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This study aims to investigate the control of arsenic distribution by biogeochemical processes in the Indian Sundarban mangrove ecosystem and the importance of this ecosystem as an arsenic source for surrounding coastal water. The As(V)/As(III) ratio was found to be significantly lower in both surface and pore waters compared to sea water, which could be attributed to biogeochemical interconversion of these arsenic forms. The biological uptake of arsenic due to primary and benthic production occurs during the post-monsoon season, and is followed by the release of arsenic during the biochemical degradation and dissolution of plankton in the pre-monsoon season. These results suggest that arsenic is immobilized during incorporation into the arsenic-bearing initial phase, and unlikely to be released into pore water until the complete microbial degradation of arsenic-bearing organic compounds.

Methyl-coenzyme M reductase (MCR) catalyzes the terminal step in the formation of biological methane from methyl-coenzyme M (Me-SCoM) and coenzyme B (CoBSH). The active site in MCR contains a Ni-F(430) cofactor, which can exist in different oxidation states. The catalytic mechanism of methane formation has remained elusive despite intense spectroscopic and theoretical investigations. On the basis of spectroscopic and crystallographic data, the first step of the mechanism is proposed to involve a nucleophilic attack of the Ni(I) active state (MCR(red1)) on Me-SCoM to form a Ni(III)-methyl intermediate, while computational studies indicate that the first step involves the attack of Ni(I) on the sulfur of Me-SCoM, forming a CH(3)(*) radical and a Ni(II)-thiolate species. In this study, a combination of Ni K-edge X-ray absorption spectroscopic (XAS) studies and density functional theory (DFT) calculations have been performed on the Ni(I) (MCR(red1)), Ni(II) (MCR(red1-silent)), and Ni(III)-methyl (MCR(Me)) states of MCR to elucidate the geometric and electronic structures of the different redox states. Ni K-edge EXAFS data are used to reveal a five-coordinate active site with an open upper axial coordination site in MCR(red1). Ni K-pre-edge and EXAFS data and time-dependent DFT calculations unambiguously demonstrate the presence of a long Ni-C bond ( approximately 2.04 A) in the Ni(III)-methyl state of MCR. The formation and stability of this species support mechanism I, and the Ni-C bond length suggests a homolytic cleavage of the Ni(III)-methyl bond in the subsequent catalytic step. The XAS data provide insight into the role of the unique F(430) cofactor in tuning the stability of the different redox states of MCR.

Kinase Gcn2 is activated by amino acid starvation and downregulates translation initiation by phosphorylating the alpha subunit of translation initiation factor 2 (eIF2alpha). The Gcn2 kinase domain (KD) is inert and must be activated by tRNA binding to the adjacent regulatory domain. Previous work indicated that Saccharomyces cerevisiae Gcn2 latency results from inflexibility of the hinge connecting the N and C lobes and a partially obstructed ATP-binding site in the KD. Here, we provide strong evidence that a network of hydrophobic interactions centered on Leu-856 also promotes latency by constraining helix alphaC rotation in the KD in a manner relieved during amino acid starvation by tRNA binding and autophosphorylation of Thr-882 in the activation loop. Thus, we show that mutationally disrupting the hydrophobic network in various ways constitutively activates eIF2alpha phosphorylation in vivo and bypasses the requirement for a key tRNA binding motif (m2) and Thr-882 in Gcn2. In particular, replacing Leu-856 with any nonhydrophobic residue activates Gcn2, while substitutions with various hydrophobic residues maintain kinase latency. We further provide strong evidence that parallel, back-to-back dimerization of the KD is a step on the Gcn2 activation pathway promoted by tRNA binding and autophosphorylation. Remarkably, mutations that disrupt the L856 hydrophobic network or enhance hinge flexibility eliminate the need for the conserved salt bridge at the parallel dimer interface, implying that KD dimerization facilitates the reorientation of alphaC and remodeling of the active site for enhanced ATP binding and catalysis. We propose that hinge remodeling, parallel dimerization, and reorientation of alphaC are mutually reinforcing conformational transitions stimulated by tRNA binding and secured by the ensuing autophosphorylation of T882 for stable kinase activation.

This article examines the effects of changing parameters in the test which was proposed by the FDA at the October 2005 Advisory Committee meeting for confirming delivered dose uniformity in orally inhaled and nasal drug products. This article is an extension of the characterization study presented in an accompanying article (Part 1). The goal of this study is to understand how parameters of the test affect the test performance. The effects of changing test parameters such as target interval, maximum allowable proportion in the tail area, and sample size are examined. The results show that changing the maximum allowable tail area and/or the target interval have the largest impact on the test outcomes, i.e., probability of acceptance for a given batch mean and standard deviation. The presented information may provide potential users of the test with a set of tools for optimizing the test characteristics for a particular product.

The robustness of the parametric tolerance interval test, which was proposed by the Food and Drug Administration for control of delivered dose uniformity in orally inhaled and nasal drug products, is investigated in this article using different scenarios for deviations from a univariate normal distribution. The studied scenarios span a wide range of conditions, the purpose of which is to provide an understanding of how the test performs depending on the nature and degree of the deviation from normality. Operating characteristic curves were generated to compare the performance of the test for different types of distributions (normal and non-normal) having the same proportion of doses in the tails (on one or both sides) outside the target interval. The results show that, in most cases, non-normality does not increase the probability of accepting a batch of unacceptable quality (i.e., the test is robust) except in extreme situations, which do not necessarily represent commercially viable products. The results also demonstrate that, in the case of bimodal distributions where the life-stage means differ from each other by up to 24% label claim, the test's criterion on life-stage means does not affect pass rates because the tolerance interval portion of the test reacts to shifting means as well.

The FDA proposed a parametric tolerance interval (PTI) test at the October 2005 Advisory Committee meeting as a replacement of the attribute (counting) test for delivered dose uniformity (DDU), published in the 1998 draft guidance for metered dose inhalers (MDIs) and dry powder inhalers (DPIs) and the 2002 final guidance for inhalation sprays and intranasal products. This article (first in a series of three) focuses on the test named by the FDA "87.5% coverage." Unlike a typical two-sided PTI test, which controls the proportion of the DDU distribution within a target interval (coverage), this test is comprised of two one-sided tests (TOST) designed to control the maximum amount of DDU values in either tail of the distribution above and below the target interval. Through simulations, this article characterizes the properties and performance of the proposed PTI-TOST under different scenarios. The results show that coverages of 99% or greater are needed for a batch to have acceptance probability 98% or greater with the test named by the FDA "87.5% coverage" (95% confidence level), while batches with 87.5% coverage have less than 1% probability of being accepted. The results also illustrate that with this PTI-TOST, the coverage requirement for a given acceptance probability increases as the batch mean deviates from target. The accompanying articles study the effects of changing test parameters and the test robustness to deviations from normality.

