Biomedical subjects

K T Jones

Publications and source records attributed to K T Jones.

36 records · Page 2Linked to original sources

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↗

PubMed1995

Intracellular calcium as a second messenger following growth stimulation of human keratinocytes.

The mitogenic effect of the neuropeptide substance P and bombesin was investigated in normal human keratinocytes in serum-free culture, both with and without the presence of epidermal growth factor (EGF). Although both neuropeptides induced a small increase in cell numbers in the presence of EGF, the response was much greater in its absence, and cell numbers increased to 200% of controls at 5 days. Changes in intracellular free calcium are frequently seen following mitogenic stimulation of cells, and this phenomenon was studied in individual keratinocytes. Epidermal growth factor (10 ng/ml) induced calcium transients in 57% (n = 21) of cells. The mean intracellular free calcium was 97 +/- 11 nM (mean +/- SEM) in quiescent cells, and the calcium transients reached approximately 250 nM for 3-4 min. In the presence of EGF, calcium transients were never observed with the addition of either substance P or bombesin. For EGF-deprived cultures, 20% of keratinocytes (n = 10) showed a large calcium transient following the addition of 500 nM bombesin, and 63% (n = 12) of cells gave calcium transients following the addition of 700 nM of substance P. Studies in calcium-free medium, and following depletion of intracellular calcium stores with thapsigargin, showed that all of the calcium transients were dependent on the presence of intracellular stores, but also partially mediated by an influx of extracellular calcium. These studies demonstrate the mitogenic effect of substance P and bombesin on human keratinocytes in the absence of EGF. The ability of the neuropeptides to increase keratinocyte growth in culture suggests a possible in wound healing.

Bombesin↗

PubMed1995

Repetitive sperm-induced Ca2+ transients in mouse oocytes are cell cycle dependent.

Mature mouse oocytes are arrested at metaphase of the second meiotic division. Completion of meiosis and a block to polyspermy is caused by a series of repetitive Ca2+ transients triggered by the sperm at fertilization. These Ca2+ transients have been widely reported to last for a number of hours but when, or why, they cease is not known. Here we show that Ca2+ transients cease during entry into interphase, at the time when pronuclei are forming. In fertilized oocytes arrested at metaphase using colcemid, Ca2+ transients continued for as long as measurements were made, up to 18 hours after fertilization. Therefore sperm is able to induce Ca2+ transients during metaphase but not during interphase. In addition metaphase II oocytes, but not pronuclear stage 1-cell embryos showed highly repetitive Ca2+ oscillations in response to microinjection of inositol trisphosphate. This was explored further by treating in vitro maturing oocytes at metaphase I for 4-5 hours with cycloheximide, which induced nuclear progression to interphase (nucleus formation) and subsequent re-entry to metaphase (nuclear envelope breakdown). Fertilization of cycloheximide-treated oocytes revealed that continuous Ca2+ oscillations in response to sperm were observed after nuclear envelope breakdown but not during interphase. However interphase oocytes were able to generate Ca2+ transients in response to thimerosal. This data suggests that the ability of the sperm to trigger repetitive Ca2+ transients in oocytes is modulated in a cell cycle-dependent manner.

Animals↗

PubMed1994

Staurosporine, a non-specific PKC inhibitor, induces keratinocyte differentiation and raises intracellular calcium, but Ro31-8220, a specific inhibitor, does not.

The responsiveness of normal human keratinocytes to different modulators of protein kinase C (PKC) was investigated. The PKC agonist TPA, staurosporine (a non-specific inhibitor), and Ro31-8220 (a specific inhibitor) were studied for effect on cell morphology, growth rate, involucrin expression, and intracellular calcium levels. Surprisingly the response to nanomolar concentrations of staurosporine was similar to TPA and induced a fusiform morphology, inhibited growth, increased involucrin levels, and raised intracellular calcium. Staurosporine also increased the number of cornified envelopes, and its action therefore appeared identical to TPA. In contrast, Ro31-8220 had little effect on morphology or growth and blocked both the TPA-induced growth inhibition and calcium rise. Ro31-8220 had no effect on staurosporine-induced growth inhibition but partially reduced its associated calcium rise. These results suggest PKC activation is required for keratinocyte differentiation and that staurosporine acts like a PKC agonist to give a similar effect as TPA. Specific inhibition of PKC by Ro31-8220 inhibits TPA-induced differentiation.

