pathway of the binding process and allows trapping a new intermediate, which is not accumulated in the binding process.     

Combined expression of S-VSPalpha in two different organelles enhances its accumulation and total lysine production in leaves of transgenic tobacco plants

Soybean vegetative storage proteins (S-VSPs) accumulate to high levels in vacuoles of both wild types and heterologous plants. Here it is shown that directing S-VSPalpha to two different organelles-chloroplasts and vacuoles-in a single transgenic plant significantly increased its accumulation. Accumulation of S-VSPalpha in heterologous plants correlated with total soluble lysine. Using this approach with essential amino-acid-rich transgene proteins may lead to a breakthrough in improving plant nutritional quality.