Plant natural products remain a good resource for the discovery of novel pharmaceuticals. A mouse macrophage-based quantitative, reverse transcription polymerase chain reaction (qRT-PCR) system was optimized to screen plant extracts for antiinflammatory activities using three well known genetic markers of inflammation. Plants used for extraction were taxonomically identified and vouchered species from two Central Asian countries, Uzbekistan and Kyrgyzstan, collected through the International Cooperative Biodiversity Groups (ICBG) program. The mRNA expression of cyclooxygenase-2, interleukin 1beta and inducible nitric oxide synthase genes in RAW macrophages was determined quantitatively in response to treatment with plant extracts applied at 100 microg/mL. The screening of 1000 extracts from 449 plant species belonging to 68 plant families resulted in 75 extracts (7.5%) showing strong (75% or higher inhibition) activity against at least one target gene. Many extracts showed qualitative and quantitative differences in the levels of activities against each target gene. Extracts identified from this screen were able to reduce inflammatory symptoms in vivo, thereby validating the screening approach.

Background: Double-stranded (ds) RNA, generated during viral infection, binds and activates the mammalian anti-viral protein kinase PKR, which phosphorylates the translation initiation factor eIF2alpha leading to the general inhibition of protein synthesis. Although PKR-like activity has been described in fish cells, the responsible enzymes eluded molecular characterization until the recent discovery of goldfish and zebrafish PKZ, which contain Z-DNA-binding domains instead of dsRNA-binding domains (dsRBDs). Fish and amphibian PKR genes have not been described so far., Results: Here we report the cloning and identification of 13 PKR genes from 8 teleost fish and amphibian species, including zebrafish, demonstrating the coexistence of PKR and PKZ in this latter species. Analyses of their genomic organization revealed up to three tandemly arrayed PKR genes, which are arranged in head-to-tail orientation. At least five duplications occurred independently in fish and amphibian lineages. Phylogenetic analyses reveal that the kinase domains of fish PKR genes are more closely related to those of fish PKZ than to the PKR kinase domains of other vertebrate species. The duplication leading to fish PKR and PKZ genes occurred early during teleost fish evolution after the divergence of the tetrapod lineage. While two dsRBDs are found in mammalian and amphibian PKR, one, two or three dsRBDs are present in fish PKR. In zebrafish, both PKR and PKZ were strongly upregulated after immunostimulation with some tissue-specific expression differences. Using genetic and biochemical assays we demonstrate that both zebrafish PKR and PKZ can phosphorylate eIF2alpha in yeast., Conclusion: Considering the important role for PKR in host defense against viruses, the independent duplication and fixation of PKR genes in different lineages probably provided selective advantages by leading to the recognition of an extended spectrum of viral nucleic acid structures, including both dsRNA and Z-DNA/RNA, and perhaps by altering sensitivity to viral PKR inhibitors. Further implications of our findings for the evolution of the PKR family and for studying PKR/PKZ interactions with viral gene products and their roles in viral infections are discussed.

Methyl-coenzyme M reductase (MCR) catalyzes the terminal step in methanogenesis by using N-7-mercaptoheptanolyl-threonine phosphate (CoBSH) as the two-electron donor to reduce 2-(methylthiol)ethane sulfonate (methyl-SCoM) to methane, and producing the heterodisulfide, CoBS-SCoM. The active site of MCR includes a noncovalently bound Ni tetrapyrrolic cofactor called coenzyme F430, which is in the Ni(I) state in the active enzyme (MCRred1). Bromopropanesulfonate (BPS) is a potent inhibitor and reversible redox inactivator that reacts with MCRred1 to form an EPR-active state called MCRPS, which is an alkyl-nickel species. When MCRPS is treated with free thiol containing compounds, the enzyme is reconverted to the active MCRred1 state. In this paper, we demonstrate that the reactivation of MCRPS to MCRred1 by thiols involves formation of a thioether product. MCRPS also can be converted to active MCRred1 by treatment with sodium borohydride. Reactivation is highest with the smallest free thiol HS-. Interestingly, MCRPS can also be reductively activated with analogues of CoBSH such as CoB8SH and CoB9SH, but not CoBSH itself. Unambiguous demonstration of the formation of a methylthioether product from thiolysis of an alkyl-Ni species provides support for a methyl-Ni intermediate in the MCR-catalyzed last step in methanogenesis and the first proposed step in anaerobic methane oxidation.

Four wild berry species, Amelanchier alnifolia, Viburnum trilobum, Prunus virginiana, and Shepherdia argentea, all integral to the traditional subsistence diet of Native American tribal communities, were evaluated to elucidate phytochemical composition and bioactive properties related to performance and human health. Biological activity was screened using a range of bioassays that assessed the potential for these little-known dietary berries to affect diabetic microvascular complications, hyperglycemia, pro-inflammatory gene expression, and metabolic syndrome symptoms. Nonpolar constituents from berries, including carotenoids, were potent inhibitors of aldose reductase (an enzyme involved in the etiology of diabetic microvascular complications), whereas the polar constituents, mainly phenolic acids, anthocyanins, and proanthocyanidins, were hypoglycemic agents and strong inhibitors of IL-1beta and COX-2 gene expression. Berry samples also showed the ability to modulate lipid metabolism and energy expenditure in a manner consistent with improving metabolic syndrome. The results demonstrate that these berries traditionally consumed by tribal cultures contain a rich array of phytochemicals that have the capacity to promote health and protect against chronic diseases, such as diabetes.

Cytotoxic chemotherapy that induces lymphopenia is predicted to ablate the benefits of active antitumor immunization. Temozolomide is an effective chemotherapeutic agent for patients with glioblastoma multiforme, but it induces significant lymphopenia. Although there is monthly fluctuation of the white blood cell count, specifically the CD4 and CD8 counts, there was no cumulative decline in the patient described in this case report. Depriving patients of this agent, in order to treat with immunotherapy, is controversial. Despite conventional dogma, we demonstrated that chemotherapy and immunotherapy can be delivered concurrently without negating the effects of immunotherapy. In fact, the temozolomide-induced lymphopenia may prove to be synergistic with a peptide vaccine secondary to inhibition of regulatory T cells or their delayed recovery.