Alkaloids↗

PubMed1994

Thapsigargin raises intracellular free calcium levels in human keratinocytes and inhibits the coordinated expression of differentiation markers.

Thapsigargin raises intracellular free calcium ([Ca2+]i) by potently inhibiting the endoplasmic reticulum Ca-ATPase, which sequesters calcium from the cytosol. In human keratinocytes a rise in [Ca2+]i has been associated with differentiation and therefore we investigated the action of thapsigargin on this process. At concentrations above 3 nM thapsigargin inhibited keratinocyte proliferation. Thapsigargin induced an immediate transient [Ca2+]i rise in calcium-free or 70 microM calcium medium but a more prolonged rise in 2 mM calcium. For keratinocytes cultured in 70 microM calcium medium a late [Ca2+]i rise was also observed, after 6 h, similar to the effect of known differentiation stimuli. However, immunohistochemical techniques did not show any expression of the differentiation-specific protein involucrin, a component of the cornified envelope. When keratinocyte differentiation was induced by an increase in the extracellular calcium from 70 microM to 2 mM abundant involucrin and desmoplakin, a component of desmosomes, were synthesised. Both proteins gave staining patterns which suggested incorporation into structural proteins, but thapsigargin disrupted the calcium-induced pattern of involucrin and desmoplakin synthesis. Thapsigargin did not induce differentiation, possibly due to its inability to activate protein kinase C and raise inositol trisphosphate levels. We conclude that a rise in [Ca2+]i does not alone induce keratinocyte differentiation but may act with other intracellular signals to promote differentiation.

Biomarkers↗

PubMed1994

Ni2+ blocks the Ca2+ influx in human keratinocytes following a rise in extracellular Ca2+.

In keratinocytes a rise in extracellular Ca2+ induces differentiation and is associated with a sustained increase in intracellular Ca2+, due to Ca2+ entry across the plasma membrane. We have investigated the mechanism of Ca2+ entry in human keratinocytes following this rise, using Fura-2-loaded cells and the cations Ni2+, Co2+, Mn2+, and La3+ and Ca2+ channel blocker verapamil. Keratinocytes were permeable to La3+, Mn2+, and Co2+; Fura-2 fluorescence was quenched by Mn2+ and Co2+. Verapamil was unable to block Ca2+ entry. Ni2+ did not enter keratinocytes, but blocked the influx of extracellular Ca2+ and Mn2+. Thapsigargin depleted Ca2+ stores, inducing a large transient intracellular rise, and the efflux of this Ca2+ was not blocked by Ni2+. We conclude that keratinocytes are permeable to a number of cations, but not Ni2+, which may be used to block the entry of the other cations during the study of cation flux into cells. The data presented are consistent with calcium entry by a nonspecific cation channel recently described on keratinocytes.

Calcium↗

PubMed1994

Intracellular free calcium and growth changes in single human keratinocytes in response to vitamin D and five 20-epi-analogues.

Vitamin D, 1,25(OH)2D3, decreases proliferation and promotes differentiation of keratinocytes, and other keratinocyte differentiation stimuli have been associated with an early rise in intracellular free calcium, [Ca2+]i. We therefore investigated the effect of 1,25(OH)2D3, its precursor D3 and five 20-epi-analogues (EB1089, KH1060, KH1139, MC1288, MC1301) on growth and [Ca2+]i levels of normal human keratinocytes. Cells were cultured in medium MCDB153 with an extracellular calcium concentration of 70 microM or 1 mM. All the analogues were more potent than 1,25(OH)2D3 at inducing the morphological changes of differentiation, but D3 was inactive. At concentrations down to 10(-8) M 1,25(OH)2D3, caused significant inhibition of growth, as assessed by counting cells and measurement of thymidine labelling. At 5 days 50% inhibition of growth occurred with 64 nM 1,25(OH)2D3 and 3330 nM D3. All the analogues were more potent than 1,25(OH)2D3, and KH1060 inhibited growth at 10(-10) M. In single keratinocytes [Ca2+]i was measured by microspectrofluorimetric techniques using the dye fura-2. No immediate rise in [Ca2+]i was observed following addition of 1,25(OH)2D3 or the analogues up to 10(-6) M. However 10(-7) M 1,25(OH)2D3 or the analogues induced a gradual increase in [Ca2+]i, significant at 4 h (P < 0.001), which increased further over 2-3 days. D3 had no effect on [Ca2+]i. Increases in [Ca2+]i following the differentiation stimuli of either 2 mM extracellular calcium or 1,25(OH)2D3 were similar at 48 h, increasing from 100 +/- 3 nM (mean +/- SEM) in control cells to 150 +/- 3 nM with 2 mM calcium and 144 +/- 6 nM with 10(-7) M 1,25(OH)2D3. The effect of extracellular calcium in raising [Ca2+]i within minutes was more rapid than 1,25(OH)2D3, but in combination the two were not additive.

