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Biomedical subjects

K T Jones

Publications and source records attributed to K T Jones.

At least 19 recordsLinked to original sources
PubMed2000

Injections of porcine sperm extracts trigger fertilization-like calcium oscillations in oocytes of a marine worm.

The precise mechanisms by which sperm trigger calcium transients in eggs or oocytes during fertilization remain unknown. Based on time-lapse confocal microscopy, we show that intracellular injections of porcine sperm extracts cause the oocytes of a marine nemertean worm to undergo repetitive calcium oscillations resembling those obtained during normal fertilizations. Such findings are consistent with the view that fertilization involves a soluble sperm factor (SF) which is capable of eliciting calcium transients without binding to externally situated receptors on the oocyte plasmalemma. This study also describes for the first time the wave-like propagation patterns of SF-induced calcium transients that are generated in a heterologous combination of gametes obtained from different phyla of animals. Such cross-reactivity between distantly related taxa suggests that the intracellular signaling pathways triggered by sperm factors can be well conserved.

Animals↗
PubMed2000

Different Ca2+-releasing abilities of sperm extracts compared with tissue extracts and phospholipase C isoforms in sea urchin egg homogenate and mouse eggs.

A soluble phospholipase C (PLC) from boar sperm generates InsP(3) and hence causes Ca(2+) release when added to sea urchin egg homogenate. This PLC activity is associated with the ability of sperm extracts to cause Ca(2+) oscillations in mammalian eggs following fractionation. A sperm PLC may, therefore, be responsible for causing the observed Ca(2+) oscillations at fertilization. In the present study we have further characterized this boar sperm PLC activity using sea urchin egg homogenate. Consistent with a sperm PLC acting on egg PtdIns(4,5)P(2), the ability of sperm extracts to release Ca(2+) was blocked by preincubation with the PLC inhibitor U73122 or by the addition of neomycin to the homogenate. The Ca(2+)-releasing activity was also detectable in sperm from other species and in whole testis extracts. However, activity was not observed in extracts from other tissues. Moreover recombinant PLCbeta1, -gamma1, -gamma2, -delta1, all of which had higher specific activities than boar sperm extracts, were not able to release Ca(2+) in the sea urchin egg homogenate. In addition these PLCs were not able to cause Ca(2+) oscillations following microinjection into mouse eggs. These results imply that the sperm PLC possesses distinct properties that allow it to hydrolyse PtdIns(4,5)P(2) in eggs.

Animals↗
PubMed1999

The soluble sperm factor that causes Ca2+ release from sea-urchin (Lytechinus pictus) egg homogenates also triggers Ca2+ oscillations after injection into mouse eggs.

Cytosolic extracts of boar sperm contain a soluble phospholipase C (PLC) activity that induces Ca2+ release in sea-urchin (Lytechinus pictus) egg homogenates and an uncharacterized protein factor that causes Ca2+ oscillations when injected into mammalian eggs. In the present study we fractionated boar sperm extracts on three different FPLC chromatographic columns and found that the fractions that caused maximal Ca2+ release in sea-urchin egg homogenates were also the ones that triggered Ca2+ oscillations in mouse eggs. Our data suggests that the sperm factor which triggers Ca2+ oscillations in eggs contains a PLC and not the 33 kDa glucosamine deaminase previously suggested to be one its components.

Animals↗
PubMed1999

The antiproliferative effect of lectin from the edible mushroom (Agaricus bisporus) on human keratinocytes: preliminary studies on its use in psoriasis.