Ire1 is an ancient transmembrane sensor of ER stress with dual protein kinase and ribonuclease activities. In response to ER stress, Ire1 catalyzes the splicing of target mRNAs in a spliceosome-independent manner. We have determined the crystal structure of the dual catalytic region of Ire1at 2.4 A resolution, revealing the fusion of a domain, which we term the KEN domain, to the protein kinase domain. Dimerization of the kinase domain composes a large catalytic surface on the KEN domain which carries out ribonuclease function. We further show that signal induced trans-autophosphorylation of the kinase domain permits unfettered binding of nucleotide, which in turn promotes dimerization to compose the ribonuclease active site. Comparison of Ire1 to a topologically disparate ribonuclease reveals the convergent evolution of their catalytic mechanism. These findings provide a basis for understanding the mechanism of action of RNaseL and other pseudokinases, which represent 10% of the human kinome.

An efficient system was developed, and several variables tested, for generating a large-scale insertional-mutagenesis population of rice. The most important feature in this improved Ac/Ds tagging system is that one can conveniently carry out large-scale screening in the field and select transposants at the seedling stage. Rice was transformed with a plasmid that includes a Basta-resistance gene (bar). After the Ds element is excised during transposition, bar becomes adjacent to the ubiquitin promoter, and the rice plant becomes resistant to the herbicide Basta. In principle, one can plant up to one million plants in the field and select those plants that survive after spraying with Basta. To test the utility of this system, 4 Ds starter lines were crossed with 14 different Ac plants, and many transposants were successfully identified after planting 134,285 F2 plants in the field. Over 2,800 of these transposants were randomly chosen for PCR analysis, and the results fully confirmed the reliability of the field screening procedure.

Translational control mediated by phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2alpha) is central to stress-induced programs of gene expression. Trypanosomatids, important human pathogens, display differentiation processes elicited by contact with the distinct physiological milieu found in their insect vectors and mammalian hosts, likely representing stress situations. Trypanosoma brucei, the agent of African trypanosomiasis, encodes three potential eIF2alpha kinases (TbeIF2K1 to -K3). We show here that TbeIF2K2 is a transmembrane glycoprotein expressed both in procyclic and in bloodstream forms. The catalytic domain of TbeIF2K2 phosphorylates yeast and mammalian eIF2alpha at Ser51. It also phosphorylates the highly unusual form of eIF2alpha found in trypanosomatids specifically at residue Thr169 that corresponds to Ser51 in other eukaryotes. T. brucei eIF2alpha, however, is not a substrate for GCN2 or PKR in vitro. The putative regulatory domain of TbeIF2K2 does not share any sequence similarity with known eIF2alpha kinases. In both procyclic and bloodstream forms TbeIF2K2 is mainly localized in the membrane of the flagellar pocket, an organelle that is the exclusive site of exo- and endocytosis in these parasites. It can also be detected in endocytic compartments but not in lysosomes, suggesting that it is recycled between endosomes and the flagellar pocket. TbeIF2K2 location suggests a relevance in sensing protein or nutrient transport in T. brucei, an organism that relies heavily on posttranscriptional regulatory mechanisms to control gene expression in different environmental conditions. This is the first membrane-associated eIF2alpha kinase described in unicellular eukaryotes.

Methyl-coenzyme M reductase (MCR) from methanogenic archaea catalyzes the final step in the biological synthesis of methane. Using coenzyme B (CoBSH) as the two-electron donor, MCR reduces methyl-coenzyme M (methyl-SCoM) to methane and the mixed disulfide, CoB-S-S-CoM. MCR contains coenzyme F430, an essential redox-active nickel tetrahydrocorphin, at its active site. The active form of MCR (MCRred1) contains Ni(I)-F430. When 3-bromopropane sulfonate (BPS) is incubated with MCRred1, an alkyl-Ni(III) species is formed that elicits the MCRPS EPR signal. Here we used EPR and UV-visible spectroscopy and transient kinetics to study the reaction between MCR from Methanothermobacter marburgensis and a series of brominated carboxylic acids, with carbon chain lengths of 4-16. All of these compounds give rise to an alkyl-Ni intermediate with an EPR signal similar to that of the MCRPS species. Reaction of the alkyl-Ni(III) adduct, formed from brominated acids with eight or fewer total carbons, with HSCoM as nucleophile at pH 10.0 results in the formation of a thioether coupled to regeneration of the active MCRred1 state. When reacted with 4-bromobutyrate, MCRred1 forms the alkyl-Ni(III) MCRXA state and then, surprisingly, undergoes "self-reactivation" to regenerate the Ni(I) MCRred1 state and a bromocarboxy ester. The results demonstrate an unexpected reactivity and flexibility of the MCR active site in accommodating a broad range of substrates, which act as molecular rulers for the substrate channel in MCR.

We describe a 15-year-old girl who presented with bilateral exudative retinal detachment, a previously unreported complication, due to orbital pseudotumor. She initially responded to steroids, but subsequently became steroid dependent. Azathioprine was effective in controlling further relapses during follow-up of 22 months.

The extract of Tripterygium wilfordii Hook F. (TwHF), which showed anti-inflammatory and immunosuppressive activities in human clinical trials for rheumatoid arthritis, was subjected to the activity-guided fractionation and spectroscopic characterization of bioactives. A tetrahydrofuran lignan, tripterygiol (1), and eight known compounds, all capable of suppressing pro-inflammatory gene expression were identified. Most of the pharmacological activity of the extract can be attributed to triptolide, its most abundant and active component, with some contribution from tripdiolide.

Generation of an indexed, saturated, insertional-mutant library is an aid to understanding the functions of genes in an organism. However, 10 years of work by many investigators have not yet yielded such a library in rice. The major reason is that determining the chromosomal locations of a very large number of random insertion mutants by flanking sequence analysis is highly labor intensive, and therefore, libraries that do exist have not been indexed. We report here an efficient procedure to construct an indexed, region-specific, insertional-mutant library of rice. The procedure makes use of efficient long-PCR-based high-throughput indexing, coupled with a random but anchored population of Ds transposants. Long-PCR indexing allows rapid and simultaneous determination of the chromosomal locations of a large number of mutants that surround a particular anchor line, thus converting a random library into an indexed one. Such a library can be used directly, without the need to screen a large random library for a desired mutant plant.

