BvgS and BvgA, a two-component system, regulate virulence gene expression in Bordetella pertussis. BvgS is a transmembrane sensor protein that can autophosphorylate and phosphorylate BvgA. Phosphorylated BvgA activates transcription of virulence genes. The cytoplasmic region of BvgS contains three domains separated by alanine/proline-rich sequences--the transmitter, receiver and C-terminus. We report that the C-terminal domain, like the transmitter and receiver, is an essential part of the phosphorelay from BvgS to BvgA. The BvgS C-terminal domain is phosphorylated in trans via a phosphotransfer mechanism by the cytoplasmic portion of BvgS, and trans-phosphorylation of the C-terminal domain requires both the transmitter and receiver. We also demonstrate that phosphorylated, purified C-terminal domain alone is sufficient for phosphotransfer to BvgA. A point mutation in the C-terminal domain (His1172->>Gln) abolishes BvgS activity in vivo and eliminates detectable phosphorylation of BvgA in vitro. Activity of BvgS His 1172->>Gln could be restored by providing the wild-type C-terminal domain in trans. Our results indicate an obligatory role for an alternate phosphodonor module in the BvgAS phosphorelay.

Acidosis is associated with inflammation and ischemia and activates cation channels in sensory neurons. Inflammation also induces expression of FMRFamidelike neuropeptides, which modulate pain. We found that neuropeptide FF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe amide) and FMRFamide (Phe-Met-Arg-Phe amide) generated no current on their own but potentiated H+-gated currents from cultured sensory neurons and heterologously expressed ASIC and DRASIC channels. The neuropeptides slowed inactivation and induced sustained currents during acidification. The effects were specific; different channels showed distinct responses to the various peptides. These results suggest that acid-sensing ion channels may integrate multiple extracellular signals to modify sensory perception.

AMPA receptor GluRA subunits with mutations at position 750, a residue shown previously to control allosteric regulation by cyclothiazide, were analyzed for modulation of deactivation and desensitization by cyclothiazide, aniracetam, and thiocyanate. Point mutations from Ser to Asn, Ala, Asp, Gly, Gln, Met, Cys, Thr, Leu, Val, and Tyr were constructed in GluRAflip. The last four of these mutants were not functional; S750D was active only in the presence of cyclothiazide, and the remaining mutants exhibited altered rates of deactivation and desensitization for control responses to glutamate, and showed differential modulation by cyclothiazide and aniracetam. Results from kinetic analysis are consistent with aniracetam and cyclothiazide acting via distinct mechanisms. Our experiments demonstrate for the first time the functional importance of residue 750 in regulating intrinsic channel-gating kinetics and emphasize the biological significance of alternative splicing in the M3-M4 extracellular loop.

The delta pH-driven and Sec-related thylakoidal protein translocases recognise distinct types of thylakoid transfer signal, yet all transfer signals resemble bacterial signal peptides in structural terms. Comparison of known transfer signals reveals a single concrete difference: signals for the delta pH-dependent system contain a common twin-arginine motif immediately before the hydrophobic region. We show that this motif is critical for the delta pH-driven translocation process; substitution of the arg-arg by gln-gln or even arg-lys totally blocks translocation across the thylakoid membrane, and replacement by lys-arg reduces the rate of translocation by>> 100-fold. The targeting information in this type of signal thus differs fundamentally from that of bacterial signal peptides, where the required positive charge can be supplied by any basic amino acid. Insertion of a twin-arg motif into a Sec-dependent substrate does not alter the pathway followed but reduces translocation efficiency, suggesting that the motif may also repel the Sec-type system. Other information must help to specify the choice of translocation mechanism, but this information is unlikely to reside in the hydrophobic region because substitution by a hydrophobic section from an integral membrane protein does not affect the translocation pathway.

