The covalent binding story of the complement proteins C3 and C4 (I) 1972-1981.

Alex Law and Paul Levine recall their work to establish the covalent bond between C3 and target surfaces. It started with a naive experiment by analyzing the membrane polypeptides of sheep erythrocytes bound with 125I-labelled C3. They found complexes with molecular weight higher than the individual C3 polypeptides. These complexes survived all conditions designed to disrupt non-covalent interactions. They then showed that the bond was an ester, with an active acyl group on C3 which reacted with a hydroxyl group on the acceptor molecule. With the discovery of an internal thioester by Jim Prahl, Jamila Janatova, Brian Tack and their colleagues, it became clear that the reaction was by an acyl transfer from the thioester of C3 to the target hydroxyl group. Later on they showed that C4 also bound covalently to target molecules. By establishing a fluid phase system to study the kinetics of the binding reactions of C3 and C4, Alex was able to continue the work in the MRC Immunochemistry Unit in Oxford from 1981, to eventually determine the chemical mechanism of the binding reaction. In order to give some sense of reality, this article is written as a narrative from Alex, who did the experiments. Both Alex and Paul are retired. Pauls lives on Martha's Vineyard where he writes occasional articles on science for one of the Island's newspapers. Alex lives in Hong Kong and tries to make some sense of the local politics.

Cigarette smoking and glaucoma in the United States population.

PurposeTo evaluate the association between cigarette smoking and glaucoma in the United States population.Patients and methodsUS civilian, non-institutionalized population from 2005 to 2008 administrations of the National Health and Nutrition Examination Survey that were ≥40 years of age with visual fields and optic disc photographs were included. Diagnosis of glaucoma was based on the Rotterdam criteria. Logistic regression modeling was performed to assess the association between glaucoma and smoking history, while controlling for age, gender, ethnicity, household income, alcohol consumption, diabetes, and hypertension.ResultsIn 3864 participants, 212 (5.5%) had glaucoma (corresponds to a population weighted glaucoma prevalence of 3.7% in a total of 83 570 127 subjects). Population weighted proportion of current smokers was 20.6% and ex-smokers was 28.3%. Participants with glaucoma were older (63.0±11.6 vs 56.1±11.2, P=0.002), likely to be male (57.1% vs 49.2%, P=0.03), to be Black (36.3% vs 20.7%, P<0.001), and to have diabetes (18.9% vs 12.4%, P=0.006) and hypertension (50.5% vs 39.7%, P=0.003). Current smokers had a lower odds of glaucoma compared to non-smokers (OR=0.61, 95% CI=0.41-0.88, P=0.009), and ex-smokers (OR=0.46, 95% CI=0.28-0.76, P=0.002). The effect estimates were similar in adjusted models, but not statistically significant. Among smokers, greater pack/day of smoking history was associated with statistically significantly higher odds of glaucoma (OR=1.70, 95% CI=1.08-2.67, P=0.02).ConclusionsAmong cigarette smokers, heavy smoking defined by greater number of pack of cigarettes smoked per day is associated with higher odds of glaucoma. Health care providers should include this association when counseling patients on their smoking habit.

Function and conformation analyses of an aspartate substitution of the invariant glycine in the integrin ßI domain α1-α1' helix.

We showed that the αLß2 integrin with the non-functional mutation G150D cannot be induced with Mg/EGTA to express the mAb KIM127 epitope, which reports the leg-extended conformation. We extended the study to the αIIbß3, an integrin without an αI domain. The equivalent mutation, i.e. G161D, also resulted in an expressible, but non-adhesive αIIbß3 integrin. An NMR study of synthetic peptides spanning the α1-α1' helix of the ß3 I domain shows that both wild-type and mutant peptides are α-helical. However, whereas in the wild-type peptide this helix is continuous, the mutant presents a discontinuity, or kink, precisely at the site of mutation G161D. Our results suggest that the mutation may lock integrin heterodimers in a bent conformation that prevents integrin activation via conformational extension.

The integrin αL leg region controls the Mg/EGTA mediated activation of LFA-1.

