Bicycle saddle shape affects penile blood flow.

The purpose of this study was to evaluate the effect of bicycle saddle shape on penile blood flow during cycling. Penile blood flow was measured using a laser Doppler flowmeter in 20 potent male volunteers. In a counterbalanced, crossover design, measurements were taken in the standing and sitting positions, on either a narrow unpadded or wide unpadded saddle, before and after cycling for 5 min. Before cycling, penile blood flow (ml/min/100 g tissue) was significantly decreased from 1.6+/-0.7 to 1.5+/-0.7 (P=0.010) on the wide saddle and from 1.7+/-0.6 to 1.0+/-0.5 (P<0.001) on the narrow saddle. After 5 min of cycling, the changes in penile blood flow on the wide and narrow saddles were 0.34+/-0.49 and -0.38+/-0.49, respectively (P<0.001). The narrow saddle is associated with more significant reductions in penile blood flow and could be a source of blunt perineal trauma, potentially leading to erectile dysfunction.

Diabetes mellitus induces vaginal tissue fibrosis by TGF-beta 1 expression in the rat model.

The commonly reported sexual problem in women with diabetes mellitus is lack of vaginal lubrication. It is our hypothesis that reduced vaginal lubrication in diabetic women may result from the structural changes of the vagina. The aim of this study was to investigate in the diabetic rat model the vaginal structures using histochemistry and the expression of TGF-beta 1 using immunohistochemistry. Twenty female Sprague-Dawley rats (200-210 g) were divided into two groups: control and experimental. The experimental group (n = 10) received intravenous injection of streptozotocin (50 mg/kg). After 4 weeks, blood glucose levels were measured, and the vagina of the rat was excised. Serial sections of the vagina were used to perform hematoxylin and eosin (H & E) and Masson's trichrome stains, and for immunohistochemistry to identify TGF-beta 1 expression. The mean blood glucose concentrations were 67 +/- 11 mg/dL (range; 50-85) in the control group and 522 +/- 61 mg/dl (range; 429-590) in the experimental group. In the diabetic animals, vaginal tissue revealed reduced epithelial layers and decreased vaginal submucosal vasculatures compared to the control animals. The collagen connective tissue in the submucosal area of the diabetic animal tissue showed a dense and irregular, distorted arrangement. The immunoreactivity of TGF-beta 1 in the diabetic animals was prominent in the collagen connective tissue, fibroblasts, and smooth muscle fibers, whereas no immunoactivity was detected in the vaginal structures of the control animals. Diabetes mellitus may induce vaginal tissue fibrosis by TGF-beta 1 expression in the rat model. This implies that reduced vaginal lubrication in the diabetic women may result from the structural changes of the vagina.

A new potential of blood oxygenation level dependent (BOLD) functional MRI for evaluating cerebral centers of penile erection.

It is well known that penile erection is dependent on commands from the central nervous system. However, there has been little research on the central control of penile erection. The aim of this study was to evaluate, for the first time, the cerebral centers of penile erection using BOLD-functional MRI. Functional magnetic resonance imaging (fMRI) on a 1.5T MR scanner was performed in 12 sexually potent male volunteers (mean age: 23) and two hypogonadal impotent patients. In this study, blood oxygenation level dependent (BOLD) technique was utilized to create fMRI reflecting local brain activities. Real-time visual stimulation was performed with an alternatively combined erotic and non-erotic film to identify and quantify the activated brain regions associated with sexual response. Subjective sexual arousal and penile erection responses were assessed using 5-point scales ranging from 1 (no change) to 5 (maximal increase). In normal volunteers, the mean scores on subjective sexual arousal and penile erection by sexual stimulation with erotic film were 3.0 and 3.3 respectively, whereas there were no changes by non-erotic stimulation. During the visual stimulation the occipital cortex was activated by either an erotic or non-erotic film, the erotic film gave 150-200% stronger activation. However, more than seven of the 12 healthy subjects were significantly activated in the areas of inferior frontal lobe, cingulate gyrus, insula gyrus, corpus callosum, thalamus, caudate nucleus, globus pallidus, and inferior temporal lobe by erotic stimulation. In the hypogonadal patients, brain activation in response to the erotic film decreased compared to normal volunteers, however, it was restored by testosterone supplementation. These results are the first demonstration to show the functional neuroanatomy of the brain associated with sexual arousal by visual sexual stimulation using BOLD-based fMRI. Further studies are needed to verify that fMRI provides an important new tool in evaluating the cerebral center of the penile erection.