In order to assess the relative importance of polar versus steric interactions, infrared spectra and overall CO binding properties were measured at room temperature for 41 different recombinant myoglobins containing mutations at His64(E7), Val68(E11), Phe43(CD1), Arg45(CD3), Phe46(CD4), and Leu29(B10). The results were compared to the crystal structures of wild-type, Phe29, Val46, Ala68, Phe68, GlnGln, Met, and Trp substitutions at position 64, but in each case there is a net increase in the intensity of this high-frequency component. Replacement of Val68 with Ala, Leu, Ile, and Phe produces little effect on the IR spectrum, whereas these mutations cause 20-fold changes in KCO, presumably due to steric effects. Replacement of Val68 with Thr decreases KCO 4-5-fold, whereas the position of the major IR band increases from 1945 to 1961 cm-1. Replacement of Val68 with Asn also causes a large decrease in KCO, but in this case, the peak position of the major IR band decreases from 1945 to 1916 cm-1. Nine replacements were made in the CD corner at positions 43, 45, and 46. All of the resultant mutants show increased stretching frequencies that can be correlated with movement of the imidazole side chain of His64 away from the bound ligand. All five substitutions at position 29 cause changes in the IR spectra. The Leu29->>Phe mutation had the largest effect, producing a single band centered at 1932 cm-1. Together these data demonstrate that there is little direct correlation between affinity, vCO, and Fe-C-O geometry. The major factor governing vCO appears to be the electrostatic potential surrounding the bound ligand and not steric hindrance. The presence of positive charges from proton donors, such as N epsilon of His64 and N delta of Asn68, cause a decrease in the bond order and stretching frequency of bound CO. In contrast, the negative portion of the Thr68 dipole points directly toward the bound ligand and increases the C-O bond order and stretching frequency. Movement of His64 away from the bound ligand or replacement of this residue with aliphatic amino acids prevents hydrogen-bonding interactions, causing vCO to increase.(ABSTRACT TRUNCATED AT 250 WORDS)

The nucleotide sequence of the mRNA encoding the fusion (F0) protein of a virulent strain of Newcastle disease virus was determined. A single open reading frame in the sequence encodes a protein of 553 amino acids with a calculated molecular weight of 59058. The amino acid sequence predicted several structural features involving the fusion-inducing hydrophobic stretch (residues 117-142) and the cleavage-activation site (residues 112-116) to generate the disulfide-linked F1 and F2 subunits. The cleavage-activation site as well as a part of the fusion-inducing sequence were compared among a series of virulent and avirulent strains by the chain-termination method using a synthetic oligonucleotide primer. It was found that without exception, the cleavage-activation site of virulent strains consisted of two dibasic residues with an intervening glutamine, Arg-Arg-Gln-Arg-Arg, whereas the corresponding region of avirulent strains was made of a sequence with single basic residues scattered among uncharged residues, Gly-LysArg-Gln-GlySer-Arg. On the basis of these observations and the previous results showing a strict correlation between the pathogenicity and the cleavability of the fusion protein of NDV (Y. Nagai, H-D. Klenk, and R. Rott, Virology, 72, 494-508, 1976), we propose the importance of the dibasic residues for efficient proteolytic activation of the fusion protein and for the pantropic property of NDV. Some strains were found to have Leu-Ile-Gly as the N-terminus of F1, whereas others contained Phe-Ile-Gly, indicating that Phe-X-Gly is not always conserved at F1 N-terminus of paramyxovirus.

