Observation of the waveguide resonance in a periodically patterned high refractive index broadband antireflection coating.

Grating waveguide structures have been prepared by the deposition of a high refractive index broadband antireflection coating onto a patterned fused silica substrate. Aluminum oxide and hafnium oxide as well as mixtures thereof have been used as coating materials. Optical reflection measurements combined with atomic force microscopy have been used to characterize the structures. Upon illumination with a TE wave, the best structure shows a narrow reflection peak located at 633 nm at an incidence angle of about 17°. The peak reflectance of that sample accounts for more than 89%. Off-resonance interference structures appear strongly suppressed in the spectrum between 450 and 800 nm because of the characteristics of the designed antireflection layer. The structure thus possesses a notch filter spectral characteristic in a broad spectral range.

The injury patterns, management and outcomes of retroperitoneal haemorrhage caused by lumbar arterial bleeding at a Level-1 Trauma Centre: A 10-year retrospective review.

PURPOSE: Haemorrhagic shock remains a leading preventable cause of death amongst trauma patients. Failure to identify retroperitoneal haemorrhage (RPH) can lead to irreversible haemorrhagic shock. The arteries of the middle retroperitoneal region (i.e., the 1st to 4th lumbar arteries) are complicit in haemorrhage into the retroperitoneal space. However, predictive injury patterns and subsequent management implications of haemorrhage secondary to bleeding of these arteries is lacking. MATERIALS AND METHODS: We performed a retrospective cohort study of patients diagnosed with retroperitoneal haemorrhage who presented to our Level-1 Trauma Centre (2009-2019). We described the associated injuries, management and outcomes relating to haemorrhage of lumbar arteries (L1-4) from this cohort to assess risk and management priorities in non-cavitary haemorrhage compared to RPH due to other causes. RESULTS: Haemorrhage of the lumbar arteries (LA) is associated with a higher proportion of lumbar transverse process (TP) fractures. Bleeding from branches of these vessels is associated with lower systolic blood pressure, increased incidence of massive transfusion, higher shock index, and a higher Injury Severity Score (ISS). A higher proportion of patients in the LA group underwent angioembolisation when compared to other causes of RPH. CONCLUSION: This study highlights the injury patterns, particularly TP fractures, in the prediction, early detection and management of haemorrhage from the lumbar arteries (L1-4). Compared to other causes of RPH, bleeding of the LA responds to early, aggressive haemorrhage control through angioembolisation. These injuries are likely best treated in Level-1 or Level-2 trauma facilities that are equipped with angioembolisation facilities or hybrid theatres to facilitate early identification and management of thoracolumbar bleeds.

Mitotic checkpoints.

Entry into mitosis is triggered by activation of maturation promoting factor and a complex of p34cdc2 kinase and cyclin B. Activation induces nuclear lamina breakdown, chromosome condensation and mitotic spindle assembly. Exit from mitosis is initiated by the degradation of cyclin B and the subsequent inactivation of maturation-promoting factor. A more thorough understanding of the checkpoints for initiation of and exit from mitosis has evolved during the past few years.

Mechanisms of phosphatidylserine exposure, a phagocyte recognition signal, on apoptotic T lymphocytes.

The appearance of phosphatidylserine (PS) on the cell surface during apoptosis in thymocytes and cytotoxic T lymphocyte cell lines provokes PS-dependent recognition by activated macrophages. Flow cytometric analysis of transbilayer lipid movements in T lymphocytes undergoing apoptosis reveals that downregulation of the adenosine triphosphate-dependent amino-phospholipid translocase and activation of a nonspecific lipid scramblase are responsible for PS reaching the surface from its intracellular location. Both mechanisms are expressed at the same time, and precede DNA degradation, zeiosis, and cell lysis in the apoptotic pathway.

Effect of streptolysin O on erythrocyte membranes, liposomes, and lipid dispersions. A protein-cholesterol interaction.

