The apoptosis-associated protein BNIPL interacts with two cell proliferation-related proteins,MIF and GFER

Bcl-2/adenovirus E1B 19 kDa interacting protein 2-like, BNIP-2-like (BNIPL) is a recently cloned and characterized apoptosis-associated protein that shares 72% homology with BNIP-2. It is highly expressed in human placenta and lung. A yeast two-hybrid system was used to obtain two BNIPL-interacting proteins, MIF (macrophage migration inhibitory factor) and GFER (growth factor erv1 (Saccharomyces cerevisiae)-like). The interactions were confirmed by glutathione S-transferase pull-down assay in vitro and co-immunoprecipitation assay in vivo. Colony formation assay and cell proliferation test suggest that overexpression of BNIPL could inhibit the growth of BEL-7402 cells. These findings suggest that BNIPL may physically bind to cell proliferation-related proteins, MIF and GFER.     

Modulation of steroidogenesis by selenium in a novel adrenal cell line developed using targeted tumorigenesis

Glutathione peroxidase (GPx-1) is a selenoenzyme that metabolizes H(2)O(2), a source of potentially toxic free radicals. Steroidogenesis is markedly inhibited by H(2)O(2) in vitro. OBJECTIVE: to study the effects of selenium deficiency on GPx activity and adrenal steroidogenesis in a novel adrenal cell line developed using targeted tumorigenesis. METHODS: AN4Rppc7 cells were grown for 7 days in serum-free medium. 8-Br-cAMP-stimulated concentrations of steroid hormones were measured by RIA. StAR (Steroid Acute Reactive Protein) mRNA was measured by Northern blot. RESULTS: selenium deficiency caused a 99% There was a 51%, progesterone, corticosterone and aldosterone production, respectively (p<0.05 by ANOVA). StAR mRNA was not affected by selenium. CONCLUSIONS: selenium deficiency causes a marked decrease in GPx activity. Decreased steroid hormone production occurs for selenium concentrations equal or lower than 5 nM. The absence of changes in StAR mRNA content suggests that selenium deficiency does not affect cholesterol access to the mitochondria.     

Design, synthesis, and biological evaluation of HIV/FIV protease inhibitors incorporating a conformationally constrained macrocycle with a small P3' residue

A series of norstatine-based HIV/FIV protease inhibitors incorporating a 15-membered macrocycle as a mimic of the tripeptide (Ala-Val-Phe), a motif with a small P3' residue elective against the FIV protease and the drug-resistant HIV proteases, has been synthesized. It was found that the macrocycle is important to the overall activity of the inhibitors. Certain inhibitors were developed expressing low nanomolar inhibitory activity against the HIV/FIV proteases and they are also effective against some drug-resistant as well as TL3-resistant HIV proteases.     

Structure-activity studies of FIV and HIV protease inhibitors containing allophenylnorstatine

The interaction of P1 and P3 side chains with the combining S1 and S3 hydrophobic subsites of HIV and FIV proteases has been explored using asymmetric competitive inhibitors. The inhibitors evaluated contained (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid (allophenylnorstatine) as the hydroxymethylcarbonyl isostere, (R)-5,5-dimethyl-1, 3-thiazolidine-4-carbonyl as P1', Val as P2 and P2' residues, and a variety of amino acids at the P3 and P3' positions. All inhibitors showed competitive inhibition of both enzymes with higher potency against the HIV protease in vitro. Within this series, 31 (VLE776) is the most effective inhibitor against FIV protease, and it contains Phe at P3, but no P3' residue. VLE776 also exhibited potent antiviral activities against the drug-resistant HIV mutants (G48V and V82F) and the TL3-resistant HIV mutants. Explanation of the inhibition activities was described. In addition, a new strategy was described for development of bifunctional inhibitors, which combine the protease inhibitor and another enzyme inhibitor in one molecule.     

