Minimization and stabilization of the Mycobacterium tuberculosis recA intein

Many naturally occurring inteins consist of two functionally independent domains, a protein-splicing domain and an endonuclease domain. In a previous study, a 168 amino acid residue mini-intein was generated by removal of the central endonuclease domain of the 440 residue Mycobacterium tuberculosis (Mtu) recA intein. In addition, directed evolution experiments identified a mutation, V67L, that improved the activity of the mini-intein significantly. A recent crystal structure shows that the loop connecting two beta-strands from the N-terminal and C-terminal intein subdomains of the mini-intein is disordered. The goals of the present study were to generate smaller mini-intein derivatives and to understand the basis for reversal of the splicing defect by the V67L mutation. Guided by the structural information, we generated a number of derivatives 135 to 152 residues in length, with V67 or L67. All of the new minimal inteins are functional in splicing. In vivo selection experiments for function showed that by removal of the loop region, 137 residues may be the lower limit for full protein-splicing activity. In addition, the activation effect of the V67L mutation was observed to be universal for mini-inteins longer than 137 residues. Structural and functional analyses indicate that the role of the mutation is in stabilization of the mini-intein core.     

Global food insecurity. treatment of major food crops with elevated carbon dioxide or ozone under large-scale fully open-air conditions suggests recent models may have overestimated future yields

Predictions of yield for the globe's major grain and legume arable crops suggest that, with a moderate temperature increase, production may increase in the temperate zone, but decline in the tropics. In total, global food supply may show little change. This security comes from inclusion of the direct effect of rising carbon dioxide (CO2) concentration, [CO2], which significantly stimulates yield by decreasing photorespiration in C3 crops and transpiration in all crops. Evidence for a large response to [CO2] is largely based on studies made within chambers at small scales, which would be considered unacceptable for standard agronomic trials of new cultivars or agrochemicals. Yet, predictions of the globe's future food security are based on such inadequate information. Free-Air Concentration Enrichment (FACE) technology now allows investigation of the effects of rising [CO2] and ozone on field crops under fully open-air conditions at an agronomic scale. Experiments with rice, wheat, maize and soybean show smaller increases in yield than anticipated from studies in chambers. Experiments with increased ozone show large yield losses (20%), which are not accounted for in projections of global food security. These findings suggest that current projections of global food security are overoptimistic. The fertilization effect of CO2 is less than that used in many models, while rising ozone will cause large yield losses in the Northern Hemisphere. Unfortunately, FACE studies have been limited in geographical extent and interactive effects of CO2, ozone and temperature have yet to be studied. Without more extensive study of the effects of these changes at an agronomic scale in the open air, our ever-more sophisticated models will continue to have feet of clay.     

Blood pressure maintenance in NHE3-deficient mice with transgenic expression of NHE3 in small intestine

NHE3 Na(+)/H(+) exchanger knockout (Nhe3(-/-)) mice have severe absorptive deficits in the kidney proximal tubule and intestinal tract. The resulting hypovolemia has confounded efforts to carefully evaluate the specific effects of NHE3 deficiency on kidney function. Development of mice with transgenic expression of NHE3 in the small intestine (tgNhe3(-/-)) has allowed us to analyze the role of renal NHE3 in overall maintenance of blood pressure, pressure natriuresis, and autoregulation of both glomerular filtration rate (GFR) and renal blood flow (RBF). Ambulatory blood pressure, measured by telemetry, was lower in tgNhe3(-/-) mice than in wild-type controls (tgNhe3(+/+)) when the mice were maintained on a normal NaCl diet but was normalized when they were provided with a high NaCl intake. Furthermore, administration of the AT1-receptor blocker losartan showed that circulating ANG II plays a major role in maintaining blood pressure in tgNhe3(-/-) mice fed normal NaCl but not in those receiving high NaCl. Clearance studies revealed a blunted pressure-natriuresis response in tgNhe3(-/-) mice at lower blood pressures but a robust response at higher blood pressures. Autoregulation of GFR and RBF was normal in tgNhe3(-/-) mice. These results show that dietary NaCl loading normalizes blood pressure in awake tgNhe3(-/-) mice and that alterations in NHE3 activity are not essential for normal autoregulation of GFR and RBF. Furthermore, the data strongly support the hypothesis that NHE3 plays an important role in the diuretic and natriuretic responses to increases in blood pressure but also show that mechanisms not involving NHE3 mediate pressure natriuresis in the higher range of blood pressures studied.     