The TRPC family of receptor-activated cation channels (TRPC channels) can be subdivided into four subfamilies based on sequence homology as well as functional similarities. Members of the TRPC3/6/7 subfamily share common biophysical characteristics and are activated by diacylglycerol in a membrane-delimited manner. At present, it is only poorly understood whether members of the TRPC3/6/7 subfamily are functionally redundant or whether they serve distinct cellular roles. By electrophysiological and fluorescence imaging strategies we show that TRPC3 displays considerable constitutive activity, while TRPC6 is a tightly regulated channel. To identify potential molecular correlates accounting for the functional difference, we analyzed the glycosylation pattern of TRPC6 compared with TRPC3. Two NX(S/T) motifs in TRPC6 were mutated (Asn to Gln) by in vitro mutagenesis to delete one or both extracellular N-linked glycosylation sites. Immunoblotting analysis of HEK 293 cell lysates expressing TRPC6 wild type and mutants favors a model of TRPC6 that is dually glycosylated within the first (e1) and second extracellular loop (e2) as opposed to the monoglycosylated TRPC3 channel (Vannier, B., Zhu, X., Brown, D., and Birnbaumer, L. (1998) J. Biol. Chem. 273, 8675-8679). Elimination of the e2 glycosylation site, missing in the monoglycosylated TRPC3, was sufficient to convert the tightly receptor-regulated TRPC6 into a constitutively active channel, displaying functional characteristics of TRPC3. Reciprocally, engineering of an additional second glycosylated site in TRPC3 to mimic the glycosylation status in TRPC6 markedly reduced TRPC3 basal activity. We conclude that the glycosylation pattern plays a pivotal role for the tight regulation of TRPC6 through phospholipase C-activating receptors.

The activity of Hsp70 proteins is regulated by accessory proteins, which include members of the DnaJ-like protein family. Characterized by the presence of a highly conserved 70-amino acid J domain, DnaJ homologues activate the ATPase activity of Hsp70 proteins and stabilize their interaction with unfolded substrates. DnaJ homologues have been identified in most organelles where they are involved in nearly all aspects of protein synthesis and folding. Within the endoplasmic reticulum (ER), DnaJ homologues have also been shown to assist in the translocation, secretion, retro-translocation, and ER-associated degradation (ERAD) of secretory pathway proteins. By using bioinformatic methods, we identified a novel mammalian DnaJ homologue, ERdj4. It is the first ER-localized type II DnaJ homologue to be reported. The signal sequence of ERdj4 remains uncleaved and serves as a membrane anchor, orienting its J domain into the ER lumen. ERdj4 co-localized with GRP94 in the ER and associated with BiP in vivo when they were co-expressed in COS-1 cells. In vitro experiments demonstrated that the J domain of ERdj4 stimulated the ATPase activity of BiP in a concentration-dependent manner. However, mutation of the hallmark tripeptide HPD (His ->> Gln) in the J domain totally abolished this activation. ERdj4 mRNA expression was detected in all human tissues examined but showed the highest level of the expression in the liver, kidney, and placenta. We found that ERdj4 was highly induced at both the mRNA and protein level in response to ER stress, indicating that this protein might be involved in either protein folding or ER-associated degradation.

DNA polymerase eta (Poleta) is unique among eukaryotic polymerases in its proficient ability for error-free replication through ultraviolet-induced cyclobutane pyrimidine dimers, and inactivation of Poleta (also known as POLH) in humans causes the variant form of xeroderma pigmentosum (XPV). We present the crystal structures of Saccharomyces cerevisiae Poleta (also known as RAD30) in ternary complex with a cis-syn thymine-thymine (T-T) dimer and with undamaged DNA. The structures reveal that the ability of Poleta to replicate efficiently through the ultraviolet-induced lesion derives from a simple and yet elegant mechanism, wherein the two Ts of the T-T dimer are accommodated in an active site cleft that is much more open than in other polymerases. We also show by structural, biochemical and genetic analysis that the two Ts are maintained in a stable configuration in the active site via interactions with Gln 55, Arg 73 and Met 74. Together, these features define the basis for Poleta's action on ultraviolet-damaged DNA that is crucial in suppressing the mutagenic and carcinogenic consequences of sun exposure, thereby reducing the incidence of skin cancers in humans.