The effect of the bacterial cytolytic toxin, streptolysin O (SLO), on rabbit erythrocyte membranes, liposomes, and lipid dispersions was examined. SLO produced no gross alterations in the major erythrocyte membrane proteins or lipids. However, when erythrocytes were treated with SLO and examined by electron microscopy, rings and "C"-shaped structures were observed in the cell membrane. The rings had an electron-dense center, 24 nm in diameter, and the overall diameter of the structure was 38 nm. Ring formation also occurred when erythrocyte membranes were fixed with glutaraldehyde and OsO4 before the addition of toxin. In contrast, rings were not seen when erythrocytes were treated with toxin at 0 degrees C, indicating that adsorption of SLO to the membrane is not sufficient for ring formation since toxin is known to bind to erythrocytes at that temperature. The ring structures were present on lecithin-cholesterol-dicetylphosphate liposomes after SLO treatment, but there was no release of the trapped, internal markers, K2CrO4 or glucose. The crucial role of cholesterol in the formation of rings and C's was demonstrated by the fact that these structures were present in toxin-treated cholesterol dispersions, but not in lecithin-dicetylphosphate dispersions nor in the SLO preparations alone. The importance of cholesterol was also shown by the finding that no rings were present in membranes or cholesterol dispersions which had been treated with digitonin before SLO was added. Although rings do not appear to be "holes" in the membrane, a model is proposed which suggests that cholesterol molecules are sequestered during ring and C-structure formation, and that this process plays a role in SLO-induced hemolysis.

Relation between the organization of spectrin and of membrane lipids in lymphocytes.

In lymphocytes, the cytoskeletal protein spectrin exhibits two organizational states. Because the plasma membrane lipids of lymphocytes also display two organizational states, it was asked whether there is a relation between the organization of spectrin and of membrane lipids. When mouse thymocytes were stained with merocyanine 540 (MC540), a fluorescent lipophilic probe that binds preferentially to loosely packed, disorganized lipid bilayers, some cells fluoresced brightly and some only dimly or not at all. When the same population was stained for spectrin by indirect immunofluorescence, the spectrin in some cells was uniformly distributed, while in others it was concentrated in a unipolar aggregate. Techniques enriching for mature thymocytes selected for cells displaying low MC540 fluorescence and aggregated spectrin, the same characteristics found in peripheral blood lymphocytes. Flow cytometric sorting of thymocytes based on MC540 phenotype simultaneously sorted them by spectrin phenotype. Finally, treatment with agents that alter the distribution of spectrin caused mature lymphocytes to display high MC540 fluorescence and uniform spectrin. Thus, a relation exists between the organizational states of spectrin and of membrane lipids in lymphocytes: aggregated spectrin is found in cells with tightly organized membrane lipids, uniform spectrin in those with loosely organized lipids. Spectrin may thus be involved in modulating membrane lipid organization in lymphocytes as it is in erythrocytes. Since loosely organized lipids may promote adhesion of blood cells to reticuloendothelial cells, spectrin may thereby be involved in transducing an internally generated adhesion signal to the lymphocyte surface.

Golgi alkalinization by the papillomavirus E5 oncoprotein.

The E5 oncoprotein of bovine papillomavirus type I is a small, hydrophobic polypeptide localized predominantly in the Golgi complex. E5-mediated transformation is often associated with activation of the PDGF receptor (PDGF-R). However, some E5 mutants fail to induce PDGF-R phosphorylation yet retain transforming activity, suggesting an additional mechanism of action. Since E5 also interacts with the 16-kD pore-forming subunit of the vacuolar H(+)-ATPase (V-ATPase), the oncoprotein could conceivably interfere with the pH homeostasis of the Golgi complex. A pH-sensitive, fluorescent bacterial toxin was used to label this organelle and Golgi pH (pH(G)) was measured by ratio imaging. Whereas pH(G) of untreated cells was acidic (6.5), no acidification was detected in E5-transfected cells (pH approximately 7.0). The Golgi buffering power and the rate of H(+) leakage were found to be comparable in control and transfected cells. Instead, the E5-induced pH differential was attributed to impairment of V-ATPase activity, even though the amount of ATPase present in the Golgi complex was unaltered. Mutations that abolished binding of E5 to the 16-kD subunit or that targeted the oncoprotein to the endoplasmic reticulum abrogated Golgi alkalinization and cellular transformation. Moreover, transformation-competent E5 mutants that were defective for PDGF-R activation alkalinized the Golgi lumen. Neither transformation by sis nor src, two oncoproteins in the PDGF-R signaling pathway, affected pH(G). We conclude that alkalinization of the Golgi complex represents a new biological activity of the E5 oncoprotein that correlates with cellular transformation.

Studies on the mechanism of polyethylene glycol-mediated cell fusion using fluorescent membrane and cytoplasmic probes.

The mechanism by which polyethylene glycol (PEG) mediates cell fusion has been studied by examining the movements of membrane lipids and proteins, as well as cytoplasmic markers, from erythrocytes to monolayers of cultured cells to which they have been fused. Fluorescence and freeze-fracture electron microscopy and fluorescence recovery after photobleaching have yielded the following results: (a) In the presence of both fusogenic and nonfusogenic PEG membranes are brought together at closely apposed contact regions. (b) Fluorescent lipid probes quickly spread from the membranes of erythrocytes to cultured cells in the presence of both fusogenic and nonfusogenic PEG. (c) Proteins of the erythrocyte membranes were never observed to diffuse into the cultured cell membrane. (d) Water-soluble proteins did not diffuse from the erythrocyte interior into the target cell cytoplasm until the PEG was removed. These data suggest that the coordinate action of two distinct components is necessary for fusion as mediated by PEG. Presumably, the polymer itself promotes close apposition of the adjacent cell membranes but the fusion stimulus is provided by the additives contained in commercial PEG.

