Biomedical subjects

L Llanes

Publications and source records attributed to L Llanes.

9 recordsLinked to original sources

PubMed2005

Quantitative real-time reverse transcription: polymerase chain reaction of prostate-specific antigen (PSA) for detection of circulating prostatic cells in patients with clinically localized prostate cancer.

OBJECTIVE: To evaluate the clinical utility of using the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) to quantify prostate-specific antigen (PSA) mRNA in peripheral blood samples from patients with prostate cancer as a predictor of extraprostatic extension of the disease and to assess any correlations with known predictive markers of this condition. METHODS: Immediately before radical prostatectomy, peripheral blood samples were taken from 42 men with clinically localized prostate cancer and analysed for PSA and 18S ribosomal (endogenous control) genes using real-time RT-PCR (with gene expression assays and the comparative CT-cycle threshold-method for quantifying). A total of 30 healthy male blood donors aged <50 y was taken as a control group. The relationships between PSA mRNA values, pathological and clinical features were analysed. PSA mRNA value, PSA level and biopsy Gleason score were then compared as predictors of extraprostatic extension. RESULTS: PSA gene expression was 3.73 times significantly higher in patients with clinically localized prostate cancer than in healthy men (P<0.05). There was no relationship between PSA real-time RT-PCR values and pathological stage pT2 or pT3 (P=0.5), and no association between PSA mRNA value and serum PSA level (P=0.9) or the Gleason score of the preoperative biopsy (P=0.9). CONCLUSION: There was no significant advantage in using the real-time RT-PCR assay of PSA mRNA before surgery to stage prostate cancer and to discriminate between organ-confined and extraprostatic extension.

Aged↗

PubMed2002

The clinical utility of the prostate specific membrane antigen reverse-transcription/polymerase chain reaction to detect circulating prostate cells: an analysis in healthy men and women.

OBJECTIVE: To evaluate the overall specificity of nested reverse transcriptase-polymerase chain reaction (RT-PCR) to detect prostate-specific membrane antigen (PSM) mRNA in peripheral blood samples of healthy donors. SUBJECTS AND METHODS: Peripheral blood samples were taken from 60 healthy blood-donors (30 men and 30 women aged < 50 years) and analysed for PSM-mRNA using nested RT-PCR (in 'hot-start' conditions and confirmed using nested EcoRI restriction enzyme). Intron-spanning primer pairs specific for human PSM were deduced from the GenBank sequence (M99487) using gene software. The outer primer pair for PSM was: fwd: 1368 5'-TCACCGGGACTCATGGGTGT-3'; reverse: 1860 5'-GCCTGAAGCAATTCCAAGTCGG-3'. Inner primer pair for PSM was: fwd: 1480 5'-AAGGAAGGGTGGAGACCTAG-3'; reverse: 5-ACTGAACTCTGGGGAAGGAC-3'. The integrity of cDNAs was checked using primer pairs specific for the housekeeping gene beta-actin. The specificity and false-positive rate were calculated assuming that the underlying prostate cancer incidence was nil. RESULTS: The first PCR was negative for all samples (100% specificity; 0% false-positive rate). The nested PCR detected 23 positive samples (23/60, 38%) with an overall specificity of 62% (false positive rate, 38%). CONCLUSION: Nested RT-PCR of PSM-mRNA in peripheral blood is highly unspecific. Its clinical utility in the management of prostate cancer must be low. Further development is needed of quantitative RT-PCR, primers that identify prostatic PSM or another prostate-specific marker gene to differentiate PSM mRNA from circulating prostate cells and from non-prostatic tissues.

Adult↗

PubMed2001

Prostate specific antigen density. Is there a role for this parameter when screening for prostate cancer?

The objective of this paper is to validate prostate specific antigen (PSA) density (PSAD) routine use to enhance PSA specificity in men with normal digital rectal examination and intermediate PSA values. It is a retrospective study of 235 men from a prostate cancer (PCa) screening program. All of them presented PSA values between 4 and 10 ng/ml, normal digital rectal examination, and a transrectal ultrasound (TRUS) guided biopsy available (PSA>/=4 ng/ml as the sole criterion for biopsy). Multivariate analysis failed to demonstrate higher PSAD values in men with PCa. PSAD cutoff points higher than 0.07 ng/ml per cc were considered as unacceptable, with less than 95% sensitivity. When a cutoff point of 0.15 was considered, as many as 30.6% of the cancers were missed. In conclusion we cannot recommend the use of this parameter for the above mentioned purpose.Prostate Cancer and Prostatic Diseases (2001) 4, 146-149.

Journal Article↗

PubMed2000

Detecting circulating prostate cells in patients with clinically localized prostate cancer: clinical implications for molecular staging.

