Mathematical modelling of competitive LDL/VLDL binding and uptake by hepatocytes

Elevated levels of low-density-lipoprotein cholesterol (LDL-C) in the plasma are a well-established risk factor for the development of coronary heart disease. Plasma LDL-C levels are in part determined by the rate at which LDL particles are removed from the bloodstream by hepatic uptake. The uptake of LDL by mammalian liver cells occurs mainly via receptor-mediated endocytosis, a process which entails the binding of these particles to specific receptors in specialised areas of the cell surface, the subsequent internalization of the receptor-lipoprotein complex, and ultimately the degradation and release of the ingested lipoproteins' constituent parts. We formulate a mathematical model to study the binding and internalization (endocytosis) of LDL and VLDL particles by hepatocytes in culture. The system of ordinary differential equations, which includes a cholesterol-dependent pit production term representing feedback regulation of surface receptors in response to intracellular cholesterol levels, is analysed using numerical simulations and steady-state analysis. Our numerical results show good agreement with in vitro experimental data describing LDL uptake by cultured hepatocytes following delivery of a single bolus of lipoprotein. Our model is adapted in order to reflect the in vivo situation, in which lipoproteins are continuously delivered to the hepatocyte. In this case, our model suggests that the competition between the LDL and VLDL particles for binding to the pits on the cell surface affects the intracellular cholesterol concentration. In particular, we predict that when there is continuous delivery of low levels of lipoproteins to the cell surface, more VLDL than LDL occupies the pit, since VLDL are better competitors for receptor binding. VLDL have a cholesterol content comparable to LDL particles; however, due to the larger size of VLDL, one pit-bound VLDL particle blocks binding of several LDLs, and there is a resultant drop in the intracellular cholesterol level. When there is continuous delivery of lipoprotein at high levels to the hepatocytes, VLDL particles still out-compete LDL particles for receptor binding, and consequently more VLDL than LDL particles occupy the pit. Although the maximum intracellular cholesterol level is similar for high and low levels of lipoprotein delivery, the maximum is reached more rapidly when the lipoprotein delivery rates are high. The implications of these results for the design of in vitro experiments is discussed.

MS-based clinical proteomics: biomarker discovery in men’s cancer

Importance of biomarker discovery in men’s cancer diagnosis and prognosis Each year around 10,000 men in the UK die as a result of prostate cancer (PCa) making it the 3rd most common cancer behind lung and breast cancer; worldwide more than 670,000 men are diagnosed every year with the disease [1]. Current methods of diagnosis of PCa mainly rely on the detection of elevated prostate-specific antigen (PSA) levels in serum and/or physical examination by a doctor for the detection of an abnormal prostate. PSA is a glycoprotein produced almost exclusively by the epithelial cells of the prostate gland [2]. Its role is not fully understood, although it is known that it forms part of the ejaculate and its function is to solubilise the sperm to give them the mobility to swim. Raised PSA levels in serum are thought to be due to both an increased production of PSA from the proliferated prostate cells, and a diminished architecture of affected cells, allowing an easier distribution of PSA into the wider circulatory system.

Ribose 2′-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5

The 5'-cap-structures of higher eukaryote mRNAs are ribose 2'-O-methylated. Likewise, a number of viruses replicating in the cytoplasm of eukayotes have evolved 2'-O-methyltransferases to modify autonomously their mRNAs. However, a defined biological role of mRNA 2'-O-methylation remains elusive. Here we show that viral mRNA 2'-O-methylation is critically involved in subversion of type-I-interferon (IFN-I) induction. We demonstrate that human and murine coronavirus 2'-O-methyltransferase mutants induce increased IFN-I expression, and are highly IFN-I sensitive. Importantly, IFN-I induction by 2'-O-methyltransferase-deficient viruses is dependent on the cytoplasmic RNA sensor melanoma differentiation-associated gene 5 (MDA5). This link between MDA5-mediated sensing of viral RNA and mRNA 2'-O-methylation suggests that RNA modifications, such as 2'-O-methylation, provide a molecular signature for the discrimination of self and non-self mRNA.