Lectins or agglutinins are proteins with affinity for specific sugar residues. Peanut agglutinin (PNA) and the lectin from the edible mushroom (Agaricus bisporus, ABL) both bind to the disaccharide galactosyl beta-1,3-N-acetyl galactosamine alpha-. This is expressed in keratinocytes as an O-linked chain on CD44, a polymorphic membrane glycoprotein. Many lectins are mitogens and PNA is a mitogen for colonic epithelial cells. However, ABL reversibly inhibits proliferation of colonic cancer cell lines without cytotoxicity and thus has therapeutic potential in situations such as psoriasis where proliferation is increased. We have therefore investigated the effect of ABL on the growth of normal human cultured keratinocytes and a human papilloma virus (HPV)-transformed cell line. In a 5-day dose-response study, keratinocyte growth was greatly reduced by 1.0 microg/mL ABL and completely inhibited by 3.0 microg/mL ABL (ANOVA, P < 0.0001). Exposure to 1.0 microg/mL ABL for only 8 h gave the same growth inhibition as did continued exposure for 3 days. No cytotoxic or morphological changes were observed. An HPV-immortalized cell line was relatively resistant to ABL: in a 5-day dose-response study, exposure to 30 microg/mL was required to inhibit cell growth completely. Topical application of ABL 0.01% or 0.1% to normal human skin caused no change in skin erythema, blood flow or thickness compared with vehicle or baseline (n = 6). ABL 0. 1% in white soft paraffin was compared with vehicle in 11 psoriatic patients, using comparative contralateral plaques. Twice daily application for 2 weeks showed no significant difference from vehicle-treated sites, although the skin thickness of plaques fell from 5.3 +/- 0.4 (n = 11, mean +/- SEM) to 4.1 +/- 0.3 mm. In view of the in vitro results further studies are warranted, particularly if means can be found to improve the epidermal penetration of the relatively large ABL molecule (60 kDa).

Adult↗
PubMed1998

A mammalian sperm cytosolic phospholipase C activity generates inositol trisphosphate and causes Ca2+ release in sea urchin egg homogenates.

Injection of sperm extracts triggers Ca2+ oscillations in mammalian eggs similar to those seen at fertilisation. Here, we show that addition of sperm extracts to sea urchin egg homogenates causes Ca2+ release and inositol 1,4,5-trisphosphate (InsP3) production. Furthermore depleting homogenates of phosphatidylinositol lipids using a phosphatidylinositol-specific phospholipase C blocked the sperm extract from causing InsP3 production and a Ca2+ rise. A response could be recovered by the addition of phosphatidylinositol 4,5-bisphosphate to either sperm extracts or egg homogenates. These data indicate that sperm extracts contain an InsP3-generating phospholipase C which may play a role in Ca2+ release at fertilisation.

Animals↗
PubMed1998

The passage of Ca2+ and fluorescent markers between the sperm and egg after fusion in the mouse.

Mouse sperm-egg fusion was examined using two-photon and confocal microscopy. A delay of several minutes occurred between the first observable event of fusion (which was the diffusion of Ca2+-sensitive dyes from egg into sperm) and any change in egg cytoplasmic Ca2+. When indo-1 dextran was used to obtain ratiometric two-photon images, there was no detectable local increase in egg cytoplasmic Ca2+ near the site of sperm fusion. However, the sperm underwent a Ca2+ transient which appeared to be coincident with the egg cytoplasm Ca2+ transient, which suggested that there was a high permeability pathway for Ca2+ between egg and sperm. To exclude this pathway from providing trigger Ca2+ for the egg transient, we reduced bathing [Ca2+] to approx. 18 microM and 13nM (with EGTA). In these conditions the first egg Ca2+ transient was not prevented, which makes an obligatory role for extracellular Ca2+ in the initiation of the egg Ca2+ transient unlikely. Both FITC-albumin (70 kDa) and 10 kDa dextran-linked Ca2+ indicators were able to diffuse into the sperm from the egg. In addition, phycoerythrin (240 kDa) rapidly diffused into the sperm shortly after fusion (but before any changes in Ca2+ occurred). This suggests that the 'pore(s)' that form during sperm-egg fusion must be at least 8 nm in diameter. These data are compatible with the idea that a diffusible sperm protein could trigger the observed changes in intracellular Ca2+ in the egg, but do not exclude the possibility that other second messengers are generated during sperm-egg fusion.

Animals↗
PubMed1998

Protein kinase C action at fertilization: overstated or undervalued?

