Biochemical characteristics and inhibitor selectivity of mouse indoleamine 2,3-dioxygenase-2.

The first step in the kynurenine pathway of tryptophan catabolism is the cleavage of the 2,3-double bond of the indole ring of tryptophan. In mammals, this reaction is performed independently by indoleamine 2,3-dioxygenase-1 (IDO1), tryptophan 2,3-dioxygenase (TDO) and the recently discovered indoleamine 2,3-dioxygenase-2 (IDO2). Here we describe characteristics of a purified recombinant mouse IDO2 enzyme, including its pH stability, thermal stability and structural features. An improved assay system for future studies of recombinant/isolated IDO2 has been developed using cytochrome b (5) as an electron donor. This, the first description of the interaction between IDO2 and cytochrome b (5), provides further evidence of the presence of a physiological electron carrier necessary for activity of enzymes in the "IDO family". Using this assay, the kinetic activity and substrate range of IDO2 were shown to be different to those of IDO1. 1-Methyl-D-tryptophan, a current lead IDO inhibitor used in clinical trials, was a poor inhibitor of both IDO1 and IDO2 activity. This suggests that its immunosuppressive effect may be independent of pharmacological inhibition of IDO enzymes, in the mouse at least. The different biochemical characteristics of the mouse IDO proteins suggest that they have evolved to have distinct biological roles.

Modeling of substrate specificity of the Alzheimer's disease amyloid precursor protein beta-secretase.

The enzyme BACE (beta-site APP-cleaving enzyme) has recently been identified as the beta-secretase that cleaves the amyloid precursor protein (APP) to produce the N terminus of the Abeta peptide found in plaques in the brains of Alzheimer's disease patients. BACE is an aspartic protease similar to pepsin and renin. Comparative modeling of the three-dimensional structure of BACE in complex with its substrate shows that several residues confer specificity of the enzyme for APP. In particular, Arg296 forms a salt-bridge with the P1' Asp of the APP substrate, explaining the unusual preference of BACE among aspartic proteases for a P1' residue that is negatively charged. Several hydrophobic residues in the enzyme form a pocket for the P1 hydrophobic residue (Met in wild-type APP and Leu in APP with the "Swedish mutation" associated with early-onset of Alzheimer's disease). Inhibitors that can bind to the BACE active site may prove useful for drugs to treat and prevent Alzheimer's disease.

Ancient Ethiopian genome reveals extensive Eurasian admixture throughout the African continent.

Characterizing genetic diversity in Africa is a crucial step for most analyses reconstructing the evolutionary history of anatomically modern humans. However, historic migrations from Eurasia into Africa have affected many contemporary populations, confounding inferences. Here, we present a 12.5× coverage ancient genome of an Ethiopian male ("Mota") who lived approximately 4500 years ago. We use this genome to demonstrate that the Eurasian backflow into Africa came from a population closely related to Early Neolithic farmers, who had colonized Europe 4000 years earlier. The extent of this backflow was much greater than previously reported, reaching all the way to Central, West, and Southern Africa, affecting even populations such as Yoruba and Mbuti, previously thought to be relatively unadmixed, who harbor 6 to 7% Eurasian ancestry.

Ice premelting during differential scanning calorimetry.

Premelting at the surface of ice crystals is caused by factors such as temperature, radius of curvature, and solute composition. When polycrystalline ice samples are warmed from well below the equilibrium melting point, surface melting may begin at temperatures as low as -15 degrees C. However, it has been reported (Bronshteyn and Steponkus, 1993. Biophys. J. 65:1853-1865) that when polycrystalline ice was warmed in a differential scanning calorimetry (DSC) pan, melting began at about -50 degrees C, this extreme behavior being attributed to short-range forces. We show that there is no driving force for such premelting, and that for pure water samples in DSC pans curvature effects will cause premelting typically at just a few degrees below the equilibrium melting point. We also show that the rate of warming affects the slope of the DSC baseline and that this might be incorrectly interpreted as an endotherm. The work has consequences for DSC operators who use water as a standard in systems where subfreezing runs are important.

Ammonia effects on microinvertebrates and fish in outdoor experimental streams.

Laboratory data on ammonia effects, the US EPA national water quality criteria for ammonia, and ammonia site-specific criteria were evaluated in four outdoor experimental streams (one control and three treatment streams) over a 76-week period. Calculated un-ionised ammonia concentrations varied daily and seasonally according to pH and temperature changes. Populations of four major microinvertebrate taxonomic groups (cladocerans, copepods, rotifers and protozoans) were monitored during a 4-week period early in the study, and six fish species (fathead minnows, bluegills, channel catfish, white suckers, walleyes, and rainbow trout) were tested for various time intervals, from 4 to 26 weeks, throughout the 76-week study period. Copepods and rotifers were unaffected in all three treatment streams, based on comparisons with the control stream. Cladoceran and protozoan populations were reduced in at least two treatment streams, but because of large variability, effects were considered to be inconclusive. However, complete mortality of cladocerans did occur in the high and medium treatments when placed in in situ biomonitor chambers. All six fish species were affected in one or more treatments. Generally, the fish effect values agreed with most laboratory effect values. Of 12 fish groups tested, one channel catfish group and one white sucker group were affected below the recommended protection levels of the national and site-specific criteria. The lowest effect concentrations tested for the other ten groups occurred above the criteria levels.