We have shown that Mg/EGTA (5 mM Mg(2+) and 1.5 mM EGTA) could effectively promote the adhesion of integrin αLß2 to its ligand ICAM-1 but could not promote that of the αMß2 to denatured BSA. In order to determine the structural differences between αL and αM that specifically contribute to Mg/EGTA sensitivity, a series of αL/αM chimeras were constructed. Our results showed that αLß2 with αM calf-1 domain completely lost the response to Mg/EGTA activation. In the reverse experiment, αMß2 would require the presence of both the αL calf-1 and calf-2 domain to initiate the Mg/EGTA sensitivity.

Characterization of single amino acid substitutions in the ß2 integrin subunit of patients with leukocyte adhesion deficiency (LAD)-1.

Leukocyte adhesion deficiency 1 (LAD-1) is caused by defects in the ß2 integrin subunit. We studied 18 missense mutations, 14 of which fail to support the surface expression of the ß2 integrins. Integrins with the ß2-G150D mutation fail to bind ligands, possibly due to the failure of the α1 segment of the ßI domain to assume an α-helical structure. Integrins with the ß2-G716A mutation are not maintained in their resting states, and the patient has the severe phenotype of LAD-1. The ß2-S453N and ß2-P648L mutants support the expression of integrins and adhesion functions. They should be re-classified as polymorphic variants.

Hypoxia-induced changes to integrin α 3 glycosylation facilitate invasion in epidermoid carcinoma cell line A431.

Hypoxia is a critical microenvironmental factor that drives cancer progression through angiogenesis and metastasis. Glycoproteins, especially those on the plasma membrane, orchestrate this process; however, questions remain regarding hypoxia-perturbed protein glycosylation in cancer cells. We focused on the effects of hypoxia on the integrin family of glycoproteins, which are central to the cellular processes of attachment and migration and have been linked with cancer in humans. We employed electrostatic repulsion hydrophilic interaction chromatography coupled with iTRAQ labeling and LC-MS/MS to identify and quantify glycoproteins expressed in A431. The results revealed that independent of the protein-level change, N-glycosylation modifications of integrin α 3 (ITGA3) were inhibited by hypoxia, unlike in other integrin subunits. A combination of Western blot, flow cytometry, and cell staining assays showed that hypoxia-induced alterations to the glycosylation of ITGA3 prevented its efficient translocation to the plasma membrane. Mutagenesis studies demonstrated that simultaneous mutation of glycosites 6 and 7 of ITGA3 prevented its accumulation at the K562 cell surface, which blocked integrin α 3 and ß 1 heterodimer formation and thus abolished ITGA3's interaction with extracellular ligands. By generating A431 cells stably expressing ITGA3 mutated at glycosites 6 and 7, we showed that lower levels of ITGA3 on the cell surface, as induced by hypoxia, conferred an increased invasive ability to cancer cells in vitro under hypoxic conditions. Taken together, these results revealed that ITGA3 translocation to the plasma membrane suppressed by hypoxia through inhibition of glycosylation facilitated cell invasion in A431.

Differences in the compliance with hospital infection control practices during the 2009 influenza H1N1 pandemic in three countries.

BACKGROUND: In December 2009, the World Health Organization (WHO) issued updated guidelines on the prevention of H1N1 influenza virus in healthcare settings. In 2010, the WHO pandemic influenza alert level was still at phase 6. AIM: To study the practice of infection control measures during the 2009 influenza H1N1 pandemic among healthcare workers (HCWs) in three countries. METHODS: A standardized, self-administered anonymous questionnaire survey was conducted in 2010 among doctors, nurses and allied HCWs in 120 hospital-based clinical departments in Hong Kong, Singapore and the UK. Questions were asked on demographics; previous experience and perceived severity of influenza; infection control practices; uptake of seasonal influenza vaccination and H1N1 vaccination. Multiple logistic regression was used to test the independent association with different factors. FINDINGS: A total of 2100 HCWs in the three countries participated. They reported high compliance (>80%) with infection control procedures regarded as standard for droplet-transmitted infections including wearing and changing gloves, and washing hands before and after patient contact. However, the reported use of masks with indirect or direct patient contact (surgical or N95 as required by their hospital) varied considerably (96.4% and 70.4% for Hong Kong; 82.3% and 87.7% for Singapore; 25.3% and 62.0% for the UK). Reported compliance was associated with job title, number of patient contacts and perceived severity of pandemics. There was no association between the uptake for seasonal or 2009 H1N1 vaccines and compliance. CONCLUSIONS: Compliance with infection control measures for pandemic influenza appears to vary widely depending on the setting.

