A recent paper in BMC Developmental Biology describes the development of the annulus of the mouse... more A recent paper in BMC Developmental Biology describes the development of the annulus of the mouse sperm cell, but much remains to be learnt about sperm cells despite their importance in human fertility.
The International Journal of Biochemistry & Cell Biology, 2008
The mechanisms that co-ordinate centrosome maturation and the migration of human cells remain elu... more The mechanisms that co-ordinate centrosome maturation and the migration of human cells remain elusive. Protein phosphatase 4 (Ppp4) is a ubiquitous protein serine/threonine phosphatase in eukaryotes that is enriched at centrosomes. HEK293 cells cultures depleted to 30% Ppp4c levels by lentivirus-delivered stable gene silencing were delayed in mitosis at the prometaphase/metaphase boundary and displayed cells with aberrant chromosome organisation and microtubules unconnected to the centrosomes. The levels of ␣and ␥-tubulin and aurora A were decreased; in mitotic cells, the cytological localisations of polo-like kinase 1, ␣and ␥-tubulin and aurora A were aberrant and the phosphorylation of Aurora A-Thr 288 was decreased. The novel localisation of endogenous Ppp4 regulatory subunit, R3A, to centrosomes in human mitotic cells suggests that a Ppp4c-R2-R3 trimeric complex mediates centrosome maturation. We demonstrate for the first time that human cells depleted to 30% Ppp4c showed severely decreased migration and exhibit decreased levels of both total -actin and filamentous actin in cell extensions, filopodia and lamellopodia-like structures. Our studies show that Ppp4c is required for the organisation of the actin cytoskeleton at the leading edge of human cells during migration. We also demonstrate that the active forms of the RhoGTPases, Rac1 and Cdc42, are substantially decreased in the presence and absence of growth factor in Ppp4c depleted cells, implicating Ppp4c in the regulation of these GTPases. The results suggest that Ppp4c-R2-R3 complexes may co-ordinate centrosome maturation and cell migration via regulation of RhoGTPases and that Ppp4 may be a useful anticancer target.
Altered growth and polyamine catabolism following exposure of the chocolate spot pathogen Botrytis fabae to the essential oil of Ocimum basilicum
Mycologia, 2005
Biomass of the fungal pathogen Botrytis fabae in liquid culture amended with two chemotypes of th... more Biomass of the fungal pathogen Botrytis fabae in liquid culture amended with two chemotypes of the essential oil of basil, Ocimum basilicum, was reduced significantly at concentrations of 50 ppm or less. The methyl chavicol chemotype oil increased the activity of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC), but polyamine concentrations were not significantly altered. In contrast, the linalol chemotype oil decreased AdoMetDC activity in B. fabae, although again polyamine concentrations were not altered significantly. However activities of the polyamine catabolic enzymes diamine oxidase (DAO) and polyamine oxidase (PAO) were increased significantly in B. fabae grown in the presence of the essential oil of the two chemotypes. It is suggested that the elevated activities of DAO and PAO may be responsible, in part, for the antifungal effects of the basil oil, possibly via the generation of hydrogen peroxide and the subsequent triggering of programmed cell death.
Quantitative mass spectrometry-based proteomics is a vital tool in modern life science research. ... more Quantitative mass spectrometry-based proteomics is a vital tool in modern life science research. In contrast to the popularity of approaches for relative protein quantitation, the widespread use of absolute quantitation has been hampered by inefficient and expensive production of labeled protein standards. To optimize production of isotopically labeled standards, we genetically modified a commonly employed protein expression Escherichia coli strain, BL21 (DE3), to construct an auxotroph for arginine and lysine. This bacterial strain allows low-cost, high-level expression of fully labeled proteins with no conversion of labeled arginine to proline. In combination with a fluorescence-based quantitation of standards and nontargeted LCÀMS/MS analysis of unfractionated total cell lysates, this strain was used to determine the copy number of a post-translational modifier, small ubiquitin-like modifier (SUMO-2), in HeLa, human sperm, and chronic lymphocytic leukemia cells. By streamlining and improving the generation of labeled standards, this production system increases the breadth of absolute quantitation by mass spectrometry and will facilitate a far wider uptake of this important technique than previously possible.
