Ariela Gordon-Shaag

Ca2+-Dependent Desensitization of TRPV2 Channels Is Mediated by Hydrolysis of Phosphatidylinositol 4,5-Bisphosphate

TRPV2 is a member of the transient receptor potential family of ion channels involved in chemical and thermal pain transduction. Unlike the related TRPV1 channel, TRPV2 does not appear to bind either calmodulin or ATP in its N-terminal ankyrin repeat domain. In addition, it does not contain a calmodulin-binding site in the distal C-terminal region, as has been proposed for TRPV1. We have found that TRPV2 channels transiently expressed in F-11 cells undergo Ca2+-dependent desensitization, similar to the other TRPVs, suggesting that the mechanism of desensitization may be conserved in the subfamily of TRPV channels. TRPV2 desensitization was not altered in whole-cell recordings in the presence of calmodulin inhibitors or on coexpression of mutant calmodulin but was sensitive to changes in membrane phosphatidylinositol 4,5-bisphosphate (PIP2), suggesting a role of membrane PIP2 in TRPV2 desensitization. Simultaneous confocal imaging and electrophysiological recording of cells expressing TRPV2 and a fluorescent PIP2-binding probe demonstrated that TRPV2 desensitization was concomitant with depletion of PIP2. We conclude that the decrease in PIP2 levels on channel activation underlies a major component of Ca2+-dependent desensitization of TRPV2 and may play a similar role in other TRP channels.

Prevalence and associated factors of keratoconus in Jerusalem: a cross-sectional study.

Purpose: To determine the prevalence and associated factors for keratoconus in a college student population sample in Jerusalem. Methods: Volunteers participated in this cross-sectional study. Videokeratography was performed on both eyes of each subject who also completed an anonymous questionnaire. Keratoconus was defined by cone apex ≥ 50D, inferior-superior dioptric difference ≥ 3.5 diopters, as well as positive results from the software indices KISA, KCI and KSI. The association between independent predictors and keratoconus was analyzed using multivariate logistic regression analysis. Results: Of a total of 987 volunteers, 981 (mean age 24.4) were included. The prevalence of keratoconus among all subjects was 2.34% (95% confidence interval [CI] 1.4–3.3). It was significantly higher in men (4.91%, CI 2.6–7.3) than women (1.07%, CI 0.3–1.9) but not between Israeli Arabs (3.0%, CI 0.6–5.4) and Israeli Jews (2.2%, CI 1.2–3.3). Keratoconus was significantly associated with positive family history of the disease (Odds Ratio [OR] 17.1, CI 5.0–57.8, P < 0.001), male gender (OR 5.4, CI 2.1–14.3, P = 0.001) and atopy (OR 3.0, CI 1.2–7.6, P = 0.02), but not with eye rubbing. Conclusions: The prevalence of keratoconus in Jerusalem was found to be much higher than that seen in other parts of the world, except India. This may be related to a combination of genetic and environmental factors. Positive family history, male gender and atopy were shown to be significant predictors. The results of this study signal a need for public health outreach and intervention for keratoconus.

Membrane lipid modulations remove divalent open channel block from TRP-like and NMDA channels.

Open channel block is a process in which ions bound to the inside of a channel pore block the flow of ions through that channel. Repulsion of the blocking ions by depolarization is a known mechanism of open channel block removal. For the NMDA channel, this mechanism is necessary for channel activation and is involved in neuronal plasticity. Several types of transient receptor potential (TRP) channels, including the Drosophila TRP and TRP-like (TRPL) channels, also exhibit open channel block. Therefore, removal of open channel block is necessary for the production of the physiological response to light. Because there is no membrane depolarization before the light response develops, it is not clear how the open channel block is removed, an essential step for the production of a robust light response under physiological conditions. Here we present a novel mechanism to alleviate open channel block in the absence of depolarization by membrane lipid modulations. The results of this study show open channel block removal by membrane lipid modulations in both TRPL and NMDA channels of the photoreceptor cells and CA1 hippocampal neurons, respectively. Removal of open channel block is characterized by an increase in the passage-rate of the blocking cations through the channel pore. We propose that the profound effect of membrane lipid modulations on open channel block alleviation, allows the productions of a robust current in response to light in the absence of depolarization.