OBJECTIVE: To evaluate the clinical utility of using the reverse transcriptase-polymerase chain reaction (RT-PCR) to detect prostate-specific antigen (PSA) mRNA in peripheral blood samples from patients with prostate cancer, as a predictor of extraprostatic disease, and to assess any correlations with known predictive markers of this condition. PATIENTS AND METHODS: Immediately before radical prostatectomy, peripheral blood samples were taken from 25 men with clinically localized prostate cancer and analysed for PSA mRNA using RT-PCR (in 'hot-start' conditions and confirmed using ClaI restriction enzyme). The relationships between PSA mRNA positivity, pathological and clinical features were analysed; PSA mRNA positivity, PSA level and biopsy Gleason score were then compared as predictors of extraprostatic disease. RESULTS: There was no relationship between PSA mRNA positivity and pathological stage (pT2 or pT3), and no association between PSA mRNA positivity and serum PSA level, PSA density, the findings on a digital rectal examination or transrectal ultrasonography, and perineural invasion in the prostatic biopsy. However, there was a significant correlation between the Gleason score of the preoperative biopsy and PSA mRNA positivity. The best predictors of extraprostatic disease were the biopsy Gleason score and the PSA level. CONCLUSION: There was no significant advantage in using the RT-PCR assay of PSA mRNA before surgery to stage prostate cancer and to discriminate between organ-confined and extraprostatic neoplasms.

Adult↗

PubMed1998

Role of autonomic innervation in rat prostatic structure maintenance: a morphometric analysis.

PURPOSE: To analyze the effects of major pelvic ganglion (MPG) excision on the structure of rat prostate. MATERIALS AND METHODS: We studied 80 Sprague-Dawley rats (300-350 gm. weight). Forty-two were anesthetized and the right MPG excised. After 28-30 days, the same-side prostatic ventral lobe (VL) was obtained for macroscopic, light (LM), and transmission electron microscopy (TEM) evaluation. A computerized morphometric analysis was performed on epithelial and muscle cells. Results were compared with 38 right VL of non-operated, same-aged rats. RESULTS: A 36.6% reduction (0.14 gm.) of VL fresh weight was found in the denervated group (p <0.001). Mean tissue proportions observed in the LM study were 27.9% (epithelial), 48.3% (stromal), and 51.8% (glandular) in the non-operated group, versus 14.8% (p <0.001), 55.7%, and 44.4% (not significant) respectively, after MPG excision. No difference was found regarding the vascular pattern. In the denervated rats, TEM analysis found a significant reduction in total and supranuclear cell height (change in cell polarity), as well as in cytoplasm, Golgi and endoplasmic reticulum areas. Secretory granule count, total area (p <0.001), and density of apical microvilli were also reduced. On the other hand, only an increase in the area of cytoplasm ribosomal aggregates was detected in the smooth muscle cell analysis. CONCLUSIONS: Our study demonstrated a rat prostatic VL atrophy in the denervated side, due to a shrinkage in the epithelial component of the gland. Ultrastructural findings also suggest an overall decrease of epithelial cell secretory activity. Finally, the increase of ribosomal aggregates found in stromal smooth muscle could reflect an activation of these cells after denervation.

Animals↗

PubMed1996

Interleukin-1 beta stimulates sphingomyelin hydrolysis in cultured granulosa cells: evidence for a regulatory role of ceramide on progesterone and prostaglandin biosynthesis.

In granulosa cells labeled to isotopic steady-state with [3H]serine, addition of interleukin-1 beta (IL1 beta) or bacterial sphingomyelinase (SMase) induced a rapid decrease (approximately 60% by 10 min) in cellular [3H]Sphingomyelin content and a prolonged generation (up to 60 min) of [3H]ceramide, the immediate lipid-moiety generated in response to sphingomyelin hydrolysis. In FSH-treated cells, IL1 beta (0.3-30 ng/ml) inhibited progesterone biosynthesis in a dose-dependent manner, an effect that was also observed in cells exposed to increasing concentrations of bacterial SMase (0.003-0.3 U/ml) or the membrane-permeable ceramide analogue N-hexanoylsphingosine (C6-cer:0.1-10 microM). Abrogation of progesterone biosynthesis was not a sole consequence of inadequate cAMP biosynthesis because cyclic nucleotide levels remained elevated (3- to 4-fold over untreated cultures) after addition of IL1 beta, SMase, or two different cell permeable ceramide analogues (C2-cer and C6-cer) to gonadotropin-stimulated granulosa cells. Moreover, taken into account that exogenous SMase or C6-cer partially abolished progesterone biosynthesis induced by But2cAMP (0.5 mM) or cholera toxin (CTX: 1 microgram/ml), the above mentioned results support the notion that activation of the sphingomyelin pathway exerts its inhibitory effects on granulosa cell steroidogenic activity at site(s) of action both proximal and distal to cAMP generation. As determined by RT-PCR analysis, the inhibitory effect of IL1 beta, SMase, or C6-cer on gonadotropin-stimulated steroidogenesis was accompanied by arrested transcription of the mitochondrial cholesterol side chain cleavage enzyme (P450scc) and 3 beta-hydroxysteroid dehydrogenase/delta 5-4isomerase, the two FSH-inducible steps involved in progesterone biosynthesis. Although bacterial SMase or the ceramide analogue C6-cer alone did not exactly reproduce the effect of IL1 beta on granulosa cell prostaglandin E2 (PGE2) biosynthesis, both agents augmented net PGE2 production and messenger RNA levels of the inducible prostaglandin endoperoxide synthase/cyclooxygenase (PGHS-2) in cytokine-treated cells. Although the effect on PGHS-2 messenger RNA may account for the facilitatory role of ceramide on IL1 beta-induced PGE2 biosynthesis, neither SMase nor the membrane-permeant ceramide analogue were able to augment prostaglandin accumulation in the presence of exogenously added arachidonate precursor. Collectively, whereas these results show that ceramide triggers a negative-effector pathway that is both necessary and sufficient to reproduce the inhibitory effect of IL1 beta on FSH-stimulated granulosa cell steroidogenesis, they also support the notion that sphingomyelin hydrolysis may be important for cytokine-induced PGHS-2 expression but not sufficient to reproduce IL1 beta-stimulated PGE2 biosynthesis.

