Incorporation Of Radioactivity Research Articles

7,3′,4′-Trihydroxyisoflavone, a Metabolite of the Soy Isoflavone Daidzein, Suppresses Ultraviolet B-induced Skin Cancer by Targeting Cot and MKK4

Nonmelanoma skin cancer is one of the most frequently occurring cancers in the United States. Chronic exposure to UVB irradiation is a major cause of this cancer. Daidzein, along with genistein, is a major isoflavone found in soybeans; however, little is known about the chemopreventive effects of daidzein and its metabolites in UVB-induced skin cancer. Here, we found that 7,3',4'-trihydroxyisoflavone (THIF), a major metabolite of daidzein, effectively inhibits UVB-induced cyclooxygenase 2 (COX-2) expression through the inhibition of NF-κB transcription activity in mouse skin epidermal JB6 P+ cells. In contrast, daidzein had no effect on COX-2 expression levels. Data from Western blot and kinase assays showed that 7,3',4'-THIF inhibited Cot and MKK4 activity, thereby suppressing UVB-induced phosphorylation of mitogen-activated protein kinases. Pull-down assays indicated that 7,3',4'-THIF competed with ATP to inhibit Cot or MKK4 activity. Topical application of 7,3',4'-THIF clearly suppressed the incidence and multiplicity of UVB-induced tumors in hairless mouse skin. Hairless mouse skin results also showed that 7,3',4'-THIF inhibits Cot or MKK4 kinase activity directly, resulting in suppressed UVB-induced COX-2 expression. A docking study revealed that 7,3',4'-THIF, but not daidzein, easily docked to the ATP binding site of Cot and MKK4, which is located between the N- and C-lobes of the kinase domain. Collectively, these results provide insight into the biological actions of 7,3',4'-THIF, a potential skin cancer chemopreventive agent.

Oostatic peptides containing d-amino acids: synthesis, oostatic activity, degradation, accumulation in ovaries and NMR study

Analogs of the H-Tyr-Asp-Pro-Ala-Pro-OH pentapeptide with D-amino acid residues either in differing or in all of the positions of the sequences were prepared and their oostatic potency was compared with that of the parent pentapeptide. The D-amino acid residue containing analogs exhibited an equal or even higher oostatic effect in the flesh fly Neobellieria bullata than the parent peptide. Contrary to the rapid incorporation of radioactivity from the labeled H-Tyr-Asp-[3H]Pro-Ala-Pro-OH pentapeptide into the ovaries of N. bullata in vitro, the radioactivity incorporation from the labeled pentapeptides with either D-aspartic acid or D-alanine was significantly delayed. As compared to the parent pentapeptide, also the degradation of both the D-amino acid-containing analogs mentioned above proceeded at a significantly lower rate. The decreased intake of radioactivity, the lower degradation and finally also the high oostatic effect may be ascribed to the decreased enzymatic degradation of the peptide bonds neighboring the D-amino acid residues in the corresponding peptides. The introduction of the non-coded D: -amino acids thus enhances the oostatic effect in N. bullata owing to the prolonged half-life of the corresponding pentapeptides, which can thus affect more ovarian cells.

Lognormal Distribution of Cellular Uptake of Radiopharmaceuticals: Implications for Biologic Response in Cancer Treatment

See page [642][1] Predicting biologic response after incorporation of radioactivity largely relies on calculating the absorbed dose. Traditionally, dosimetry has been performed using the mean absorbed dose at the organ level ([1][2]) and, more recently, at the suborgan and voxel levels ([2][3]).