Caffeine-induced uncoupling of mitosis from the completion of DNA replication in mammalian cells.

Caffeine was shown to induce mitotic events in mammalian cells before DNA replication (S phase) was completed. Synchronized BHK cells that were arrested in early S phase underwent premature chromosome condensation, nuclear envelope breakdown, morphological "rounding up," and mitosis-specific phosphoprotein synthesis when they were exposed to caffeine. These mitotic responses occurred only after the cells had entered S phase and only while DNA synthesis was inhibited by more than 70 percent. Inhibitors of protein synthesis blocked these caffeine-induced events, while inhibitors of RNA synthesis had little effect. These results suggest that caffeine induces the translation or stabilizes the protein product (or products) of mitosis-related RNA that accumulates in S-phase cells when DNA replication is suppressed. The ability to chemically manipulate the onset of mitosis should be useful for studying the regulation of this event in mammalian cells.

A subfamily of P-type ATPases with aminophospholipid transporting activity.

The appearance of phosphatidylserine on the surface of animal cells triggers phagocytosis and blood coagulation. Normally, phosphatidylserine is confined to the inner leaflet of the plasma membrane by an aminophospholipid translocase, which has now been cloned and sequenced. The bovine enzyme is a member of a previously unrecognized subfamily of P-type adenosine triphosphatases (ATPases) that may have diverged from the primordial enzyme before the separation of the known families of ion-translocating ATPases. Studies in Saccharomyces cerevisiae suggest that aminophospholipid translocation is a general function of members of this family.

The E5 transforming gene of bovine papillomavirus encodes a small, hydrophobic polypeptide.

Bovine papillomavirus (BPV-1) contains two independent transforming genes that have been mapped to the E5 and E6 open reading frames (ORF's). The E5 transforming protein was identified by means of an antiserum against a synthetic peptide corresponding to the 20 COOH-terminal amino acids of the E5 ORF. The E5 polypeptide is the smallest viral transforming protein yet characterized; it had an apparent size of 7 kilodaltons. The transforming polypeptide is encoded entirely within the second half of the E5 ORF and its predicted amino acid composition is very unusual; 68% of the amino acids are strongly hydrophobic and 34% are leucine. Cell fractionation studies localized this polypeptide predominantly to cellular membranes.

Imaging macrophages in trehalose with SIMS.

Phagocytosis is a major component of the animal immune system where apoptotic cellular material, metabolites, and waste are safely processed. Further, efficient phagocytosis by macrophages is key to maintaining healthy vascular systems and preventing atherosclerosis. Single-cell images of macrophage phagocytosis of red blood cells, RBCs, and polystyrene microspheres have been chemically mapped with TOF-SIMS. We demonstrate here cholesterol and phosphocholine localizations as relative to time and activity.

RING finger-dependent ubiquitination by PRAJA is dependent on TGF-beta and potentially defines the functional status of the tumor suppressor ELF.

In gastrointestinal cells, biological signals for transforming growth factor-beta (TGF-beta) are transduced through transmembrane serine/threonine kinase receptors that signal to Smad proteins. Smad4, a tumor suppressor, is often mutated in human gastrointestinal cancers. The mechanism of Smad4 inactivation, however, remains uncertain and could be through E3-mediated ubiquitination of Smad4/adaptor protein complexes. Disruption of ELF (embryonic liver fodrin), a Smad4 adaptor protein, modulates TGF-beta signaling. We have found that PRAJA, a RING-H2 protein, interacts with ELF in a TGF-beta-dependent manner, with a fivefold increase of PRAJA expression and a subsequent decrease in ELF and Smad4 expression, in gastrointestinal cancer cell lines (P < 0.05). Strikingly, PRAJA manifests substantial E3-dependent ubiquitination of ELF and Smad3, but not Smad4. Delta-PRAJA, which has a deleted RING finger domain at the C terminus, abolishes ubiquitination of ELF. A stable cell line that overexpresses PRAJA exhibits low levels of ELF in comparison to a Delta-PRAJA stable cell line, where ELF expression is high compared to normal controls. The alteration of ELF and/or Smad4 expression and/or function in the TGF-beta signaling pathway may be induced by enhancement of ELF degradation, which is mediated by a high-level expression of PRAJA in gastrointestinal cancers. In hepatocytes, half-life (t(1/2)) and rate constant for degradation (k(D)) of ELF is 1.91 h and 21.72 min(-1) when coupled with ectopic expression of PRAJA in cells stimulated by TGF-beta, compared to PRAJA-transfected unstimulated cells (t(1/2) = 4.33 h and k(D) = 9.6 min(-1)). These studies reveal a mechanism for tumorigenesis whereby defects in adaptor proteins for Smads, such as ELF, can undergo degradation by PRAJA, through the ubiquitin-mediated pathway.

