Kinetic analysis of bacteriophage Sf6 binding to outer membrane protein a using whole virions.

For successful infection, viruses must recognize their respective host cells. A common mechanism of host recognition by viruses is to utilize a portion of the host cell as a receptor. Bacteriophage Sf6, which infects Shigella flexneri, uses lipopolysaccharide as a primary receptor and then requires interaction with a secondary receptor, a role that can be fulfilled by either outer membrane proteins (Omp) A or C. Our previous work showed that specific residues in the loops of OmpA mediate Sf6 infection. To better understand Sf6 interactions with OmpA loop variants, we determined the kinetics of these interactions through the use of biolayer interferometry, an optical biosensing technique that yields data similar to surface plasmon resonance. Here, we successfully tethered whole Sf6 virions, determined the binding constant of Sf6 to OmpA to be 36 nM. Additionally, we showed that Sf6 bound to five variant OmpAs and the resulting kinetic parameters varied only slightly. Based on these data, we propose a model in which Sf6: Omp receptor recognition is not solely based on kinetics, but likely also on the ability of an Omp to induce a conformational change that results in productive infection. Keywords: Sf6; Shigella flexneri; OmpA; biolayer interferometry.

Confirmation of immunogenic consensus sequence HIV-1 T-cell epitopes in Bamako, Mali and Providence, Rhode Island.

The design of epitope-driven vaccines for HIV has been significantly hampered by concerns about conservation of vaccine epitopes across clades of HIV. In previous work, we have described a computer-driven method for a cross-clade HIV vaccine comprised of overlapping, highly conserved helper T-cell epitopes or "immunogenic consensus sequence epitopes" (ICS epitopes). Here, we evaluated and compared the immunogenicity of 20 ICS HIV epitopes in ELISpot assays performed using peripheral blood monocytes (PBMC) from HIV-infected donors in Providence, Rhode Island, USA and in Bamako, Mali, West Africa. Each core 9-mer HIV sequence contained in a given consensus peptide was conserved in at least 105 to as many as 2,250 individual HIV-1 strains. Nineteen of the 20 ICS epitopes (95%) were confirmed in ELISpot assays using PBMC obtained from 13 healthy, HIV-1 infected subjects in Providence, and thirteen of the epitopes (65%) were confirmed in ELISpot assays using PBMC derived from 42 discarded blood units obtained at the Central Blood Bank in Bamako. Twelve of the epitopes were confirmed in ELISpot assays performed both in Providence and Bamako. These data confirm the utility of bioinformatics tools to select and design novel vaccines containing "immunogenic consensus sequence" T-cell epitopes for a globally relevant vaccine against HIV; a similar approach may also be useful for any pathogen that exhibits high variability (influenza, HCV, or variola for example). An HIV vaccine containing these immunogenic consensus sequences is currently under development.

Analyzing Mycobacterium tuberculosis proteomes for candidate vaccine epitopes.

Secreted antigens of Mycobacterium tuberculosis (Mtb) induce strong T cell responses and interferon-gamma (IFN-gamma) secretion, both of which are integral in the defense against Mtb. We used web-based tools (SignaIP and Prosite) to identify putative secreted proteins from Mtb genomes CDC 1551 and H37Rv. We then used EpiMatrix, a proprietary pattern-matching algorithm, to do a preliminary analysis of these proteins for regions that contained a high number of class II MHC binding motif matches. The use of bioinformatics tools reduced the number of potential epitopes to be screened to 5% of the 1.3 million overlapping peptides. Peripheral blood mononuclear cells (PBMC) were obtained from healthy, asymptomatic tuberculin skin test-positive donors. Of the 17 highest-ranking peptide candidates that could be synthesized for this preliminary in vitro evaluation, 15 (88%) stimulated IFN-gamma response, and eight (47%) stimulated lymphocyte proliferation in vitro. IFN-gamma ELISpot assays were therefore a more sensitive test for T cell response to these peptides than were proliferation assays. One highly promiscuous epitope (MT2281-26-J, WRRRPLSSALLSFGLLLGGLPL) induced IFN-gamma secretion in PBMC from 11 of 25 Mtb immune subjects (44%). Overall, 15 epitopes, and MT2281-26-J in particular, are candidates for inclusion in a multi-epitope TB vaccine. These findings support the systematic application of bioinformatics tools to whole genomes when used in combination with in vitro methods for screening and confirming epitopes.