Blood-oxygenation-level-dependent functional magnetic resonance imaging for evaluating cerebral regions of female sexual arousal response.

OBJECTIVES: To evaluate, for the first time, the cerebral regions associated with female sexual arousal evoked by visual stimulation using noninvasive blood-oxygenation-level-dependent (BOLD) functional magnetic resonance imaging (fMRI). METHODS: A total of 6 healthy right-handed female volunteers (mean age 33 years, range 25 to 41) underwent fMRI on a 1.5-T MR scanner, in which the BOLD technique was used to create fMR images reflecting local brain activities. Real-time visual stimulation was performed with alternatively combined erotic and nonerotic films to identify the activated brain regions associated with sexual response. The perceived sexual arousal response was assessed using a scale ranging from 1 (no change) to 5 (maximal increase). RESULTS: The mean score for perceived sexual arousal by erotic visual stimulation was 2.7 on the 5-point scale and was unchanged by nonerotic stimulation. During the visual task, the occipital cortex was activated by both the erotic and the nonerotic films; however, the following cerebral areas were significantly (P <0.05) activated, varying from 4 of 6 to 6 of 6 women: inferior frontal lobe, cingulate gyrus, insula gyrus, corpus callosum, thalamus, caudate nucleus, globus pallidus, and inferior temporal lobe. CONCLUSIONS: This study is the first to evaluate noninvasive BOLD-fMRI in identifying cerebral regions associated with sexual arousal response evoked by visual stimulation in women.

Nitric oxide synthase gene and protein expression are upregulated by Bacille Calmette-Guérin in the rat bladder.

OBJECTIVE: We hypothesize that Bacille Calmette-Guérin (BCG) may act through the regulation of various isoforms of nitric oxide synthase (NOS) [inducible (iNOS), endothelial (eNOS), and neuronal (nNOS)] genes and protein expression in rat bladder. METHODS: Adult female Sprague-Dawley rats (200-250 g) were injected transurethrally with BCG (22 rats) and phosphate-buffered saline (22 control rats), and after 2, 4, 6, and 12 h, and 1, 2, 3, 5, 7, 10, and 14 days, the bladders were harvested. Normal and BCG-treated rat bladders were analyzed for mRNA expression for iNOS, eNOS, and nNOS by reverse-transcriptase polymerase chain reaction. Protein expression was determined by Western blotting and immunohistochemistry. RESULTS: mRNA expression for iNOS was induced after 2 h of BCG injection in the rat bladder. Gene expression for iNOS was highest at 6 h to 1 day followed by decreased expression, reaching its lowest level at 5 days. eNOS mRNA expression was detected in control bladders, but its level was higher in the BCG-treated animals. nNOS mRNA expression was present in all samples, but did not change after BCG treatment. Western blotting confirmed these findings. Immunohistochemistry analysis demonstrated that eNOS was present mainly in endothelium, while iNOS was detected in stroma and nNOS in epithelium and smooth muscle of the rat bladder. CONCLUSION: The present study demonstrates, for the first time, that BCG treatment up-regulates gene and protein expression of iNOS and eNOS in normal rat bladders, suggesting that BCG action may be mediated through NOS pathways.

Specific inhibition of rat brain phospholipase D by lysophospholipids.

Although the importance of phospholipase D (PLD) in signal transduction in mammalian cells is well documented, the negative regulation of PLD is poorly understood. This is primarily due to a lack of known specific inhibitors of PLD. We herein report that the activity of partially purified rat brain PLD is inhibited by certain lysophospholipids, such as lysophosphatidylinositol, lysophosphatidylglycerol, and lysophosphatidylserine in a highly specific manner. Inhibition of PLD by lysophospholipids was dose-dependent: the concentration of lysophosphatidylinositol required for half-maximal inhibition was about 3 micrometer. An analysis of the enzyme-kinetics suggested that lysophospholipids act as non-competitive inhibitors of PLD activity. As expected, PLD activity was stimulated by ADP-ribosylation factor (Arf) and phosphatidylinositol 4,5-bisphosphate (PIP(2)). The inhibition of PLD by lysophospholipids, however, was not affected by the presence or absence of Arf or by an increase in PIP(2) concentration. A protein-binding assay suggested that lysophospholipids bind directly to PLD. These results indicate that the observed inhibition of PLD by lysophospholipids is due to their direct interaction rather than to an interaction between lysophospholipids and either Arf or PIP(2). The present study suggests that certain lysophospholipids are specific inhibitors of rat brain PLD in a cell-free system and may provide the new opportunities to investigate mechanisms by which PLD is regulated by lysophospholipids, presumably liberated by phospholipase A(2) activation, in mammalian cells.