Induction of cyclin A gene expression by homocysteine in vascular smooth muscle cells.

Homocysteine is an important and independent risk factor for arteriosclerosis. We showed previously that homocysteine stimulates vascular smooth muscle cell proliferation, a hallmark of arteriosclerosis. We show here that homocysteine and serum increased DNA synthesis synergistically in both human and rat aortic smooth muscle cells (RASMCs). Treatment of quiescent RASMCs with 1 mM homocysteine or 2% calf serum for 36 h increased cyclin A mRNA levels by 8- and 14-fold, respectively, whereas homocysteine plus serum increased cyclin A mRNA levels by 40-fold, indicating a synergistic induction of cyclin A mRNA. Homocysteine did not increase the half-life of cyclin A mRNA (2.9 h), but it did increase the transcriptional rate of the cyclin A gene in nuclear run-on experiments. The positive effect of homocysteine on cyclin A gene transcription was confirmed by our finding that homocysteine increased cyclin A promoter activity and ATF-binding protein levels in RASMCs. Finally, 1 mM homocysteine increased cyclin A protein levels and cyclin A-associated kinase activity by threefold. This homocysteine-induced expression lesions by promoting proliferation of vascular smooth muscle cells.

Disappearance of cyclin A correlates with permanent withdrawal of cardiomyocytes from the cell cycle in human and rat hearts.

The regulated expression of cyclins controls the cell cycle. Because cardiomyocytes in adult mammals withdraw permanently from the cell cycle and thus cannot regenerate after injury, we examined cyclin expression during development by comparing cyclin A-E mRNA levels in fetal and adult human hearts. Cyclin B mRNA was detectable in adult hearts, although at a level markedly lower than that in fetal hearts. Levels of cyclin C, D1, D2, D3, and E mRNA were essentially identical in the two groups. In contrast, cyclin A mRNA was undetectable in adult hearts whereas cyclin A mRNA and protein were readily detectable in fetal hearts and cardiomyocytes, respectively. We then measured cyclin A mRNA and protein levels in rat hearts at four stages of development (fetal and 2, 14, and 28 d). Cyclin A mRNA and protein levels decreased quickly after birth (to 37% at day 2) and became undetectable within 14 d, an observation consistent with reports that cardiomyocytes stop replicating in rats by the second to third postnatal week. This disappearance of cyclin A gene expression in human and rat hearts at the time cardiomyocytes become terminally differentiated suggests that cyclin A downregulation is important in the permanent withdrawal of cardiomyocytes from the cell cycle.

44-amino-acid E5 transforming protein of bovine papillomavirus requires a hydrophobic core and specific carboxyl-terminal amino acids.

The 44-amino-acid E5 protein of bovine papillomavirus type 1 is the shortest known protein with transforming activity. To identify the specific amino acids required for in vitro focus formation in mouse C127 cells, we used oligonucleotide-directed saturation mutagenesis to construct an extensive collection of mutants with missense mutations in the E5 gene. Characterization of mutants with amino acid substitutions in the hydrophobic middle third of the E5 protein indicated that efficient transformation requires a stretch of hydrophobic amino acids but not a specific amino acid sequence in this portion of the protein. Many amino acids in the carboxyl-terminal third of the protein can also undergo substitution without impairment of focus-forming activity, but the amino acids at seven positions, including two cysteine residues that mediate dimer formation, appear essential for efficient transforming activity. These essential amino acids are the most well conserved among related fibropapillomaviruses. The small size of the E5 protein, its lack of similarity to other transforming proteins, and its ability to tolerate many amino acid substitutions implies that it transforms cells via a novel mechanism.

Membrane phase state and the rearrangement of hematopoietic cell surface receptors.