Neuroepithelial bodies in mammalian lung express functional serotonin type 3 receptor

Serotonin (5-HT) type 3 receptor (5-HT(3)-R) is a ligand-gated ion channel found primarily in the central and peripheral nervous system. We report expression and functional characterization of 5-HT(3)-R in pulmonary neuroepithelial body (NEB) cells. Using nonisotopic in situ hybridization, we demonstrate expression of 5-HT(3)-R mRNA in NEB cells in the lungs of different mammals (hamster, rabbit, mouse, and human). Dual immunocytochemistry (for 5-HT and 5-HT(3)-R) and confocal microscopy localized 5-HT(3)-R on NEB cell plasma membrane from rabbit. The electrophysiological characteristics of 5-HT(3)-R in NEB cells were studied in fresh slices of neonatal hamster lung using the whole cell patch-clamp technique. Application of the 5-HT (5-150 microM) and 5-HT(3)-R agonist 2-methyl-5-HT (5-150 microM) induced inward currents in a concentration-dependent manner. The 5-HT-induced current was blocked (76.5 +/- 5.9%) by the specific 5-HT(3)-R antagonist ICS-205-930 (50 microM), whereas katanserin and p-4-iodo-N-(2-[4-(methoxyphenyl)-1-piperazinyl]ethyl)-N-2-pyridinylbenzamide had minimal effects. Forskolin had no effect on desensitization and amplitude of the 5-HT-induced current. The reduction of Ca(2+) and Mg(2+) in the extracellular solution enhanced the amplitude of the 5-HT-induced current because of slower desensitization. Our studies suggest that 5-HT(3)-R in NEB cells may function as an autoreceptor and may potentially be involved in modulation of hypoxia signaling.     

Tumor targeting of radiometal labeled anti-CEA recombinant T84.66 diabody and t84.66 minibody: comparison to radioiodinated fragments

Recombinant antibody fragments offer potential advantages over intact monoclonal antibodies in the radioimmunoscintigraphy (RIS) of solid tumors. Due to their smaller molecular size, antibody fragments have shown rapid tumor targeting and blood clearance, a more uniform tumor distribution and a lower potential to elicit a human immune response. Previously, we have expressed two genetically engineered antibody fragments, the T84.66 diabody (scFv dimer) and the T84.66 minibody (scFv-CH3 dimer), specific to carcinoembryonic antigen (CEA). When radioiodinated, both antibody fragments exhibited rapid tumor targeting and rapid blood clearance in xenografted mice. To extend and optimize their future clinical RIS utility with radiometals, these antibody fragments were conjugated with the macrocycle 1,4,7,10-tetraazacyclododecane N,N',N' ',N' "-tetraacetic acid (DOTA) and labeled with 111In. Tumor targeting and biodistribution studies were carried out in athymic mice xenografted with a human colorectal tumor cell line, LS174T. The [111In]T84.66 diabody (55 kDa) exhibited very rapid tumor targeting with 12.5 +/- 0.4% injected dose per gram (% ID g(-1) +/- standard error) at 2 h and reached a maximum of 13.3 +/- 0.9% ID g(-1) at 6 h. However, kidney uptake was observed to reached a peak of 183.5 +/- 21.0% ID g(-1) at 6 h, a result similar to that reported by others for other low molecular weight fragments labeled with radiometals. Preadministration of an oral dose of D-lysine resulted in a 59% lowering of the renal accumulation at 6 h, but was accompanied by a 31% reduction of tumor uptake to 9.2 +/- 1.2% ID g(-1). The second recombinant antibody fragment, the [111In]T84.66 minibody (80 kDa), displayed rapid tumor targeting of 14.2 +/- 6.1% ID g(-1) at 2 h, and reached a maximum activity of 24.5 +/- 6.1% ID g(-1) by 12 h. Renal uptake achieved a plateau of 12-13% ID g(-1) which cleared to 7.2% ID g(-1) at 72 h. However, hepatic uptake was elevated and reached a maximum of 26.0 +/- 1.0% ID g(-1) at 12 h in these xenograft-bearing mice. Experiments in nontumor bearing mice showed a reduction of hepatic activity at 12 h to 16.6 +/- 1.5% ID g(-1), indicative of an intrinsic hepatic accumulation of the [111In]DOTA-T84.66 minibody or metabolites. While the anti-CEA [111In]DOTA-T84.66 diabody and T84.66 minibody retain the rapid tumor targeting properties of the radioiodinated form, the normal organ accumulation (kidneys and liver, respectively) of the [111In]DOTA forms appeared problematic for RIS and RIT applications. Development of alternative blocking strategies or new metabolizable chelates are under investigation to enhance the utility of the radiometal form of these and other promising recombinant antibody fragments.     

