Association of gelsolin with actin filaments and cell membranes of macrophages and platelets.

Recent evidence that polyphosphoinositides regulate the function of the actin-modulating protein gelsolin in vitro raises the possibility that gelsolin interacts with cell membranes. This paper reports ultrastructural immunohistochemical data revealing that gelsolin molecules localize with plasma and intracellular membranes, including rough endoplasmic reticulum, cortical vesicles and mitochondria of macrophages, and blood platelets. Anti-gelsolin gold also labeled the surface and interior of secondary lysosomes presumably representing plasma gelsolin ingested by these cells from the lung surface by endocytosis. Gelsolin molecules, visualized with colloidal gold in replicas of the cytoplasmic side of the substrate-adherent plasma membrane of mechanically unroofed and rapidly frozen and freeze-dried macrophages, associated with the ends of short actin filaments sitting on the cytoplasmic membrane surface. A generalized distribution of gelsolin molecules in thin sections of resting platelets rapidly became peripheral, and plasmalemma association increased following thrombin stimulation. At later times the distribution reverted to the cytoplasmic distribution of resting cells. These findings provide the first evidence for gelsolin binding to actin filament ends in cells and indicate that gelsolin functions in both cytoplasmic and membrane domains.

Association of profilin with filament-free regions of human leukocyte and platelet membranes and reversible membrane binding during platelet activation.

Profilin is a conserved, widely distributed actin monomer binding protein found in eukaryotic cells. Mammalian profilin reversibly sequesters actin monomers in a high affinity profilactin complex. In vitro, the complex is dissociated in response to treatment with the polyphosphoinositides, phosphatidylinositol monophosphate, and phosphatidylinositol 4,5-bisphosphate. Here, we demonstrate the ultrastructural immunolocalization of profilin in human leukocytes and platelets. In both cell types, a significant fraction of profilin is found associated with regions of cell membrane devoid of actin filaments and other discernible structures. After platelet activation, the membrane association of profilin reversibly increases. This study represents the first direct evidence for an interaction between profilin and phospholipids in vivo.

Engrailed (Gln50-->Lys) homeodomain-DNA complex at 1.9 A resolution: structural basis for enhanced affinity and altered specificity.

BACKGROUND: The homeodomain is one of the key DNA-binding motifs used in eukaryotic gene regulation, and homeodomain proteins play critical roles in development. The residue at position 50 of many homeodomains appears to determine the differential DNA-binding specificity, helping to distinguish among binding sites of the form TAATNN. However, the precise role(s) of residue 50 in the differential recognition of alternative sites has not been clear. None of the previously determined structures of homeodomain-DNA complexes has shown evidence for a stable hydrogen bond between residue 50 and a base, and there has been much discussion, based in part on NMR studies, about the potential importance of water-mediated contacts. This study was initiated to help clarify some of these issues. RESULTS: The crystal structure of a complex containing the engrailed Gln50-->Lys variant (QK50) with its optimal binding site TAATCC (versus TAATTA for the wild-type protein) has been determined at 1.9 A resolution. The overall structure of the QK50 variant is very similar to that of the wild-type complex, but the sidechain of Lys50 projects directly into the major groove and makes several hydrogen bonds to the O6 and N7 atoms of the guanines at base pairs 5 and 6. Lys50 also makes an additional water-mediated contact with the guanine at base pair 5 and has an alternative conformation that allows a hydrogen bond with the O4 of the thymine at base pair 4. CONCLUSIONS: The structural context provided by the folding and docking of the engrailed homeodomain allows Lys50 to make remarkably favorable contacts with the guanines at base pairs 5 and 6 of the binding site. Although many different residues occur at position 50 in different homeodomains, and although numerous position 50 variants have been constructed, the most striking examples of altered specificity usually involve introducing or removing a lysine sidechain from position 50. This high-resolution structure also confirms the critical role of Asn51 in homeodomain-DNA recognition and further clarifies the roles of water molecules near residues 50 and 51.

Engrailed homeodomain-DNA complex at 2.2 A resolution: a detailed view of the interface and comparison with other engrailed structures.

We report the 2.2 A resolution structure of the Drosophila engrailed homeodomain bound to its optimal DNA site. The original 2.8 A resolution structure of this complex provided the first detailed three-dimensional view of how homeodomains recognize DNA, and has served as the basis for biochemical studies, structural studies and molecular modeling. Our refined structure confirms the principal conclusions of the original structure, but provides important new details about the recognition interface. Biochemical and NMR studies of other homeodomains had led to the notion that Gln50 was an especially important determinant of specificity. However, our refined structure shows that this side-chain makes no direct hydrogen bonds to the DNA. The structure does reveal an extensive network of ordered water molecules which mediate contacts to several bases and phosphates (including contacts from Gln50), and our model provides a basis for detailed comparison with the structure of an engrailed Q50K altered-specificity variant. Comparing our structure with the crystal structure of the free protein confirms that the N and C termini of the homeodomain become ordered upon DNA-binding. However, we also find that several key DNA contact residues in the recognition helix have the same conformation in the free and bound protein, and that several water molecules also are "preorganized" to contact the DNA. Our structure helps provide a more complete basis for the detailed analysis of homeodomain-DNA interactions.