Hematologically important mutations: leukocyte adhesion deficiency (first update).

Leukocyte adhesion deficiency (LAD) is an immunodeficiency caused by defects in the adhesion of leukocytes (especially neutrophils) to the blood vessel wall. As a result, patients with LAD suffer from severe bacterial infections and impaired wound healing, accompanied by neutrophilia. In LAD-I, mutations are found in ITGB2, the gene that encodes the ß subunit of the ß(2) integrins. This syndrome is characterized directly after birth by delayed separation of the umbilical cord. In the rare LAD-II disease, the fucosylation of selectin ligands is disturbed, caused by mutations in SLC35C1, the gene that encodes a GDP-fucose transporter of the Golgi system. LAD-II patients lack the H and Lewis Le(a) and Le(b) blood group antigens. Finally, in LAD-III (also called LAD-I/variant) the conformational activation of the hematopoietically expressed ß integrins is disturbed, leading to leukocyte and platelet dysfunction. This last syndrome is caused by mutations in FERMT3, encoding the kindlin-3 protein in all blood cells that is involved in the regulation of ß integrin conformation.

NMDA receptor activation enhances inhibitory GABAergic transmission onto hippocampal pyramidal neurons via presynaptic and postsynaptic mechanisms.

N-methyl-D-aspartate (NMDA) receptors (NMDARs) are implicated in synaptic plasticity and modulation of glutamatergic excitatory transmission. Effect of NMDAR activation on inhibitory GABAergic transmission remains largely unknown. Here, we report that a brief application of NMDA could induce two distinct actions in CA1 pyramidal neurons in mouse hippocampal slices: 1) an inward current attributed to activation of postsynaptic NMDARs; and 2) fast phasic synaptic currents, namely spontaneous inhibitory postsynaptic currents (sIPSCs), mediated by GABA(A) receptors in pyramidal neurons. The mean amplitude of sIPSCs was also increased by NMDA. This profound increase in the sIPSC frequency and amplitude was markedly suppressed by the sodium channel blocker TTX, whereas the frequency and mean amplitude of miniature IPSCs were not significantly affected by NMDA, suggesting that NMDA elicits repetitive firing in GABAergic interneurons, thereby leading to GABA release from multiple synaptic sites of single GABAergic axons. We found that the NMDAR open-channel blocker MK-801 injected into recorded pyramidal neurons suppressed the NMDA-induced increase of sIPSCs, which raises the possibility that the firing of interneurons may not be the sole factor and certain retrograde messengers may also be involved in the NMDA-mediated enhancement of GABAergic transmission. Our results from pharmacological tests suggest that the nitric oxide signaling pathway is mobilized by NMDAR activation in CA1 pyramidal neurons, which in turn retrogradely facilitates GABA release from the presynaptic terminals. Thus NMDARs at glutamatergic synapses on both CA1 pyramidal neurons and interneurons appear to exert feedback and feedforward inhibition for determining the spike timing of the hippocampal microcircuit.

A novel 3' splice-site mutation and a novel gross deletion in leukocyte adhesion deficiency (LAD)-1.

A patient was diagnosed with leukocyte adhesion deficiency-1. She was born in 1996 and her parents are not known to be related. Her leukocytes expressed less than 2% of the CD18 antigens relative to normal individuals. Molecular analysis revealed that she is a compound heterozygote. She inherited a 27,703bp deletion from her father (g.43201_PTTG1IP:10890del27703), spanning from intron 11 of the gene for the ß2 integrin (ITGB2, CD18, NG_007270.2) to intron 2 of the gene for the Pituitary Tumor-Transforming Gene 1 Interacting Protein (PTTG1IP, NC_000021.8). The maternal allele has a g.23457C>A mutation at position -10 in intron 2 of the ITGB2 gene, resulting in the activation of a cryptic 3' splice site in intron 2 to include 43 intronic nucleotides (r.[59-43_59-1ins;59-10C>A]).