Bonnet monkey cervical mucus glycoproteins. Study of the minor glycoprotein components of periovulatory phase mucus.

Two glycoproteins present in small quantities, in addition to a main glycoprotein, were isolated from the periovulatory phase mucus. The glycoproteins, obtained in minute amounts, showed distinct variations in the relative proportions of sugar residues. The results of analytical as well as chemical studies suggest the existence of two distinct molecular species differing in structure. The main component of the minor glycoproteins, Fraction A, Characterized by Smith degradation and by subsequent methylation and inhibition of hemagglutination, bears similarities to the periovulatory phase main glycoprotein. The other component, Fraction B, characterized by Smith degradation followed by inhibition of hemagglutination, shows structural resemblance to the premenstrual phase glycoprotein.

Large-scale comparison of protein sequence alignment algorithms with structure alignments.

Sequence alignment programs such as BLAST and PSI-BLAST are used routinely in pairwise, profile-based, or intermediate-sequence-search (ISS) methods to detect remote homologies for the purposes of fold assignment and comparative modeling. Yet, the sequence alignment quality of these methods at low sequence identity is not known. We have used the CE structure alignment program (Shindyalov and Bourne, Prot Eng 1998;11:739) to derive sequence alignments for all superfamily and family-level related proteins in the SCOP domain database. CE aligns structures and their sequences based on distances within each protein, rather than on interprotein distances. We compared BLAST, PSI-BLAST, CLUSTALW, and ISS alignments with the CE structural alignments. We found that global alignments with CLUSTALW were very poor at low sequence identity (<25%), as judged by the CE alignments. We used PSI-BLAST to search the nonredundant sequence database (nr) with every sequence in SCOP using up to four iterations. The resulting matrix was used to search a database of SCOP sequences. PSI-BLAST is only slightly better than BLAST in alignment accuracy on a per-residue basis, but PSI-BLAST matrix alignments are much longer than BLAST's, and so align correctly a larger fraction of the total number of aligned residues in the structure alignments. Any two SCOP sequences in the same superfamily that shared a hit or hits in the nr PSI-BLAST searches were identified as linked by the shared intermediate sequence. We examined the quality of the longest SCOP-query/ SCOP-hit alignment via an intermediate sequence, and found that ISS produced longer alignments than PSI-BLAST searches alone, of nearly comparable per-residue quality. At 10-15% sequence identity, BLAST correctly aligns 28%, PSI-BLAST 40%, and ISS 46% of residues according to the structure alignments. We also compared CE structure alignments with FSSP structure alignments generated by the DALI program. In contrast to the sequence methods, CE and structure alignments from the FSSP database identically align 75% of residue pairs at the 10-15% level of sequence identity, indicating that there is substantial room for improvement in these sequence alignment methods. BLAST produced alignments for 8% of the 10,665 nonimmunoglobulin SCOP superfamily sequence pairs (nearly all <25% sequence identity), PSI-BLAST matched 17% and the double-PSI-BLAST ISS method aligned 38% with E-values <10.0. The results indicate that intermediate sequences may be useful not only in fold assignment but also in achieving more complete sequence alignments for comparative modeling.

An inhibitory monoclonal antibody binds at the turn of the helix-turn-helix motif in the N-terminal domain of HIV-1 integrase.

With the increase in our understanding of its structure and enzymatic mechanism, HIV-1 integrase (IN) has become a promising target for designing drugs to treat patients with AIDS. To investigate the structure and function of IN, a panel of monoclonal antibodies (mAbs) directed against HIV-1 IN was raised and characterized previously in this laboratory. Among them, mAbs17, -4, and -33 were found to inhibit IN activity in vitro. In this study, we investigated the interaction of N-terminal-specific mAb17 and its isolated Fab fragment with full-length HIV-1 IN(1-288) and its isolated N-terminal, Zn(2+)-binding domain IN(1-49). Our results show that binding of Zn(2+) to IN(1-49) stabilizes the mAb17-IN complex and that dimer dissociation is not required for binding of the Fab. To identify the epitope recognized by mAb17, we developed a protein footprinting technique based on controlled proteolysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Binding was mapped to a region within amino acids Asp(25)-Glu(35). This peptide corresponds to the end of a helix-turn-helix motif in the IN(1-55) NMR structure and contributes to the dimerization of the N-terminal domain. Antibody binding also appears to destabilize the N-terminal helix in this domain. A molecular model of the [IN(1-49)](2).(Fab)(1) complex shows Fab binding across the dimer protein and suggests a potential target for drug design. These data also suggest that mAb17 inhibits integrase activity by blocking critical protein-protein interactions and/or by distorting the orientation of the N-terminal alpha-helix. The relevance of our results to an understanding of IN function is discussed.