Background: Ca 2ϩ signals, elicited by cues from the oocyte and female tract, regulate human sper... more Background: Ca 2ϩ signals, elicited by cues from the oocyte and female tract, regulate human sperm behavior. Results: CatSper channel activation (flagellum) and Ca 2ϩ store mobilization (neck) caused similar [Ca 2ϩ ] i elevation but induced functionally different behaviors. Conclusion: Sperm motility pattern is determined by the site of Ca 2ϩ mobilization. Significance: Selection of Ca 2ϩ signaling components and/or regulation of their availability for activation controls human sperm behavior.
study question: Do cell penetrating peptides (CPPs) translocate into spermatozoa and, if so, coul... more study question: Do cell penetrating peptides (CPPs) translocate into spermatozoa and, if so, could they be utilized to deliver a much larger protein cargo? summary answer: Chemically diverse polycationic CPPs rapidly and efficiently translocate into spermatozoa. They exhibit differential accumulation within intracellular compartments without detrimental influences upon cellular viability or motility but they are relatively ineffective in transporting larger proteins.
study question: What is the prevalence of defects in the Ca 2+ -signalling pathways mediating hyp... more study question: What is the prevalence of defects in the Ca 2+ -signalling pathways mediating hyperactivation (calcium influx and store mobilization) among donors and sub-fertile patients and are they functionally significant, i.e. related to fertilization success at IVF? summary answer: This study identifies, for the first time, the prevalence of Ca 2+ store defects in sperm from research donors, IVF and ICSI patients. It highlights the biological role and importance of Ca 2+ signalling (Ca 2+ store mobilization) for fertilization at IVF.
PP1 (protein phosphatase 1) is a ubiquitously expressed serine/ threonine-specific protein phosph... more PP1 (protein phosphatase 1) is a ubiquitously expressed serine/ threonine-specific protein phosphatase whose activity towards different substrates appears to be mediated via binding to specific proteins that play critical regulatory and targeting roles. In the present paper we report the cloning and characterization of a new protein, termed SARP (several ankyrin repeat protein), which is shown to interact with all isoforms of PP1 by a variety of techniques. A region encompassing a consensus PP1-binding motif in SARP (K 354 VHF 357 ) modulates endogenous SARP-PP1 activity in mammalian cells. This SARP-PP1 interaction motif lies partially within the first ankyrin repeat in contrast with other proteins [53BP2 (p53 binding protein 2), MYPT1/M 110 /MBS (myosin binding protein of PP1) and TIMAP (transforming growth factor β inhibited, membrane-associated protein)], where a PP1-binding motif precedes the ankyrin repeats. Alternative mRNA splicing produces several isoforms of SARP from a single human gene at locus 11q14. SARP1 and/or SARP2 (92-95 kDa) are ubiquitously expressed in all tissues with high levels in testis and sperm, where they are shown to interact with both PP1γ 1 and PP1γ 2 . SARP3 (65 kDa) is most abundant in brain where SARP isoforms interact with both PP1α and PP1γ 1 . SARP is highly abundant in the nucleus of mammalian cells, consistent with the putative nuclear localization signal at the N-terminus. The presence of a leucine zipper near the C-terminus of SARP1 and SARP2, and the binding of mammalian DNA to SARP2, suggests that SARP1 and SARP2 may be transcription factors or DNA-associated proteins that modulate gene expression.
Sperm dysfunction is the single most common cause of infertility, yet what is remarkable is that,... more Sperm dysfunction is the single most common cause of infertility, yet what is remarkable is that, there is no drug a man can take or add to his spermatozoa in vitro to improve fertility. One reason for the lack of progress in this area is that our understanding of the cellular and molecular workings of the mature spermatazoon is limited. However, over the last few years there has been considerable progress in our knowledge base and in addressing new methods to diagnose sperm dysfunction. We review the current state of the field and provide insights for further development. We conclude that: (i) there is little to be gained from more studies identifying/categorizing various populations of men using a basic semen assessment, where an effort is required in making sure the analysis is performed in an appropriate high quality way; (ii) technological development is likely to bring the reality of sperm function testing closer to implementation into the clinical pathways. In doing this, these assays must be robust, cheap (or more appropriately termed cost effective), easy to use and clinically useful; and (iii) clinical necessity, e.g., the need to identify the highest quality spermatozoon for injection is driving basic research forward. This is an exciting time to be an andrologist and, likely, a fruitful one.
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Papers by Senga Oxenham