Jeffrey Smith

Activation of the Cdc42p GTPase by cyclin-dependent protein kinases in budding yeast

Cyclin‐dependent kinases (CDKs) trigger essential cell cycle processes including critical events in G1 phase that culminate in bud emergence, spindle pole body duplication, and DNA replication. Localized activation of the Rho‐type GTPase Cdc42p is crucial for establishment of cell polarity during G1, but CDK targets that link the Cdc42p module with cell growth and cell cycle commitment have remained largely elusive. Here, we identify the GTPase‐activating protein (GAP) Rga2p as an important substrate related to the cell polarity function of G1 CDKs. Overexpression of RGA2 in the absence of functional Pho85p or Cdc28p CDK complexes is toxic, due to an inability to polarize growth. Mutation of CDK consensus sites in Rga2p that are phosphorylated both in vivo and in vitro by Pho85p and Cdc28p CDKs results in a loss of G1 phase‐specific phosphorylation. A failure to phosphorylate Rga2p leads to defects in localization and impaired polarized growth, in a manner dependent on Rga2p GAP function. Taken together, our data suggest that CDK‐dependent phosphorylation restrains Rga2p activity to ensure appropriate activation of Cdc42p during cell polarity establishment. Inhibition of GAPs by CDK phosphorylation may be a general mechanism to promote proper G1‐phase progression.

Multiple substitution of protons by sodium ions in sodiated oligoglycines

Abstract Multiply-sodiated ions were observed by electrospraying oligoglycines and their N-acetylated and O-amidated derivatives in the presence of sodium hydroxide. These ions have all their hydrogens in the peptide linkages replaced by sodiums; one hydrogen in the C-terminal amide and the hydrogen in the C-terminal carboxylic group are also replaced. The N-terminal amine hydrogens are unreactive. These results are consistent with earlier postulations [Int. J. Mass Spectrom. 192 (1999) 303, J. Am. Soc. Mass Spectrom. 11 (2000) 967], and apparently confirm the gas-phase origin of these ions (formed in the electrospray source and/or the lens region). Collision-induced dissociation of multiply-sodiated oligoglycines showed that the major product ions are C-terminal ions.

Identification of lysophosphatidylcholine (LPC) and platelet activating factor (PAF) from PC12 cells and mouse cortex using liquid chromatography/multi‐stage mass spectrometry (LC/MS3)

Lipids play essential roles in cellular structural support, energy storage and signal transduction. Recently, mass spectrometry (MS) has been used to produce three-dimensional maps that elucidate the lipid composition of complex cellular lysates. The identification of individual lipids within these maps is slow and requires the synthesis and spiking of each candidate lipid. We present a novel MS-based technique that rapidly elucidates the atomic connectivity of the fatty acid/alcohol substituent on the sn-1 position of several different families of glycerophosphocholine-containing lipids within the confines of a chromatographic separation. Sodiated lipid species were fragmented to produce radical cations which lost successive methylene groups upon further collisional activation to reveal the identity of the parent molecule. This approach was demonstrated to be effective on isobaric members of the lysophosphatidylcholine (LPC) and platelet activating factor (PAF) families of glycerophospholipids. We demonstrate the application of this technique to unambiguously identify these species within complex cellular lysates and tissue extracts.

Current status and prospects for development of a vaccine against Trichomonas vaginalis infections.

Trichomonas vaginalis is a sexually transmitted pathogen with an annual worldwide incidence of over 276 million infections, the highest of all curable and non-viral STI. A large proportion of cases are asymptomatic and under-diagnosed with conventional diagnostic tools. Infection has important maternal and fetal health consequences and can lead to a higher probability of HIV transmission and susceptibility. Lack of affordable accurate diagnostic tests globally and metronidazole resistance hinder T. vaginalis control efforts. Based on data from current vaccination studies in animal models, a human vaccine is achievable to intervene on the substantial incidence of infection.

Meganuclease targeting of PCSK9 in macaque liver leads to stable reduction in serum cholesterol

Clinical translation of in vivo genome editing to treat human genetic diseases requires thorough preclinical studies in relevant animal models to assess safety and efficacy. A promising approach to treat hypercholesterolemia is inactivating the secreted protein PCSK9, an antagonist of the LDL receptor. Here we show that single infusions in six non-human primates of adeno-associated virus vector expressing an engineered meganuclease targeting PCSK9 results in dose-dependent disruption of PCSK9 in liver, as well as a stable reduction in circulating PCSK9 and serum cholesterol. Animals experienced transient, asymptomatic elevations of serum transaminases owing to the formation of T cells against the transgene product. Vector DNA and meganuclease expression declined rapidly, leaving stable populations of genome-edited hepatocytes. A second-generation PCSK9-specific meganuclease showed reduced off-target cleavage. These studies demonstrate efficient, physiologically relevant in vivo editing in non-human primates, and highlight safety considerations for clinical translation.

Inducing Immune Protection Against Trichomonas vaginalis: A Novel Vaccine Approach to Prevent HIV Transmission

Human immunodeficiency virus (HIV) infection is a pandemic that affects all parts of the globe. Current treatments fail to cure disease and for this reason there is significant interest in producing a vaccine to prevent HIV transmission, but clinical trials have proved disappointing. For this reason it is important to consider alternative measures to control incidence and prevalence of the disease. Trichomonas vaginalis is a highly prevalent and under-diagnosed sexually transmitted infection that facilitates transmission of and susceptibility to HIV infection (McClelland et al., 2007; Mavedzenge et al., 2010). Although current treatment is effective the disease is still poorly controlled and there are concerns about increasing levels of drug resistance (Upcroft & Upcroft, 2001). Efforts to research disease mechanisms and immune response with consideration to a rational vaccine design approach should be investigated as a potential method to reduce global incidence of T. vaginalis infection. A reliable murine model of T. vaginalis infection has been established with symptoms in female mice mimicking those seen in women (from vaginitis/vulvitis and discharge to asymptomatic but culture-positive for infection). Using this model it has been shown that vaccinating mice (by injection of trichomonad cells with adjuvant) protects the animals from subsequent vaginal infection (Abraham et al., 1996). By studying immune responses in mice, factors that are critical for immunological protection can be elucidated to create a “blueprint” for an effective human vaccine. Herein we provide an overview of the current understanding of T. vaginalis infection and epidemiology, methods of diagnosis and treatment, and implications of animal models for understanding disease mechanisms . We discuss how and why current T. vaginalis treatment protocols fail to control infection incidence with consideration as to how a T. vaginalis vaccine could overcome these obstacles and reduce disease burden. The association between T. vaginalis and HIV is examined and the potential for reducing HIV infection rates by lowering T. vaginalis incidence is elucidated. A vaccine against T. vaginalis would provide long term protection that could be more successful than treatment in controlling the spread of this very common disease. As T. vaginalis infection is a clear risk factor for HIV acquisition, it is our belief that this approach would also be effective in ameliorating HIV incidence and prevalence, especially in areas such as South and Southeast Asia and sub-Saharan Africa where HIV and T. vaginalis are endemic (United Nations, 2009; World Health Organization, 2001).