A biopsychosocial model of glycemic control in diabetes: stress, coping and regimen adherence.

This study examines stress, coping, and regimen adherence as determinants of chronic and transient metabolic control in diabetes. We also examine the interaction of biologic vulnerability and psychosocial risk factors to see if Type 1 (insulin dependent diabetes mellitus) or Type 2 (noninsulin dependent diabetes mellitus) diabetes had greater responsiveness to psychosocial risk factors. Analyses of data from insulin-treated adults with Type 1 (N = 57) and Type 2 (N = 61) diabetes supported the biopsychosocial model. For Type 1 diabetes, self-controlling persons had better glycemic control and emotional persons had worse (because of differences in stress). All of these associations were mediated by regimen compliance. For Type 2 diabetes, self-controlling persons had better glycemic control for reasons other than regimen compliance. There was an interaction between biologic and psychosocial factors, with psychosocial factors accounting for more variance in glycemic control within Type 1 patients. Stable psychosocial resources (i.e., education, being married, and positive coping styles) were associated with better chronic glycemic control, while stress and regimen nonadherence were associated with worse transient glycemic control.

An artificial transmembrane segment directs SecA, SecB, and electrochemical potential-dependent translocation of a long amino-terminal tail.

Many integral membrane proteins contain an amino-terminal segment, often referred to as an N-tail, that is translocated across a membrane. In many cases, translocation of the N-tail is initiated by a cleavable, amino-terminal signal peptide. For N-tail proteins lacking a signal peptide, translocation is initiated by a transmembrane segment that is carboxyl to the translocated segment. The mechanism of membrane translocation of these segments, although poorly understood, has been reported to be independent of the protein secretion machinery. In contrast, here we describe alkaline phosphatase mutants containing artificial transmembrane segments that demonstrate that translocation of a long N-tail across the membrane is dependent upon SecA, SecB, and the electrochemical potential in the absence of a signal peptide. The corresponding mutants containing signal peptides also use the secretion machinery but are less sensitive to inhibition of its components. We present evidence that inhibition of SecA by sodium azide is incomplete even at high concentrations of inhibitor, which suggests why SecA-dependent translocation may not have been detected in other systems. Furthermore, by varying the charge around the transmembrane segment, we find that in the absence of a signal peptide, the orientation of the membrane-bound alkaline phosphatase is dictated by the positive inside rule. However, the presence of a signal peptide is an overriding factor in membrane orientation and renders all mutants in an Nout-Cin orientation.

Stress buffering and glycemic control. The role of coping styles.

OBJECTIVE: To test the hypotheses that chronic psychosocial stress is associated with worse glycemic control and that coping moderates (buffers) this effect. RESEARCH DESIGN AND METHODS: Subjects consisted of 105 insulin-treated adults from the Diabetes Division of Henry Ford Hospital who filled out questionnaires on stress and coping and received an HbA1 test at a clinic appointment. Six coping styles were examined, including both emotion- and problem-focused styles. Two standardized stress inventories were administered. Ineffective coping was defined as scoring below the median for stress-dampening coping styles and above the median for stress-exacerbating styles. RESULTS: Stress was significantly (P less than 0.05) correlated with higher HbA1 in all but one ineffective coping subgroup. Conversely, none of 12 correlations between stress and glycemic control was significant in the effective coping subgroups. CONCLUSIONS: Chronic psychosocial stress is associated with worse glycemic control among those who do not cope effectively with stress. Effective coping can protect individuals from the deleterious effects of stress.

Insulin therapy in diabetes. The importance of good glucose control.

Good blood glucose control is desirable to prevent or retard the chronic complications of diabetes mellitus. In diabetics who have a nearly normal hemoglobin A1c (glycohemoglobin) concentration and who can adjust their diet and activities to avoid hypoglycemia, one insulin injection daily may be adequate. In most insulin-dependent diabetics, however, a more complicated insulin regimen, tailored to the patient's needs, is required. The best treatment regimen is one that causes minimal disruption in the patient's life-style, costs little, yet produces good results. Glucose levels should be assessed frequently and therapy adjusted as needed. Although good glucose control is difficult to achieve, new monitoring techniques and insulin delivery systems have made the goal more attainable than ever before. By following the motto "Recheck often and change PRN," physicians can achieve the goal in more patients and thus help reduce the chronic complications of diabetes mellitus.