Transformed murine hematopoietic cells of several lineages bound the fluorescent membrane probe merocyanine 540, whereas their normal counterparts did not. Similar selective binding was reproduced in artificial liposomes which bound this probe above their phase transition temperature, but not below it. The regions of the membrane to which merocyanine 540 binds along with the receptors for the lectin concanavalin A, but not the receptors for the lectin wheat germ agglutinin, were rearranged during the course of induced differentiation of erythroleukemia cells. Based on these findings, we propose a model of hematopoietic cell surface differentiation in which proteins such as concanavalin A receptors, which are destined for removal from the plasma membrane, are specifically associated with disordered, liquid-like lipid domains which can be visualized with merocyanine 540. For the specific case of erythroid differentiation, these domains and their associated proteins are collected at the region of the membrane where nuclear extrusion occurs and are eliminated from the reticulocyte plasma membrane by the enucleation event.

Multiple systems for recognition of apoptotic lymphocytes by macrophages.

In vivo, apoptotic lymphocytes are recognized and phagocytosed by macrophages well before the final stages of DNA degradation and cell lysis. The recognition process is apparently triggered by the exposure of phosphatidylserine (PS) on the cell surface, an event which precedes cell lysis by several hours. However, multiple receptors appear to respond to this event. We demonstrate here that both activated and unactivated macrophages recognize PS, but with different receptor systems. Phagocytosis of apoptotic lymphocytes by activated (but not by unactivated) macrophages is inhibited by pure PS vesicles as well as by N-acetylglucosamine, implicating involvement of a lectin-like receptor in this case. Conversely, uptake of apoptotic lymphocytes by unactivated (but not by activated) macrophages is inhibited by PS on the surface of erythrocytes as well as by the tetrapeptide RGDS and cationic amino acids and sugars, implicating involvement of the vitronectin receptor in this case. Recognition by both classes of macrophages is blocked by the monocyte-specific monoclonal antibody 61D3. The signal recognized by activated macrophages appears to develop on the lymphocyte prior to assembly of the signal recognized by unactivated macrophages. Collectively, these results suggest that PS exposure on the surface of apoptotic lymphocytes generates a complex and evolving signal recognized by different receptor complexes on activated and unactivated macrophages.

Elevation of cell cycle control proteins during spontaneous immortalization of human keratinocytes.

A human line of spontaneously immortalized keratinocytes (SIK cells) has been derived from ostensibly normal epidermis and has proven useful in dissecting molecular changes associated with immortalization. The original cultures had a normal karyotype and a colony forming efficiency of approximately 3% through 10 passages. At passage 15, after which normal strains ordinarily senesce, these cells continued vigorous growth and gradually increased in colony forming efficiency, stabilizing at approximately 30% by passage 40. During the early stage of increasing colony forming efficiency, the cells acquired a single i(6p) chromosomal aberration and 5- to 10-fold increases in expression of the cell-cycle control proteins cyclin A, cyclin B, and p34cdc2. Additional chromosomal aberrations accumulated at later passages (i(8q) and +7), but the i(6p) and the increased expression of cell-cycle proteins were maintained, raising the possibility that these features were important for immortalization. Regulation of cell growth and differentiation in the cultures appeared minimally altered compared with normal keratinocytes as judged by their microscopic appearance and generation of abortive colonies, sensitivity to growth suppression by transforming growth factor-beta and tetradecanoylphorbol acetate, and dependence upon epidermal growth factor for progressive growth.

Maternal hypomagnesemia alters hippocampal NMDAR subunit expression and programs anxiety-like behaviour in adult offspring.

It is well established that maternal undernutrition and micronutrient deficiencies can lead to altered development and behaviour in offspring. However, few studies have explored the implications of maternal Mg deficiency and programmed behavioural and neurological outcomes in offspring. We used a model of Mg deficiency (prior to and during pregnancy and lactation) in CD1 mice to investigate if maternal Mg deficiency programmed changes in behaviour and NMDAR subunit expression in offspring. Hippocampal tissue was collected at postnatal day 2 (PN2), PN8, PN21 and 6 months, and protein expression of NMDAR subunits GluN1, GluN2A and GluN2B was determined. At 6 months of age, offspring were subject to behavioural tasks testing aspects of anxiety-like behaviour, memory, and neophobia. Maternal hypomagnesemia was associated with increased GluN1, GluN2A and GluN2B subunit expression in female offspring at 6 months, but decreased GluN1 and GluN2A expression in males. The GluN2B:GluN2A expression ratio was increased in both sexes. Male (but not female) offspring from Mg-deficient dams showed anxiety-like behaviour, with reduced head dips (Suok test), and reduced exploration of open arms (elevated plus maze). Both male and female offspring from Mg-deficient dams also showed impaired recognition memory (novel object test). These findings suggest that maternal Mg deficiency can result in behavioural deficits in adult life, and that these changes may be related to alterations in hippocampal NMDA receptor expression.