Studies into the role of the SEF14 fimbrial antigen in the pathogenesis of Salmonella enteritidis

To investigate the role of the SEF14 fimbrial antigen in pathogenesis, a single defined sefA (SEF14(-)) inactivated mutant of Salmonella enteritidis strain LA5 was constructed and tested in a number of biological assay systems. There was no significant difference between the wild-type strain and the isogenic SEF14(-) mutant in their abilities to adhere to and invade HEp-2 epithelial cells or their survival in mouse peritoneal macrophages, whereas the SEF14(-) mutant was ingested more rapidly by isolated human PMN. Both the strains colonized the intestine, invaded and spread systemically in 1 day-old chicks, laying hens and BALB/c mice equally well. A significantly greater number of chicks excreted the wildtype SEF14(+) strain during the first week following infection as compared to those infected with the SEF14(-) mutant. However, similar numbers of chicks excreted the two strains between 2 and 7 weeks after infection. These results indicate that possession of SEF14 fimbriae alone do not appear to play a significant role in the pathogenesis of S. enteritidis although its contribution to virulence may be dependent on the host species infected. (C) 1996 Academic Press Limited

Characterization of the Salmonella typhimurium proteome by semi-automated two dimensional HPLC-mass spectrometry: detection of proteins implicated in multiple antibiotic resistance

The proteome of Salmonella enterica serovar Typhimurium was characterized by 2-dimensional HPLC mass spectrometry to provide a platform for subsequent proteomic investigations of low level multiple antibiotic resistance (MAR). Bacteria (2.15 +/- 0.23 x 10(10) cfu; mean +/- s.d.) were harvested from liquid culture and proteins differentially fractionated, on the basis of solubility, into preparations representative of the cytosol, cell envelope and outer membrane proteins (OMPs). These preparations were digested by treatment with trypsin and peptides separated into fractions (n = 20) by strong cation exchange chromatography (SCX). Tryptic peptides in each SCX fraction were further separated by reversed-phase chromatography and detected by mass spectrometry. Peptides were assigned to proteins and consensus rank listings compiled using SEQUEST. A total of 816 +/- 11 individual proteins were identified which included 371 +/- 33, 565 +/- 15 and 262 +/- 5 from the cytosolic, cell envelope and OMP preparations, respectively. A significant correlation was observed (r(2) = 0.62 +/- 0.10; P < 0.0001) between consensus rank position for duplicate cell preparations and an average of 74 +/- 5% of proteins were common to both replicates. A total of 34 outer membrane proteins were detected, 20 of these from the OMP preparation. A range of proteins (n = 20) previously associated with the mar locus in E. coli were also found including the key MAR effectors AcrA, TolC and OmpF.

Modulation of hTREK-1 by carbon monoxide

TREK-1 is a background K channel important in the regulation of neuronal excitability. Here, we demonstrate that recombinant human TREK-1 is activated by low concentrations of carbon monoxide (CO) and nitric oxide (NO), applied via their respective donor molecules. Related channels hTASK-1 and hTASK-3 were unaffected by CO. Effects of both CO and NO were prevented by preincubation of cells with the protein kinase G inhibitor, Rp-8-Br-PET-cGMPS. The effects of CO were independent of NO formation. At higher concentrations, both NO and CO were inhibitory. As both NO and CO are important neuronal gasotransmitters and TREK is crucial in regulating neuronal excitability, our results provide a novel means by which these gases may modulate neuronal activity.

