A Protein-Conjugate Approach to Develop a Monoclonal Antibody-Based Antigen Detection Test for the Diagnosis of Human Brucellosis

Human brucellosis is most commonly diagnosed by serology based on agglutination of fixed Brucella abortus as antigen. Nucleic acid amplification techniques have not proven capable of reproducibly and sensitively demonstrating the presence of Brucella DNA in clinical specimens. We sought to optimize a monoclonal antibody-based assay to detect Brucella melitensis lipopolysaccharide in blood by conjugating B. melitensis LPS to keyhole limpet hemocyanin, an immunogenic protein carrier to maximize IgG affinity of monoclonal antibodies. A panel of specific of monoclonal antibodies was obtained that recognized both B. melitensis and B. abortus lipopolysaccharide epitopes. An antigen capture assay was developed that detected B. melitensis in the blood of experimentally infected mice and, in a pilot study, in naturally infected Peruvian subjects. As a proof of principle, a majority (7/10) of the patients with positive blood cultures had B. melitensis lipopolysaccharide detected in the initial blood specimen obtained. One of 10 patients with relapsed brucellosis and negative blood culture had a positive serum antigen test. No seronegative/blood culture negative patients had a positive serum antigen test. Analysis of the pair of monoclonal antibodies (2D1, 2E8) used in the capture ELISA for potential cross-reactivity in the detection of lipopolysaccharides of E. coli O157:H7 and Yersinia enterocolitica O9 showed specificity for Brucella lipopolysaccharide. This new approach to develop antigen-detection monoclonal antibodies against a T cell-independent polysaccharide antigen based on immunogenic protein conjugation may lead to the production of improved rapid point-of-care-deployable assays for the diagnosis of brucellosis and other infectious diseases.     

Characterization of two type 1 Cu sites of Hyphomicrobium denitrificans nitrite reductase: a new class of copper-containing nitrite reductases

We report (1) the amino acid sequence of Hyphomicrobium denitrificans nitrite reductase (HdNIR), containing two type 1 Cu sites and one type 2 Cu site; (2) the expression and preparation of wild-type HdNIR and two mutants replacing the Cys ligand of each type 1 Cu with Ala; and (3) their spectroscopic and functional characterization. The open-reading frame of 50-kDa HdNIR is composed of the 15-kDa N-terminal domain having a type 1 Cu-binding motif like cupredoxins and the 35-kDa C-terminal domain having type 1 Cu-binding and type 2 Cu-binding motifs such as common nitrite reductases (NIRs). Moreover, the amino acid sequences of the N- and C-terminal domains are homologous to those of plastocyanins and NIRs, respectively. The point mutation of the Cys ligand of each type 1 Cu with Ala gives two mutants, C114A and C260A, possessing one type 1 Cu and one type 2 Cu. The spectroscopic data of C114A reveal that the C-terminal NIR-like domain has the green type 1 Cu (type 1 Cu(C)), showing two intense absorption peaks at 455 (epsilon = 2600 M(-1) cm(-1)) and 600 nm (epsilon = 2800 M(-1) cm(-1)) and a rhombic EPR signal like those of the green type 1 Cu of Achromobacter cycloclastes NIR (AcNlR). The spectroscopic data of C260A elucidate that the N-terminal Pc-like domain in HdNIR contains the blue type 1 Cu (type 1 Cu(N)), exhibiting an intense absorption band at 605 nm (epsilon = 2900 M(-1) cm(-1)) and an axial EPR signal like those of the blue type 1 Cu of Alcaligenes xylosoxidans NIR (AxNIR). The sum of the visible absorption or EPR spectra of C114A and C260A is almost equal to the corresponding spectrum of wild-type HdNIR. The spectroscopic characterization of the type 1 Cu indicates that the geometries of the type 1 Cu(N) and Cu(C) sites are slightly distorted tetrahedral (or axially elongated bipyramidal) and flattened tetrahedral, respectively. In the cyclic voltammograms, the midpoint potentials (E(1/2)), probably because of the type 1 Cu ions of C114A and C260A, are observed at +321 and +336 mV versus normal hydrogen electrode (NHE) at pH 7.0, respectively. These values, which are close to each other, are more positive than those ( approximately +0.24-0.28 V at pH 7.0) of the type 1 Cu sites of AcNIR and AxNIR. The electron-accepting capability of C114A from cytochrome c(550) is almost similar to that of wild-type HdNIR, whereas that of C260A is very low. This suggests that the type 1 Cu(C) in the C-terminal domain is essential for the enzyme functions of HdNIR.     