The protein kinases PKR, GCN2, and PERK phosphorylate translation initiation factor eIF2alpha to regulate general and genespecific protein synthesis under various cellular stress conditions. Recent x-ray crystallographic structures of PKR and GCN2 revealed distinct dimeric configurations of the kinase domains. Whereas PKR kinase domains dimerized in a back-to-back and parallel orientation, the GCN2 kinase domains displayed an antiparallel orientation. The dimerization interfaces on PKR and GCN2 were localized to overlapping surfaces on the N-terminal lobes of the kinase domains but utilized different intermolecular contacts. A key feature of the PKR dimerization interface is a salt bridge interaction between Arg(262) from one protomer and Asp(266) from the second protomer. Interestingly, these two residues are conserved in all eIF2alpha kinases, although in the GCN2 structure, the two residues are too remote to interact. To test the importance of this potential salt bridge interaction in PKR, GCN2, and PERK, the residues constituting the salt bridge were mutated either independently or together to residues with the opposite charge. Single mutations of the Asp (or Glu) and Arg residues blocked kinase function both in yeast cells and in vitro. However, for all three kinases, the double mutation designed to restore the salt bridge interaction with opposite polarity resulted in a functional kinase. Thus, the salt bridge interaction and dimer interface observed in the PKR structure is critical for the activity of all three eIF2alpha kinases. These results are consistent with the notion that the PKR structure represents the active state of the eIF2alpha kinase domain, whereas the GCN2 structure may represent an inactive state of the kinase.

Methyl-coenzyme M reductase (MCR) catalyzes the final step in methane biosynthesis by methanogenic archaea and contains a redox-active nickel tetrahydrocorphin, coenzyme F430, at its active site. Spectroscopic and computational methods have been used to study a novel form of the coenzyme, called F330, which is obtained by reducing F430 with sodium borohydride (NaBH4). F330 exhibits a prominent absorption peak at 330 nm, which is blue shifted by 100 nm relative to F430. Mass spectrometric studies demonstrate that the tetrapyrrole ring in F330 has undergone reduction, on the basis of the incorporation of protium (or deuterium), upon treatment of F430 with NaBH4 (or NaBD4). One- and two-dimensional NMR studies show that the site of reduction is the exocyclic ketone group of the tetrahydrocorphin. Resonance Raman studies indicate that elimination of this pi-bond increases the overall pi-bond order in the conjugative framework. X-ray absorption, magnetic circular dichroism, and computational results show that F330 contains low-spin Ni(II). Thus, conversion of F430 to F330 reduces the hydrocorphin ring but not the metal. Conversely, reduction of F430 with Ti(III) citrate to generate F380 (corresponding to the active MCR(red1) state) reduces the Ni(II) to Ni(I) but does not reduce the tetrapyrrole ring system, which is consistent with other studies [Piskorski, R., and Jaun, B. (2003) J. Am. Chem. Soc. 125, 13120-13125; Craft, J. L., et al. (2004) J. Biol. Inorg. Chem. 9, 77-89]. The distinct origins of the absorption band shifts associated with the formation of F330 and F380 are discussed within the framework of our computational results. These studies on the nature of the product(s) of reduction of F430 are of interest in the context of the mechanism of methane formation by MCR and in relation to the chemistry of hydroporphinoid systems in general. The spectroscopic and time-dependent DFT calculations add important insight into the electronic structure of the nickel hydrocorphinate in its Ni(II) and Ni(I) valence states.

Winter cress (Barbarea verna) seed preparations rich in phenethylisothiocyanate (PEITC) had strong in vivo and in vitro anti-inflammatory activity, significantly reducing the size of carrageenan-induced rat paw edema. This in vivo effect was comparable with that of the nonsteroidal anti-inflammatory drug aspirin. The seed preparation, in a concentration-dependent manner, reduced the mRNA levels of inflammation-related genes such as the inducible forms of cyclooxygenase and nitric-oxide synthase and the proinflammatory cytokine interleukin in lipopolysaccharide-stimulated mouse macrophage cell line RAW 264.7. Activity of the seed preparation was similar to that of the synthetic PEITC. PEITC was the most active of five different forms of isothiocyanate tested for their effects on in vitro proinflammatory gene expression. In vitro activity of the seed preparation was also compared with that of two known anti-inflammatory drugs. We conclude that Barbarea verna seed preparation may function as a potent anti-inflammatory agent, interfering with the transcription of proinflammatory genes.

The antiviral protein kinase PKR inhibits protein synthesis by phosphorylating the translation initiation factor eIF2alpha on Ser51. Binding of double-stranded RNA to the regulatory domains of PKR promotes dimerization, autophosphorylation, and the functional activation of the kinase. Herein, we identify mutations that activate PKR in the absence of its regulatory domains and map the mutations to a recently identified dimerization surface on the kinase catalytic domain. Mutations of other residues on this surface block PKR autophosphorylation and eIF2alpha phosphorylation, while mutating Thr446, an autophosphorylation site within the catalytic-domain activation segment, impairs eIF2alpha phosphorylation and viral pseudosubstrate binding. Mutational analysis of catalytic-domain residues preferentially conserved in the eIF2alpha kinase family identifies helix alphaG as critical for the specific recognition of eIF2alpha. We propose an ordered mechanism of PKR activation in which catalytic-domain dimerization triggers Thr446 autophosphorylation and specific eIF2alpha substrate recognition.

Four stress-responsive protein kinases, including GCN2 and PKR, phosphorylate eukaryotic translation initiation factor 2alpha (eIF2alpha) on Ser51 to regulate general and gene-specific protein synthesis. Phosphorylated eIF2 is an inhibitor of its guanine nucleotide exchange factor, eIF2B. Mutations that block translational regulation were isolated throughout the N-terminal OB-fold domain in Saccharomyces cerevisiae eIF2alpha, including those at residues flanking Ser51 and around 20 A away in the conserved motif K79GYID83. Any mutation at Glu49 or Asp83 blocked translational regulation; however, only a subset of these mutations impaired Ser51 phosphorylation. Substitution of Ala for Asp83 eliminated phosphorylation by GCN2 and PKR both in vivo and in vitro, establishing the critical contributions of remote residues to kinase-substrate recognition. In contrast, mutations that blocked translational regulation but not Ser51 phosphorylation impaired the binding of eIF2B to phosphorylated eIF2alpha. Thus, two structurally distinct effectors of eIF2 function, eIF2alpha kinases and eIF2B, have evolved to recognize the same surface and overlapping determinants on eIF2alpha.

Phytohormones are well-known regulators of the symbiotic Rhizobium-legume association in the plant host. The enod40 nodulin gene is associated with the earliest phases of the nodule organogenesis programme in the legume host and modifying its expression resulted in perturbations of nodule development in Medicago truncatula. Therefore in our pursuit to mimic the initial signal transduction steps of legume nodulation in the alien physiological set-up of a rice plant, we have expressed the Mtenod40 gene in rice. Molecular data confirm the stable integration, inheritance and transcription of the foreign gene in this non-legume. We have compared the phytohormonal responses of Mtenod40-overexpressing and control plants in a homologous legume background (M. truncatula) and in the non-legume rice. An enod40-mediated root growth response, induced by inhibition of ethylene biosynthesis, was observed in both plants. On the other hand, a significant differential effect of cytokinins was observed only in rice plants. This suggests that ethylene inhibits enod40 action both in legumes and non-legumes and reinforces that some of the early signal transduction steps of the nodule developmental programme may function in rice.