Observations of diurnal and spatial variability of radiative forcing by equatorial deep convective clouds

From geostationary satellite observations of equatorial Africa and the equatorial east Atlantic during May and June 2000 we explore the radiative forcing by deep convective cloud systems in these regions. Deep convective clouds (DCCs) are associated with a mean radiative forcing relative to non–deep convective areas of −39 W m−2 over the Atlantic Ocean and of +13 W m−2 over equatorial Africa (±10 W m−2 in both cases). We show that over land the timing of the daily cycle of convection relative to the daily cycle in solar illumination and surface temperature significantly affects the mean radiative forcing by DCCs. Displacement of the daily cycle of DCC coverage by 2 hours changes their overall radiative effect by ∼10 W m−2, with implications for the simulation of the radiative balance in this region. The timing of the minimum DCC cover over land, close to noon local time, means that the mean radiative forcing is nearly maximized.

Heme oxygenase-1 regulates cell proliferation via carbon monoxide-mediated inhibition of T-type Ca2+ channels

Induction of the antioxidant enzyme heme oxygenase-1 (HO-1) affords cellular protection and suppresses proliferation of vascular smooth muscle cells (VSMCs) associated with a variety of pathological cardiovascular conditions including myocardial infarction and vascular injury. However, the underlying mechanisms are not fully understood. Over-expression of Cav3.2 T-type Ca2+ channels in HEK293 cells raised basal [Ca2+]i and increased proliferation as compared with non-transfected cells. Proliferation and [Ca2+]i levels were reduced to levels seen in non-transfected cells either by induction of HO-1 or exposure of cells to the HO-1 product, carbon monoxide (CO) (applied as the CO releasing molecule, CORM-3). In the aortic VSMC line A7r5, proliferation was also inhibited by induction of HO-1 or by exposure of cells to CO, and patch-clamp recordings indicated that CO inhibited T-type (as well as L-type) Ca2+ currents in these cells. Finally, in human saphenous vein smooth muscle cells, proliferation was reduced by T-type channel inhibition or by HO-1 induction or CO exposure. The effects of T-type channel blockade and HO-1 induction were non-additive. Collectively, these data indicate that HO-1 regulates proliferation via CO-mediated inhibition of T-type Ca2+ channels. This signalling pathway provides a novel means by which proliferation of VSMCs (and other cells) may be regulated therapeutically.

Heme oxygenase-1 protects against Alzheimer’s amyloid-β1-42 induced toxicity via carbon monoxide production

Heme oxygenase-1 (HO-1), an inducible enzyme up-regulated in Alzheimer‟s disease (AD), catabolises heme to biliverdin, Fe2+ and carbon monoxide (CO). CO can protect neurones from oxidative stress-induced apoptosis by inhibiting Kv2.1 channels, which mediate cellular K+ efflux as an early step in the apoptotic cascade. Since apoptosis contributes to the neuronal loss associated with amyloid β peptide (Aβ) toxicity in AD, we investigated the protective effects of HO-1 and CO against Aβ1-42 toxicity in SH-SY5Y cells, employing cells stably transfected with empty vector or expressing the cellular prion protein, PrPc, and rat primary hippocampal neurons. Aβ1-42 (containing protofibrils) caused a concentrationdependent decrease in cell viability, attributable at least in part to induction of apoptosis, with the PrPc expressing cells showing greater susceptibility to Aβ1-42 toxicity. Pharmacological induction or genetic over-expression of HO-1 significantly ameliorated the effects of Aβ1-42. The CO-donor CORM-2 protected cells against Aβ1-42 toxicity in a concentration-dependent manner. Electrophysiological studies revealed no differences in the outward current pre- and post-Aβ1-42 treatment suggesting that K+ channel activity is unaffected in these cells. Instead, Aβ toxicity was reduced by the L-type Ca2+ channel blocker nifedipine, and by the CaMKKII inhibitor, STO-609. Aβ also activated the downstream kinase, AMP-dependent protein kinase (AMPK). CO prevented this activation of AMPK. Our findings indicate that HO-1 protects against Aβ toxicity via production of CO. Protection does not arise from inhibition of apoptosis-associated K+ efflux, but rather by inhibition of AMPK activation, which has been recently implicated in the toxic effects of Aβ. These data provide a novel, beneficial effect of CO which adds to its growing potential as a therapeutic agent.