Herpes simplex virus 1-encoded protein kinase UL13 phosphorylates viral Us3 protein kinase and regulates nuclear localization of viral envelopment factors UL34 and UL31

UL13 and Us3 are protein kinases encoded by herpes simplex virus 1. We report here that Us3 is a physiological substrate for UL13 in infected cells, based on the following observations. (i) The electrophoretic mobility, in denaturing gels, of Us3 isoforms from Vero cells infected with wild-type virus was slower than that of isoforms from cells infected with a UL13 deletion mutant virus (DeltaUL13). After treatment with phosphatase, the electrophoretic mobility of the Us3 isoforms from cells infected with wild-type virus changed, with one isoform migrating as fast as one of the Us3 isoforms from DeltaUL13-infected cells. (ii) A recombinant protein containing a domain of Us3 was phosphorylated by UL13 in vitro. (iii) The phenotype of DeltaUL13 resembles that of a recombinant virus lacking the Us3 gene (DeltaUs3) with respect to localization of the viral envelopment factors UL34 and UL31, whose localization has been shown to be regulated by Us3. UL34 and UL31 are localized in a smooth pattern throughout the nuclei of cells infected with wild-type virus, whereas their localization in DeltaUL13- and DeltaUs3-infected cells appeared as nuclear punctate patterns. These results indicate that UL13 phosphorylates Us3 in infected cells and regulates UL34 and UL31 localization, either by phosphorylating Us3 or by a Us3-independent mechanism.     

Human leptospirosis caused by a new, antigenically unique Leptospira associated with a Rattus species reservoir in the Peruvian Amazon

As part of a prospective study of leptospirosis and biodiversity of Leptospira in the Peruvian Amazon, a new Leptospira species was isolated from humans with acute febrile illness. Field trapping identified this leptospire in peridomestic rats (Rattus norvegicus, six isolates; R. rattus, two isolates) obtained in urban, peri-urban, and rural areas of the Iquitos region. Novelty of this species was proven by serological typing, 16S ribosomal RNA gene sequencing, pulsed-field gel electrophoresis, and DNA-DNA hybridization analysis. We have named this species "Leptospira licerasiae" serovar Varillal, and have determined that it is phylogenetically related to, but genetically distinct from, other intermediate Leptospira such as L. fainei and L. inadai. The type strain is serovar Varillal strain VAR 010(T), which has been deposited into internationally accessible culture collections. By microscopic agglutination test, "Leptospira licerasiae" serovar Varillal was antigenically distinct from all known serogroups of Leptospira except for low level cross-reaction with rabbit anti-L. fainei serovar Hurstbridge at a titer of 1:100. LipL32, although not detectable by PCR, was detectable in "Leptospira licerasiae" serovar Varillal by both Southern blot hybridization and Western immunoblot, although on immunoblot, the predicted protein was significantly smaller (27 kDa) than that of L. interrogans and L. kirschneri (32 kDa). Isolation was rare from humans (2/45 Leptospira isolates from 881 febrile patients sampled), but high titers of MAT antibodies against "Leptospira licerasiae" serovar Varillal were common (30%) among patients fulfilling serological criteria for acute leptospirosis in the Iquitos region, and uncommon (7%) elsewhere in Peru. This new leptospiral species reflects Amazonian biodiversity and has evolved to become an important cause of leptospirosis in the Peruvian Amazon.     

