Transduction of envelope stress in Escherichia coli by the Cpx two-component system

Disruption of normal protein trafficking in the Escherichia coli cell envelope (inner membrane, periplasm, outer membrane) can activate two parallel, but distinct, signal transduction pathways. This activation stimulates the expression of a number of genes whose products function to fold or degrade the mislocalized proteins. One of these signal transduction pathways is a two-component regulatory system comprised of the histidine kinase CpxA and the response regulator, CpxR. In this study we characterized gain-of-function Cpx* mutants in order to learn more about Cpx signal transduction. Sequencing demonstrated that the cpx* mutations cluster in either the periplasmic, the transmembrane, or the H-box domain of CpxA. Intriguingly, most of the periplasmic cpx* gain-of-function mutations cluster in the central region of this domain, and one encodes a deletion of 32 amino acids. Strains harboring these mutations are rendered insensitive to a normally activating signal. In vivo and in vitro characterization of maltose-binding-protein fusions between the wild-type CpxA and a representative cpx* mutant, CpxA101, showed that the mutant CpxA is altered in phosphotransfer reactions with CpxR. Specifically, while both CpxA and CpxA101 function as autokinases and CpxR kinases, CpxA101 is devoid of a CpxR-P phosphatase activity normally present in the wild-type protein. Taken together, the data support a model for Cpx-mediated signal transduction in which the kinase/phosphatase ratio is elevated by stress. Further, the sequence and phenotypes of periplasmic cpx* mutations suggest that interactions with a periplasmic signaling molecule may normally dictate a decreased kinase/phosphatase ratio under nonstress conditions.     

Human monocyte-derived macrophages express an approximately 120-kD Ox-LDL binding protein with strong identity to CD68

A protein that specifically binds oxidized LDL (Ox-LDL) has recently been characterized in mouse peritoneal macrophages and identified as macrosialin, a protein with a molecular weight of 95 kD. First, the present work shows that human monocyte-derived macrophages express a membrane protein with a molecular weight of approximately 120 kD that selectively binds Ox-LDL. Second, we tested whether this approximately 120-kD Ox-LDL binding protein had any relation to CD68, the human homologue of macrosialin. The following evidence was obtained to support the role of CD68 as an Ox-LDL binding protein: (1) Ligand blots with Ox-LDL and Western blots with Ki-M6, an anti-human CD68 monoclonal antibody, revealed a single band with a molecular weight of approximately 120 kD under reducing and nonreducing condition. (2) The expression patterns of the approximately 120-kD Ox-LDL binding membrane protein and of CD68 paralleled each other during monocyte/macrophage differentiation. (3) Digestion with N-glycosidase F demonstrated that both CD68 and the Ox-LDL binding protein are glycoproteins; both showed a similar shift of approximately 18 kD in apparent molecular weight. (4) CD68, probed with monoclonal antibody Ki-M6, and the approximately 120-kD Ox-LDL binding protein were coprecipitated with EMB11, another anti-CD68 antibody. About 5000 molecules of CD68 are expressed on the cell surface of human macrophages. Ligation of 125I-Ki-M6 to cells leads to its internalization and degradation. This capacity would be sufficient to allow for the specific uptake and degradation of Ox-LDL. Taken together, these data support a role for CD68 as a specific Ox-LDL binding protein in human monocyte-derived macrophages.     

Detection of flunixin in greyhound urine by a kinetic enzyme-linked immunosorbent assay

Anxiety levels in a sample of 65 long-term cancer survivors were assessed in a study of the effects of a planned discharge from an oncology clinic. Thirty-one percent of patients scored > or = 8, and 12% > or = 11 on the anxiety subscale of the Hospital Anxiety and Depression Scale (HADS), indicating that anxiety rates in patients in long-standing remission do not greatly differ from patients with active disease. Despite the provision of continued support and guaranteed fast-access return to the clinic if necessary, 28% of patients refused to be discharged. Fear that recurrence would not be detected was the reason most frequently cited. Seventy-five percent of these patients were HADS anxiety cases. A second assessment 4-5 months later of the 41 patients who were discharged showed a slight, but non-significant increase in anxiety rates suggesting that anxiety in cancer survivors may be persistent and not related to clinic attendance.     