At fertilization, the spermatozoon is generally held to generate two important second messengers, inositol trisphosphate and diacylglycerol. A similar situation arises when these signalling molecules are generated after a hormone binds to its plasma membrane receptor. This signalling mechanism releases intracellular Ca2+ which causes cortical granule release and initiates meiotic resumption. This review will examine the role played at fertilization by protein kinase C which is a primary target of diacylglycerol. The pharmacological agents phorbol esters, which mimic the action of diacylglycerol, when added to mammalian oocytes induce cortical granule release and may cause meiotic resumption. However, the originally accepted mechanism of fertilization is now questioned with the recent finding of a soluble sperm Ca2+-releasing factor expelled directly into the oocyte cytoplasm, bypassing any membrane receptor. Therefore, it is timely to re-evaluate the role played by protein kinase C at fertilization in light of a mechanism that may produce Ca2+ without producing diacylglycerol concomitantly. This article will examine the evidence implicating activation of protein kinase C in Ca2+ oscillations, cortical granule release and meiotic resumption. It will contend that pharmacological studies relying on the specificity of phorbol esters and other agonists, as well as inhibitors of protein kinase C, have produced conflicting interpretations of the role of this kinase at fertilization.

Calcium↗
PubMed1998

MYCIN II: design and implementation of a therapy reference with complex content-based indexing.

We describe the construction of MYCIN II, a prototype system that provides for content-based markup and search of a forthcoming clinical therapeutics textbook, Antimicrobial Therapy and Vaccines. Existing commercial search technology for digital references utilizes generic tools such as textword-based searches with geographical or statistical refinements. We suggest that the drawbacks of such systems significantly restrict their use in everyday clinical practice. This is in spite of the fact that there is a great need for the information contained within these same references. The system we describe is intended to supplement keyword searching so that certain important questions can be asked easily and can be answered reliably (in terms of precision and recall). Our method attacks this problem in a restricted domain of knowledge-clinical infectious disease. For example, we would like to be able to answer the class of questions exemplified by the following query: "What antimicrobial agents can be used to treat endocarditis caused by Eikenella corrodens?" We have compiled and analyzed a list of such questions to develop a concept-based markup scheme. This scheme was then applied within an HTML markup to electronically "highlight" passages from three textbook chapters. We constructed a functioning web-based search interface. Our system also provides semi-automated querying of PubMed using our concept markup and the user's actions as a guide.

Abstracting and Indexing↗
PubMed1997

A cytosolic sperm protein factor mobilizes Ca2+ from intracellular stores by activating multiple Ca2+ release mechanisms independently of low molecular weight messengers.

Ca2+ oscillations can be induced in mammalian eggs and somatic cells by microinjection of a cytosolic sperm protein factor. The nature of the sperm factor-induced Ca2+ signaling was investigated by adding sperm protein extracts to homogenates of sea urchin eggs, which contain multiple classes of Ca2+ release mechanisms. We show that the sperm factor mobilizes Ca2+ from non-mitochondrial Ca2+ stores in egg homogenates after a distinct latency. This latency is abolished by preincubation of sperm extracts with egg cytosol. The preincubation step is highly temperature-dependent and generates a high molecular weight, protein-based Ca2+-releasing agent that can also mobilize Ca2+ from purified egg microsomes. This Ca2+ release appears to be mediated via both inositol 1,4,5-trisphosphate and ryanodine receptors, since homologous desensitization of these two release mechanisms by their respective agonists inhibits further release by the sperm factor. However, sperm factor-induced Ca2+ release by these channels is independent of inositol 1,4, 5-trisphosphate or cADPR since antagonists of either of these two messengers did not block the Ca2+ release effected by the sperm factor. The sperm protein factor may cause Ca2+ release via an enzymatic step that generates a protein-based Ca2+-releasing agent.

Animals↗
PubMed1997

Unique protein kinase C profile in mouse oocytes: lack of calcium-dependent conventional isoforms suggested by rtPCR and Western blotting.

rtPCR and Western blotting were used to determine which members of the PKC family are present in both immature and mature mouse oocytes. Using isoform-specific PCR primers and antibodies PKC-delta and -lambda were detected while such techniques failed to observe the conventional isoforms of PKC-alpha, -beta, -gamma. This isoform profile was confirmed using an alternative PCR strategy, which allowed discrimination of PCR products derived from conventional and novel PKC isoforms. In addition PKC-epsilon, -eta, -theta and -zeta were not detected by rtPCR. These results suggest that the predominant isoforms in oocytes are PKC-delta and -lambda.

Animals↗
PubMed1997

Meiotic and mitotic Ca2+ oscillations affect cell composition in resulting blastocysts.