Decreased HIV-associated T cell apoptosis by HIV protease inhibitors.

Antiretroviral treatment of patients infected with HIV results in improvements in CD4+ T cell number. Emerging evidence suggests that some of the improvements in CD4+ T cell number that occur in response to protease inhibitor (PI) therapy may not be accounted for solely by enhanced viral suppression, implying that PI may directly affect T cell survival. Since HIV T cell depletion is associated with enhanced apoptosis, we analyzed the effect of PIs on T cell apoptosis. In vitro treatment of peripheral blood lymphocytes (PBLs) from HIV-infected but untreated patients with reverse transcriptase inhibitors (RTIs) does not alter apoptosis, whereas PI treatment rapidly reduces CD4+ and CD8+ T cell apoptosis. In contrast, PI treatment does not alter apoptosis in PBL blasts from HIV-negative patients, or in Jurkat T cells. Consistent with this observation, 8 days of PI therapy in HIV-infected patients does not significantly alter plasma viremia, yet results in significant inhibition of CD4+ and CD8+ T cell apoptosis. The inhibitory effects of PI on apoptosis have implications concerning the treatment of HIV and its pathogenesis.

Home health care. Clinical pathways and quality integration.

Though clinical pathways in home health care facilitate monitoring of patients' progress and use of resources, literature on home health care is scant. This article shows how quality integration is incorporated into pathways by developing the outcomes, with contributions from both clinical team and patient. Variances in a pathway, caused by either clinician or patient, can identify trends and problems that can be addressed by staff, either through patient education or changes in policy and procedures.

The DNA-binding domain of p53 contains the four conserved regions and the major mutation hot spots.

Mutations in the p53 tumor suppressor gene are the most commonly observed genetic alterations in human cancer. The majority of these mutations occur in the conserved central portion of the gene, but there has been little information about the function of this region. Using proteolytic digestion of the 393-amino-acid human p53 protein, we have identified a 191-amino-acid protease-resistant fragment (residues 102-292) that corresponds to the central portion of p53, and we show that this core fragment is the sequence-specific DNA-binding domain of the protein. DNA binding is inhibited by metal chelating agents, and we find that the core domain contains zinc. Proteolytic digests also reveal a 53-amino-acid carboxy-terminal domain which we show to be the tetramerization domain of p53.

Disruption of MAP kinase activation and nuclear factor binding to the IL-12 p40 promoter in HIV-infected myeloid cells.

Progressive immunodeficiency in HIV infection is paralleled by a decrease in IL-12 production, a cytokine crucial for cellular immune function. Here we examine the molecular mechanisms by which HIV infection suppresses IL-12 p40 expression. HIV infection of THP-1 myeloid cells resulted in decreased LPS-induced nuclear factor binding to the NF-kappaB, AP-1, and Sp1 sites of the IL-12 p40 promoter. By site-directed mutagenesis we determined that each of these sites was necessary for transcriptional activation of the IL-12 p40 promoter. Binding of NF-kappaB p50, c-Rel, p65, Sp1, Sp3, c-Fos, and c-Jun proteins to their cognate nuclear factor binding sites was somewhat impaired by HV infection, although a role for other as yet unidentified factors cannot be dismissed. The cellular levels of these transcription factors were unaffected by HIV infection, with the exception of a decrease in expression of NF-kappaB p65, consistent with the observed decrease in its binding to the IL-12 p40 promoter following HIV infection. Analysis of regulation of upstream LPS-induced MAP kinases demonstrated impaired phosphorylation of JNK and p38 MAPK, and suppressed phosphorylation and degradation of IkappaBalpha following HIV infection. These results suggest that alterations in nuclear factor binding to numerous sites in the IL-12 p40 promoter, together may contribute to the suppression in IL-12 p40 transcription previously reported. These effects on nuclear factor binding may be a direct effect of HIV infection on the IL-12 p40 promoter, or may occur indirectly as a consequence of altered MAP kinase activation.

The management of patient encounter time in a high-stakes assessment using standardized patients.

