Development of the mammalian tooth has been intensively studied as a model system for epithelial/mesenchymal interactions during organogenesis, and progress has been made in identifying key molecules involved in this signaling. We show that activin betaA is expressed in presumptive tooth-germ mesenchyme and is thus a candidate for a signaling molecule in tooth development. Analysis of tooth development in activin betaA mutant embryos shows that incisor and mandibular molar teeth fail to develop beyond the bud stage. Activin betaA is thus an essential component of tooth development. Development of maxillary molars, however, is unaffected in the mutants. Using tissue recombination experiments we show that activin is required in the mesenchyme prior to bud formation and that although activin signaling from mesenchyme to epithelium takes place, mutant epithelium retains its ability to support tooth development. Implantation of beads soaked in activin A, into developing mandibles, is able to completely rescue tooth development from E11.5, but not E12.5 or E13.5, confirming that activin is an early, essential mesenchyme signal required before tooth bud formation. Normal development of maxillary molars in the absence of activin shows a position specific role for this pathway in development of dentition. Functional redundancy with activin B or other TGFbeta family members that bind to activin receptors cannot explain development of maxillary molars in the mutants since the activin-signaling pathway appears not to be active in these tooth germs. The early requirement for activin signaling in the mesenchyme in incisor and mandibular molar tooth germs must be carried-out in maxillary molar mesenchyme by other independent signaling pathways.

We present a computational method that identifies and extracts mutation data from the scientific literature. We focused on the extraction of single point mutations for the GPCR and NR superfamilies. After validation by plausibility filters, the mutation data is integrated into the corresponding MCSIS where it is combined with structural and sequence information already stored in these databases. We extracted and validated 2736 true point mutations from 914 articles on GPCRs and 785 true point mutations from 1094 articles on NRs. The current version of our automated extraction algorithm identifies 49.3% of the GPCR point mutations with a specificity of 87.9%, and 64.5% of the NR point mutations with a specificity of 85.8%. MuteXt routinely analyzes 100 electronic articles in approximately 1 h.

Falcipain-2 (FP2) is a papain family cysteine protease and important hemoglobinase of erythrocytic Plasmodium falciparum parasites. Inhibitors of FP2 block hemoglobin hydrolysis and parasite development, suggesting that this enzyme is a promising target for antimalarial chemotherapy. FP2 and related plasmodial cysteine proteases have an unusual 14-aa motif near the C terminus of the catalytic domain. Recent solution of the structure of FP2 showed this motif to form a ␤-hairpin that is distant from the enzyme active site and protrudes out from the protein. To evaluate the function of this motif, we compared the activity of the wild-type enzyme with that of a mutant lacking 10 aa of the motif ( ⌬10 FP2). ⌬10 FP2 had nearly identical activity to that of the wild-type enzyme against peptide substrates and the protein substrates casein and gelatin. However, ⌬10 FP2 demonstrated negligible activity against hemoglobin or globin. FP2 that was inhibited with trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (FP2 E-64 ) formed a complex with hemoglobin, but ⌬10 FP2 E-64 did not, indicating that the motif mediates binding to hemoglobin independent of the active site. A peptide encoding the motif blocked hemoglobin hydrolysis, but not the hydrolysis of casein. Kinetics for the inhibition of ⌬10 FP2 were very similar to those for FP2 with peptidyl and protein inhibitors, but ⌬10 FP2 was poorly inhibited by the inhibitory prodomain of FP2. Our results indicate that FP2 utilizes an unusual motif for two specific functions, interaction with hemoglobin, its natural substrate, and interaction with the prodomain, its natural inhibitor.M alaria is one of the most important infectious diseases in the world. Plasmodium falciparum, the most virulent human malaria parasite, is believed to cause hundreds of millions of illnesses and over a million deaths each year (1). The control of malaria has been hindered by increasing resistance of malaria parasites to available drugs (2). New antimalarial drugs, ideally directed against new targets, are urgently needed (3). Among potential new targets for antimalarial therapy are proteases that hydrolyze hemoglobin. Intraerythrocytic malaria parasites break down hemoglobin in an acidic food vacuole to supply amino acids for parasite protein synthesis and to maintain osmotic stability (4, 5). Multiple proteases appear to participate in hemoglobin processing (6). Cysteine protease inhibitors block hemoglobin hydrolysis, indicating that cysteine proteases play a key role in this process (7). Falcipain-2 (FP2) and falcipain-3 (FP3) are papain-family cysteine proteases of erythrocytic stages of P. falciparum that localize to the food vacuole and readily hydrolyze hemoglobin (8, 9). Disruption of the FP2 gene led to the accumulation of undegraded hemoglobin in trophozoites, confirming a critical role for this enzyme in hemoglobin hydrolysis (10). Inhibition of FP2 and related proteases led to a block in parasite development (11, 12) and cured mice with murine malaria (13, 14), and efforts to develop...