Intestinal cell conversion ofdl-2-hydroxy-(4-methylthio)butanoic acidin vitro: dietary up-regulation by this methionine precursor

DL-2-Hydroxy-(4-methylthio)butanoic acid (HMTBA) is a synthetic source of dietary methionine (Met) widely used in poultry nutrition. HMTBA is transported in the intestinal epithelium by the monocarboxylate transporter 1, after which its biological utilisation relies on its conversion to L-Met. This process involves stereospecific HMTBA oxidation to 2-keto-(4-methylthio)butanoic acid (KMB) and transamination to L-Met. In the present study, we examined HMTBA conversion to L-Met, further incorporation into cellular proteins and the regulation of both processes by HMTBA supplementation in differentiated intestinal Caco-2 cells. The results showed D- and L-HMTBA oxidation in the enterocytes, this process being up-regulated by HMTBA. The data also revealed that KMB transamination is not linked to a specific amino group donor. However, the branched-chain amino acid L-leucine is the preferred amino group donor. Furthermore, transamination was not affected by HMTBA availability. The incorporation of radioactivity from HMTBA into cellular proteins was not significantly different from that of L-Met and was not affected by HMTBA supplementation. In conclusion, the results reveal the capacity of Caco-2 cells to convert HMTBA to L-Met and the up-regulation of conversion by nutritional HMTBA supplementation, thus highlighting the contribution of the intestinal epithelium in the utilisation of HMTBA as a dietary source of Met.

Effect of water storage and heat treatment on the cytotoxicity of soft liners

To evaluate the effect of water storage time on the cytotoxicity of soft liners. Sample discs of soft liners Dentusoft, Dentuflex, Trusoft, Ufi-Gel-P and denture base acrylic resin Lucitone-550 were prepared and divided into four groups: GN: No treatment, G24: Stored in water at 37°C for 24 h; G48: Stored in water at 37°C for 48 h, GHW: Immersed in water at 55°C for 10 min. To analyse the cytotoxic effect, three samples of each group were placed in tubes with Dubelcco's Modified Eagle Mediums and incubated at 37°C for 24 h. During this period, the toxic substances were leached to the culture medium. The cytotoxicity was analysed quantitatively by the incorporation of radioactivity (3)H-thymidine checking the number of viable cells (synthesis of DNA). The data were statistically analysed using two-way anova and Tukey's honestly significant difference tests (α = 0.05). Treatments did not reduce the cytotoxicity effect of the soft liners (p > 0.05). It was found that Ufi-Gel-P had a non-cytotoxic effect, Trusoft had a slightly cytotoxic effect, Dentuflex had a moderated cytotoxic effect, Dentusoft alternated between slightly and non-cytotoxic effect, and Lucitone-550 had non-cytotoxic effect when stored in water for 48 h. The effect of water storage and the heat treatment did not reduce the cytotoxicity of the soft liners.

Recombinant coagulation factor VIIa labelled with the fac‐99 mTc(CO)3‐core: synthesis and in vitro evaluation of a putative new radiopharmaceutical for imaging in acute bleeding lesion

AbstractCoagulation in blood is initiated when coagulation factor VII (FVII) binds to exposed TF and is activated to FVIIa, and the TF/FVIIa complex may therefore provide a marker of vascular injury potentially applicable in diagnostic imaging of acute gastrointestinal (GI) bleeding. Methods: Recombinant FVIIa (rFVIIa) was radiolabeled with technetium‐99 m in a direct labeling reaction using the ‘carbonyl approach’ using the IsoLink® carbonyl labeling agent. The properties of 99 mTc(CO)3‐rFVIIa complex was analyzed by TCA precipitation, HPLC and FVIIa functional integrity was tested in in vitro assays. Results: Labeling of rFVIIa was possible without tagging with a chelater. Incorporation of radioactivity depended strongly on rFVIIa concentration and temperature. More than 95% incorporation was achieved after 30 min at 45°C with 0.76 mg/ml rFVIIa. 99 mTc(CO)3‐rFVIIa was obtained in 46% radiochemical yield and in >95% radiochemical purity. Pull down experiments showed that the biological activity (binding to tissue factor and to anti‐FVII antibody) of the radiolabelled product remained intact in the formulation mixture as well as in human serum. By computer modeling analysis, two candidate sites for stabilizing the 99 mTc(CO)‐ligand structure in FVIIa were identified. Conclusion: Radiolabelled rFVIIa derivatives may represent a novel tool for the diagnosis of acute gastrointestinal bleeding lesions. Copyright © 2010 John Wiley & Sons, Ltd.

