Although it is known that all fertilization events are due to elevated intracellular calcium ([Ca2+]i), it is not known how sperm induce an increase in zygote [Ca2+]i. We report that sperm can increase inositol 1,4,5-trisphosphate in Xenopus laevis eggs that have been preinjected with calcium buffers to prevent the increase in [Ca2+]i (both the initial increase and the subsequent wave) after fertilization. After buffering [Ca2+]i to levels well below basal, IP3 production was not blocked, whereas IP3 metabolism may be inhibited. Also, heparin (an inhibitor of inositol 1,4,5-trisphosphate action and the fertilization response) does not prevent a normal increase in IP3 after fertilization. Conditions that produce various levels of polyspermy are associated with IP3 increases similar to those noted after monospermic fertilization. These data suggest a specific order of fertilization events: sperm utilize an initial production of inositol 1,4,5-trisphosphate to produce the [Ca2+]i increase at fertilization.

Animals↗

PubMed1993

Protein kinases are required for embryonic neural crest cell galvanotaxis.

Embryonic quail neural crest cells migrate towards the negative pole of an imposed dc electric field as small as 7 mV/mm (0.4 mV per average cell length). The involvement of protein kinases in the mechanism utilized by these cells to detect and respond to such imposed fields was tested through the use of several kinase inhibitors. Evidence for the involvement of protein kinase C (PKC) included: (1) inhibition of the directed motility by 1 microM sphingosine that was reversed by the addition of the phorbol ester, PMA; (2) stimulation of a faster response to the imposed field by PMA; and (3) inhibition of the directed translocation by 5 microM H-7. However, another PKC inhibitor, staurosporine, did not inhibit the directed translocation (1 nM-1 microM). We also found evidence for the involvement of either cAMP- or cGMP-dependent protein kinase. The galvanotactic response was partially inhibited by the addition of 10 microM H-9 and the response was enhanced in the presence of the phosphodiesterase inhibitor, IBMX. However, the adenylate cyclase stimulant, forskolin, had no significant influence on the directed motility, although it reduced the average cell velocity. While these experiments suggest that cAMP- or cGMP-dependent protein kinase or PKC may be involved in the galvanotaxis response, two other protein kinases appeared not to be required. The myosin light chain kinase inhibitor, ML-7, had no effect on the directed motility in an imposed field, so myosin light chain kinase may not be required for galvanotaxis. Similarly, 5 microM W-7 had no significant effect on the directed translocation, suggesting that calmodulin-dependent protein kinase is not involved. Interestingly, the continuous activity of a protein kinase is apparently not required for the directed translocation response. The addition of the PKC and cAMP-dependent protein kinase inhibitor, H-7, after the cells had been exposed to the field for 1 hour, had no effect on the subsequent directed translocation. Thus, for these inhibitors to block the directed translocation, they must be present at the same time as the initial field application. This implies that an integral step in the cellular response mechanism for galvanotaxis involves the stimulation of a protein kinase whose effect is long lasting.

Animals↗

PubMed1993

The sperm-induced Ca2+ wave following fertilization of the Xenopus egg requires the production of Ins(1, 4, 5)P3.

We used fluorescence ratio imaging of fura-2 in the egg of Xenopus laevis to study the initiation and propagation of the wave of increased free Ca2+ that is normally triggered at fertilization. Naturally matured, jellied eggs were injected with fura-2 and ratio-imaged with fluorescence excitation at 350 and 385 nm while sperm were added. The [Ca2+]i rise normally begins as a small spot near the surface of the egg, remains fairly localized for 20-60 sec, and then spreads more rapidly across the egg at 7.5 +/- 0.05 microns/sec to reach the antipode about 5 min after fertilization. The [Ca2+]i wave velocity is slowed by increasing the concentration of fura-2 in the cytoplasm to 100 microM, and 250 microM fura-2 blocks wave propagation. The peak [Ca2+]i in a sperm-activated wave is 2.2 +/- 0.1 microM and [Ca2+]i returns to preactivation levels within 21 +/- 0.7 min after fertilization. We further studied the mechanism by which sperm trigger the Ca2+ wave by injecting substances that interfere with Ins(1,4,5)P3-induced Ca2+ release or Ca(2+)-induced Ca2+ release (CICR). Heparin (3 kDa) inhibits sperm-induced egg activation in a manner that is linearly proportional to its cytoplasmic concentration. At 130 microM (390 micrograms/ml), sperm-induced activation is completely blocked and at 75 microM (225 micrograms/ml) activation of half of the eggs is inhibited. All eggs injected with 130 microM heparin are polyspermic as verified using Hoechst dye to label nuclear DNA. Imaging eggs injected with 75 microM heparin revealed multiple, transient "spots" of increased [Ca2+]i that failed to spread across the egg. Injection of a monoclonal antibody to PIP2 (0.2 microM), kt3g, blocked sperm-induced egg activation in 73% of the 30 eggs injected, suggesting that activation requires the hydrolysis of PIP2 in the membranes of the egg rather than simply the introduction of Ins(1,4,5)P3 from the sperm. This sperm-induced egg activation is not blocked by either of two CICR inhibitors, procaine (10 mM) or ruthenium red (30 microM), and egg activation is not triggered by either of two stimulators of CICR, caffeine (10 mM) or ryanodine (50 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Transrectal sonography for serial evaluation of prostatic malignancy.