Characteristics of five bacteriophages of yellow-pigmented enterobacteria

Four bacteriophages (C2, C2F, E3, and E16P) belonging to morphological group C3 and one belonging to morphological group A3 (E16B) were purified by deuterium oxide gradient centrifugation and cesium chloride gradient centrifugation. Morphological group C3 phages had a densityd=1.534–1.541 and group A3 phage (E16B) had a densityd=1.492 in CsCl. Phages of morphological group C3 isolated onEnterobacter sakazakii (C2, C2F) and onErwinia herbicola (E3, E16P) were compared withSalmonella newport phage 7-11 with respect to host-range, genome size, antigenic relatedness, and ultraviolet and heat susceptibility. Phages C2 and C2F could multiply inEnterobacter cloacae, E. sakazakii, Erwinia herbicola, E. rhapontici, andLevinea malonatica; whereas phages E3, E16P, and 7-11 could multiply on these same species and onEscherichia coli and severalSalmonella serotypes. Molecular weights of phage DNAs were determined to be 58×10⁶ (C2), 60×10⁶ (7-11), 67×10⁶ (E3), and 39×10⁶ (E16B).All studied phages of morphological group C3 (includingSalmonella newport phage 7-11) were neutralized by anti-phage C2 serum. Despite differences in neutralization kinetics and in ultraviolet and heat sensitivities, these phages of morphological group C3 constitute one phage species. Phage E16B (morphological group A3) had a host-range limited toEnterobacter cloacae, Erwinia herbicola, andE. rhapontici; it was antigenically unrelated to the preceding phage group C3, and showed ultraviolet and heat susceptibility close to that of coliphage T4.     

The complete mitochondrial genome of the yeast Pichia sorbitophila

Here, we report the complete nucleotide sequence of the 39 107-bp mitochondrial genome of the yeast Pichia sorbitophila . This genome is closely related to those of Candida parapsilosis and Debaryomyces hansenii , as judged from sequence similarities and synteny conservation. It encodes three subunits of cytochrome oxidase ( COX1, COX2 and COX3 ), three subunits of ATP synthase ( ATP6, ATP8 and ATP9 ), the seven subunits of NADH dehydrogenase ( NAD1-6 and NAD4L ), the apocytochrome b ( COB ), the large and small rRNAs and a complete set of tRNAs. Although the mitochondrial genome of P. sorbitophila contains the same core of mitochondrial genes observed in the ascomycetous yeasts, those coding for the RNAse P and the ribosomal protein VAR1p are missing. Moreover, the mtDNA of P. sorbitophila contains several introns in its genes and has the particularity of possessing an intron, which is not linked to any upstream exon.     

The Memory Cytotoxic T-Lymphocyte (CTL) Response to Human Cytomegalovirus Infection Contains Individual Peptide-Specific CTL Clones That Have Undergone Extensive Expansion In Vivo

Human cytomegalovirus (HCMV)-specific CD8⁺ cytotoxic T lymphocytes (CTL) appear to play an important role in the control of virus replication and in protection against HCMV-related disease. We have previously reported high frequencies of memory CTL precursors (CTLp) specific to the HCMV tegument protein pp65 in the peripheral blood of healthy virus carriers. In some individuals, the CTL response to this protein is focused on only a single epitope, whereas in other virus carriers CTL recognized multiple epitopes which we identified by using synthetic peptides. We have analyzed the clonal composition of the memory CTL response to four of these pp65 epitopes by sequencing the T-cell receptors (TCR) of multiple independently derived epitope-specific CTL clones, which were derived by formal single-cell cloning or from clonal CTL microcultures. In all cases, we have observed a high degree of clonal focusing: the majority of CTL clones specific to a defined pp65 peptide from any one virus carrier use only one or two different TCRs at the level of the nucleotide sequence. Among virus carriers who have the same major histocompatibility complex (MHC) class I allele, we observed that CTL from different donors that recognize the same peptide-MHC complex often used the same Vβ segment, although other TCR gene segments and CDR3 length were not in general conserved. We have also examined the clonal composition of CTL specific to pp65 peptides in asymptomatic human immunodeficiency virus-infected individuals. We have observed a similarly focused peptide-specific CTL response. Thus, the large population of circulating HCMV peptide-specific memory CTLp in virus carriers in fact contains individual CTL clones that have undergone extensive clonal expansion in vivo.