In the course of our studies on transfer RNA involvement in chlorophyll biosynthesis, we have determined the structure of chloroplast glutamate tRNA species. Barley chloroplasts contain in addition to a tRNA(Glu) species at least two other glutamate-accepting tRNAs. We now show that the sequences of these tRNAs differ significantly: they are differentially modified forms of tRNA(Gln) (as judged by their UUG anticodon). These mischarged Glu-tRNA(Gln) species can be converted in crude chloroplast extracts to Gln-tRNA(Gln). This reaction requires a specific amidotransferase and glutamine or asparagine as amide donors. Aminoacylation studies show that chloroplasts, plant and animal mitochondria, as well as cyanobacteria, lack any detectable glutaminyl-tRNA synthetase activity. Therefore, the requirement for glutamine in protein synthesis in these cells and organelles is provided by the conversion of glutamate attached to an 'incorrectly' charged tRNA. A similar situation has been described for several species of Gram-positive bacteria. Thus, it appears that the occurrence of this pathway of Gln-tRNA(Gln) formation is widespread among organisms and is a function conserved during evolution. These findings raise questions about the origin of organelles and about the evolution of the mechanisms maintaining accuracy in protein biosynthesis.

The formation of aminoacyl-transfer RNA is a crucial step in ensuring the accuracy of protein synthesis. Despite the central importance of this process in all living organisms, it remains unknown how archaea and some bacteria synthesize Asn-tRNA and Gln-tRNA. These amide aminoacyl-tRNAs can be formed by the direct acylation of tRNA, catalysed by asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase, respectively. A separate, indirect pathway involves the formation of mis-acylated Asp-tRNA(Asn) or Glu-tRNA(Gln), and the subsequent amidation of these amino acids while they are bound to tRNA, which is catalysed by amidotransferases. Here we show that all archaea possess an archaea-specific heterodimeric amidotransferase (encoded by gatD and gatE) for Gln-tRNA formation. However, Asn-tRNA synthesis in archaea is divergent: some archaea use asparaginyl-tRNA synthetase, whereas others use a heterotrimeric amidotransferase (encoded by the gatA, gatB and gatC genes). Because bacteria primarily use transamidation, and the eukaryal cytoplasm uses glutaminyl-tRNA synthetase, it appears that the three domains use different mechanisms for Gln-tRNA synthesis; as such, this is the only known step in protein synthesis where all three domains have diverged. Closer inspection of the two amidotransferases reveals that each of them recruited a metabolic enzyme to aid its function; this provides direct evidence for a relationship between amino-acid metabolism and protein biosynthesis.

Botulinum neurotoxin serotype F contains the zinc binding motif of zinc endopeptidases. Atomic adsorption analysis of highly purified toxin preparation revealed the presence of one atom of zinc per molecule of toxin, which could be removed with EDTA or o-phenanthroline. The light chain of the neurotoxin was shown to have a zinc-dependent protease activity specific for VAMP/synaptobrevin, an integral membrane protein of synaptic vesicles. Both isoforms of rat VAMP were cleaved at the same site corresponding to the single Gln-Lys peptide bond present in their sequences. This proteolytic activity was inhibited by EDTA, o-phenanthroline, and captopril as well as by VAMP peptides spanning the cleavage site.

The cerebral tricarboxylic acid (TCA) cycle rate and the rate of glutamine synthesis were measured in rats in vivo under normal physiological and hyperammonemic conditions using 13C NMR spectroscopy. In the hyperammonemic animals, blood ammonia levels were raised from control values of approximately 0.05 mM to approximately 0.35 mM by an intravenous ammonium acetate infusion. Once a steady-state of cerebral metabolites was established, a [1-13C]glucose infusion was initiated, and 13C NMR spectra acquired continuously on a 7-tesla spectrometer to monitor 13C labeling of cerebral metabolites. The time courses of glutamate and glutamine C-4 labeling were fitted to a mathematical model to yield TCA cycle rate (V(TCA)) and the flux from glutamate to glutamine through the glutamine synthetase pathway (V(gln)). Under hyperammonemia the value of V(TCA) was 0.57 +/- 0.16 micromol/min per g (mean +/- SD, n = 6) and was not significantly different (unpaired t test; P>> 0.10) from that measured in the control animals (0.46 +/- 0.12 micromol/min per g, n = 5). Therefore, the TCA cycle rate was not significantly altered by hyperammonemia. The measured rate of glutamine synthesis under hyperammonemia was 0.43 +/- 0.14 micromol/min per g (mean +/- SD, n = 6), which was significantly higher (unpaired t test; P < 0.01) than that measured in the control group (0.21 +/- 0.04 micromol/ min per g, n = 5). We propose that the majority of the glutamine synthetase flux under normal physiological conditions results from neurotransmitter substrate cycling between neurons and glia. Under hyperammonemia the observed increase in glutamine synthesis is comparable to the expected increase in ammonia transport into the brain and reported measurements of glutamine efflux under such conditions. Thus, under conditions of elevated plasma ammonia an increase in the rate of glutamine synthesis occurs as a means of ammonia detoxification, and this is superimposed on the constant rate of neurotransmitter cycling through glutamine synthetase.