Serial changes in the prostatic bed as identified by transrectal sonography in 31 patients with prostatic carcinoma were correlated with systemic change in disease. Seventy-three sonograms were performed, allowing interval change to be evaluated in 42 instances. Sonography demonstrating regression correlated with clinical response in 89 percent (8/9) of studies and with disease progression in 91 percent (10/11). Stable disease was correctly evaluated by sonography in only 56 percent (10/18) of studies. Six of those 8 incorrectly identified as stable were in men with prostatic irradiation prior to inclusion in this study. If they are excluded, stability was accurately gauged in 83 percent (10/12). Stability of the prostate on serial sonography may not accurately reflect clinical status in men with prior prostatic irradiation. In 1 patient abscess was superimposed on and could not be differentiated from tumor. Nodules of increased, decreased, and mixed echogenicity showed the same response to treatment as the total prostate volume.

Abscess↗

PubMed1986

Etoposide in prostatic cancer: experimental studies and phase II trial in patients with bidimensionally measurable disease.

Etoposide, a semisynthetic derivative of podophyllotoxin, was evaluated concurrently in vitro against a human derived hormone-resistant cell line, PC-3, and in vivo in bidimensionally measurable hormone-resistant human prostatic cancer. In vitro, a dose-response relationship was observed, with 74% inhibition at 10 micrograms/ml (1 h incubation) and greater than 99% inhibition at 90 micrograms/ml, both in the range of clinically achievable concentrations. In vivo, 1 PR (5%, 95% confidence limits 0-12%) of 18+ months was observed in 20 adequately treated patients. The results confirm the limited role of etoposide in hormone-refractory disease and the need for new model systems for evaluation of potential chemotherapeutic compounds in this disease.

Adenocarcinoma↗

PubMed1985

Public health and economic policy: financing the NHS.

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Financing, Government↗

PubMed1984

Metabolism of 2,4-dichlorophenol by isolated perfused rat liver.

The uptake and metabolism of 2,4-[14C]dichlorophenol (2,4-[14C]DCP) was studied in the isolated perfused rat liver. The uptake of radioactivity in liver increased 6.6% (25.3-31.9%) in the presence of adenosine 5'-triphosphate (ATP) and 14.9% (25.3-40.2%) in the presence of ATP and galactosamine. This increase in the uptake of radioactivity was indicative of maintaining the integrity of liver cells. The glucuronide conjugate of 2,4-DCP in bile was derivatized by permethylation and characterized by gas chromatography-mass spectrometry (GC-MS). Two unusual metabolites were isolated from the liver and perfusate by extracting with hexane. The gas chromatogram of these metabolites gave peaks at retention times 12.0 and 15.5 min, and were characterized by mass spectrometry. The fragmentation pattern of these metabolites confirmed their identity as dichloromethoxyphenols.

Adenosine Triphosphate↗

PubMed1974

Social science and health policy in the United Kingdom: some contribution of the social sciences to the bureaucratization of the National Health Service.

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Costs and Cost Analysis↗

PubMed1968

Behavioral effects in monkeys of racemates of two biologically active marijuana constituents.

Both dl-Delta(8)- and dl-Delta(9)-tetrahydrocannabinol produced marked alterations of behavior in rhesus and squirrel monkeys. Squirrel monkeys appeared to have visual hallucinations. Continuous avoidance behavior of squirrel monkeys was stimulated by both drugs, but high doses of dl-Delta(9)-tetrahydrocannabinol also caused depression after the stimulant phase. Complex behavior involving memory and visual discrimination in rhesus monkeys was markedly disrupted by both drugs.

Animals↗