Effect of cell-seeding density on the proliferation and gene expression profile of human umbilical vein endothelial cells within ex vivo culture.

BACKGROUND AIMS: Characterization of endothelial cell-biomaterial interaction is crucial for the development of blood-contacting biomedical devices and implants. However, a crucial parameter that has largely been overlooked is the cell-seeding density. METHODS: This study investigated how varying cell-seeding density influences human umbilical vein endothelial cell (HUVEC) proliferation on three different substrata: gelatin, tissue culture polystyrene (TCPS) and poly-l-lactic acid (PLLA). RESULTS: The fastest proliferation was seen on gelatin, followed by TCPS and PLLA, regardless of seeding density. On both TCPS and gelatin, maximal proliferation was attained at an initial seeding density of 1000 cells/cm(2). At seeding densities above and below 1000 cells/cm(2), the proliferation rate decreased sharply. On PLLA, there was a decrease in cell numbers over 7 days of culture, below a certain threshold seeding density (c. 2500-3000 cells/cm(2)), which meant that some of the cells were dying off rather than proliferating. Above this threshold seeding density, HUVEC displayed slow proliferation. Subsequently, quantitative real-time polymerase chain reaction (RT-qPCR) analysis of eight gene markers associated with adhesion and endothelial functionality (VEGF-A, integrin-α5, VWF, ICAM1, ICAM2, VE-cadherin, endoglin and PECAM1) was carried out on HUVEC seeded at varying densities on the three substrata. A significant downregulation of gene expression was observed at an ultralow cell-seeding density of 100 cells/cm(2). This was accompanied by an extremely slow proliferation rate, probably because of an acute lack of intercellular contacts and paracrine signaling. CONCLUSION: Hence, this study demonstrates that seeding density has a profound effect on the proliferation and gene expression profile of endothelial cells seeded on different biomaterial surfaces.

Comparison of long-term surgical success of Ahmed Valve implant versus trabeculectomy in open-angle glaucoma.

AIM: To compare the long-term intraocular pressure (IOP) outcomes of Ahmed Glaucoma Valve (AGV) implantation to trabeculectomy with mitomycin C (MMC) in open-angle glaucoma (OAG). METHODS: 78 OAG patients who underwent AGV implantation were matched with respect to age, preoperative surgery, preoperative IOP and preoperative medicines to 88 OAG patients who underwent trabeculectomy with MMC with a minimum of 3 years' follow-up. The cumulative probability of success between the two groups with different criteria was analysed: (1) an IOP < or =21 mm Hg and a reduction of IOP>/=15% from baseline; and (2) an IOP < or =18 mm Hg and a reduction of IOP > or =20% from baseline. No loss of light perception, no additional glaucoma surgery and no hypotony were also required. RESULTS: The 5-year cumulative probability of success was not statistically significant between eyes that had an AGV or trabeculectomy with MMC when success was defined as criteria A (p = 0.094). However, when success was defined according to criteria B, eyes undergoing trabeculectomy with MMC had a higher rate of success (p = 0.024). CONCLUSIONS: Trabeculectomy with MMC has a significantly higher 5-year cumulative probability of success compared with AGV implants when greater reduction IOP is necessary.

Transmembrane helices that form two opposite homodimeric interactions: an asparagine scan study of alphaM and beta2 integrins.

Integrins are alpha/beta heterodimers, but recent in vitro and in vivo experiments also suggest an ability to associate through their transmembrane domains to form homomeric interactions. While the results of some in vitro experiments are consistent with an interaction mediated by a GxxxG-like motif, homo-oligomers observed after in vivo cross-linking are consistent with an almost opposite helix-helix interface. We have shown recently that both models of interaction are compatible with evolutionary conservation data, and we predicted that the alpha-helices in both models would have a similar rotational orientation. Herein, we have tested our prediction using in vitro asparagine scan of five consecutive residues along the GxxxG-like motif of the transmembrane domain of alpha and beta integrins, alphaM and beta2. We show that Asn-mediated dimerization occurs twice for every turn of the helix, consistent with two almost opposite forms of interaction as suggested previously for alphaIIb and beta3 transmembrane domains. The orientational parameters helix tilt and rotational orientation of each of these two Asn-stabilized dimers were measured by site-specific infrared dichroism (SSID) in model lipid bilayers and were found to be consistent with our predicted computational models. Our results highlight an intrinsic tendency for integrin transmembrane alpha-helices to form two opposite types of homomeric interaction in addition to their heteromeric interactions and suggest that integrins may form complex and specific networks at the transmembrane domain during function.