Aminoacyl-tRNAs (aa-tRNAs) are the essential substrates for translation. Most aa-tRNAs are formed by direct aminoacylation of tRNA catalyzed by aminoacyl-tRNA synthetases. However, a smaller number of aa-tRNAs (Asn-tRNA, Gln-tRNA, Cys-tRNA and Sec-tRNA) are made by synthesizing the amino acid on the tRNA by first attaching a non-cognate amino acid to the tRNA, which is then converted to the cognate one catalyzed by tRNA-dependent modifying enzymes. Asn-tRNA or Gln-tRNA formation in most prokaryotes requires amidation of Asp-tRNA or Glu-tRNA by amidotransferases that couple an amidase or an asparaginase to liberate ammonia with a tRNA-dependent kinase. Both archaeal and eukaryotic Sec-tRNA biosynthesis and Cys-tRNA synthesis in methanogens require O-phosophoseryl-tRNA formation. For tRNA-dependent Cys biosynthesis, O-phosphoseryl-tRNA synthetase directly attaches the amino acid to the tRNA which is then converted to Cys by Sep-tRNA: Cys-tRNA synthase. In Sec-tRNA synthesis, O-phosphoseryl-tRNA kinase phosphorylates Ser-tRNA to form the intermediate which is then modified to Sec-tRNA by Sep-tRNA:Sec-tRNA synthase. Complex formation between enzymes in the same pathway may protect the fidelity of protein synthesis. How these tRNA-dependent amino acid biosynthetic routes are integrated into overall metabolism may explain why they are still retained in so many organisms.

The binding sites for four monoclonal antibodies, rho 1D4, rho 3C2, rho 3A6, and rho 1C5, have been localized within the C-terminal region of bovine rhodopsin: Asp18'-Glu-Ala16'-Ser-Thr-Thr-Val12'-Ser-Lys-Thr-Gl u8'-Thr-Ser-Gln-Val4'-Ala-Pr o -Ala1'. Antibody binding sites were localized by using synthetic C-terminal peptides in conjunction with solid-phase competitive inhibition assays and limited proteolytic digestion of rhodopsin in conjunction with electrophoretic immunoblotting techniques. Binding of the rho 1D4 and rho 3C2 antibodies to immobilized rhodopsin was inhibited with peptides of length 1'-8' and longer. Antibody rho 1D4 binding was not inhibited by peptides 2'-13' or 3'-18', indicating that the C-terminal alanine residue of rhodopsin was required. Similar competitive inhibition studies indicated that the antibody rho 3A6 required peptides of length 1'-12' and longer whereas rho 1C5 required peptide 1'-18'. Peptide 3'-18' was as effective as 1'-18' in inhibiting rho 3A6 binding to rhodopsin, but replacement of glutamic acid in position 8' with glutamine abolished competition. This substitution had little effect on the binding of antibody rho 1C5. Thus, Glu8' was essential for rho 3A6 binding but not for the binding of the rho 1C5 antibody. Cleavage of the seven amino acid C-terminus from rhodopsin and further cleavage to F1 (Mr 25 000) and F2 (Mr 12 000) fragments with Staphylococcus aureus V8 protease abolished binding of rho 1D4 antibody to the membrane-bound rhodopsin fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequence analysis was performed of an ovine hypothalamic 41-residue polypeptide that had been postulated to be a putative corticotropin-releasing factor (CRF) because of its high intrinsic corticotropin releasing activity. The NH2-terminal 39 residues of CRF were determined by Edman degradation of 0.6-3.5 nmol of peptide in a Wittmann-Liebold modified Beckman 890C spinning cup sequencer with reverse-phase high-pressure liquid chromatography for the identification of amino acid phenylthiohydantoins (direct micro-sequence analysis). Evidence for residue 40 (isoleucine) was provided by direct micro-sequence analysis of 2.0 nmol of acetylated CRF selectively cleaved at its arginine residues by trypsin prior to analysis. The thermolytic COOH-terminal fragment isoleucyl-alanineamide was characterized as its dansyl derivative. Based on the analytical data, the following primary structure is proposed for ovine hypothalamic CRF: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Leu-Asp -Ile-Ala-NH2. In agreement with this proposal, the synthetic replicate of CRF is highly potent in stimulating secretion of both corticotropin and beta-endorphin-like immunoactivities.