The proteasome inhibitor bortezomib drastically affects inflammation and bone disease in adjuvant‐induced arthritis in rats

To explore the effect of bortezomib in splenocytes and fibroblast-like synoviocytes (FLS) and its in vivo potency in a rat model of adjuvant-induced arthritis (AIA), which resembles human rheumatoid arthritis (RA). AIA was induced with Freund's complete adjuvant. Splenocyte and FLS proliferation and apoptosis were measured by radioactivity incorporation and flow cytometry, respectively. The invasiveness of FLS from rats with AIA was tested in a Transwell system. The pattern of cytokine secretion was evaluated by cytometric bead array in splenocyte supernatants. Bortezomib was administered prophylactically or therapeutically, and arthritis was assessed clinically and histologically. Immunohistochemistry was performed for markers of inflammation and angiogenesis in joints. Hematologic and biochemical parameters were tested in peripheral blood (PB). Representative animals were examined by computed tomography (CT) scanning before and after bortezomib administration. The expression of Toll-like receptor 2 (TLR-2), TLR-3, and TLR-4 in PB and FLS was measured by real-time polymerase chain reaction, and alterations in specific cell populations in PB and spleen were determined by flow cytometry. In vitro, bortezomib exhibited significant inhibitory and proapoptotic activity in splenocytes and FLS from rats with AIA, altered the inflammatory cytokine pattern, and reduced the invasiveness of FLS from rats with AIA. In vivo, bortezomib significantly ameliorated disease severity. Remission was associated with improved histology and decreased expression of CD3, CD79a, CD11b, cyclooxygenase 1, and factor VIII in target tissues as well as down-regulation of TLR expression in PB and cultured FLS. CT scanning demonstrated a bone healing effect after treatment. Our findings suggest that bortezomib affects AIA in a pleiotropic manner and that this drug may be effective in RA.

Upregulation of thymidine kinase activity compensates for loss of thymidylate synthase activity in Mycoplasma pneumoniae

Thymidylate, an essential building block of DNA, is synthesized either from deoxyuridylate by thymidylate synthase (TS) or thymidine (dT) by thymidine kinase (TK). Thymidylate kinase (TMPK) phosphorylates dTMP to dTTP. Thymidine phosphorylase (TP) catalyses reversible phosphorolysis of dT. Using transposon mutagenesis M. pneumoniae TS gene (thyA/MPN320) was interrupted and requirement of these enzymes was studied. We found that TK activity and transcript levels and TP activity, but not TMPK or TS activity, are growth-phase-regulated, with induction at the exponential growth phase and a decline after the stationary phase. Inactivation of thyA results in upregulation of TK transcript and a 10-fold increase in TK activity, reduced TMPK level and it had no effect on TP activity. The level of [3H]-dT uptake and incorporation into DNA in the thyA mutant correlates with increases in TK activity, suggesting that dT uptake and metabolism is TK-dependent and that upregulation of TK activity in the thyA mutant compensates for the lack of ThyA activity. [3H]-dU uptake was low compared with dT, and incorporation of radioactivity into DNA in the thyA mutant indicates the presence of an alternative TS. Our results suggest that TK and TMPK are potential targets for the development of Mycoplasma-specific antibiotics.

Identification of a Coiled Coil in Werner Syndrome Protein That Facilitates Multimerization and Promotes Exonuclease Processivity