CD8⁺ cytotoxic T lymphocytes (CTL) recognize virus-infected cells via the T-cell receptor (TCR), an αβ heterodimer that has specificity for the peptide antigen presented by major histocompatibility complex (MHC) class I molecules. During T-cell development in the thymus, the TCR β-chain is constructed by rearrangement of variable (V), diversity (D), and joining (J) gene segments, and the α-chain by rearrangement of V and J segments. Additional diversity is generated by imperfect joining of these segments, exonucleotide nibbling at the joins, and addition of non-germ line-encoded N-region nucleotides (25). The regions spanning the V-D-J and V-J joins constitute the hypervariable CDR3 regions which are thought to interact with the middle of the bound peptide and to account for approximately 50% of the TCR’s interaction with peptide (14, 15, 20). The α- and β-chain complementarity determining regions CDR1, which reside within the TCR V segments, are thought to interact with the N and C termini of a peptide that is bound to MHC. By contrast, Vα and Vβ CDR2s are thought to interact predominantly with the MHC itself (14, 15).Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that infects between 60 and 90% of individuals, depending on the population studied. After primary HCMV infection, the virus persists lifelong in a latent state in cells of the myeloid lineage and under the control of the immune system (5). HCMV reactivation can, however, cause serious disease in immunocompromised individuals, such as patients with advanced human immunodeficiency virus (HIV) infection (30) and patients who have undergone bone marrow transplantation (33). Evidence from animal models (32) and from studies of immunosuppressed humans (39) indicates that virus-specific CD8⁺ CTL have a role in protection against CMV disease.We previously studied in detail the HCMV-specific CTL response in healthy virus carriers. All seropositive donors had high frequencies of MHC-restricted HCMV-specific memory CTL precursors in peripheral blood and strongly recognized one of the viral tegument proteins, pp65. In some donors, the CTL response to this protein was highly focused, recognizing only a single epitope within pp65, whereas in others the CTL recognized multiple pp65 peptides (41 and unpublished data).The aim of this study was to examine the clonal composition of the memory CTL response to HCMV pp65 by determining how many different CTL clones are involved in the recognition of a given pp65 peptide. In order to do this, we analyzed the TCR α- and β-chain usage of multiple independently derived peptide-specific CTL clones from healthy virus carriers.Previous studies have examined the heterogeneity of the CTL response to other human virus infections within single subjects (2, 8, 11, 18, 19, 22, 38) or between different donors (2, 6, 8, 11, 23, 38). In the most extreme cases, a very high degree of TCR focusing has been seen: in a study of one HIV-positive individual’s CTL response to an HLA-B14-restricted HIV env peptide, the same TCR was used by 9 of 10 peptide-specific CTL clones, each derived at different time points over the course of 36 months (22). Similarly, multiple independent CTL clones specific to an HLA-B8-restricted Epstein-Barr virus (EBV) peptide derived from one virus carrier at one time point all used the same TCR (2). The CTL response to different human T-lymphotropic virus type 1 (HTLV-1) peptides has been observed to be oligoclonal within individual donors (38). However, in a variety of other human and mouse viral infections within a given individual, the repertoire of CTL specific for a given peptide has been highly heterogeneous (8, 11, 18, 19).The TCRs of CTL obtained from different donors that recognize the same peptide-MHC complex often show some conservation of gene segment usage, although they differ in hypervariable sequence. For example, Vβ segments and certain β-chain CDR3 motifs were conserved between TCR that recognized an HLA-A2-restricted influenza virus peptide in CTL clones derived from different donors (23); the same phenomenon has been seen for an HLA-B27 restricted influenza virus peptide (6) and an HLA-A11-restricted EBV peptide (8). A much higher degree of TCR conservation has also been seen; the same TCR α- and β-chain protein sequences were used by CTL clones from four of five unrelated donors that recognized an HLA-B8 restricted EBV peptide (2). In the case of HTLV-1, CTL from different donors that were specific to the same peptide used largely unrelated TCR (38).For all of the human viruses so far studied, the clonal composition of virus-specific CTL has only been examined for a very few viral peptide-MHC combinations, sometimes in only one donor or at only one time point. In this study, we have therefore examined multiple CTL clones specific to a total of four pp65 peptides, all restricted by three different HLA alleles. We have derived these clones from six healthy virus carriers at one to four time points up to 18 months apart. To identify CTL clonotypes for longitudinal studies and to determine whether HIV infection modifies the clonal composition of HCMV-specific CTL, we have also examined pp65-specific memory CTL in two asymptomatic HIV-infected subjects who are HCMV seropositive. For any given individual, whether HIV seropositive or seronegative, our results indicate that the memory CTL response to individual HCMV pp65 epitopes is highly focused and contains CTL clones that have undergone extensive expansion in vivo.     