Calcitriol↗

PubMed1994

Representational momentum for a spiral path.

When a ball is shot through a spiral tube at high speed, the ball emerges in a straight path tangent to the ball's point of departure from the tube. However, past research has shown that many Ss believe the ball follows a curved pathway. In the 3 experiments described in this article, a ball traveling through a spiral tube was animated on a computer graphics screen. Ss' memory distortions for positions of the ball along each of 3 pathways were measured using a representational momentum paradigm. Retention interval was varied across the 3 experiments. The results from the 3 experiments reported here replicate retention interval results previously reported for representational momentum effects, and they suggest that the representational pathway of a ball exiting a spiral tube is spiral in shape. These findings may shed some light on why people have demonstrated naiveté on the spiral tube problem in past research.

Humans↗

PubMed1994

Proliferating cell nuclear antigen decreases in normal human keratinocytes with differentiation stimuli but not in an HPV immortalised cell line.

Proliferating cell nuclear antigen (PCNA) is a co-factor for DNA polymerase delta, which replicates genomic DNA during cell growth and division. Using a monoclonal antibody to PCNA (PC10) and conventional immunofluorescent techniques, we have compared the effect of differentiation stimuli on PCNA expression in normal and HPV immortalised keratinocytes. Two positive nuclear staining patterns were observed, a strong speckled form characteristic of proliferating cells and a weaker diffuse form. Strong nuclear staining was present in 44 +/- 4% (mean +/- SEM) of normal keratinocytes proliferating as a monolayer in 70 microM calcium serum-free medium but decreased to 13 +/- 3% after the differentiation stimulus of 2 mM calcium medium for 2 days. An even greater reduction was observed following other differentiation agents, 1,25 dihydroxyvitamin D3, the phorbol ester TPA and the non-specific protein kinase C inhibitor staurosporine. Transforming growth factor-beta, which slows keratinocyte growth without inducing differentiation, reduced strong staining to 17 +/- 3% of cells, but with an increase in the diffuse pattern of staining from 39 +/- 4 to 57 +/- 3%. HPV immortalised cells were resistant to the above agents except staurosporine, which inhibited growth and reduced the strong nuclear staining from 44 +/- 5% to 15 +/- 2%.

Alkaloids↗

PubMed1993

Age differences in memory-span errors: speed or inhibitory mechanisms?

This study investigated age differences in errors on the digit-span task. Protocols of 119 men and women, aged 18-99 years, were scored for the occurrence of three types of errors derived from the speed-of-processing and inhibition-deficit frameworks: omission errors, intrusion errors, and transposition errors. The types of errors made on the digit-span task varied with span size. At larger span sizes, participants were more likely to omit digits or introduce nonstimulus digits than to transpose those in storage. No age differences in intrusion errors were found, however, old-old women (75+ years) were significantly more likely than young (18-25 years) and old (60-70 years) adults of either sex to exhibit transposition errors. Consequently, old-old women were significantly less likely to exhibit omission errors. The results indicate that the digit-span task may involve two parallel processes: digit storage/recall and serial/position storage-recall. Age differences in the serial-processing component, rather than in the digit storage/recall component, may be age sensitive. These results are discussed within an inhibition-deficit framework of cognitive aging.

Adolescent↗