Werner syndrome (WS) is a rare progeroid disorder characterized by genomic instability, increased cancer incidence, and early onset of a variety of aging pathologies. WS is unique among early aging syndromes in that affected individuals are developmentally normal, and phenotypic onset is in early adulthood. The protein defective in WS (WRN) is a member of the large RecQ family of helicases but is unique among this family in having an exonuclease. RecQ helicases form multimers, but the mechanism and consequence of multimerization remain incompletely defined. Here, we identify a novel heptad repeat coiled coil region between the WRN nuclease and helicase domains that facilitates multimerization of WRN. We mapped a novel and unique DNA-dependent protein kinase phosphorylation site proximal to the WRN multimerization region. However, phosphorylation at this site affected neither exonuclease activity nor multimeric state. We found that WRN nuclease is stimulated by DNA-dependent protein kinase independently of kinase activity or WRN nuclease multimeric status. In addition, WRN nuclease multimerization significantly increased nuclease processivity. We found that the novel WRN coiled coil domain is necessary for multimerization of the nuclease domain and sufficient to multimerize with full-length WRN in human cells. Importantly, correct homomultimerization is required for WRN function in vivo as overexpression of this multimerization domain caused increased sensitivity to camptothecin and 4-nitroquinoline 1-oxide similar to that in cells lacking functional WRN protein.

Synthesis and initial tumor affinity testing of iodine-123 labelled EGFR-affine agents as potential imaging probes for hormone-refractory prostate cancer

The epidermal growth factor receptor (EGFR) is over-expressed in a variety of human cancers, including in hormone-refractory prostate carcinomas, in which the EGFR has been associated with advanced disease stage, resistance to standard treatment and poor prognosis. Therefore, the EGFR is considered to be a promising molecular target for molecular imaging and therapy for hormone-refractory prostate cancer. This work describes the synthesis and initial tumor affinity testing of the EGFR antagonist 123I-mAb425 and the EGF receptor tyrosine kinase (EGFR-TK) inhibitor 123I-PD153035 as potential imaging probes for studying EGFR-expressing prostate cancer using single photon emission tomography. Methods 123I-mAb425 and 123I-PD153035 were prepared, starting from the IgG2a antibody and EGFR antagonist mAb425, that binds to the external domain of the EGF receptors, and from the EGFR-TK inhibitor PD153035, targeting the intra-endothelial tyrosine kinase domain of the EGFR, respectively. The potential of 123I-mAb425 and 123I-PD153035 to target EGFR-positive prostate carcinoma was tested on androgen-insensitive PC-3 and DU-145 prostate carcinoma cell lines, and on the androgen-sensitive LNCaP prostate cancer cell line for comparison. In vivo, the capability of 123I-mAb425 and 123I-PD153035 to target hormone-refractory prostate cancer was assessed in RNU rats or nu/nu mice bearing human PC3 prostate cancer xenografts. Results 123I-mAb425 was obtained in >90% radiochemical yield using the IODO-GEN ® method. 123I-PD153035 was synthesized by a non-isotopic [ 123I]iodo-debromination of PD153035 in 50–60% radiochemical yield in a total synthesis time including HPLC separation of 70 min. In vitro 123I-mAb425 and 123I-PD153035 accumulated highly in human PC-3 and DU-145 prostate cancer cells. Radioactivity incorporation into PC-3 and DU-145 tumor cells following 15-min incubation at 37 °C varied from 25% to 48% of the total loaded activity per 10 6 tumor cells (560–1230 cpm/1000 cells). In comparison, the uptake of the EGFR-affine probes into LNCaP prostate carcinoma cells was significantly low (105 ± 25 cpm/1000 cells). Inhibition experiments revealed that 123I-mAb425 is taken up into tumor cells via the same pathway as the naturally occurring epidermal growth factor (EGF), while 123I-PD153035 accumulation in prostate cancer cells occurs presumably via the same pathway as the selective EGFR-Tyrosine kinase antagonist AG1418. In vivo, the human prostate cancer xenografts in mouse war accurately visualized after i.v. administration of 123I-mAb425 by a gamma camera. Conclusion These data suggest that 123I-mAb425 and 123I-PD153035 are promising candidates as imaging probes for EGFR-positive prostate cancer and warrant further in vivo validations to ascertain their potential as imaging agents for clinical used.