Cellular Transcription Factor Sp1 Recruits Simian Virus 40 Capsid Proteins to the Viral Packaging Signal, ses

ABSTRACT Simian virus 40 (SV40) capsid assembly occurs in the nucleus. All three capsid proteins bind DNA nonspecifically, raising the dilemma of how they attain specificity to the SV40 minichromosome in the presence of a large excess of genomic DNA. The SV40 packaging signal, ses, which is required for assembly, is composed of multiple DNA elements that bind transcription factor Sp1. Our previous studies showed that Sp1 participates in SV40 assembly and that it cooperates in DNA binding with VP2/3. We hypothesized that Sp1 recruits the capsid proteins to the viral minichromosome, conferring upon them specific DNA recognition. Here, we have tested the hypothesis. Computer analysis showed that the combination of six tandem GC boxes at ses is not found at cellular promoters and therefore is unique to SV40. Cooperativity in DNA binding between Sp1 and VP2/3 was not abolished at even a 1,000-fold excess of cellular DNA, providing strong support for the recruitment hypothesis. Sp1 also binds VP1 and cooperates with VP1 in DNA binding. VP1 pentamers (VP15) avidly interact with VP2/3, utilizing the same VP2/3 domain as described for polyomavirus. We conclude that VP15-VP2/3 building blocks are recruited by Sp1 to ses, where they form the nucleation center for capsid assembly. By this mechanism the virus ensures that capsid formation is initiated at a single site around its minichromosome. Sp1 enhances the formation of SV40 pseudovirions in vitro, providing additional support for the model. Analyses of Sp1 and VP3 deletion mutants showed that Sp1 and VP2/3 bind one another and cooperate in DNA binding through their DNA-binding domains, with additional contacts outside these domains. VP1 contacts Sp1 at residues outside the Sp1 DNA-binding domain. These and additional data allowed us to propose a molecular model for the VP15-VP2/3-DNA-Sp1 complex.

The Genetic and Environmental Factors for Keratoconus

Keratoconus (KC) is the most common cornea ectatic disorder. It is characterized by a cone-shaped thin cornea leading to myopia, irregular astigmatism, and vision impairment. It affects all ethnic groups and both genders. Both environmental and genetic factors may contribute to its pathogenesis. This review is to summarize the current research development in KC epidemiology and genetic etiology. Environmental factors include but are not limited to eye rubbing, atopy, sun exposure, and geography. Genetic discoveries have been reviewed with evidence from family-based linkage analysis and fine mapping in linkage region, genome-wide association studies, and candidate genes analyses. A number of genes have been discovered at a relatively rapid pace. The detailed molecular mechanism underlying KC pathogenesis will significantly advance our understanding of KC and promote the development of potential therapies.

Aberrations and Topography in Normal, Keratoconus-Suspect, and Keratoconic Eyes

Purpose. This study was designed to compare higher order aberrations of the cornea and of the eye with inferior-superior (I-S) corneal topographic values in keratoconic eyes. Methods. We studied 92 eyes from 78 subjects: 21 eyes of 14 subjects with suspected keratoconus, 23 eyes of 16 subjects with manifest keratoconus, and 48 eyes of 48 subjects without keratoconus using the L80 wave+, an instrument which can measure corneal topography and aberrations simultaneously with a large dynamic range making it possible to evaluate higher order aberrations to the seventh order of the Zernike polynomial function series. Results. All ocular and corneal higher order aberrations were found to be significantly higher for keratoconic than normal eyes, but for suspected keratoconus the results were mixed. Corneal aberrations were higher than ocular aberrations due to compensation from the internal aberrations. For manifest keratoconus, the corneal and ocular vertical coma displayed the largest difference being 38.6 and 78.5 times higher, respectively, than normal eyes, while the largest differences for suspected keratoconus were only 5.3 and 4.0 times higher, respectively. On the other hand, inferior-superior dioptric asymmetry was 9.4 and 37.3 times higher for suspected keratoconus and keratoconic eyes, respectively, than normal eyes. The separation of normality curves between suspected keratoconus and normal eyes was 28.6% for I-S and 14.3% for both corneal vertical coma and corneal total coma. Conclusions. Although corneal vertical coma and, to a lesser extent, ocular vertical coma were found to be good indicators for the detection of keratoconic eyes, the traditional corneal topographic value such as the inferior-superior dioptric asymmetry remains an important predictor for identifying suspected keratoconus. However, ocular vertical coma and ocular higher order total root mean square also represent a good means of identifying suspected keratoconus.