Permissive transmembrane helix heterodimerization is required for the expression of a functional integrin.

The current paradigm is that integrin is activated via inside-out signalling when its cytoplasmic tails and TMs (transmembrane helices) are separated by specific cytosolic protein(s). Perturbations of the helical interface between the alpha- and beta-TMs of an integrin, as a result of mutations, affect its function. Previous studies have shown the requirement for specific pairing between integrin subunits by ectodomain-exchange analyses. It remains unknown whether permissive alpha/beta-TM pairing of an integrin is also required for pairing specificity and the expression of a functionally regulated receptor. We performed scanning replacement of integrin beta2-TM with a TM of other integrin beta-subunits. With the exception of beta4 substitution, others presented beta2-integrins with modified phenotypes, either in their expression or ligand-binding properties. Subsequently, we adopted alphaLbeta2 for follow-on experiments because its conformation and affinity-state transitions have been well defined as compared with other members of the beta2-integrins. Replacement of beta2- with beta3-TM generated a chimaeric alphaLbeta2 of an intermediate affinity that adhered to ICAM-1 (intercellular adhesion molecule 1) but not to ICAM-3 constitutively. Replacing alphaL-TM with alphaIIb-TM, forming a natural alphaIIb/beta3-TM pair, reversed the phenotype of the chimaera to that of wild-type alphaLbeta2. Interestingly, the replacement of alphaLbeta2- with beta3-TM showed neither an extended conformation nor the separation of its cytoplasmic tails, which are well-reported hallmarks of an activated alphaLbeta2, as determined by reporter mAb (monoclonal antibody) KIM127 reactivity and FRET (fluorescence resonance energy transfer) measurements respectively. Collectively, our results suggest that TM pairing specificity is required for the expression of a functionally regulated integrin.

Reversion mutations in patients with leukocyte adhesion deficiency type-1 (LAD-1).

Leukocyte adhesion deficiency type-1 (LAD-1) is an autosomal recessive immunodeficiency caused by mutations in the beta2 integrin, CD18, that impair CD11/CD18 heterodimer surface expression and/or function. Absence of functional CD11/CD18 integrins on leukocytes, particularly neutrophils, leads to their incapacity to adhere to the endothelium and migrate to sites of infection. We studied 3 LAD-1 patients with markedly diminished neutrophil CD18 expression, each of whom had a small population of lymphocytes with normal CD18 expression (CD18(+)). These CD18(+) lymphocytes were predominantly cytotoxic T cells, with a memory/effector phenotype. Microsatellite analyses proved patient origin of these cells. Sequencing of T-cell subsets showed that in each patient one CD18 allele had undergone further mutation. Interestingly, all 3 patients were young adults with inflammatory bowel disease. Somatic reversions of inherited mutations in primary T-cell immunodeficiencies are typically associated with milder clinical phenotypes. We hypothesize that these somatic revertant CD18(+) cytotoxic T lymphocytes (CTLs) may have altered immune regulation. The discovery of 3 cases of reversion mutations in LAD-1 at one center suggests that this may be a relatively common event in this rare disease.

Acupuncture for glaucoma.