Deficiencies in the MutS protein disrupt methyl-directed mismatch repair (MMR), generating a mutator phenotype typified by high mutation rates and promiscuous recombination. How such deficiencies might arise in the natural environment was determined by analysing pathogenic strains of Escherichia coli. Quantitative Western immunoblotting showed that the amount of MutS in a wild-type strain of the enterohaemorrhagic pathogen E. coli O157 : H7 decreased about 26-fold in stationary-phase cells as compared with the amount present during exponential-phase growth. The depletion of MutS in O157 : H7 is significantly greater than that observed for a laboratory-attenuated E. coli K-12 strain. In the case of stable mutators, mutS defects in strains identified among natural isolates were analysed, including two E. coli O157 : H7 strains, a diarrhoeagenic E. coli O55 : H7 strain, and a uropathogenic strain from the E. coli reference (ECOR) collection. No MutS could be detected in the four strains by Western immunoblot analyses. RNase T2 protection assays showed that the strains were either deficient in mutS transcripts or produced transcripts truncated at the 3' end. Nucleotide sequence analysis revealed extensive deletions in the mutS region of three strains, ranging from 7.5 to 17.3 kb relative to E. coli K-12 sequence, while the ECOR mutator contained a premature stop codon in addition to other nucleotide changes in the mutS coding sequence. These results provide insights into the status of the mutS gene and its product in pathogenic strains of E. coli.

Two forms of poly(A) polymerase (PAPI and PAPII) from germinated wheat embryos have been resolved on DEAE-cellulose ion-exchange chromatography by a linear gradient of 0-500 mM (NH(4))(2)SO(4). Further purification shows that both forms are monomeric in nature with an identical molecular weight, approximately 65 kDa. The phosphoprotein nature of PAPI and PAPII has been established by in vivo labelling with (32)P-orthophosphate. Acid hydrolysis of both (32)P-labelled purified PAPI and PAPII has revealed that phosphorylations generally take place in serine and threonine residues. PAPI and PAPII have also been characterised with respect to V(max) and K(m) for poly(A). The V(max) and K(m) values of PAPI are 28.57 and 11.37 microg, respectively, whereas 34.48 and 7.04 microg of PAPII. In vitro dephosphorylation of the purified enzyme by alkaline phosphatase leads to a significant loss of the enzyme activity, which is regained upon phosphorylation by a 65 kDa protein kinase (PK) purified from wheat embryos. The extent of phosphorylation by protein kinase shows that PK has similar affinity towards both PAPI and PAPII, whereas the phosphate incorporation in PAPII is twofold higher than PAPI suggesting their distinct chemical nature.

The subject area of this review provides extraordinary challenges and opportunities. The challenges relate to the fact that the integration of various fields such as microbiology, biochemistry, plant physiology, eukaryotic as well as bacterial genetics, and applied plant sciences are required to assess the disposition of rice, an alien host, for establishing such a unique phenomenon as biological nitrogen fixation. The opportunities signify that, if successful, the breakthrough will have a significant impact on the global economy and will help improve the environment. This review highlights the literature related to the area of legume-rhizobia interactions, particularly those aspects whose understanding is of particular interest in the perspective of rice. This review also discusses the progress achieved so far in this area of rice research and the possibility of built-in nitrogen fixation in rice in the future. However, it is to be borne in mind that such research does not ensure any success at this point. It provides a unique opportunity to broaden our knowledge and understanding about many aspects of plant growth regulation in general.

In the present study, the effects of orally-administered lithium on testicular morphology were examined in the spotted munia (Lonchura punctulata), a seasonally breeding sub-tropical finch. Adult males were procured from natural populations during the month of August, a time when these birds begin to show seasonal reproductive maturity in an annual cycle. Both during the period of acclimation, and throughout the subsequent experimental period, the birds were maintained in an open aviary simulating natural environmental conditions. Lithium was dissolved in distilled water and was administered via the oral route by means of a commercially available stomach-tube. A total of five experimental groups were utilized. The first group (Group A) served as control and received lithium-free distilled water in a similar manner. In the remaining four groups, lithium was administered daily as follows: Group B (2.5 mEq/Kg body weight for 5 days); Group C (2.5 mEq/Kg for 10 days); Group D (5.0 mEq/Kg for 5 days) and Group E (5.0 mEq/Kg for 10 days). All lithium administrations were carried out between 14:00 and 15:00h. Twenty-four hours after the last oral lithium, final body weights were recorded, blood samples were obtained (by brachial vein puncture for the measurement of serum lithium) and the animals were sacrificed, and testes were collected for histological studies. Our results indicated that lithium treatment led to a significant reduction in testicular weight and seminiferous tubular diameter, and a marked degenerative changes in germ cells in that most of the spermatids and mature spermatozoa showed necrotic changes and were sloughed off from the seminiferous tubular epithelium. Complete desquamation and loss of germ cells, and their clump formation were also noted within many seminiferous tubular lumen. Notably these adverse effects were observed when serum lithium levels were within the therapeutic range for human. These results confirm our earlier report on lithium's adverse effects on testicular function, and extend further to show that lithium indeed has a significant adverse effect on the histomorphology, and, thus, the function of the testis in birds.

An Escherichia coli O157:H7 strain isolated from a patient with hemorrhagic colitis was found to exhibit two slightly different colony morphology types on differential medium. Each morphological type, designated TT12A and TT12B, was isolated, and serological testing using various assays confirmed that both strains carried the O157 and the H7 antigens. Biochemical testing showed that the strains had identical profiles on AP120E analysis and, like typical O157:H7 strains, did not ferment sorbitol or exhibit beta-glucuronidase activity. Analysis with a multiplex PCR assay showed that TT12B did not carry the gene for either Shiga toxin 1 (Stx1) or Stx2, whereas these genes were present in TT12A and the toxins were produced. Apart from that, both strains carried the +93 gusA mutation, the cluster I ehxA gene for enterohemolysin, and the eae gene for gamma-intimin, which are all characteristics of the O157:H7 serotype. Phenotypic assays confirmed that both strains exhibited enterohemolysin activity and the attachment and effacing lesion on HeLa cells. Multilocus enzyme electrophoresis analysis showed that the strains are closely related genetically and belong in the same clonal group. Pulsed-field gel electrophoresis (PFGE) typing of XbaI-digested genomic DNA revealed that the two strains differed by two bands but shared 90% similarity and clustered in the same clade. All other non-Stx-producing O157:H7 strains examined clustered in a major clade that was distinct from that of Stx-producing O157:H7 strains. The findings that TT12B was identical to TT12A, except for Stx production, and its PFGE profile is also more closely related to that of Stx-producing O157:H7 strains suggest that TT12B was derived from TT12A by the loss of both stx genes.