Morphological characterization of periodontium-derived human stem cells

The aim of this study has been to characterize adult human somatic periodontium-derived stem cells (PDSCS) isolated from human periodontium and to follow their differentiation after cell culture. PDSCS were isolated from human periodontal tissue and cultured as spheres in serum-free medium. After 10 days the primary spheres were dissociated and the secondary spheres sub-cultured for another 1-2 weeks. Cells from different time points were analyzed, and immunohistochemical and electron microscopic investigations carried out. Histological analysis showed differentiation of spheres deriving from the PDSCS with central production of extracellular matrix beginning 3 days after sub-culturing. Isolated PDSCS developed pseudopodia which contained actin. Tubulin was found in the central portion of the cells. Pseudopodia between different cells anastomosed, indicating intercellular transport. Immunostaining for osteopontin demonstrated a positive reaction in primary spheres and within extracellular matrix vesicles after sub-culturing. In cell culture under serum-free conditions human PDSCS form spheres which are capable of producing extracellular matrix. Further investigations have do be carried out to investigate the capability of these cells to differentiate into osteogenic progenitor cells.

Supra-molecular assembly of a lumican-derived peptide amphiphile enhances its collagen-stimulating activity

C16-YEALRVANEVTLN, a peptide amphiphile (PA) incorporating a biologically active amino acid sequence found in lumican, has been examined for its influence upon collagen synthesis by human corneal fibroblasts in vitro, and the roles of supra-molecular assembly and activin receptor-like kinase ALK receptor signaling in this effect were assessed. Cell viability was monitored using the Alamar blue assay, and collagen synthesis was assessed using Sirius red. The role of ALK signaling was studied by receptor inhibition. Cultured human corneal fibroblasts synthesized significantly greater amounts of collagen in the presence of the PA over both 7-day and 21-day periods. The aggregation of the PA to form nanotapes resulted in a notable enhancement in this activity, with an approximately two-fold increase in collagen production per cell. This increase was reduced by the addition of an ALK inhibitor. The data presented reveal a stimulatory effect upon collagen synthesis by the primary cells of the corneal stroma, and demonstrate a direct influence of supra-molecular assembly of the PA upon the cellular response observed. The effects of PA upon fibroblasts were dependent upon ALK receptor function. These findings elucidate the role of self-assembled nanostructures in the biological activity of peptide amphiphiles, and support the potential use of a self-assembling lumican derived PA as a novel biomaterial, intended to promote collagen deposition for wound repair and tissue engineering purposes

3-D volume rendering maxillofacial analysis of angular measurements by multislice CT

Objective. This study was designed to determine the precision and accuracy of angular measurements using three-dimensional computed tomography (3D-CT) volume rendering by computer systems. Study design. The study population consisted of 28 dried skulls that were scanned with a 64-row multislice CT, and 3D-CT images were generated. Angular measurements, (n = 6) based upon conventional craniometric anatomical landmarks (n = 9), were identified independently in 3D-CT images by 2 radiologists, twice each, and were then performed by 3D-CT imaging. Subsequently, physical measurements were made by a third examiner using a Beyond Crysta-C9168 series 900 device. Results. The results demonstrated no statistically significant difference between interexaminer and intraexaminer analysis. The mean difference between the physical and 3-D-based angular measurements was -1.18% and -0.89%, respectively, for both examiners, demonstrating high accuracy. Conclusion. Maxillofacial analysis of angular measurements using 3D-CT volume rendering by 64-row multislice CT is established and can be used for orthodontic and dentofacial orthopedic applications.