Molecular cloning, expression, and functional analysis of a cis-prenyltransferase from Arabidopsis thaliana. Implications in rubber biosynthesis.

cis-Prenyltransferase catalyzes the sequential condensation of isopentenyl diphosphate with allylic diphosphate to synthesize polyprenyl diphosphates that play vital roles in cellular activity. Despite potential significance of cis-prenyltransferase in plant growth and development, no gene of the enzyme has been cloned from higher plants. Using sequence information of the conserved region of cis-prenyltransferase cloned recently from Escherichia coli, Micrococcus luteus, and yeast, we have isolated and characterized the first plant cis-prenyltransferase from Arabidopsis thaliana. Sequence analysis revealed that the protein is highly homologous in several conserved regions to cis-prenyltransferases from M. luteus, E. coli, and yeast. In vitro analyses using the recombinant protein overexpressed in E. coli revealed that the enzyme catalyzed the formation of polyprenyl diphosphates ranging in carbon number from 100 to 130 with a predominance of C(120). The enzyme exhibited a higher affinity for farnesyl diphosphate than for geranylgeranyl diphosphate, with the K(m) values being 0.13 and 3.62 micrometer, respectively, but a lower affinity for isopentenyl diphosphate, with a K(m) value of 23 micrometer. In vitro rubber biosynthesis analysis indicated that the Arabidopsis cis-prenyltransferase itself could not catalyze the formation of higher molecular weight polyprenyl diphosphates similar to natural rubber. A reverse transcriptase-polymerase chain reaction analysis showed that the gene was expressed at low levels in Arabidopsis plant, in which expression of the cis-prenyltransferase in leaf and root was higher than that in stem, flower, and silique. These results indicate the tissue-specific expression of cis-prenyltransferase and suggest a potential role and significance of the enzyme in the polyisoprenoid biosynthesis in plants.

Late presentation of ureteral injury after laparoscopic surgery.

OBJECTIVE: To describe clinical presentation, etiology, and treatment of ureteral injuries recognized late in women who had gynecologic laparoscopies. METHODS: We reviewed the charts of 12 women who had delayed recognition of ureteral injuries between January 1991 and December 1998. RESULTS: Patients presented with fever, hematuria, flank pain, or peritonitis between 3 and 33 days postoperatively. The mechanism of ureteral injuries was electrocoagulation in seven women, laser ablation in one, and stapler ligation in four. The sites of injury were near the inferior margin of the sacroiliac joint on excretory urogram in eight women and near the ureterovesical junction in four. Three women initially treated with internal ureteral stents were subsequently treated with ureteroneocystostomy because of progression of urinary ascites in two and a delayed ureteral stricture in one. In nine patients, attempts at ureteral stenting were unsuccessful and immediate ureteral reconstruction was done. Outcomes were good in all cases. CONCLUSION: Delayed recognition of ureteral injury after gynecologic laparoscopy was associated with serious complications, and initial treatment with ureteral stenting was not useful. We advocate early open repair for those injuries.

Frequent genotype changes at -308, and 488 regions of the tumor necrosis factor-alpha (TNF-alpha) gene in patients with prostate cancer.

