SCB1, a BURP-domain protein gene,from developing soybean seed coats

We describe a gene, SCB1 (Seed Coat BURP-domain protein 1), that is expressed specifically within the soybean (Glycine max [L.] Merrill) seed coat early in its development. Northern blot analysis and mRNA in situ hybridization revealed novel patterns of gene expression during seed development. SCB1 mRNA accumulated first within the developing thick-walled parenchyma cells of the inner integument and later in the thick- and thin-walled parenchyma cells of the outer integument. This occurred prior to the period of seed coat maturation and seed filling and before either of the layers started to degrade. SCB1 may therefore play a role in the differentiation of the seed coat parenchyma cells. In addition, the protein product appears to be located within cell walls. The SCB1 gene codes for a new member of a class of modular proteins that possess a carboxy-terminal BURP domain and a variety of different repeated sequences. The sequence of the genomic clone revealed the insertion of a Tgm transposable element in the upstream promoter region but it is not certain whether it contributes to the tissue-specific pattern of SCB1 expression.     

Identification of SNARE and cell trafficking regulatory proteins in the salivary glands of the lone star tick,Amblyomma americanum (L.)

Prostaglandin E(2) (PGE(2)) stimulates secretion of tick salivary gland proteins via a phosphoinositide signaling pathway and mobilization of intracellular Ca(2+) (). Highly conserved intracellular SNARE (soluble NSF attachment protein receptors) complex proteins are associated with the mechanism of protein secretion in vertebrate and invertebrate neuronal and non-neuronal cells. Proteins in the salivary glands of partially fed female lone star ticks cross-react individually with antibodies to synaptobrevin-2 (vesicle (v)-SNARE), syntaxin-1A, syntaxin-2 and SNAP-25 (target (t)-SNAREs), cytosolic alpha/beta SNAP and NSF (N-ethylmaleimide-sensitive fusion protein), Ca(2+) sensitive synaptotagmin, vesicle associated synaptophysin, and regulatory cell trafficking GTPases Rab3A and nSec1. V-SNARE and t-SNARE proteins form an SDS-resistant, boiling sensitive core complex in the salivary glands. Antibodies to SNARE complex proteins inhibit PGE(2)-stimulated secretion of anticoagulant protein in permeabilized tick salivary glands. We conclude that SNARE and cell trafficking regulatory proteins are present and functioning in the process of PGE(2)-stimulated Ca(2+) regulated protein secretion in tick salivary glands.     

Novel strategies for targeting the dimerization interface of HIV protease with cross-linked interfacial peptides

As the prevalence of AIDS continues to grow, and current therapeutic agents begin to lose efficacy, the need for alternative treatments to combat HIV has become significantly greater. Targeting the highly conserved dimerization interface of HIV protease (PR) with interfacial peptides has been shown to reduce the activity of the enzyme due to generation of inactive monomers. The potency of these peptide-based inhibitors has been dramatically increased by cross-linking the interfacial sequences derived from HIV PR. This review focuses on a variety of strategies to develop potent, low-molecular-weight dimerization inhibitors of HIV PR.     

A chemical,genetic, and structural analysis of the nuclear bile acid receptor FXR

The farnesoid X receptor (FXR) functions as a bile acid (BA) sensor coordinating cholesterol metabolism, lipid homeostasis, and absorption of dietary fats and vitamins. However, BAs are poor reagents for characterizing FXR functions due to multiple receptor independent properties. Accordingly, using combinatorial chemistry we evolved a small molecule agonist termed fexaramine with 100-fold increased affinity relative to natural compounds. Gene-profiling experiments conducted in hepatocytes with FXR-specific fexaramine versus the primary BA chenodeoxycholic acid (CDCA) produced remarkably distinct genomic targets. Highly diffracting cocrystals (1.78 A) of fexaramine bound to the ligand binding domain of FXR revealed the agonist sequestered in a 726 A(3) hydrophobic cavity and suggest a mechanistic basis for the initial step in the BA signaling pathway. The discovery of fexaramine will allow us to unravel the FXR genetic network from the BA network and selectively manipulate components of the cholesterol pathway that may be useful in treating cholesterol-related human diseases.     

cDNA cloning and expression of the mouse Na/H antiporter (NHE-1) and a potential splice variant

A cDNA for the Mus musculus Na/H exchanger-isoform 1 (NHE-1) was identified in a BALB/c myoblast library by its hybridization to rat NHE-1 sequences. Analysis of the clone showed it to display extensive homology with NHE-1 clones from other mammalian species; however, the region of interspecific homology was abruptly interrupted in the midst of the open reading frame by 166 bp of unrelated sequence. This extra sequence is likely to be an unspliced intron 9. Aside from the retained intron 9, the NHE-1 cDNA clone is otherwise fully processed, with all of the other ten introns removed and containing a poly(A) tract. From PCR results this variant represents a significant but minor population of NHE-1 RNAs. The variant message does associate with polysomes thereby suggesting it to be translated into protein. The location of the retained intron in the carboxy terminus of the protein is such that its translation would produce a protein predicted to be still capable of effecting Na and H translocation but whose regulatory features would be markedly altered.Amino acid sequence comparison of the mouse NHE-1 (derived from the fully processed message) with that of other mammalian species demonstrated two exceptionally divergent regions; the C-terminal cytoplasmic tail (residues 750-790), containing a region of 6-8 contiguous acidic amino acids variably composed of aspartate and glutamate residues, and the N-terminal extracellular domain that includes an N-linked glycosylation site (residues 60-80).     