3-Hydroxysteroid Dehydrogenases↗

PubMed1995

Ceramide mediates tumor necrosis factor effects on P450-aromatase activity in cultured granulosa cells.

In [3H]serine-labelled granulosa cells treatment with TNF alpha (10 ng/ml) resulted in a transient decrease in cellular [3H]sphingomyelin and generation of [3H]ceramide that remained elevated 60 min later. In cells labelled with [methyl-14C]choline, TNF alpha induced a similar reduction in [14C]sphingomyelin content that was accompanied by a sustained elevation in [14C]phosphorylcholine levels. In FSH-primed cells, TNF alpha inhibited P450-AROM activity in a dose-dependent manner, an effect that was also observed in cells treated with bacterial sphingomyelinase (SMase 0.003-0.3 U/ml) or increasing concentrations (0.1-10 microM) of N-acetylsphingosine (C2-cer) a membrane-permeable analogue of ceramide. These results support the notion that sphingomyelin degradation to a bioeffector molecule ceramide, may be an early event involved in TNF alpha-induced signal transduction in granulosa cells.

Animals↗

PubMed1993

Effects of tetrandrine on Ca(2+)- and Na(+)-currents of single bullfrog cardiomyocytes.

Tetrandrine is a natural alkaloid classified as a calcium antagonist. However, its precise actions on Ca(2+)-currents in cardiac cells have not been fully characterized. In the present study, we have investigated the mechanism of action of tetrandrine on the Ca(2+)-currents of single bullfrog cardiac cells, using the patch-clamp technique. Tetrandrine slightly increased ICaL from negative holding potentials (-100 mV) at low concentrations (10 nM-1 microM) and inhibited it at higher concentrations. At depolarized holding potentials (-50 mV) only an enhanced inhibition was seen. Tetrandrine blockade of the L-type Ca(2+)-current (ICaL) was mostly tonic. This is similar to ICaL blockade by nifedipine but not by verapamil, the latter being mostly use-dependent. Use-dependent effects of tetrandrine and nifedipine were evident at high rates. Availability curves were shifted leftwards (10-12 mV) by tetrandrine (10 microM) and nifedipine (1 microM). The T-type Ca(2+)-current (ICaT), although less sensitive, was decreased by both agents in a voltage-independent way. Tetrandrine (10-30 microM) but not nifedipine (1-10 microM), depressed the Na(+)-current (INa) in tonic, use- and voltage-dependent manners. We conclude that tetrandrine and nifedipine share some common actions on cardiac Ca(2+)-channels, while showing differences in their actions on Na(+)-channels. The depression of INa by tetrandrine suggests it could be effective on supraventricular tachycardias.

Alkaloids↗

PubMed1989

The androgen receptor does not mediate progestin regulation of progesterone biosynthesis in cultured rat granulosa cells.

In the present investigation the influence of androgens and progestins on the FSH modulation of progesterone biosynthesis was studied in cultured rat granulosa cells. Cells obtained from the ovaries of immature estrogen treated rats were cultured for three days in serum free medium or in medium supplemented with FSH or CPA, with or without reduced androgen DHT or the synthetic progestin R5020 alone or in combination with the anti-androgen CPA. Treatment with FSH increased pregnenolone, progesterone and 20 alpha-OHP accumulation in the culture medium 20-, 14- and 7-fold, respectively. Furthermore FSH increased the activity of the enzyme 3 beta-HSD. Concurrent treatment with DHT or R5020 augmented the FSH stimulated steroidogenesis of cultured cells. The androgen enhancement of FSH stimulated steroidogenesis of cultured granulosa cells was blocked by concomitant treatment with CPA, whereas treatment of cultures with anti-androgen did not affect the stimulatory effect of the synthetic progestin R5020.

20-alpha-Dihydroprogesterone↗