The liver is an essential organ that produces several serum proteins, stores vital nutrients, and detoxifies many carcinogenic and xenobiotic compounds. Various growth factors positively regulate liver growth, but only a few negative regulators are known. Among the latter are the transforming growth factor ␤ (TGF-␤) superfamily members TGF-␤1 and activin A. To study the function of novel activin family members, we have cloned and generated mice deficient in the activin ␤C and ␤E genes. Expression analyses demonstrated that these novel genes are liver specific in adult mice. Here, we show by RNase protection that activin ␤C transcripts are present in the liver beginning at embryonic day 11.5 (E11.5) whereas activin ␤E expression is detected starting from E17.5. Gene targeting in embryonic stem cells was used to generate mice with null mutations in either the individual activin ␤C and ␤E genes or both genes. In contrast to the structurally related activin ␤A and ␤B subunits, which are necessary for embryonic development and pituitary follicle-stimulating hormone homeostasis, mice deficient in activin ␤C and ␤E were viable, survived to adulthood, and demonstrated no reproductive abnormalities. Although activin ␤C and ␤E mRNAs are abundantly expressed in the liver of wild-type mice, the single and double mutants did not show any defects in liver development and function. Furthermore, in the homozygous mutant mice, liver regeneration after >70% partial hepatectomy was comparable to that in wild-type mice. Our results suggest that activin ␤C and ␤E are not essential for either embryonic development or liver function.Growth factors and hormones play an extremely important role in regulating biological processes from patterning of the early embryo to regulating the function of tissues and organs. The largest family of growth factors is the transforming growth factor ␤ (TGF-␤) superfamily of secreted dimeric proteins (12). Members of this family include activins, TGF-␤s, bone morphogenetic proteins (BMPs), and growth differentiation factors and demonstrate diverse functions including roles in left-right asymmetry, skeletal development, reproduction, and oncogenesis (29, 52). Both activins (␤-␤ dimers) and inhibins (␣-␤ dimers) have historically been shown to be regulators of follicle-stimulating hormone (FSH) secretion from the pituitary gland (54). These earlier results were later confirmed by in vivo analysis of activin ␤B-, activin receptor type IIA (ActRIIA)-, and ␣-inhibin-deficient mice (32-34). In studies involving Xenopus laevis oocytes (36), activins were tested for their ability to induce mesoderm formation. However, this observation is not true for mice (35), suggesting that the results obtained with X. laevis oocyte injection experiments may be due to nonphysiological effects of the activin ligands.The adult liver detoxifies the blood through the actions of various enzymes, synthesizes normal serum proteins such as the acute-phase proteins and albumin, and produces bile, which is critical for normal fat absor...

On our initial discovery that prion protein (PrP)-derived peptides were capable of capturing the pathogenic prion protein (PrP Sc ), we have been interested in how these peptides interact with PrP Sc . After screening peptides from the entire human PrP sequence, we found two peptides (PrP 19 -30 and PrP100-111) capable of binding full-length PrP Sc in plasma, a medium containing a complex mixture of other proteins including a vast excess of the normal prion protein (PrP C ). The limit of detection for captured PrP Sc was calculated to be 8 amol from a Ϸ10 5 -fold dilution of 10% (wt/vol) human variant Creutzfeldt-Jakob disease brain homogenate, with >3,800-fold binding specificity to PrP Sc over PrP C . Through extensive analyses, we show that positively charged amino acids play an important, but not exclusive, role in the interaction between the peptides and PrP Sc . Neither hydrophobic nor polar interactions appear to correlate with binding activity. The peptide-PrP Sc interaction was not sequence-specific, but amino acid composition affected binding. Binding occurs through a conformational domain that is only present in PrP Sc , is species-independent, and is not affected by proteinase K digestion. These and other findings suggest a mechanism by which cationic domains of PrP C may play a role in the recruitment of PrP C to PrP Sc .plasma ͉ Creutzfeldt-Jakob disease ͉ detection ͉ cationic interaction ͉ diagnostic

No abstract

Oocyte-somatic cell communication is necessary for normal ovarian function. However, the identities of the majority of oocyte-secreted proteins remain unknown. A novel cDNA encoding mouse oocyte-secreted protein 1 (OOSP1) was identified using a modified subtractive hybridization screen. The Oosp1 cDNA encodes a 202-amino acid protein that contains a 21-amino acid signal peptide sequence, 5 putative N-linked glycosylation consensus sequences, and 6 cysteines that are predicted to form 3 disulfide bonds. OOSP1 shares amino acid identity with placental-specific protein 1 (PLAC1), a secreted protein expressed in the placenta and the ectoplacental cone. The Oosp1 mRNA is approximately 1.0 kb and is present at high levels in the oocytes of adult ovaries and at lower levels in the spleen. The mouse Oosp1 gene is 5 exons, spans greater than 16.4 kb, and localizes to chromosome 19 at a position that shares synteny with human chromosome 11q12-11q13. The identification of OOSP1 as a new oocyte-secreted protein permits future in vitro and in vivo functional analyses to define its role in ovarian folliculogenesis.

No abstract