Abstract 1649: Pim kinase inhibitor, SGI-1776, induces apoptosis in acute myeloid leukemia primary cells

Abstract Small molecule SGI-1776 was found to be potent and selective inhibitor of Pim family serine-threonine kinases [IC50: Pim-1 (7 nM), Pim-2 (363 nM) and Pim-3 (69 nM)] without effects on other kinases except Flt-3 and haspin. In preclinical studies in chronic lymphocytic leukemia, SGI-1776 treatment resulted in apoptosis induction that was associated with a decrease in RNA synthesis, Mcl-1 protein level, and c-Myc phosphorylation (Chen, LS et al, Blood 2009, 114: 4150). Increased levels of Pim-2 are reported in acute myeloid leukemia (AML) as well as Flt-3 activating mutation, providing a rationale to test SGI-1776 in AML. We evaluated SGI-1776 using FLT3-ITD AML cell lines (MOLM-13, MV4-11), xenograft models, and in primary cells from AML patients, and our results indicate that SGI-1776 is effective in AML. In MOLM-13 and MV4-11 cell lines, the IC50 for viability was < 10 nM after treatment with SGI-1776 for 72 h, which was associated apoptosis induction and reduction in phosphoBad (Ser112) and Bad protein. In xenograft models of both cell lines, treatment with 135mg/kg SGI-1776 resulted in a consistent decline in tumor volume after 14 days. To further these investigations in primary cells, we obtained cells from AML patients including FLT-3 positive and mutated FLT3-ITD patients, and evaluated the potential for SGI-1776 to induce cell death. In vitro incubation of AML cells (n = 4) with 1, 3, and 10 μM SGI-1776 for 24 h resulted in an average increase in apoptosis of 5%,12% and 19% respectively, measured using Annexin-staining compared with untreated cells. Apoptosis induction was verified via PARP cleavage immunoblot, and apoptosis was observed in cells from FLT3 negative or mutated FLT3-ITD patients. In the samples evaluated, there was disparity in cytogenetic factors, Flt-3 status and prior chemotherapeutic treatment history. To elucidate its mechanism of action, we evaluated the effect of SGI-1776 on Pim kinase function. There was no observed change in phosphorylation of Pim targets histone H3 (Ser10) or p21 (Thr145), however protein levels of both total and phospho-Bad(Ser112) decreased following SGI-1776 treatment. In contrast, there was no change in anti-apoptotic proteins Bcl-2, Bcl-xL, and Mcl-1, and levels of pro-apoptotic proteins, Bak and Bax, were also unaffected. However, there was a decrease in phospho-c-Myc(Ser 62), which is a Pim target and associated with stabilization of c-Myc protein. Consequently, we evaluated the potential inhibition of c-Myc driven transcription by SGI-1776 by measuring total RNA synthesis. Following treatment with 1, 3 or 10 μM SGI-1776, there was a decrease in total RNA synthesis to 75%, 42% and 25% of control respectively, measured using a radioactive uridine incorporation assay (n = 4). Taken together, SGI-1776 was demonstrated to be an effective agent against AML in cell lines, xenograft models, and primary cells, and may be an effective new agent in the treatment of AML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1649.

Investigation of the direct effects of salmon calcitonin on human osteoarthritic chondrocytes

BackgroundCalcitonin has been demonstrated to have chondroprotective effects under pre-clinical settings. It is debated whether this effect is mediated through subchondral-bone, directly on cartilage or both in combination. We investigated possible direct effects of salmon calcitonin on proteoglycans and collagen-type-II synthesis in osteoarthritic (OA) cartilage.MethodsHuman OA cartilage explants were cultured with salmon calcitonin [100 pM-100 nM]. Direct effects of calcitonin on articular cartilage were evaluated by 1) measurement of proteoglycan synthesis by incorporation of radioactive labeled 35SO4 [5 μCi] 2) quantification of collagen-type-II formation by pro-peptides of collagen type II (PIINP) ELISA, 3) QPCR expression of the calcitonin receptor in OA chondrocytes using four individual primer pairs, 4) activation of the cAMP signaling pathway by EIA and, 5) investigations of metabolic activity by AlamarBlue.ResultsQPCR analysis and subsequent sequencing confirmed expression of the calcitonin receptor in human chondrocytes. All doses of salmon calcitonin significantly elevated cAMP levels (P < 0.01 and P < 0.001). Calcitonin significantly and concentration-dependently [100 pM-100 nM] induced proteoglycan synthesis measured by radioactive 35SO4 incorporation, with a 96% maximal induction at 10 nM (P < 0.001) corresponding to an 80% induction of 100 ng/ml IGF, (P < 0.05). In alignment with calcitonin treatments [100 pM-100 nM] resulted in 35% (P < 0.01) increased PIINP levels.ConclusionCalcitonin treatment increased proteoglycan and collagen synthesis in human OA cartilage. In addition to its well-established effect on subchondral bone, calcitonin may prove beneficial to the management of joint diseases through direct effects on chondrocytes.