In vivo measurements of protein synthesis using isotope-labeled amino acids (AAs) are hampered by the heterogeneity of AA pools and, for slow turnover proteins, the difficulty and expense of long-term labeling. Continuous oral heavy water (2H2O) labeling can safely maintain stable body water 2H enrichments for weeks or months. 2H is metabolically incorporated into C-H bonds of nonessential AAs (NEAAs) and hence into proteins. No posttranslational label exchange occurs, so 2H incorporation into protein NEAAs, in principle, reports on protein synthesis. Here, we show by mass isotopomer distribution analysis (MIDA) of 2H2O-labeled rodent tissue proteins that metabolic 2H flux into C-H bonds of Ala, Gly, or Gln used for protein synthesis is nearly complete. By 2H2O labeling of rodents, turnover of bone and muscle mixed proteins was quantified and stimulation of liver collagen synthesis by CCl4 was detected. Kinetics of several human serum proteins were also measured, reproducing published t1/2 estimates. Plateau enrichments in Ala varied among different proteins. Moderate amounts of protein, isolated chromatographically or electrophoretically, sufficed for kinetic analyses. In conclusion, 2H2O labeling permits sensitive, quantitative, operationally simple measurements of protein turnover in vivo by the rise-to-plateau approach, especially for proteins with slow constitutive turnover.

A novel trypsin-like protease associated with rat bronchiolar epithelial Clara cells, named Tryptase Clara, was purified to homogeneity from rat lung by a series of standard chromatographic procedures. The enzyme has apparent molecular masses of 180 +/- 16 kDa on gel filtration and 30 +/- 1.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Its isoelectric point is pH 4.75. Studies with model peptide substrates showed that the enzyme preferentially recognizes a single arginine cleavage site, cleaving Boc-Gln-Ala-Arg-4-methylcoumaryl-7-amide most efficiently and having a pH optimum of 7.5 with this substrate. The enzyme is strongly inhibited by aprotinin, diisopropylfluorophosphate, antipain, leupeptin, and Kunitz-type soybean trypsin inhibitor, but inhibited only slightly by Bowman-Birk soybean trypsin inhibitor, benzamidine, and alpha 1-antitrypsin. Immunohistochemical studies indicated that the enzyme is located exclusively in the bronchiolar epithelial Clara cells and colocalized with surfactant. An immunoreactive protein with a molecular mass of 28.5 kDa was also detected in airway secretions by Western blotting analyses, suggesting that the 30-kDa protease in Clara cells is processed before or after its secretion. Proteolytic cleavage of the hemagglutinin of influenza virus is a prerequisite for the virus to become infectious. Tryptase Clara was shown to cleave the hemagglutinin and activate infectivity of influenza A virus in a dose-dependent way. These results suggest that the enzyme is a possible activator of inactive viral fusion glycoprotein in the respiratory tract and thus responsible for pneumopathogenicity of the virus.

