Morphologic characterization of osteoblast-like cell cultures isolated from newborn rat calvaria

Summary Two methods for harvesting osteoblast-like cell populations from newborn (10 days) rat calvaria were compared. The first one consisted in culturing the periosteum-free bones and then trypsinizing the cells on the bone surface. The second one involved the migration of the osteoblasts on glass fragments before trypsinization. Since the plating efficiency, the proportion of alkaline phosphatase-positive cells, the population doubling time, and the calcium deposition were more adequate, the second method was used to further characterize the behavior of the cultures. During the first week of culture, the cells featured shapes similar to those observedin vivo on the surface of periosteum-free calvaria. They formed multilayers and, in the presence of ascorbic acid, synthetized an organic matrix containing exclusively type I collagen. Later, small amounts of type III collagen appeared. The cells were embedded in the matrix and progressively acquired the morphologic phenotype of osteocyte-like cells. The matrix mineralized in the presence of β-glycerophosphate. The technique of dropinoculation (high concentration of cells in a small volume of medium) promoted the multilayer formation and the achievement of large mineralized plates (about 1 cm²) in 3 weeks of culture.     

Catheter drainage of symptomatic postoperative mediastinal effusion guided by computed tomography. A nonoperative approach

Postoperative mediastinal effusion after cardiac operations is a common phenomenon and has a potential for serious complications, such as tamponade necessitating urgent drainage. Computed tomography of the chest provides excellent postoperative visualization of the mediastinum. Catheter insertion or paracentesis guided by computed tomography has been used to accomplish nonoperative drainage of symptomatic postoperative mediastinal effusion in six cases. This technique offers simplicity, safety, and cost effectiveness.     

Percutaneous transluminal coronary angioplasty - Histopathological analysis of nine necropsy cases

Summary From 1982 to 1984 nine of 300 patients undergoing transluminal coronary angioplasty died. The nine coronary arteries and one saphenous aorto-coronary by-pass graft affected by angioplasty were studied by light microscopy. The following types of lesions were found, frequently in association: rupture of the plaque, circumscribed or reaching to the intimal layer or extending beyond it, dissections (fissures) between arterial layers, intra-plaque haemorrhage, plaque emboli and thrombosis. In two cases the therapeutic approach was considered to be clinically and pathologically successful; the patients survived 24 h (case 6) and forty days (case 4). Case 6 which presented recent lesions indicative of success showed, in contrast with the other non-successful cases, rupture affecting not only the initimal layer but also deeper structures of the arterial wall. There were also more extensive fissures. Case 4 which presented late alterations indicative of success showed a plaque fracture whose borders were kept apart by fibrous tissue. In conclusion, we believe that angioplasty allows the re-establishment of arterial blood flow by provoking deep intimal and medial rupture producing a small fissure between the arterial layers and a widening of the lumen; in cases with good late results these alterations cicatrize leaving a wider arterial lumen.     

Normally unused positional cues guide ectopic afferents in the leech CNS.

Central projections from peripheral sensory neurons segregate into distinct, ventrally positioned longitudinal tracts within the segmental ganglia of the leech Hirudo medicinalis. As documented here, there is an additional tract in the neuropils of the fifth and sixth body ganglia, located at the lateral margin and formed by afferent axons (the "sex afferents") originating from sensory neurons located in the male and female genitalia. Ablation of the genitalia results in the complete absence of this additional tract. We asked (1) whether segmental differences exist in the distribution of pathway cues available to the sex afferents, and (2) whether central pathway selection by these axons is specific. We transplanted the primordia of the male genitalia to several ectopic positions posterior to the sixth body segment and labeled the ectopic sex afferents in order to examine their paths in the CNS. In about 50% of the experimental animals, afferent axons originating in the transplanted tissue segregated into a distinct lateral fiber bundle within the neuropil of a nearby ganglion, in a position corresponding to their normal one in the sex ganglia. The sex afferents therefore find their normal pathways even in the ganglia of inappropriate segments, although these pathways are not used by any other afferents in these ganglia. We propose, therefore, that the positional cues employed by afferent axons to select appropriate pathways in the ganglionic neuropil are expressed in all segments of the leech CNS, regardless of whether such cues are normally used by afferent axons in each segment.     