Photoactivable sphingosine as a tool to study membrane microenvironments in cultured cells

Human fibroblasts from normal subjects and Niemann-Pick A (NPA) disease patients were fed with two labeled metabolic precursors of sphingomyelin (SM), [(3)H]choline and photoactivable sphingosine, that entered into the biosynthetic pathway allowing the synthesis of radioactive phosphatidylcholine and SM, and of radioactive and photoactivable SM ([(3)H]SM-N(3)). Detergent resistant membrane (DRM) fractions prepared from normal and NPA fibroblasts resulted as highly enriched in [(3)H]SM-N(3). However, lipid and protein analysis showed strong differences between the two cell types. After cross-linking, different patterns of SM-protein complexes were found, mainly associated with the detergent soluble fraction of the gradient containing most cell proteins. After cell surface biotinylation, DRMs were immunoprecipitated using streptavidin. In conditions that maintain the integrity of domain, SM-protein complexes were detectable only in normal fibroblasts, whereas disrupting the membrane organization, these complexes were not recovered in the immunoprecipitate, suggesting that they involve proteins belonging to the inner membrane layer. These data suggest that differences in lipid and protein compositions of these cell lines determine specific lipid-protein interactions and different clustering within plasma membrane. In addition, our experiments show that photoactivable sphingolipids metabolically synthesized in cells can be used to study sphingolipid protein environments and sphingolipid-protein interactions.

Evidence of altered DNA integrity in the brain regions of suicidal victims of Bipolar Depression

Deoxyribonucleic acid (DNA) integrity plays a significant role in cell function. There are limited studies with regard to the role of DNA damage in bipolar affective disorder (BP). In the present study, we have assessed DNA integrity, conformation, and stability in the brain region of bipolar depression (BD) patients (n=10) compared to age-matched controls (n=8). Genomic DNA was isolated from 10 postmortem BD patients’ brain regions (frontal cortex, Pons, medulla, thalamus, cerebellum, hypothalamus, Parietal, temporal, occipital lobe, and hippocampus) and from the age-matched control subjects. DNA from the frontal cortex, pons, medulla, and thalamus showed significantly higher number of strand breaks in BD (P<0.01) compared to the age-matched controls. However, DNA from the hippocampus region was intact and did not show any strand breaks. The stability studies also indicated that the melting temperature and ethidium bromide binding pattern were altered in the DNA of BD patients’ brain regions, except in the hippocampus. The conformation studies showed B-A or secondary B-DNA conformation (instead of the normal B-DNA) in BD patients’ brain regions, with the exception of the hippocampus. The levels of redox metals such as Copper (Cu) and Iron (Fe) were significantly elevated in the brain regions of the sufferers of BD, while the Zinc (Zn) level was decreased. In the hippocampus, there was no change in the Fe or Cu levels, whereas, the Zn level was elevated. There was a clear correlation between Cu and Fe levels versus strand breaks in the brain regions of the BD. To date, as far as we are aware, this is a new comprehensive database on stability and conformations of DNA in different brain regions of patients affected with BD. The biological significance of these findings is discussed here.