The supportive care needs of newly diagnosed cancer patients attending a regional cancer center

BACKGROUND: The objective of this study was to examine the physical and emotional health status, self-perceived problems, and needs of newly diagnosed cancer patients to determine and plan supportive care strategies. METHODS: A cross-sectional survey of newly diagnosed cancer patients attending a regional cancer center during a 6-month period was performed. Patients with breast, colorectal, head and neck, lung, and prostate carcinoma as well as nonmelanoma of the skin were selected randomly. Patients were interviewed prior to their first appointment at the clinic. Physical health status was assessed using the Symptom Distress Scale, psychologic health status was assessed with the General Health Questionnaire (GHQ), day-to-day functioning with the Rapid Disability Scale, and social support with the modified Sarason's Social Support Scale. Perceived needs were assessed in a number of ways, including identification of patients' specific social concerns and informational needs, and by asking them to list their current problems or concerns. RESULTS: Of 156 eligible patients, 134 completed the interview. One hundred and twenty-nine patients (96%) reported current symptoms that included fatigue (66%), worried outlook (61%), difficulty sleeping (48%), and pain (42%). Forty-four patients (33%) were identified as psychologically distressed with a GHQ score of > or = 6. One hundred and fourteen patients (85%) had informational needs, 89 (66%) indicated > or = 1 social concerns, and 55 (41%) reported a need for assistance with day-to-day living. CONCLUSIONS: Patients with newly diagnosed cancer commonly report symptoms related to fatigue, pain, and psychologic distress. Other frequently reported issues relate to the need for information and social concerns regarding the patients' ability to take care of their home and maintain family and other relationships. Awareness of these issues is important for planning supportive care interventions for newly diagnosed cancer patients.     

Characterization of the elastin binding domain in the cell-surface 25-kDa elastin-binding protein of staphylococcus aureus (EbpS)

Our previous studies have established that a cell-surface 25-kDa elastin-binding protein of Staphylococcus aureus (EbpS) mediates binding of this pathogen to the extracellular matrix protein elastin. Results from binding assays examining the activity of various EbpS fragments suggested that the elastin recognition domain is contained within the first 59 amino acids. In this report, we have used functional analyses with synthetic peptides and recombinant truncated forms of EbpS to localize the elastin binding domain to a 21-amino acid region contained within residues 14-34 of EbpS. Further evidence for the importance of this domain was obtained by demonstrating that the inhibitory activity of anti-EbpS antibodies on staphylococcal elastin binding was neutralized when these antibodies were pre-absorbed with a truncated recombinant EbpS construct containing residues 1-34. Overlapping synthetic peptides corresponding to EbpS residues 14-36 were then generated and tested for elastin binding activity to define further the elastin binding domain, and results from these studies showed that sequences spanning amino acids Gln14-Asp23, Asp17-Asp23, and Thr18-Glu34 inhibit binding of Staphylococcus aureus to elastin. Our analyses indicate that the hexameric sequence Thr18-Asn-Ser-His-Gln-Asp23 is the minimal sequence common to all active synthetic peptides, proteolytic fragments, and recombinant constructs of EbpS. Furthermore, substitution of Asp23 with Asn abrogated the blocking activity of the synthetic peptides, demonstrating the requirement for a charged amino acid at this location. The composite data indicate that staphylococcal elastin binding is mediated by a discrete domain defined by short peptide sequences in the amino-terminal extracellular region of EbpS.     

Nuclear magnetic resonance analysis of solution conformations in C4-V3 hybrid peptides derived from human immunodeficiency virus (HIV) type 1 gp120: relation to specificity of peptide-induced anti-HIV neutralizing antibodies

Immunogenic peptides containing epitopes of the gp120 C4 and V3 regions from human immunodeficiency virus strains MN and EV91 have been studied by nuclear magnetic resonance and molecular modeling and used as immunogens in rhesus monkeys. The results, combined with those for other peptides, suggest a correlation between solution conformation and immunologic cross-reactivity.