Mechanism of Ca2+-dependent desensitization in TRP channels

The 30+ members of the family of TRP channels are diverse in their physiological roles, yet the mechanisms that regulate their gating may be conserved. In particular, all TRP channels show an activity-dependent inhibition which is mediated by Ca2+. The mechanism by which Ca2+ inhibits TRP channels is currently a matter of intense debate, with Ca2+-regulated kinases, phosphatases, phospholipases, and calmodulin all proposed to be involved. In this review, we will discuss different mechanisms for Ca2+-dependent desensitization in TRP channels. We will conclude with a model that focuses on Ca2+-dependent activation of phospholipase C and Ca2+ binding to calmodulin and propose that the phospholipase C and calmodulin pathways are structurally and functionally coupled.

The Abundant Nuclear Enzyme PARP Participates in the Life Cycle of Simian Virus 40 and Is Stimulated by Minor Capsid Protein VP3

ABSTRACT The abundant nuclear enzyme poly(ADP-ribose) polymerase (PARP) functions in DNA damage surveillance and repair and at the decision between apoptosis and necrosis. Here we show that PARP binds to simian virus 40 (SV40) capsid proteins VP1 and VP3. Furthermore, its enzymatic activity is stimulated by VP3 but not by VP1. Experiments with purified mutant proteins demonstrated that the PARP binding domain in VP3 is localized to the 35 carboxy-terminal amino acids, while a larger peptide of 49 amino acids was required for full stimulation of its activity. The addition of 3-aminobenzamide (3-AB), a known competitive inhibitor of PARP, demonstrated that PARP participates in the SV40 life cycle. The titer of SV40 propagated on CV-1 cells was reduced by 3-AB in a dose-dependent manner. Additional experiments showed that 3-AB did not affect viral DNA replication or capsid protein production. PARP did not modify the viral capsid proteins in in vitro poly(ADP-ribosylation) assays, implying that it does not affect SV40 infectivity. On the other hand, it greatly reduced the magnitude of the host cytopathic effects, a hallmark of SV40 infection. Additional experiments suggested that the stimulation of PARP activity by VP3 leads the infected cell to a necrotic pathway, characterized by the loss of membrane integrity, thus facilitating the release of mature SV40 virions from the cells. Our studies identified a novel function of the minor capsid protein VP3 in the recruitment of PARP for the SV40 lytic process.

Characteristics of 244 patients with keratoconus seen in an optometric contact lens practice

The purpose of this study was to describe the characteristics of keratoconic patients seen in a specialised contact lens practice from a general population with a high prevalence of the disease.

Is consanguinity a risk factor for keratoconus

Purpose To determine whether consanguinity is a risk factor for keratoconus (KC). Methods A questionnaire was distributed to all patients presenting to St. John Eye Hospital, Jerusalem, Israel. Questionnaire included data on demographic characteristics and potential risk factors. Patients were divided into two groups: cases with KC, in at least one eye, who were diagnosed by the attending ophthalmologist on the basis of abnormal corneal topography and at least one of the common signs of the disease; and controls presenting for problems other than KC and free of systemic and ocular conditions associated with KC. Multivariate logistic analyses were performed to identify risk factors for KC. Results Seventy cases and 140 controls participated in the study. Groups were similar with respect to sex and age. Univariate analyses found a significant association between KC and parental first-cousin consanguinity, eye rubbing, allergy, positive family history, education (>12 years), and sunglass wear, whereas asthma, eczema, smoking, and second-cousin consanguinity were not. Multivariate analyses showed that total consanguinity (first-cousin and second-cousin) (adjusted odds ratio, 3.96; p = 0.001), eye rubbing and absence of sunglass wear were significant risk factors. Education was also associated with KC, but family history was not so in the multivariate analysis. Conclusions This study supports the hypothesis that consanguinity is a significant risk factor for KC and provides strong support for a genetic contribution to the disease. Wearing sunglasses in this environment is beneficial, and the study confirmed that eye rubbing, allergy, and education are also significantly associated with KC after adjusting for other predictors.