Effects of daily evening (just before the onset of darkness in a 24 h light dark cycle) administration of graded doses (25, 50, or 100 microg/100 g body wt./day for 30 days) of melatonin on the concentrations of blood glucose and adrenal catecholamines were studied in sexually active male roseringed parakeets under natural (NP; approximately 12L: 12D) and artificial long (LP; 16L: 8D) and short (SP; 8L: 16D) photoperiods. Blood samples and adrenal glands were collected from each bird during the mid-day on the following day of the last treatment. The concentrations of glucose in blood and epinephrine (E) and norepinephrine (NE) in the adrenals were measured. The results of the study indicated that exogenous melatonin induces hypo- or hyperglycemia depending on the dose of hormone administered as well as to the length of photoperiod to which birds were exposed. The levels of E and NE in the adrenals were shown also to vary in relation to photoperiod and the dose of melatonin administered. But the nature of the influence of melatonin becomes different under altered photoperiodic conditions. It appears that short photoperiods are more effective than long photoperiods as a modulator of glycemic and adrenal catecholaminergic responses to exogenous melatonin. A statistically significant correlation between the levels of blood glucose and that of E and NE in the adrenals was found in the control birds, but not in the melatonin treated birds. The results suggested that the responses of blood glucose and adrenal catecholamines to the treatment with melatonin in the roseringed parakeets may not be dependent on each other.

Effects of daily (one hour prior to onset of darkness) injection of melatonin (25 micrograms/100 g body wt. for 30 days) on concentrations of blood glucose and adrenal catecholamines were studied in adult male roseringed parakeets, P. krameri under both natural (NP; about 12L:12D) and artificial long (LP; 16L:8D; lights were available in between 0600 and 2200 hrs) or short (SP; 8L:16D; lights were available between 0600 and 1400 hrs) photoperiodic conditions. The results indicate that neither LP, nor SP as such exerts any significant effect on blood glucose titre of control (vehicle of hormone administered) birds. Treatment with melatonin, however, induced hyperglycemia in both NP and LP bird groups, but hypoglycemia in SP birds. Unlike glycemic levels, amount of epinephrine (E) and norepinephrine (NE) in adrenals of control birds exhibited significant changes under altered photoperiods. A decrease in E and an increase in NE were noted in adrenals of both LP and SP birds. Exogenous melatonin in NP birds also caused a decrease in E and concomittant rise in NE levels. On the other hand, treatment of melatonin in both LP and SP bird groups resulted in an increase in the quantity of both E and NE compared to respective values in adrenals of melatonin injected NP birds. However, relative to the amount of E and NE in adrenals of placebo treated LP and SP birds, significant effect of melatonin treatment was observed only in SP birds. The results suggest that influences of exogenous melatonin on the levels of both blood glucose and adrenal catecholamines are largely modulated by short rather than long photoperiods.

Recent advances have added substantially to our understanding of the biology of estrogens. Estrogens are no longer considered to differ only in potency. Two estrogens can have similar effects in one tissue and very different effects in another. Additionally, an estrogen can have different effects in different tissues. It is now recognized that there are at least two estrogen receptors, ER-alpha and ER-beta, and that it is quite likely that estrogens also work through non-genomic mechanisms. The development of new methods of chromatographic separation has aided substantially in our ability to characterize the composition of Premarin, including the identification of estradiene, the fourth-most abundant estrogen in Premarin. Recent studies have contributed to our understanding of the unique profile of Delta(8,9) dehydroestrone sulfate, another of the Premarin estrogens. It was found that Delta(8,9) dehydroestrone sulfate is an active estrogen with a distinct pharmacological profile that results in significant clinical activity in vasomotor, neuroendocrine and bone preservation parameters. However, it displayed little or no efficacy, at the dose studied, on other peripheral parameters normally affected by classical estrogens. Increasing knowledge of the unique profiles of the Premarin components, as well as their complex interaction, will help to increase our understanding of the clinical profile of Premarin.

The discovery of kilohertz quasi-periodic oscillations (kHz QPOs) in low-mass X-ray binaries (LMXBs) with the Rossi X-Ray Timing Explorer has stimulated extensive studies of these sources. Recently, Osherovich & Titarchuk suggested a new model for kHz QPOs and the related correlations between kHz QPOs and low-frequency features in LMXBs. Here we use their results to study the mass-radius relation for the atoll source 4U 1728-34. We find that, if this model is correct, 4U 1728-34 is possibly a strange star rather than a neutron star.

Recent basic and clinical advances have consolidated the concept of tissue-selective estrogens, i.e. molecules that express different degrees of partial agonist, full agonist or antagonist activity in different tissues or cells. Delta8,9-Dehydroestrone sulfate (delta8,9-DHES) is a conjugated estrogen and a component of conjugated equine estrogens (CEE). It is metabolized in the human in at least a 1:1 ratio to its 17beta form, 17beta-delta8,9-DHES. To evaluate its activity in different clinical and biochemical parameters, a clinical research study was conducted with delta8,9-DHES and estrone sulfate as a comparator in postmenopausal women. Delta8,9-DHES was given orally at a daily dose of 0.125 mg for 12 weeks in a group of 10 women. Two additional groups of women received either estrone sulfate alone (1.25 mg/day) or the combination of delta8,9-DHES and estrone sulfate at the previously specified doses. A significant and consistent suppression of hot flushes (number, severity, and total score) was observed with delta8,9-DHES, reaching more than 95% suppression in all parameters of vasomotor symptoms. This level of activity was equal to that obtained with the much higher dose of estrone sulfate, and it was sustained for the duration of the treatment period (12 weeks). Measurements of a bone resorption marker, i.e. urinary excretion of N-telopeptide, demonstrated that delta8,9-DHES at 8 weeks produced a degree of suppression (40%) similar to that observed with the higher dose of estrone sulfate. Gonadotropin secretion (FSH and LH) was significantly suppressed in women receiving delta8,9-DHES, similar to that observed with estrone sulfate alone or with the combination of the two. Other parameters, such as total cholesterol, low density lipoprotein cholesterol and high density lipoprotein cholesterol were not modified significantly, whereas serum globulins (sex hormone-binding globulin and corticosteroid-binding globulin) showed only marginal increases after delta8,9-DHES administration. Taken together with preclinical data, it is found that delta8,9-DHES is an active estrogen with a distinct pharmacological profile that results in significant clinical activity in vasomotor, neuroendocrine (gonadotropin and PRL) and bone preservation parameters, whereas displaying little or no efficacy, at the dose tested, on other peripheral parameters normally affected by estrogens. Collectively, this information supports the concept that delta8,9-DHES is an integral component of CEE, with distinct tissue selectivity contributing to the CEE's overall clinical activity, and places this estrogen as a distinct member of a novel class of centrally active molecules with unique peripheral tissue selectivity.