At fertilization periodic Ca2+ oscillations release oocytes from meiotic arrest. The present study examined whether these oscillations have a long-term role in pre- and postimplantation development, independent of their immediate effect. Sr(2+)-containing medium was used to induce oscillations during exit from meiosis and first embryonic mitosis and Sr(2+)-activated parthenotes were compared to ethanol-activated parthenotes and embryos generated by in vitro fertilization. After embryo culture, blastocysts were differentially stained for the inner cell mass and trophectoderm. It was found that oscillations both during exit from meiosis and during mitosis acted to increase the number of inner cell mass cells. In contrast, the trophectoderm cell number was largest in ethanol-activated parthenotes and smallest in fertilized embryos. Postimplantation development was also modestly improved by extending the time of exposure to Sr(2+)-containing medium. Together these data suggest that Ca2+ oscillations have a role in long-term embryonic events and that they provide more than merely a stimulus for meiotic resumption.

Animals↗
PubMed1996

A comparison of sperm- and IP3-induced Ca2+ release in activated and aging mouse oocytes.

Ca2+ release mechanisms in oocytes are highly sensitive and a number of agents including sperm and inositol trisphosphate (IP3) generate Ca2+ transients. Recently it was shown that this sensitivity decreases after fertilization and subsequent entry into the first mitotic cell cycle (Jones et al., Development 121, 3259-3266, 1995). In this study a similar decrease in the ability of IP3 to cause repetitive Ca2+ transients was observed in parthenogenetic embryos following activation with Sr2+, ethanol, or cycloheximide. This indicates that the decline in sensitivity of the Ca2+ releasing mechanism after oocyte activation is not associated with the fertilizing sperm. A similar decline in IP3-induced Ca2+ release was observed in metaphase II oocytes at 24 hr post hCG or later, although repetitive Ca2+ transients were induced in the aged oocytes after in vitro fertilization. Sperm-induced Ca2+ transients in aged oocytes were similar in duration and peak amplitude to younger oocytes, 15-18 hr post hCG. However, they showed a much reduced rate of rise which was also observed in younger oocytes after the intracellular stores had been depleted by thapsigargin. The results suggest that factors within the oocyte, such as store size, are important in enabling sperm to generate repetitive Ca2+ transients. Also, the Ca2+ release processes decline as the oocyte ages as well as after activation.

Animals↗
PubMed1996

A cell cycle-associated change in Ca2+ releasing activity leads to the generation of Ca2+ transients in mouse embryos during the first mitotic division.

We have used Ca2+-sensitive fluorescent dyes to monitor intracellular Ca2+ during mitosis in one-cell mouse embryos. We find that fertilized embryos generate Ca2+ transients at nuclear envelope breakdown (NEBD) and during mitosis. In addition, fertilized embryos arrested in metaphase using colcemid continue to generate Ca2+ transients. In contrast, parthenogenetic embryos produced by a 2-h exposure to strontium containing medium do not generate detectable Ca2+ transients at NEBD or in mitosis. However, when parthenogenetic embryos are cultured continuously in strontium containing medium Ca2+ transients are detected in mitosis but not in interphase. This suggests that mitotic Ca2+ transients are detected in the presence of an appropriate stimulus such as fertilization or strontium. The Ca2+ transient detected in fertilized embryos is not necessary for inducing NEBD since parthenogenetic embryos undergo nuclear envelope breakdown (NEBD). Also the first sign that NEBD is imminent occurs several minutes before the Ca2+ transient. The Ca2+ transient at NEBD appears to be associated with the nucleus since nuclear transfer experiments show that the presence of a karyoplast from a fertilized embryo is essential. Finally, we show that the intracellular Ca2+ chelator Bapta inhibits NEBD in fertilized and parthenogenetic embryos in a dose-dependent manner. These studies show that during mitosis there is an endogenous increase in Ca2+ releasing activity that leads to the generation of Ca2+ transients specifically during mitosis. The ability of Ca2+ buffers to inhibit NEBD regardless of the presence of global Ca2+ transients suggests that the underlying cell cycle-associated Ca2+ releasing activity may take the form of localized Ca2+ transients.

Animals↗
PubMed1996

Ca2+ release and the development of Ca2+ release mechanisms during oocyte maturation: a prelude to fertilization.