Regulation of cytoplasmic calcium levels by two nitric oxide receptors

We examined the effects of dissolved nitric oxide (NO) gas oncytoplasmic calcium levels ([Ca²⁺]_i) in C6glioma cells under anoxic conditions. The maximum elevation (27 ± 3 nM) of [Ca²⁺]_i was reached at 10 µM NO. Asecond application of NO was ineffective if the first was >0.5 µM.The NO donor diethylamine/NO mimicked the effects of NO. Acute exposureof the cells to low calcium levels was without effect on the NO-evokedresponse. Thapsigargin (TG) increased [Ca²⁺]_iand was less effective if cells were pretreated with NO. Hemoglobin inhibited the effects of NO at a molar ratio of 10:1. 8-Bromo-cGMP waswithout effect on the NO-evoked response. If cells were pretreated withTG or exposed chronically to nominal amounts of calcium, NO decreased[Ca²⁺]_i. The results suggest that C6 gliomacells have two receptors for NO. One receptor (NO_A)elevates [Ca²⁺]_i and resides on theendoplasmic reticulum (ER). The other receptor (NO_B)decreases [Ca²⁺]_i and resides on theplasmalemma or the ER. The latter receptor dominates when the level ofcalcium within intracellular stores is diminished.

     

A comparative structural analysis of the ADF/cofilin family

Actin-depolymerizing factor (ADF) and cofilin define a family of actin-binding proteins essential for the rapid turnover of filamentous actin in vivo. Here we present the 2.0 A crystal structure of Arabidopsis thaliana ADF1 (AtADF1), the first plant crystal structure from the ADF/cofilin (AC) family. Superposition of the four AC isoform structures permits an accurate sequence alignment that differs from previously reported data for the location of vertebrate-specific inserts and reveals a contiguous, vertebrate-specific surface opposite the putative actin-binding surface. Extending the structure-based sequence alignment to include 30 additional isoforms indicates three major groups: vertebrates, plants, and "other eukaryotes." Within these groups, several structurally conserved residues that are not conserved throughout the entire AC family have been identified. Residues that are highly conserved among all isoforms tend to cluster around the tryptophan at position 90 and a structurally conserved kink in alpha-helix 3. Analysis of surface character shows the presence of a hydrophobic patch and a highly conserved acidic cluster, both of which include several residues previously implicated in actin binding.     

Technical, genetic, and ethical issues in screening and testing of African-Americans for hemochromatosis

To define more precisely populations in which hemochromatosis is frequent to rare, problems of racial classification are introduced, with particular reference to Europeans and African-Americans. Because the category "Caucasian" includes a multitude of dissimilar peoples, the categories Europeans and European-Americans have been substituted for Caucasian, which is archaic. The background of discrimination in sickle hemoglobin programs for African-Americans are then analyzed, including, discrimination by employers, life insurance, and selective mandatory testing. Discrimination and selective testing of African-American employees of the Lawrence Livermore Laboratory continues today without prior consent, as it has since the 1970s. Dissimilarities between the genetics of hemochromatosis in Europeans and their descendants, Africans, and African-Americans are briefly analyzed. Finally, it is concluded that because hemochromatosis is unlike sickle hemoglobin in that it is potentially preventable and treatable, prevention and treatment principles should apply as in other diseases. Furthermore, because hemochromatosis is so common in European-Americans, discrimination, if practiced, would not be selective for African-Americans.     

The microbial composition of three limnologically disparate hypersaline Antarctic lakes

16S rRNA clone library analysis was used to examine the biodiversity and community structure within the sediments of three hypersaline Antarctic lakes. Compared to sediment of low to moderate salinity Antarctic lakes the species richness of the hypersaline lake sediments was 2-20 times lower. The community of Deep Lake (32% salinity, average sediment temperature -15 degrees C) was made up almost entirely of halophilic Archaea. The sediment communities of two meromictic hypersaline lakes, Organic Lake (20% salinity, -7 degrees C) and Ekho Lake (15% salinity, 15 degrees C) were more complex, containing phylotypes clustering within the Proteobacteria and Cytophagales divisions and with algal chloroplasts. Many phylotypes of these lakes were related to taxa more adapted to marine-like salinity and perhaps derive from bacteria exported into the sediment from the lower salinity surface waters. The Ekho Lake clone library contained several major phylotypes related to the Haloanaerobiales, the growth of which appears to be promoted by the comparatively high in situ temperature of this lake.