A large proportion of the beneficial effects that oestrogens demonstrate on the vasculature are believed to be mediated via direct effects on the vascular wall. In this study we compared a number of oestrogenic compounds isolated from pregnant mare's urine including 17beta-oestradiol and oestrone, in terms of their abilities to inhibit stimulated endothelin-1 release from normal human coronary artery endothelial cells (CAEC). We also examined their ability to stimulate expression of constitutive endothelial nitric oxide synthase (eNOS) and explored their effects on cellular angiotensin converting enzyme (ACE). All the oestrogens tested were able to inhibit serum-stimulated ET-1 release. Oestrone and 17alpha-dihydroequilenin failed to significantly affect cellular eNOS levels. 17Beta-oestradiol and oestrone significantly increased cellular ACE levels while 17beta,delta(8,9)-dehydroestradiol decreased cellular ACE. We discuss these observations in terms of their potential clinical relevance and use as a means of screening novel oestrogen-like compounds.

In the present study, we tested the hypothesis that the neuropeptides, vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY), which are present in the thyroid nerves, act as physiological neurotransmitters involved in the regulation of thyroid hormone secretion and thyroid blood flow. Specifically, we examined whether these neuropeptides can be released into thyroid blood vessels by electrical stimulation of the major thyroid nerves or whether their expression is altered by changes in iodine intake. Sprague-Dawley rats were used in this study. The cervical sympathetic trunk or the superior laryngeal nerve was stimulated by bipolar electrodes in anesthetized rats. During nerve stimulation, blood samples were withdrawn from the thyroid vein. Thyroid blood flow was monitored by laser Doppler blood flowmetry. Sympathetic stimulation caused a marked decrease in thyroid blood flow, which was associated with a significant increase in release of norepinephrine. However, these effects were not accompanied by any change in NPY release into the thyroid vein. Stimulation of the superior laryngeal nerve was not associated with changes in thyroid blood flow or VIP release into the thyroid vein. In a separate experiment, rats were fed a diet containing low-, high-, or normal iodine concentrations. Triiodothyronine (T3) and thyroxine (T4) levels in thyroid venous plasma were significantly reduced in rats fed a low-iodine diet but not in a separate group of rats fed a high iodine diet. However, these treatments had no effect on VIP or NPY concentrations in thyroid venous plasma or in thyroid ganglia. Thus, our results indicate that VIP and NPY, which are present in the thyroid nerves, may not be directly involved in the regulation of thyroid function.

Purified bovine milk proteins that were added to cultures of murine spleen cells significantly increased cell proliferation and production of immunoglobulin M. Casein and a whey mixture consisting of alpha-lactalbumin, bovine serum albumin, bovine gamma globulin, and beta-lactoglobulin (beta-LG) were stimulatory. Of the three beta-LG preparations that are commercially available (beta-LG containing variants A and B, purified variant A, and purified variant B), the unseparated mixture containing both the A and B variants showed the most immunomodulatory activity. Both alkaline treatment and trypsin digestion of the beta-LG preparation markedly reduced its effectiveness. Polymyxin B, while greatly diminishing the stimulatory effect of lipopolysaccharide, had no significant effect on either the enhancement of cell proliferation or the enhancement of immunoglobulin production by beta-LG. In the presence of S-(n-butyl)-homocysteine sulfoxamine, the stimulatory effect of beta-LG on cell proliferation and IgM production in vitro was markedly reduced.

Studies on the occurrence of protein polymorphism are of importance not only in determining interspecific relationships but also in revealing genetically controlled variants among the populations of the same species. This study deals with the analysis of the isozyme pattern of three dehydrogenases in seven species of Indian frogs. The results correlate with the respective habitats and it is suggested that protein polymorphism is adaptively important and is maintained by natural selection.

The testes in different groups of whitethroated munia (Lonchura malabarica) were studied following exogenous treatment of testosterone propionate (1 mg/100 g body wt./day) at 06.00 h or 14.00 h or 22.00 h for 15 consecutive days during the quiescent phase of the annual testicular cycle. The testicular conditions in each group of testosterone treated and appropriate control (administered only with the vehicle of hormone at corresponding time-points) group of birds were analyzed by the studies of paired testicular weight, seminiferous tubular diameter, spermatogenetic index, and cytology of the testicular germ cells. The results of the study revealed that exogenous testosterone was stimulatory to the testicular functions irrespective of the time of treatment. The gametokinetic response of the testes to exogenous testosterone also did not show any statistical difference among the birds treated at different hours of the day. The present study demonstrates for the first time that the responses of the testes to exogenous testosterone during the post-breeding phase in an annual testicular cycle of a wild bird does not vary with the time of administration.

A new decelerating technique that places dc potentials on the orthogonal excitation and receiver plates as well as the rear trapping plate (conductance limit) of the source cell of a dual cubic cell has been applied to the standard matrix-assisted laser desorption/ionization Fourier transform mass spectrometry technique. When this five-plate trapping method is applied, high-mass ions with large translational kinetic energies can be trapped efficiently and detected. Using this approach, low-resolution spectra of carbonic anhydrase (MW = 29,000), egg albumin (MW = 45,000), and bovine albumin (MW = 66,000) have been obtained. Because the new decelerating method requires no modification to the existing cell, it is also possible to obtain high-resolution spectra for compounds with masses of ca. 14,000 Da and lower. Utilizing the five-plate trapping method, a bovine insulin spectrum with a resolving power of 20,000 was obtained. It is not yet possible to obtain higher resolution for the higher mass proteins. The reasons for this difficulty are currently being investigated.