Recombinant immunotoxins are hybrid proteins composed of an Fv that binds to a tumor antigen fused to a bacterial or plant toxin. Immunotoxin BL22 targets CD22 positive malignancies and is composed of an anti-CD22 Fv fused to a 38-kDa fragment of Pseudomonas exotoxin A (PE38). BL22 has produced many complete remissions in drug-resistant Hairy cell leukemia, where many treatment cycles can be given, because neutralizing antibodies do not form. In marked contrast, only minor responses have been observed in trials with immunotoxins targeting solid tumors, because only a single treatment cycle can be given before antibodies develop. To allow more treatment cycles and increase efficacy, we have produced a less immunogenic immunotoxin by identifying and eliminating most of the B cell epitopes on PE38. This was accomplished by mutation of specific large hydrophilic amino acids (Arg, Gln, Glu, Lys) to Ala, Ser, or Gly. The new immunotoxin (HA22-8X) is significantly less immunogenic in three strains of mice, yet retains full cytotoxic and anti-tumor activities. Elimination of B-cell epitopes is a promising approach to the production of less immunogenic proteins for therapeutic purposes.

Phenotypic heterogeneity in the repetitive portion of a human malaria circumsporozoite (CS) protein, a major target of candidate vaccines, has been found. Over 14% of clinical cases of uncomplicated Plasmodium vivax malaria at two sites in western Thailand produced sporozoites immunologically distinct from previously characterized examples of the species. Monoclonal antibodies to the CS protein of other P. vivax isolates and to other species of human and simian malarias did not bind to these nonreactive sporozoites, nor did antibodies from monkeys immunized with a candidate vaccine made from the repeat portion of a New World CS protein. The section of the CS protein gene between the conserved regions I and II of a nonreactive isolate contained a nonapeptide repeat, Ala-Asn-Gly-Ala-Gly-Asn-Gln-Pro-Gly, identical at only three amino acid positions with published nonapeptide sequences. This heterogeneity implies that a P. vivax vaccine based on the CS protein repeat of one isolate will not be universally protective.

The human beta-adrenoceptor is a member of the seven-transmembrane family of receptors, encoded by a gene on chromosome 5. beta-Adrenoceptors have been classified into beta1, beta2, and beta3 subgroups, with beta2-receptors being widely distributed in the respiratory tract, particularly in airway smooth muscle. Intracellular signaling following beta2-adrenoceptor activation is largely affected through a trimeric Gs protein coupled to adenylate cyclase. Cyclic AMP (cAMP) induces airway relaxation through phosphorylation of muscle regulatory proteins and attenuation of cellular Ca2+ concentrations. Alternative cAMP-independent pathways involving activation of membrane maxi-K+ channels and coupling through Gi to the MAP kinase system have also been described. Site-directed mutagenesis has identified Asp 113 and Ser 204/207 within the third and fourth membrane domains as the active site of the beta2-receptor, critical for beta2-agonist binding and activity. beta2-Agonists have been characterized as those that directly activate the receptor (albuterol), those that are taken up into a membrane depot (formoterol), and those that interact with a receptor-specific auxiliary binding site (salmeterol). These differences in mechanism of action are reflected in the kinetics of airway smooth muscle relaxation and bronchodilation in patients with asthma. beta-Adrenoceptor desensitization associated with beta2-agonist activation is a consequence of phosphorylation by beta-ARK and uncoupling of the receptor from Gs following beta-arrestin binding, of internalization and recycling of the receptor through processes of sequestration and resensitization and downregulation, modulated by an effect on receptor gene expression. The degree of receptor desensitization appears to differ, depending on the cell or tissue type, and is reflected in the different profiles of clinical tolerance to chronic beta2-agonist therapy. A number of polymorphisms of the beta2-receptor have been described that appear to alter the behavior of the receptor following agonist exposure. These include Arg-Gly 16, Glu-Gln 27, and Thr-lle 164. The Gly 16 receptor downregulates to a greater extent and is associated with increased airway hyperreactivity, nocturnal symptoms, and more severe asthma. The Glu 27 form appears to protect against downregulation and is associated with less reactive airways. An individual can be homozygous or heterozygous for given polymorphisms, and large populations will have to be studied to determine their importance to the asthma phenotype.