Immunological evaluation of urinary trypsin inhibitors in blood and urine: Role of N- & O-linked glycoproteins

Urinary trypsin inhibitors (uTi) suppress serine proteases during inflammation. After liberation from proinhibitors (P-alpha-I and I-alpha-I) by the white blood cell (WBC) response, uTi readily pass through the kidneys into urine. A key uTi, bikunin, is attached to O-linked and N-linked glycoconjugates. Recently, uTi inhibitors, called uristatins, were found to lack the O-linked glycoconjugates. Monoclonal antibodies were produced using purified uristatin and screened for binding differences to uristatin, bikunin, P-α-I, and I-α-I. Antibody-binding patterns were characterized using immunoaffinity binding onto protein-chip surfaces and analysis by Surface Enhanced Laser Desorption/Ionization mass spectrometry (SELDI), using specimens from patients and from purified uTi standards. Antibodies were developed and used in an enzyme-linked immunosorbent assay (ELISA) method for uTi measurement in urine and plasma specimens. ELISA was performed on specimens from normal, presumed healthy, controls and from patients who had been screened for inflammation using a high sensitivity C-reactive protein (CRP) test and a complete blood count (CBC). Polyclonal antibody against uTi showed cross-reactivity with the Tamm–Horsfall protein (THP) and with proinhibitors. Screening of anti-uTi monoclonal antibodies (Mab) revealed antibodies that did not cross-react with either of the above, thus providing a tool to measure both uristatin and bikunin in urine with Mab 3G5 and in plasma with Mab 5D11. The monoclonal antibody 5D11 cross-reacts with specific N-linked glycoconjugates of uristatin present in plasma. In ca 96% of healthy adults, uTi were present at <12 mg/l in urine and <4 mg/l in plasma. We also found that patients with an inflammation and a CRP of >2.0 mg/l had higher urinary concentrations of uTi than the control population in every subject. Free uristatin and bikunin pass readily into urine and are primarily bound to heavy chains that constitute the proinhibitor form in plasma.     

Mercury inputs and outputs at a small lake in northern Minnesota

Storages and cycling of total mercury (Hg_T), methylmercury (MeHg), and Hg⁰ are described for Spring Lake, a small bog lake in the Marcell Experimental Forest in north-central Minnesota. We quantified photoredox transformations, MeHg photolysis, burial to the sediments, and internal and external loadings of Hg_T and MeHg. Atmospheric deposition was the main input of Hg_T; MeHg was supplied by a combination of atmospheric, near-shore wetland, and biotic (methylation) sources. Hg_T outputs were dominated by burial (67%), and Hg⁰ evasion accounted for 26% of Hg_T outputs. The watershed of Spring Lake is small (3.7× lake surface area), and accordingly, bog and upland runoff were minor contributors to both Hg_T and MeHg inputs. Wet deposition was ∼9% of total MeHg input, and other external inputs (runoff, sediment porewater) provided only an additional 7%, indicating that internal production of MeHg was occurring in the lake. Photolysis of MeHg, measured in the field and laboratory, removed ∼3× the lake mass of MeHg (20 mg) annually, and was the dominant sink for MeHg. Residence times of MeHg and Hg_T in the lake were 48 and 61 days, respectively, during the open-water season, compared with only 8 days for the residence time of MeHg on settling particles (seston). Photoreduction of Hg²⁺ to Hg⁰ was greater than the reverse reaction (Hg⁰ photooxidation), and the residence time of Hg⁰ in the photic zone was short (hours). Data from this study show active cycling of all the measured species of mercury (Hg_T, MeHg, and Hg⁰) and the importance of MeHg photolysis and photo-redox processes.     

Systematic cross-selectivity study of the factors influencing template receptor interactions in molecularly imprinted nitrogen heterocycles

A systematic cross-selectivity study involving a series of structurally related N-methylated and non-methylated substituted pyridines was performed with the aim of evaluating the parameters responsible for template receptor binding in molecularly imprinted polymers. Variation in binding of substrate structure permitted evaluation of the steric restraints of the imprinted cavity. The electrostatic effects, primarily hydrogen-bonding, were investigated through rebinding in chloroform and acetonitrile. All species were non-covalently imprinted in thermoinduced methacrylic acid-ethylene glycol dimethacrylate co-polymers. Evaluation of template properties indicate that a correlation exists between non-specific binding and template basicity for a series of structural isomers. A correlation between non-specific binding and hydrophobicity was also identified for templates increasing in alkyl character. However no overall correlation was observed, as it was speculated that these factors may be competing. All species imprinted, with the exception of 2-dimethylaminopyridine, produced a selective response for the template species. Varying degrees of cross-selectivity were observed for each imprinted polymer. Polymers imprinted with templates of higher basicity demonstrated a greater degree of cross-selectivity relative to those of lower basicity. While overall binding was reduced in acetonitrile relative to chloroform, specificity was increased. This highlights the intrinsic difference in binding sites within imprinted and non-imprinted sites of the polymer. Finally, while the ability of the template species to form a co-operative interaction may be advantageous in producing a selective imprint it is not a prerequisite. For species based on this co-operative interaction the steric environment in the immediate proximity to the binding functionalities are critical to recognition. Steric hindrance of non-functionally active groups can dramatically impair the formation of interactions.