Psychosocial factors in diabetes control: adjustment of insulin-treated adults.

Twenty insulin-treated diabetic adults were studied to identify psychosocial factors important in diabetic (blood glucose) control. Diabetic control was assessed by glycosylated hemoglobin, a measure of long-term glucose control. Subjects were equally divided between "good" and "poor" glucose control groups with sex balanced in each group. A multifactorial biopsychosocial model was proposed and tested. This model incorporated both direct (psychophysiologic) and indirect (behavioral) components. The behavioral variables investigated included predisposing (orientational), enabling (resource/barrier), and conditioning (inhibiting and motivating) factors. The psychophysiologic variables investigated were stress-response factors (elevating and dampening). Univariate and multivariate analysis demonstrated significant relationships between glucose control and each category of variables, using measures of diabetes knowledge and attitudes, health locus of control, and coping styles. The findings support both the stress-coping-illness and health-belief/illness-behavior models of diabetic adjustment and control.

Synthesis of a tricyclic aphidicolin analogue that inhibits DNA synthesis in vitro.

We have hypothesized that the biological activity of the antiviral antitumor diterpene aphidicolin requires a specific stereochemical relationship between two rigidly held hydroxyl groups on the alpha face of the molecule. The complex tetracyclic carbon skeleton is not necessary but appears to serve only as a framework on which to hold the hydroxyls. In support of this theory, we have prepared a simple tricyclic triol analogue (7) whose activity approaches that of the natural product in inhibiting in vitro DNA synthesis.

Addison's disease with adrenal enlargement on computed tomographic scanning. Report on two cases of tuberculosis and review of the literature.

Two patients with Addison's disease related to urogenital tuberculosis had enlargement of one or both adrenal glands detected with computed tomographic scanning. Review of reports of adrenal size on computed tomographic examination suggests that adrenal enlargement in the presence of Addison's disease demands further investigation about the cause of the adrenal insufficiency.

Wound healing with diabetes mellitus. Better glucose control for better wound healing in diabetes.

Wound healing is impaired in diabetic patients with infection or hyperglycemia. Several approaches to glucose management are discussed for patients whose diabetic history has been stable and also for those whose diabetic condition is labile or poorly controlled.

A high-wheat bran diet in insulin-treated diabetes mellitus: assessment with the artificial pancreas.

The acute effects of adding a large amount of wheat bran to a mixed meal was examined with the artificial pancreas in seven insulin-dependent diabetic patients. The timing of the peak blood glucose concentration and its magnitude were similar with both low- and high-wheat fiber meals. The total and net areas under the glucose curve were comparable. After adjusting for individual basal needs, the insulin requirements for the test meals were nearly identical. We observed no beneficial effect of added wheat bran from this study. We could not demonstrate any need for diabetic patients to adjust insulin dosage for occasional differences in meal wheat bran content.

Oskar Hirsch and surgery for exophthalmos.

Despite a better appreciation of the relationship between the eye and the thyroid gland in Graves' disease, the treatment of moderate to severe exophthalmos remains surgical. For many years, standard textbooks recommended a risky, transfrontal approach for exophthalmos that had to be performed by a neurosurgeon. A preferable transantral procedure, which can be performed by a plastic surgeon, was originally described by Oskar Hirsch, a Viennese otorhinolaryngologist, in 1930. Although his technique offers the advantage of causing less morbidity, it was several decades before its merits were widely recognized by practitioners.

Pharmacodynamics of propylthiouracil in normal and hyperthyroid subjects after a single oral dose.

Serum levels of propylthiouracil were measured in 8 normal persons and in 7 patients with hyperthyroidism after a single, 300 mg, oral dose of 6-n-propyl-2-thiouracil (PTU). The patients with hyperthyroidism were restudied after 3, 6, and 9 weeks of individualized treatment with PTU. The serum half-life of the drug in normal subjects was 1.65 h. In patients with hyperthyroidism the serum half-life was similar, and it did not change significantly as the euthyroid state was achieved.