BACKGROUND: Glaucoma is a multifactorial optic neuropathy in which there is an acquired loss of retinal ganglion cells at levels beyond normal age-related loss and corresponding atrophy of the optic nerve. Although there are many existing treatments, glaucoma is a chronic condition. Some patients may seek complementary or alternative medicine such as acupuncture to supplement their regular treatment. The underlying plausibility of acupuncture is that disorders related to the flow of Chi (the traditional Chinese concept translated as vital force or energy) can be prevented or treated by stimulating the relevant points on the body surface. OBJECTIVES: The objective of this review was to assess the effectiveness and safety of acupuncture in people with glaucoma. SEARCH STRATEGY: We searched the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, LILACS, ZETOC, CINAHL, AMED (Allied and Complementary Medicine Database), TCMLARS (Traditional Chinese Medical Literature Analysis and Retrieval System), CBM (Chinese Biological Database), the Chinese Acupuncture Trials Register and the National Center for Complementary and Alternative Medicine web site (http://nccam.nih.gov/) in February 2006. We ran update searches of CENTRAL, MEDLINE, EMBASE, LILACS and ZETOC in July 2007. We also handsearched Chinese medical journals at Peking Union Medical College Library in April 2007. SELECTION CRITERIA: We planned to include randomized and quasi-randomized clinical trials in which one arm of the study involved acupuncture treatment. DATA COLLECTION AND ANALYSIS: Two authors independently evaluated the search results against the inclusion and exclusion criteria. Discrepancies were resolved by discussion. MAIN RESULTS: We found no randomized clinical trials and subsequently no meta-analysis was conducted. Evidence was limited to a few case series of small sample size. AUTHORS' CONCLUSIONS: At this time, it is impossible to draw reliable conclusions from the available data to support the use of acupuncture for the treatment of glaucoma. Since most glaucoma patients currently cared for by ophthalmologists do not use non-traditional therapy, the clinical practice decisions will have to be based on physician judgement and patients' value given this lack of data in the literature.

A structural hypothesis for the transition between bent and extended conformations of the leukocyte beta2 integrins.

Integrins mediate cell adhesion in response to activation signals that trigger conformational changes within their ectodomain. It is thought that a compact bent conformation of the molecule represents its physiological low affinity state and extended conformations its active state. We have determined the structure of two integrin fragments of the beta2 subunit. The first structure, consisting of the plexin-semaphorin-integrin domain, hybrid, integrin-epidermal growth factor 1 (I-EGF1), and I-EGF2 domains (PHE2), showed an L-shaped conformation with the bend located between the I-EGF1 and I-EGF2 domains. The second structure, which includes, in addition, the I-EGF3 domain, showed an extended conformation. The major reorientation of I-EGF2 with respect to the other domains in the two structures is accompanied by a change of torsion angle of the disulfide bond between Cys(461)-Cys(492) by 180 degrees and the conversion of a short alpha-helix (residues Ser(468)-Cys(475)) into a flexible coil. Based on the PHE2 structure, we introduced a disulfide bond between the plexin-semaphorin-integrin domain and I-EGF2 domains in the beta2 subunit. The resultant alphaLbeta2 integrin (leukocyte function-associated antigen-1) variant was locked in a bent state and could not be detected with the monoclonal antibody KIM127 in Mg(2+)/EGTA. However, it retained the binding activity to ICAM-1. These results provide a structural hypothesis for our understanding of the transition between the resting and active states of leukocyte function-associated antigen-1.

The cytosolic protein talin induces an intermediate affinity integrin alphaLbeta2.

The integrin alphaLbeta2 mediates leukocyte adhesion and migration that are required for a functional immune system. It is known that inside-out signaling triggers alphaLbeta2 conformational changes, which affect its ligand-binding affinity. At least three alphaLbeta2 affinity states (low, intermediate, and high) were described. The cytosolic protein talin connects alphaLbeta2 to the actin filament. The talin head domain is also known to activate alphaLbeta2 ligand binding. However, it remains to be determined whether talin promotes an intermediate or high affinity alphaLbeta2. In this study using transfectants and T cells, we showed that talin induced an intermediate affinity alphaLbeta2 that adhered constitutively to its ligand intercellular adhesion molecule (ICAM)-1 but not ICAM-3. Adhesion to ICAM-3 was induced when an additional exogenous activating agent was included. Similar profiles were observed with soluble ICAMs. In addition, the intermediate affinity alphaLbeta2 induced by talin allowed adhesion and migration of T cells on immobilized ICAMs.