PURPOSE: Tumor necrosis factor-alpha (TNF-alpha) is involved in oncogenesis of several cancers. The purpose of this study was to investigate whether genotype changes of TNF-alpha promoter regions (-238, -308) and at the 488 region are associated with human prostate cancer. MATERIALS AND METHODS: The DNA from 73 cases of human prostate cancer was analyzed by allele-specific polymerase chain reaction to characterize the genotype changes of three regions of the TNF-alpha gene in prostate cancer patients. We also determined the genotype frequency in these patients. The relative risk of variant genotype was calculated by comparing with our previous data from healthy controls. RESULTS: Genetic changes were detected in 15.1% (11/73) of prostate cancer samples at 488 region of TNF-alpha. Seventy-three percent (53/73) of the patients showed genotype GA at -308 region of TNF-alpha. Genotype GA at 488 region in TNF-alpha was observed in 73% (53/73) of the cancer and 71% (52/73) of the normal tissue. The relative risks of incidence for prostate cancer was 14-fold higher in people with genotype GA at -308 region of TNF-alpha. The relative incidence for prostate cancer was a 17-fold higher in-patient with genotype GA at 488 region of TNF-alpha. Genotype GA at -308 of TNF-alpha was related to higher clinical tumor stage of prostate cancer than genotype G (p <0.05). CONCLUSIONS: The present study demonstrates, for the first time, that the genotype changes in -308 and 488 regions of TNF-alpha are associated with prostate cancer.

Increase in free linolenic and linoleic acids associated with phospholipase D-mediated hydrolysis of phospholipids in wounded castor bean leaves.

Stimulus-induced release of polyunsaturated fatty acids from membranes has been proposed to couple the processes of stimulus perception and oxylipin synthesis in the octadecanoid signaling pathway. This study investigated wound-induced changes in free fatty acids, diacylglycerol, and phospholipids at the site of wounding and at an unwounded area of the same wounded leaf in castor bean (Ricinus communis L.). Increases in free fatty acids and diacylglycerol and decreases in phospholipids were relatively large and continuous at the site of wounding. The changes at the unwounded area were selective and transient, suggesting a regulated activation of lipid turnover in response to wounding. In unwounded cells, the free fatty acids that increased in the early phase of wounding were linolenate and linoleate, which peaked within 5 min after wounding. Diacylglycerols that increased in unwounded cells were the species containing linolenate and linoleate, not those with oleate and stearate. Within 5 min of wounding, the levels of phosphatidylcholine and phosphatidylglycerol, but not other phospholipids, decreased in unwounded cells. These results provide evidence for the wound-induced selective increase in linolenate and linoleate in unwounded cells. The varied susceptibility of different phospholipids to hydrolysis after wounding indicates that phosphatidylcholine and phosphatidylglycerol may serve as substrates that lead to the increase in linolenate and linoleate in the early phase of wound response. The pattern of increases in polyunsaturated fatty acids, diacylglycerol, and phosphatidic acid and of decreases in phospholipids suggests the activation of a PLD-initiated signaling pathway in response to wounding in castor bean.

Association of benign prostatic hyperplasia with male pattern baldness.

OBJECTIVES: Both benign prostatic hyperplasia (BPH) and male pattern baldness (androgenic alopecia) share the pathogenesis of an androgen-dependent disorder and afflict a large population of elderly men with chronobiologic progress. However, it is unclear whether these diseases are related epidemiologically. We evaluated the association of frequency and severity of male pattern baldness between patients with BPH and a control group. METHODS: A total of 225 patients with BPH (mean age 69.3 +/- 6.5 years) and 1 60 controls (mean age 68.5 +/- 6.4 years), all over 60 years of age, were included in this study. The estimation of baldness severity was based on Norwood's classification (grade I to VII). The International Prostate Symptom Score (IPSS) and genetic tendency for baldness were also evaluated. The difference between IPSS and grade of baldness between the two groups was analyzed by the Mann-Whitney test and the frequency of inherited baldness was compared by the chi-square test. Correlation between severity of baldness and IPSS in each group was estimated by Spearman's rank correlation method. RESULTS: The patients with BPH had an apparently higher grade of male pattern baldness in comparison with that of controls (median value of grade IV versus III, P <0.001). The proportion of men with male pattern baldness of grade IV or higher in the BPH group was significantly larger than that of controls (53.8% versus 36.9%, P <0.01). There was a greater frequency of inherited baldness in the BPH group than in the controls (31.6% versus 12.5%, P <0.001). No significant correlation was noted between baldness severity and IPSS in either group. CONCLUSIONS: This study demonstrates a strong association of BPH with male pattern baldness.

A case of adrenal cavernous hemangioma.