Central versus peripheral impact of estradiol on the impaired glucose metabolism in ovariectomized mice on a high-fat diet

Age-related loss of ovarian function promotes adiposity and insulin resistance in women. Estrogen (E(2)) directly enhances insulin sensitivity and suppresses lipogenesis in peripheral tissues. Recently, the central actions of E(2) in the regulation of energy homeostasis are becoming clearer; however, the functional relevance and degree of contribution of the central vs. peripheral actions of E(2) are currently unknown. Therefore, we prepared and analyzed four groups of mice. 1) Control: sham-operated mice fed a regular diet, 2) OVX-HF: ovariectomized (OVX) mice fed a 60% high-fat diet (HF), 3) E2-SC: OVX-HF mice subcutaneously treated with E(2), and 4) E2-ICV: OVX-HF mice treated with E(2) intracerebroventricularly. OVX-HF mice showed increased body weight with both visceral and subcutaneous fat volume enlargement, glucose intolerance, and insulin resistance. Both E2-SC and E2-ICV equally ameliorated these abnormalities. Although the size of adipocytes and number of CD11c-positive macrophages in perigonadal fat in OVX-HF were reduced by both E(2) treatments, peripherally administered E(2) decreased the expression of TNFα, lipoprotein lipase, and fatty acid synthase in the white adipose tissue (WAT) of OVX-HF. In contrast, centrally administered E(2) increased hormone-sensitive lipase in WAT, decreased the hepatic expression of gluconeogenic enzymes, and elevated core body temperature and energy expenditure with marked upregulation of uncoupling proteins in the brown adipose tissue. These results suggest that central and peripheral actions of E(2) regulate insulin sensitivity and glucose metabolism via different mechanisms, and their coordinated effects may be important to prevent the development of obesity and insulin resistance in postmenopausal women.     

The Galectin-1 level in serum as a novel marker for stress

Galectin-1(Gal-1), a carbohydrate-binding protein with an affinity for β-galactoside, is widely expressed in various normal and pathological tissues and it also plays an important role in regulating immune cell homeostasis and tumorigenesis. This study investigated the effects of restraint stress on serum Gal-1 by Western blot analyses and enzyme-linked immunosorbent assays. The Gal-1 levels of the restraint-stress group were significantly higher than those of the control group. However, this increase by stress was not obvious in adolescent rats. The pattern of these changes was similar to that of corticosterone. Furthermore, this Gal-1 increase in the serum was prevented by pre-treatment with a neurotoxin 6-hydroxydopamine (6-OHDA), which destroys the noradrenergic nerve terminals. However, a bilateral adrenalectomy (ADX) had no effect on the Gal-1 increase. These results suggest that Gal-1 is a candidate stress marker protein and that the stress-induced increase of Gal-1 in serum is regulated by the sympathetic nervous system under stress conditions.     

Synthesis of N-Aryl Uracils and Hypoxanthines and Their Biological Properties

Abstract

1-Benzyluracils 2a,b were treated with iodobenzene in the presence of cuprous oxide in 2,4,6-trimethylpyridine at 180°C to give the N ¹-phenyl derivatives 3a and 3b in 47% and 55%, respectively. Similar reaction of 2a with 2-bromopyridine at 120°C gave the 3-(2-pyridinyl)uracil 4a in 42% yield. However, unusual product 5 as well as 3-(2-pyridinyl) derivative 4b were obtained in the case of 2b. The structure of 5 was identified as 1-(2,6-difluorobenzyl)-3-[(2,4-dimethyl-2-pyridinyl)methyl]uracil from spectroscopic data. Reaction of the hypoxanthines 7a,b with 2-bromopyridine gave the 1-(2-pyridinyl)hypoxanthines 8a,b in low yields. But N-phenylation of 7a,b were unsuccessful.