A conserved asparagine (Asn 16) buried in the interface of the GCN4 leucine zipper selectively favours the parallel, dimeric, coiled-coil structure. To test if other polar residues confer oligomerization specificity, the structural effects of Gln and Lys substitutions for Asn 16 were characterized. Like the wild-type peptide, the Asn 16Lys mutant formed exclusively dimers. In contrast, Gln 16, despite its chemical similarity to Asn, allowed the peptide to form both dimers and trimers. The Gln 16 side chain was accommodated by qualitatively different interactions in the dimer and trimer crystal structures. These findings demonstrate that the structural selectivity of polar residues results not only from the burial of polar atoms, but also depends on the complementarity of the side-chain stereochemistry with the surrounding structural environment.

The DNA damage response depends on the concerted activity of protein serine/threonine kinases and modular phosphoserine/threonine-binding domains to relay the damage signal and recruit repair proteins. The PIKK family of protein kinases, which includes ATM/ATR/DNA-PK, preferentially phosphorylate Ser-Gln sites, while their basophilic downstream effecter kinases, Chk1/Chk2/MK2 preferentially phosphorylate hydrophobic-X-Arg-X-X-Ser/Thr-hydrophobic sites. A subset of tandem BRCT domains act as phosphopeptide binding modules that bind to ATM/ATR/DNA-PK substrates after DNA damage. Conversely, 14-3-3 proteins interact with substrates of Chk1/Chk2/MK2. FHA domains have been shown to interact with substrates of ATM/ATR/DNA-PK and CK2. In this review we consider how substrate phosphorylation together with BRCT domains, FHA domains and 14-3-3 proteins function to regulate ionizing radiation-induced nuclear foci and help to establish the G(2)/M checkpoint. We discuss the role of MDC1 a molecular scaffold that recruits early proteins to foci, such as NBS1 and RNF8, through distinct phosphodependent interactions. In addition, we consider the role of 14-3-3 proteins and the Chk2 FHA domain in initiating and maintaining cell cycle arrest.

Human influenza viruses are more efficiently isolated by inoculating patient samples into the amniotic rather than the allantoic cavity of embryonated chicken eggs. This type of cultivation selects virus variants with mutations around the hemagglutinin (HA) receptor binding site. To understand the molecular basis of these phenomena, we investigated the abundances of sialic acid (SA) linked to galactose (Gal) by the alpha-2,3 linkage (SA alpha2,3Gal) and SA alpha2,6Gal in egg amniotic and allantoic cells and in Madin-Darby canine kidney (MDCK) cells. Using SA-Gal linkage-specific lectins (Maackia amurensis agglutinin specific for SA alpha2,6Gal and Sambucus nigra agglutinin specific for SA alpha2,3Gal), we found SA alpha2,3Gal in both allantoic and amniotic cells and SA alpha2,6Gal in only the amniotic cells. MDCK cells contained both linkages. To investigate how this difference in abundances of SA alpha2,3Gal and SA alpha2,6Gal in allantoic and amniotic cells affects the appearance of host cell variants in eggs, we determined the receptor specificities and HA amino acid sequences of two different patient viruses which were isolated and passaged in the amnion or in the allantois and which were compared with MDCK cell-grown viruses. We found that the viruses maintained high SA alpha2,6Gal specificities when grown in MDCK cells or following up to two amniotic passages; however, further passages in either the amnion or allantois resulted in the acquisition of, or a complete shift to, SA alpha2,3Gal specificity, depending on the virus strain. This change in receptor specificity was accompanied by the appearance of variants in the population with Leu-to-Gln mutations at position 226 in their HA. These findings suggest that lack of SA alpha2,6Gal linkages in the allantois of chicken eggs is a selective pressure for the appearance of host cell variants with altered receptor specificities and amino acid changes at position 226.