Oogenesis involves the production of an oocyte that can undergo fertilization and support early development. The stimulus that initiates embryogenesis is an increase in the concentration of intracellular Ca2+ in the cytoplasm of the oocyte at the time of fertilization. The development of the ability of the oocyte to release Ca2+ in response to the fertilizing spermatozoon is an essential step in the process of oogenesis. Mammalian oocytes are particularly useful for studying the development of Ca2+ signalling systems, owing to the series of Ca2+ oscillations generated at fertilization, compared with the monotonic Ca2+ increase seen in nonmammalian species. Recent evidence has revealed that Ca2+ release mechanisms are modified during oogenesis. The maximal sensitivity of Ca2+ release is reached in the final stages of oocyte maturation, just before the optimal time for fertilization. In this review, we consider the mechanism underlying Ca2+ release in mammalian oocytes and discuss how the release mechanisms are modified during oocyte maturation. The tight co-ordination of the differentiation of the Ca2+ signalling system with the development of the oocyte provides a means of ensuring successful activation at the time of fertilization. Finally, we consider the consequences for embryo development in circumstances in which the co-ordination is lost.

Animals↗
PubMed1995

Ionomycin, thapsigargin, ryanodine, and sperm induced Ca2+ release increase during meiotic maturation of mouse oocytes.

Fertilization of mature mouse oocytes triggered highly repetitive Ca2+ oscillations lasting 2-3 h. However, immature oocytes generated only two or three oscillations, which ceased within 1 h. Development of repetitive Ca2+ transients to sperm occurred late in oocyte maturation and was dependent on cytoplasmic modifications that were independent of cell cycle progression from metaphase I to metaphase II. Immature oocytes released significantly less Ca2+ from stores than mature oocytes in response to ionomycin and thapsigargin. Ryanodine had no effect on intracellular Ca2+ in maturing oocytes but stimulated an increase in Ca2+ in mature oocytes. The ability of ryanodine to increase Ca2+ levels was, however, strain-dependent. Preincubation of oocytes with thapsigargin or ryanodine significantly attenuated the normal fertilization Ca2+ response, causing a decrease in the number and the rate of rise of the transients. The inhibition of sperm-induced Ca2+ transients by ryanodine was independent of its ability to cause an immediate Ca2+ increase. Low concentrations of ryanodine had no effect on resting Ca2+ levels but inhibited Ca2+ oscillations at fertilization. Similarly Ca2+ oscillations were blocked in oocytes from a strain of mouse that showed no immediate Ca2+ increase with ryanodine. These results suggest that modifications in Ca2+ stores and ryanodine-sensitive Ca2+ release mechanisms during oocyte maturation play an important role in Ca2+ oscillations at fertilization.

Animals↗
PubMed1995

Intracellular calcium modulates the responses of human melanocytes to melanogenic stimuli.

Ultraviolet radiation (UVR), the synthetic diacyglycerol (DAG), 1-oleoyl-2-acetylglycerol (OAG), and cyclic AMP (cAMP) stimulants, including cholera toxin (CT) have all been shown to increase melanogenesis in cultured human melanocytes. Indirect evidence suggests that an increase in intracellular free Ca2+ ([Ca2+]i) may be important in stimulated melanogenesis. Therefore, to determine whether melanogenic responses are modulated by [Ca2+]i, the Ca2+ in the culture medium of melanocytes ([Ca2+]o) was raised from 70 microM to 1 mM. This switch in [Ca2+]o was associated with a biphasic increase in [Ca2+]i, with an early transient rise, over minutes, and a delayed sustained rise in [Ca2+]i, over hours. The early increase was blocked by nickel chloride (NiCl2), but not affected by depletion of [Ca2+]i stores by thapsigargin, suggesting that this [Ca2+]i rise was due to Ca2+ entry across the plasma membrane. Melanocytes cultured in the absence of CT had a reduced basal melanin content following the switch to 1 mM [Ca2+]o, but in the presence of CT, which acts by stimulating cAMP synthesis, the basal level was increased. Raising [Ca2+]o resulted in enhanced melanogenic responses to UVR and OAG, in the presence or absence of CT, suggesting that Ca(2+)-dependent mechanisms are important. UVR also stimulated a delayed rise in [Ca2+]i, over 24 h, but OAG did not. These results indicate that while [Ca2+]i is not essential for melanogenesis, it plays an important role in modulating the responses of melanocytes to melanogenic stimuli.

Calcium↗