Office Systems and Their Influence on Mammography Use in Rural and Urban Primary Care

CONTEXT: Breast cancer screening rates are lower in rural communities. Although studies have addressed barriers to mammography for rural residents, physician practice barriers have received less attention. PURPOSE: Controlled clinical trials have shown that the use of office reminder systems in primary care practices is related to increased clinical care rates. Therefore, we compared office systems use in primary care practices located in rural and urban communities and assessed the impact of these systems on rural-urban differences in mammography utilization. METHODS: We identified female Kansas Medicare beneficiaries aged 65 to 79 from Medicare claims data (N = 24,030) and determined which beneficiaries received a mammogram between April 1, 1999, and March 31, 2001. We linked beneficiaries to their primary care providers and obtained surveys from 180 primary care practices on their use of office reminder systems. FINDINGS: Mammography rates ranged from 20% to 92% (mean = 65%) among the 180 practices. Flowsheets with a mammography prompt were used by 33% of the practices, 38% utilized nonphysician staff to identify women due for mammograms, and 15% used computerized reminder systems. Urban practices used flowsheets more often than rural practices (44% versus 16%, P < 0.001). A multivariable regression model demonstrated higher mammography rates in urban practices, group practices, and practices using mammography flowsheets. CONCLUSIONS: Despite success in randomized controlled trials, reminder systems are not used often by primary care providers and are used even less often in rural compared to urban practices. Consistent implementation may be a major barrier to the successful adaptation of flowsheets by primary care offices.     

Rapamycin limits CD4+ T cell proliferation in simian immunodeficiency virus–infected rhesus macaques on antiretroviral therapy

Proliferation of latently infected CD4⁺ T cells with replication-competent proviruses is an important mechanism contributing to HIV persistence during antiretroviral therapy (ART). One approach to targeting this latent cell expansion is to inhibit mTOR, a regulatory kinase involved with cell growth, metabolism, and proliferation. Here, we determined the effects of chronic mTOR inhibition with rapamycin with or without T cell activation in SIV-infected rhesus macaques (RMs) on ART. Rapamycin perturbed the expression of multiple genes and signaling pathways important for cellular proliferation and substantially decreased the frequency of proliferating CD4⁺ memory T cells (TM cells) in blood and tissues. However, levels of cell-associated SIV DNA and SIV RNA were not markedly different between rapamycin-treated RMs and controls during ART. T cell activation with an anti-CD3LALA antibody induced increases in SIV RNA in plasma of RMs on rapamycin, consistent with SIV production. However, upon ART cessation, both rapamycin and CD3LALA–treated and control-treated RMs rebounded in less than 12 days, with no difference in the time to viral rebound or post-ART viral load set points. These results indicate that, while rapamycin can decrease the proliferation of CD4⁺ TM cells, chronic mTOR inhibition alone or in combination with T cell activation was not sufficient to disrupt the stability of the SIV reservoir.     

Specificity in transmembrane helix–helix interactions can define a hierarchy of stability for sequence variants

The folding, stability, and oligomerization of helical membrane proteins depend in part on a precise set of packing interactions between transmembrane helices. To understand the energetic principles of these helix-helix interactions, we have used alanine-scanning mutagenesis and sedimentation equilibrium analytical ultracentrifugation to quantitatively examine the sequence dependence of the glycophorin A transmembrane helix dimerization. In all cases, we found that mutations to alanine at interface positions cost free energy of association. In contrast, mutations to alanine away from the dimer interface showed free energies of association that are insignificantly different from wild-type or are slightly stabilizing. Our study further revealed that the energy of association is not evenly distributed across the interface, but that there are several "hot spots" for interaction including both glycines participating in a GxxxG motif. Inspection of the NMR structure indicates that simple principles of protein-protein interactions can explain the changes in energy that are observed. A comparison of the dimer stability between different hydrophobic environments suggested that the hierarchy of stability for sequence variants is conserved. Together, these findings imply that the protein-protein interaction portion of the overall association energy may be separable from the contributions arising from protein-lipid and lipid-lipid energy terms. This idea is a conceptual simplification of the membrane protein folding problem and has implications for prediction and design.