Activation of mitogen-activated protein kinases (MAPKs) was examined in the A7r5 rat vascular smooth muscle cell line. Treatment of A7r5 cells with vasopressin, phorbol ester (PMA), or serum resulted in activation of two MAPKs, Erk-1 and Erk-2. Phosphatidylinositol-specific phospholipase C was activated in response to vasopressin but not to PMA. Vasopressin and PMA both caused maximal activation of PLD within 5 minutes. Application of bacterial phospholipase D (PLD) to A7r5 cells increased phosphatidic acid to levels similar to those seen with vasopressin or PMA. Acute exposure of the cells to vasopressin, PMA, or PLD increased phosphorylation of many of the same cytosolic and membrane proteins. However, bacterial PLD did not promote significant activation of Erk-1 and Erk-2. Phosphatidic acid and lysophosphatidic acid (LPA) likewise did not stimulate MAPK activity in A7r5 cells. Serum and vasopressin stimulated DNA synthesis when present for more than 30 min, while PLD, PMA, phosphatidic acid, and LPA were not mitogenic. These data suggest that activations of MAPKs and PLD are concurrent but independent responses to vasopressin in A7r5 cells. Acute activation of these enzymes is not sufficient to simulate DNA synthesis.

The pinealocytes in male roseringed parakeets (Psittacula krameri) were studied following bilateral castration and/or therapeutic administration of testosterone during the preparatory (June-July), progressive (Nov.-Dec.), pre-breeding (Jan.-Feb.) and breeding (March-April) phases of the annual testicular cycle. The responses of the pineal to either treatment were found to be almost identical throughout the investigation. In each reproductive phase, the pineal appeared to be hypertrophied following castration and the effect was reversed by therapeutic administration of testosterone, while hormonal treatment to the intact parakeets induced regressive changes in the pinealocytes. Collectively, the results of the current study support the hypothesis that the testis through its hormone testosterone exerts inhibitory influences on the activity of pineal, and may thus be considered as being involved in the determination of an inverse relationship between the pineal and the testis during the annual cycle of free-living parakeets.

Vasoactive intestinal peptide (VIP) is present in thyroid parasympathetic nerves. To assess the involvement of endogenous VIP in the regulation of thyroid function, blood levels of thyroid hormones and thyroid blood flows (TBF) were measured after systemic immunization against VIP or after transection of the superior laryngeal nerves in male rats, which reduced the thyroid content of VIP but did not affect blood levels of thyroid hormones or TBF. Anti-VIP monoclonal antibody or anti-VIP serum was used for immunization against VIP in normal rats. In addition, VIP antibody was given to rats fed an iodine-deficient diet for 5 days to examine the involvement of this peptide in iodine deficiency-induced increases in TBF. Effects were measured at different times (90 s, 30 min, 1 h, and 5 days) after immunoneutralization, but none of these treatments changed blood levels of thyroid hormones or TBF in normal or iodine-deficient rats. However, passive immunization against VIP was associated with a high binding capacity of rat plasma to VIP, and this treatment reduced blood levels of prolactin as well as blood flows to the duodenum, stomach, and lung. These findings suggest that the VIP present in thyroid nerves is not involved in maintaining basal thyroid hormone secretion or TBF and that this neuropeptide does not mediate thyroid vascular adjustments to dietary iodine deficiency.

Sympathetic nerve fibers to thyroid blood vessels contain both norepinephrine (NE) and neuropeptide Y (NPY). To assess the involvement of endogenous NPY in the sympathetic neural control of thyroid blood flow, appropriate doses of a selective NPY antagonist, alpha-trinositol, and an NPY antiserum (NPY-AS) were used during cervical sympathetic trunk stimulation in anesthetized rats. During all experiments, thyroid blood flow was continuously monitored by laser Doppler blood flowmetry. Neither alpha-trinositol nor NPY-AS blocked the thyroidal vasoconstriction evoked by either the first or second stimulation of the cervical sympathetic trunks. Our results suggest that NPY is not involved either directly or indirectly during acute sympathetic vasoconstriction in the rat thyroid gland.

The dissolution and bioavailability of etodolac from capsules exposed to high relative humidity and temperature were compared to those from capsules stored at room temperature (RT). Dissolution of stressed and control capsules was evaluated using a USP basket apparatus at 100 rpm with 900 mL pH 7.5 phosphate buffer (0.05 M) at 37 degrees C. The dissolution of etodolac from capsules exposed to stressed conditions was also evaluated with enzymes (pancreatin, 1%, w/v) added to the dissolution medium. The bioavailability of etodolac from capsules exposed to stressed conditions was compared in both dogs and humans to capsules stored at RT conditions. Capsules, 200 and 300 mg, exposed to stressed conditions failed the dissolution (without enzymes) specification [not less than 85% released (80% Q) in 30 min]. However, upon enzyme addition, all capsules met the specification. The rate and extent of absorption from these 200 and 300 mg etodolac capsules in dogs were equivalent to those from capsules stored at RT conditions that passed the dissolution specification. Similarly, the bioavailability of etodolac from 300 mg capsules that failed the dissolution specification upon exposure to stressed conditions was equivalent to that of control capsules in 24 adult male volunteers. Thus, an in vitro dissolution test with enzymes provides a better indication of stressed capsule performance in vivo.

It has been suggested that thyroid blood flow is regulated by both sympathetic and parasympathetic nerves. The purpose of our experiments was to study the role of neuropeptide Y (NPY) in the sympathetic neural control of thyroid blood flow. Sympathetic nerve fibers to the thyroid contain both norepinephrine (NE) and NPY. Therefore, NE (15 nmol iv bolus) and NPY (12 or 1.7 nmol/kg body wt iv infusion; 4 min) were administered to anesthetized male rats (250-300 g) either alone or together, with or without an alpha-adrenergic receptor blocker (phentolamine; 10 mg/kg body wt iv bolus). Experiments were also performed in which the cervical sympathetic trunks were stimulated (30 Hz, 10 V; 0.5 ms; 2 min) with or without phentolamine. Thyroid blood flow was monitored continuously by laser-Doppler blood flowmetry. Results are expressed as thyroid vascular conductance (TVC). NE or NPY at both doses decreased TVC relative to that in control saline-infused rats (P < 0.05). No potentiation of the NE effect by NPY was observed when the first dose of NE was injected 2 min after a high or low dose of NPY. However, the effect of a second dose of NE, injected 15 min after the end of the low dose of NPY, was prolonged compared with the effect of a second dose of NE in saline-infused rats. Phentolamine blocked the effect of NE but not that of NPY. Stimulation of the cervical sympathetic trunks decreased TVC (P < 0.01 vs. sham), and this effect was completely blocked by phentolamine.(ABSTRACT TRUNCATED AT 250 WORDS)