Adrenal hemangiomas are rare tumor. Only 29 surgical cases have been reported. Although rare, adrenal hemangiomas should be included in the differential diagnosis of adrenal neoplasms. We report an additional case of adrenal hemangioma that was removed surgically, and the pertinent literature is reviewed.

Inhibition of phospholipase D by lysophosphatidylethanolamine, a lipid-derived senescence retardant.

Phospholipid signaling mediated by lipid-derived second messengers or biologically active lipids is still new and is not well established in plants. We recently have found that lysophosphatidylethanolamine (LPE), a naturally occurring lipid, retards senescence of leaves, flowers, and postharvest fruits. Phospholipase D (PLD) has been suggested as a key enzyme in mediating the degradation of membrane phospholipids during the early stages of plant senescence. Here we report that LPE inhibited the activity of partially purified cabbage PLD in a cell-free system in a highly specific manner. Inhibition of PLD by LPE was dose-dependent and increased with the length and unsaturation of the LPE acyl chain whereas individual molecular components of LPE such as ethanolamine and free fatty acid had no effect on PLD activity. Enzyme-kinetic analysis suggested noncompetitive inhibition of PLD by LPE. In comparison, the related lysophospholipids such as lysophosphatidylcholine, lysophosphatidylglycerol, and lysophosphotidylserine had no significant effect on PLD activity whereas PLD was stimulated by lysophosphatidic acid and inhibited by lysophosphatidylinositol. Membrane-associated and soluble PLD, extracted from cabbage and castor bean leaf tissues, also was inhibited by LPE. Consistent with acyl-specific inhibition of PLD by LPE, senescence of cranberry fruits as measured by ethylene production was more effectively inhibited according to the increasing acyl chain length and unsaturation of LPE. There are no known specific inhibitors of PLD in plants and animals. We demonstrate specific inhibitory regulation of PLD by a lysophospholipid.

Activation of phospholipase D and the possible mechanism of activation in wound-induced lipid hydrolysis in castor bean leaves.

Hydrolysis of membrane lipids has been suggested to provide messengers mediating defense gene expression in the wound signaling process. It is, however, unknown which lipolytic enzyme is involved in the signaling pathway. This study investigated the temporal and spatial activation of phospholipase D (PLD; EC 3.1.4.4) and the possible activation mechanism in response to wounding in castor bear (Ricinus communis L.) leaves. Wounding triggered a rapid activation of PLD-mediated phospholipid hydrolysis, as indicated by the in vivo increase in phosphatidic acid and free choline, at not only the site of wounding but also the undamaged area of wounded leaves RNA blotting analysis indicated that PLD gene expression was not involved in the early phase of wounding-activation of PLD. Measurements of PLD by activity assay and immunoblotting suggest that the wounding-activation of PLD at unwounded cells results from intracellular translocation of PLD from cytosol to membranes. A similar translocation pattern of PLD was also obtained as a function of increased free calcium at physiological concentrations in a homogenization buffer. Based on the above results, it is proposed that wounding induces activation of PLD leading to phospholipid hydrolysis, and that the activation results from translocation of PLD to membranes, which is mediated by an increase in cytoplasmic calcium upon wounding.

Intracellular Localization of Phospholipase D in Leaves and Seedling Tissues of Castor Bean.

The intracellular distribution of phospholipase D (PLD; EC 3.1.4.4) in castor bean (Ricinus communis L.) tissues was investigated by subcellular fractionation and by immuno-electron microscopy. Centrifugal fractionation revealed that most PLD in young leaves was soluble, whereas in mature leaves a majority of PLD was associated with microsomal membranes. Further separation of microsomal membranes by a two-phase partitioning system indicated that PLD was associated with both plasma and intracellular membranes. Sucrose gradient separation of intracellular membranes showed PLD present in the endoplasmic reticulum, a submicrosomal band, and in soluble fractions but not in mitochondria and glyoxysomes of postgermination endosperm. Immunocytochemical studies found high gold labeling in vacuoles in young leaves, suggesting that the high level of soluble PLD in young leaves is due to release of PLD from vacuoles during tissue disruption. In addition to the labeling in vacuoles, gold particles were also found in the cytoplasmic matrices and plasma membrane in leaves and in 2-d postgermination seedlings. Collectively, these results show that PLD in castor bean leaf and seedling tissues is localized in the vacuole and is associated with the endoplasmic reticulum and plasma membrane and that the relative distribution between the soluble and membrane compartments changes during castor bean leaf development.