Combine-ARMS: a rapid and cost-effective protocol for molecular characterization of beta-thalassemia in Malaysia

Beta-thalassemia major patients have chronic anemia and are dependent on blood transfusions to sustain life. Molecular characterization and prenatal diagnosis of beta3-thalassemia is essential in Malaysia because about 4.5% of the population are heterozygous carriers for beta-thalassemia. The high percentage of compound heterozygosity (47.62%) found in beta-thalassemia major patients in the Thalassaemia Registry, University of Malaya Medical Centre (UMMC), Malaysia, also supports a need for rapid, economical, and sensitive protocols for the detection of beta-thalassemia mutations. Molecular characterization of beta-thalassemia mutations in Malaysia is currently carried out using ARMS, which detects a single beta-thalassemia mutation per PCR reaction. We developed and evaluated Combine amplification refractory mutation system (C-ARMS) techniques for efficient molecular detection of two to three beta-thalassemia mutations in a single PCR reaction. Three C-ARMS protocols were evaluated and established for molecular characterization of common beta-thalassemia mutations in the Malay and Chinese ethnic groups in Malaysia. Two C-ARMS protocols (cd 41-42/IVSII #654 and -29/cd 71-72) detected the beta-thalassemia mutations in 74.98% of the Chinese patients studied. The CARMS for cd 41-42/IVSII #654 detected beta-thalassemia mutations in 72% of the Chinese families. C-ARMS for cd 41-42/IVSI #5/cd 17 allowed detection of beta-thalassemia mutations in 36.53% of beta-thalassemia in the Malay patients. C-ARMS for cd 41-42/IVSI #5/cd 17 detected beta-thalassemia in 45.54% of the Chinese patients. We conclude that C-ARMS with the ability to detect two to three mutations in a single reaction provides more rapid and cost-effective protocols for beta-thalassemia prenatal diagnosis and molecular analysis programs in Malaysia.     

Intraductal laser photocoagulation of the bilateral parotid ducts for reduction of drooling in patients with cerebral palsy

Patients with cerebral palsy who experience drooling are often isolated from social interaction. Surgical treatment is effective in reducing abnormal, profuse drooling in patients who have low cognitive function, but it has a risk of complications. In this study, a new, simple procedure using laser intervention that minimizes surgical complications is described. Forty-eight patients with cerebral palsy and persistent drooling after more than 6 months of conservative treatment were enrolled in this study. An Nd:YAG laser (1064 nm) was used for intraductal laser photocoagulation of the bilateral parotid ducts at 7 to 10 W for 10 seconds. The outcome was evaluated by questionnaire-based, semiquantitative assessments of drooling severity and frequency, collection and measurement of stimulated saliva, and salivary amylase measurement. The entire procedure was completed in 25 to 65 minutes, with a mean duration of 38.4 minutes. Early complications included transient facial swelling in all patients. Swelling persisted for 6 to 37 days (mean, 11 days). One hematoma (2.1 percent of patients), two infections (4.2 percent of patients), and two cystic formations (4.2 percent of patients) also occurred. No obvious xerostomia or visible scar was noted after the procedure. In the final assessment, a significant improvement in drooling severity (p < 0.05) and frequency (p < 0.05) was noted in the majority of cases. Forty patients (83.3 percent) demonstrated remarkable improvement in drooling severity, seven patients (14.6 percent) showed significant improvement, and one patient (2.1 percent), who was also autistic, continued to experience severe drooling after the laser procedure. The decrease in the amount of saliva produced ranged from 20 to 60 percent at 12 weeks after surgery. The decrease in the amount of salivary amylase measured ranged from 4 to 97 percent at 12 weeks after surgery (p < 0.05). In conclusion, the intraductal laser photocoagulation of bilateral parotid ducts is a simple, effective procedure for reducing drooling in patients who have cerebral palsy. This procedure minimizes risks and complications, compared with those associated with conventional surgery.