Morpho-anatomical variations during stem development in some epiphytic Cactaceae

LEMOS, R. C. C. AND G. F. A. MELO-DE-PINNA (Departamento de Botanica, Instituto de Biociencias, Universidade de Sao Paulo, Rua do Matao 277, Travessa 14, Cidade Universitaria, Butanta, Caixa Postal 11461, 05422-970, Sao Paulo, SP, Brasil). Morpho-anatomical variations during stem development in some epiphytic Cactaceae. J. Torrey Bot. Soc. 138: 16-25. 2011. In this study, the morpho-anatomical features of Hatiora salicornioides (Harworth) Britton & Rose, Rhipsalis floccosa Salm-Dyck Pfeiffer, Rhipsalis elliptica G. Lindb. ex K. Schum. and Epiphyllum phyllanthus (L.) Haworth. were studied during different phases of stem development. Primary (more developed) and terminal (less developed) segments showed variations of anatomical features as exhibited by the epidermal cells in surface view and transverse section. Features of the vascular system, e.g., the occurrence of non-lignified parenchyma in bands (H. salicornioides) or in small groups (R. floccosa and R. elliptica), as well as pericycle fibers and lignified cells in the medullar region, were only observed on the primary segments. Nevertheless, based on our anatomical analysis of stem segments in different developmental phases, we conclude that some characters described and used in systematic interpretations should be revised, mainly in the vascular (secondary xylem; non-xylematic vascular fibers) and dermal systems (epidermis in surface view and transverse section).

Glucose-Induced Regulation of NHEs Activity and SGLTs Expression Involves the PKA Signaling Pathway

The effect of glucose on the intracellular pH (pH(i)) recovery rate (dpH(i)/dt) and Na(+)-glucose transporter (SGLT) localization was investigated in HEK-293 cells, a cell line that expresses endogenous NHE1, NHE3, SGLT1, and SGLT2 proteins. The activity of the Na(+)/H(+) exchangers (NHEs) was evaluated by using fluorescence microscopy. The total and membrane protein expression levels were analyzed by immunoblotting. In cells cultivated in 5 mM glucose, the pH(i) recovery rate was 0.169 +/- A 0.020 (n = 6). This value did not change in response to the acute presence of glucose at 2 or 10 mM, but decreased with 25 mM glucose, an effect that was not observed with 25 mM mannitol. Conversely, the chronic effect of high glucose (25 mM) increased the pH(i) recovery rate (similar to 40%, P < 0.05), without changes in the total levels of NHE1, NHE3, or SGLT1 expression, but increasing the total cellular (similar to 50%, P < 0.05) and the plasma membrane (similar to 100%, P < 0.01) content of SGLT2. Treatment with H-89 (10(-6) M) prevented the stimulatory effect of chronic glucose treatment on the pH(i) recovery rate and SGLT2 expression in the plasma membrane. Our results indicate that the effect of chronic treatment with a high glucose concentration is associated with increased NHEs activity and plasma membrane expression of SGLT2 in a protein kinase A-dependent way. The present results reveal mechanisms of glucotoxicity and may contribute to understanding the diabetes-induced damage of this renal epithelial cell.

The effect of an adventure race on lymphocyte and neutrophil death

The effect of an adventure race (Ecomotion Pr), which lasted for 4-5 days, on neutrophil and lymphocyte death from elite athletes was investigated. Blood was collected from 11 athletes at rest and after the adventure race. The following parameters of cell death were measured in neutrophils and lymphocytes: cell membrane integrity, DNA fragmentation, mitochondrial transmembrane depolarization and reactive oxygen species (ROS) production. Phagocytosis capacity was also evaluated in neutrophils. The adventure race raised the proportion of cells with the loss of membrane integrity; lymphocytes by 14% and neutrophils by 16.4%. The proportion of lymphocytes with DNA fragmentation (2.9-fold) and mitochondrial transmembrane depolarization (1.5-fold) increased. However, these parameters did not change in neutrophils. ROS production remained unchanged in lymphocytes, whereas an increase by 2.2-fold was found in neutrophils due to the race. Despite these changes, the phagocytosis capacity did not change in neutrophils after the race. In conclusion, the Ecomotion Pr race-induced neutrophil death by necrosis (as indicated by the loss of membrane integrity) and led to lymphocyte death by apoptosis (as indicated by increase DNA fragmentation and depolarization of mitochondrial membrane).