Determination of the Dissociation Constants for Ca2+ and Calmodulin from the Plasma Membrane Ca2+ Pump by a Lipid Probe That Senses Membrane Domain Changes

The purpose of this work was to obtain information about conformational changes of the plasma membrane Ca(2+)-pump (PMCA) in the membrane region upon interaction with Ca(2+), calmodulin (CaM) and acidic phospholipids. To this end, we have quantified labeling of PMCA with the photoactivatable phosphatidylcholine analog [(125)I]TID-PC/16, measuring the shift of conformation E(2) to the auto-inhibited conformation E(1)I and to the activated E(1)A state, titrating the effect of Ca(2+) under different conditions. Using a similar approach, we also determined the CaM-PMCA dissociation constant. The results indicate that the PMCA possesses a high affinity site for Ca(2+) regardless of the presence or absence of activators. Modulation of pump activity is exerted through the C-terminal domain, which induces an apparent auto-inhibited conformation for Ca(2+) transport but does not modify the affinity for Ca(2+) at the transmembrane domain. The C-terminal domain is affected by CaM and CaM-like treatments driving the auto-inhibited conformation E(1)I to the activated E(1)A conformation and thus modulating the transport of Ca(2+). This is reflected in the different apparent constants for Ca(2+) in the absence of CaM (calculated by Ca(2+)-ATPase activity) that sharply contrast with the lack of variation of the affinity for the Ca(2+) site at equilibrium. This is the first time that equilibrium constants for the dissociation of Ca(2+) and CaM ligands from PMCA complexes are measured through the change of transmembrane conformations of the pump. The data further suggest that the transmembrane domain of the PMCA undergoes major rearrangements resulting in altered lipid accessibility upon Ca(2+) binding and activation.

T Cells Are Required for the Production of Blister-Inducing Autoantibodies in Experimental Epidermolysis Bullosa Acquisita

Epidermolysis bullosa acquisita is a prototypical organ-specific autoimmune disease caused by autoantibodies against type VII collagen of the dermal-epidermal junction. Although mechanisms of autoantibody-induced blister formation were extensively characterized, the initiation of autoantibody production in autoimmune blistering diseases is still poorly defined. In the current study, we addressed the role of T cells for the production of blister-inducing autoantibodies in mice immunized with type VII collagen. To detect autoreactive type VII collagen-specific T cells, lymph node cells from immunized SJL mice were stimulated in vitro with recombinant Ag, and their proliferation was measured by radioactive thymidine incorporation and flow cytometry analysis of CFSE-labeled cells. Interestingly, using synthetic peptides of the immunogen, partly different T and B cell epitopes in mice immunized with type VII collagen were demonstrated. In contrast to wild-type mice, immunization with type VII collagen of SJL athymic nude mice lacking T cells did not induce an autoimmune response and blistering phenotype. Importantly, SJL nude mice repleted with T cells from immunized wild-type mice showed a robust and durable autoantibody production resulting in subepidermal blistering disease in the recipients. Our present results demonstrate that T cells are required for the initiation of autoimmunity against type VII collagen in experimental epidermolysis bullosa acquisita and provide a basis for developing T cell-directed immunomodulatory strategies for this and related autoimmune diseases.

Pyridine salvage and nicotinic acid conjugate synthesis in leaves of mangrove species

The metabolic fate of [carbonyl- 14C]nicotinamide was surveyed in leaf disks of seven mangrove species, Bruguiera gymnorrhiza, Rhizophora stylosa, Kandelia obovata, Sonneratia alba, Pemphis acidula, Lumnitzera racemosa and Avicennia marina, with and without 250 mM NaCl. Uptake of [ 14C]nicotinamide by leaf disks was stimulated by 250 mM NaCl in K. candel, R. stylosa, A. marina and L. racemosa. [Carbonyl- 14C]nicotinamide was converted to nicotinic acid and was utilised for the synthesis of nucleotides and nicotinic acid conjugates. Formation of nicotinic acid by the deaminase reaction was rapid; there was little accumulation of nicotinamide in the disks 3 h after administration. Radioactivity from [carbonyl- 14C]nicotinamide was incorporated into pyridine nucleotides (mainly NAD and NADP) in all mangrove leaves, and the rates varied from 2% (in L. racemosa) to 15% ( S. alba) of the total radioactivity taken up. NaCl generally reduced nicotinic acid salvage for NAD and NADP. In all mangrove leaf disks, the most heavily labelled compounds (up to 70% of total radioactivity) were trigonelline ( N-methylnicotinic acid) and/or nicotinic acid N-glucoside. Trigonelline was formed in all mangrove plants, but N-glucoside synthesis was found only in leaves of A. marina and K. obovata. In A. marina, incorporation of radioactivity into N-glucoside (51%) was much greater than incorporation into trigonelline (2%). In general, NaCl stimulates the synthesis of these pyridine conjugates. The rate of decarboxylation of nicotinic acid in roots of A. marina seedlings was much greater than for the corresponding reaction observed in leaves.