Expression of Phospholipase D during Castor Bean Leaf Senescence.

Membrane deterioration in plant senescence is commonly associated with progressive decreases in membrane phospholipid content. This study investigated the expression and regulation of phospholipase D (PLD; EC 3.1.4.4) during senescence in castor bean (Ricinus communis L. cv Hale) leaf discs. The rate of leaf senescence was accelerated by 50 [mu]M abscisic acid and was attenuated by 50 [mu]M cytokinin during incubation at 23[deg]C for up to 5 d. Leaf senescence was indicated by decreases in the content of total proteins, chlorophyll, and phospholipids. PLD activity in both membrane-associated and cytosolic fractions showed a gradual increase in the absence of phytohormones. Abscisic acid stimulated an increase in membrane-associated PLD and had little effect on the soluble form. On the other hand, cytokinin retarded the increase in membrane-associated PLD. Immunoblotting analysis using PLD-specific antibodies revealed that the changes in PLD activity were correlated with those of PLD protein. Analysis of PLD by nondenaturing PAGE showed the appearance of a PLD structural variant, PLD 3, in abscisic acid-treated leaf discs. Northern blotting analysis using a PLD cDNA probe revealed an increase in PLD mRNA in senescing leaf discs. These data indicate complex mechanisms for the regulation of PLD during senescence, which include increases in membrane-associated PLD, differential expression of PLD isoforms, and changes in amounts of PLD protein and mRNA. Such controlled expression points to a role for PLD in membrane deterioration and plant senescence.

Multiple Forms of Phospholipase D following Germination and during Leaf Development of Castor Bean.

Multiple molecular forms of phospholipase D (PLD; EC 3.1.4.4) were identified and partially characterized in endosperm of germinated seeds and leaves of castor bean (Ricinus communis L. var Hale). The different PLD forms were resolved by nondenaturing polyacrylamide gel electrophoresis, isoelectric focusing, and size-exclusion chromatography. PLD was detected with both a PLD activity assay and immunoblots with PLD-specific antibodies. There were three major forms of PLD, designated types 1, 2, and 3, based on their mobility during nondenaturing polyacrylamide gel electrophoresis. Molecular masses of the PLD variants were estimated at 330, 230, and 270 kD for the types 1, 2, and 3, respectively. Isoelectric points of the native type 1, 2, and 3 PLDs were approximately 6.2, 4.9, and 4.8. Under the in vitro assay conditions used, the three forms of PLD exhibited the same substrate specificity, hydrolyzing phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) but not phosphatidylserine (PS) and phosphatidylinositol (PI). The three forms of PLD differed in their substrate preferences, and the order of activities was: PLD 1, PE > PG = PC; PLD 2, PE > PG > PC; PLD 3, PE = PG = PC. The Km values of PLDs 1, 2, and 3 for PC were 1.92, 2.62, and 5.18 mM, respectively. These PLDs were expressed differentially following seed germination and during leaf development. Type 1 was found in the early stages of seedling growth and in young leaves, type 2 was present in all the tissues and growth stages examined, and type 3 was expressed in senescent tissues. The PLDs shifted from largely cytosolic to predominantly membrane-associated forms during leaf development. The present studies demonstrate the structural heterogeneity of plant PLD and growth stage-specific expression of different molecular forms. The possible role for the occurrence of multiple molecular forms of PLD in cellular metabolism is discussed.

An enzyme-immunoassay of abscisic acid in potato (Solanum commersonii) cultured cells.

Estimation of abscisic acid (ABA) content in potato (Solanum commersonii) suspension-cultured cells with an enzyme-immunoassay (EIA) was investigated. In crude extracts of potato cultured cells or even after simple clean-up using C18 cartridge, EIA based on commercial monoclonal antibodies (Idetek, Inc) failed to detect any ABA content. An interference could be removed by partitioning against ethyl acetate after the C18 cartridge so that the EIA yielded an estimate of ABA similar to that determined by high pressure liquid chromatography and gas chromatography-mass spectrometry analysis. These results demonstrate the presence of metabolites in potato cultured cell extract that prevent the binding of ABA to its binding site but not the binding of tracer.