OBJECTIVES: The purpose of this study was to gather information regarding the appropriateness of the length of time allotted for candidates to complete the history taking and physical examination tasks in a high-stakes standardized patient (SP) assessment. DESIGN: Data were collected on actual time used by 1548 examinees for each of their 10 standardized patient encounters, for which a maximum of 15 minutes was allotted, but not required. SETTING: The Clinical Skills Assessment Center of the Educational Commission for Foreign Medical Graduates (ECFMG), Philadelphia, Pennsylvania, USA. SUBJECTS: Graduates of foreign medical schools who are seeking ECFMG certification. RESULTS: The average time spent with the standardized patient was 13.3 minutes, suggesting that the 15-minute time limit was sufficient. A positive correlation was found between data-gathering scores and patient interview times. Candidates did tend to spend more time with SPs presenting with cases involving complex histories, as well as with cases of chronic conditions. CONCLUSIONS: Candidate time use varied as a function of type of clinical encounter, providing additional evidence of the content validity of the Clinical Skills Assessment.

Are interpersonal skills ratings influenced by gender in a clinical skills assessment using standardized patients?

PURPOSE: The purpose of this study was to explore possible performance differences in interpersonal skills (IPS) ratings as a function of candidate and standardized patient (SP) gender. METHODOLOGY: The IPS scores and SP characteristics for 79,999 patient encounters were studied. This included 18,325 (20.36%) female candidate to female SP, 26,872 (29.86%) male candidate to female SP, 18,281 (20.31%) female candidate to male SP, and 16,521 (29.47%) male candidate to male SP interactions. RESULTS: The analysis did not reveal a significant candidate gender by SP gender effect. There were no meaningful differences in IPS scores as a function of SP or candidate gender. CONCLUSIONS: The non-significant interaction between SP gender and candidate gender provides some evidence that male and female candidates are being assessed equivalently by male and female SPs. This result, combined with the extremely weak relationship between gender (candidate or SP) and IPS ratings, provides additional support for the fairness and defensibility of the IPS measures.

Radiation damage and immune suppression in splenic mononuclear cell populations.

We have examined alterations in all of the major splenic mononuclear cell (SMNC) populations in C57B1/6 mice following whole-body irradiation (0-700 cGy) in order to determine which populations may play a role in active immune suppression and/or haematopoietic recovery. A protocol has been established for characterization and differentiation by flow cytometric analysis (FCA) of the major MNC populations in the mouse spleen: T lymphocytes (CD4+ and CD8+ cells), B lymphocytes, natural killer (NK) cells, and monocytes/macrophages. Ionizing radiation caused decreased spleen cellularity and decreased ability of surviving SMNC to respond to mitogen. FCA revealed alterations in the relative composition of the constituent splenic cell populations following irradiation, reflecting differential radiosensitivity, with selective enrichment of NK cells (seven-fold) and CD4+ T lymphocytes (three-fold). Enrichment developed during the 7-day post-irradiation period. In addition, some MNC became activated in a dose- and time-dependent fashion following whole-body irradiation, as indicated by expression of CD71, the transferrin receptor. These cells were CD34+ and Thy 1.2+, but were CD4- or CD8- as well as CD45- (B cell). The observed increase in NK cells corresponds with a previously reported increase in natural suppressor (NS) cells following total-lymphoid irradiation (TLI). The balance of recovery-inhibiting NK cells and recovery-enhancing CD4+ T lymphocytes following irradiation may reflect or influence the degree of haematopoietic recovery, and may provide an indication of the extent of damage (biological dosimetry).

Relative alterations in blood mononuclear cell populations reflect radiation injury in mice.

Damage to the immune and hematopoietic systems following exposure to ionizing radiation, whether accidental or for therapeutic purposes, renders victims susceptible to opportunistic infections and diseases. Elucidating a reliable biological indicator or "biological dosimeter" to indicate rapidly the extent of injury sustained by an individual would be desirable. Total leukocyte count has been used historically as an indicator of immune damage, but it is not truly indicative of functional immunity post-irradiation. A flow cytometric (FCM) technique was developed to determine whether a rapid reproducible assay could be developed to assess the extent of radiation damage. To this end, peripheral blood leukocyte populations and subpopulations were monitored. C57BL/6 mice were exposed to 100-, 400-, or 700-cGy whole-body gamma-irradiation (WBI) and blood samples were collected from the retro-orbital sinus 1, 4, and 7 days post-irradiation. The blood samples were prepared for FCM by incubation with monoclonal antibodies (mAb) to various murine leukocyte CD surface markers. The results show that the proportion of CD4+ T lymphocytes increased approximately 2-fold on day 4 after 700 cGy, the proportion of B lymphocytes declined markedly at all doses relative to unirradiated controls, and natural killer (NK) cells rose dramatically (9-fold) by day 4 after 700 cGy. The patterns of alteration in the relative proportions of peripheral blood mononuclear cells (PBMC) populations observed post-irradiation, particularly in B lymphocytes and natural killer (NK) cells, seem to represent potent and consistent indicators of immune damage, allowing some inference as to the immune competence of the individual.