Distribution and biosynthesis of theanine in Theaceae plants

The theanine content of the leaves of 27 species or varieties of Theaceae plants was investigated. Theanine was present in 21 species or varieties, but in much lower amounts (<0.2 mumol/g fresh weight) than the quantity detected in Camellia sinensis var. sinensis. The major free amino acids in leaves of four species belonging to the genera Schima and Eurya, were glutamic acid, aspartic acid, glutamine, asparagine, alanine and proline and content of these amino acids is similar to or higher than theanine. Accumulation of free amino acids in these plants was generally lower than in C. sinensis var. sinensis. The biosynthetic activity of theanine, assessed by the incorporation of radioactivity from [(14)C]ethylamine, was detected in seedlings of two species of Schima. The theanine biosynthetic activity in roots was higher than that of leaves.

Ethylamine Content and Theanine Biosynthesis in Different Organs of Camellia sinensis Seedlings

We examined the distribution of ethylamine, glutamic acid and alanine, which are utilized in theanine biosynthesis, and other major amino acids in leaves, stems, cotyledons and roots of 6-week-old tea seedlings. Ethylamine and glutamic acid, which are substrates of theanine synthetase, were distributed almost uniformly in all parts of the seedlings; the contents in micromol/g fresh wt varied from 0.44-0.88 (ethylamine) and 1.6-2.4 (glutamic acid). The content of alanine, a possible precursor of ethylamine synthesis, was significantly higher in roots (3.1 micromol/g fresh wt) than in other parts. Incorporation of radioactivity from [U-14C]-alanine into theanine was also higher in roots than in other organs. In 10-week-old seedlings, [1-14C]ethylamine was converted to theanine in young and developed leaves, stems, main and lateral roots; the highest rates of conversion were detected in the main and lateral roots. These results suggest that the theanine synthesis preferentially takes place in roots but is not restricted to them; substrates and the enzymatic machinery for theanine synthesis are available in all parts of tea seedlings.

68Ga-Labeling of Biotin Analogues and their Characterization

Biotin- and (68)Ga-based tracers have been suggested as tools that could be used to monitor the survival of avidin-coated islets of Langerhans isolated from pancreas and used in transplantation, i.e., to liver. Three biotin analogues with various alkyl and poly(ethylene glycol) (PEG) chains coupled to DOTA were synthesized and labeled with (68)Ga. The (68)Ga labeling was studied at room temperature as well as elevated temperature using either conventional or microwave heating. Radioactivity incorporation reached 95% within 5 and 2 min using the, respectively, conventional and microwave heating modes. The specific activity of the tracers was improved by preconcentration and purification of the generator eluate. The binding of the labeled and nonlabeled conjugates to avidin in solution was compared to the binding of native biotin. All compounds maintained good affinity for avidin, though introducing the linkers and chelator, especially the PEG-groups, somewhat decreased the binding affinity. The extent of binding of the labeled compounds to avidin was 54-91% after 5 min. Blocking experiments were performed confirming the specificity of the binding of biotin analogues to avidin. The stability of the three labeled compounds in human serum was studied. The stability of the biotin analogue 8 (65% within 30 min) and avidin-biotin complex (80% within 120 min) might be sufficient for the monitoring of the islets of Langerhans. The tracers will be evaluated in in vitro experiments of avidin-coated islets of Langerhans and in transplantation models in vivo.