Biomedical subjects

W W Lin

Publications and source records attributed to W W Lin.

At least 55 records · Page 3Linked to original sources

PubMed1998

Unilateral testicular injury from external trauma: evaluation of semen quality and endocrine parameters.

PURPOSE: Because few studies have described the impact of unilateral testicular trauma on fertility parameters, we review the experience at the Ben Taub General Hospital during a 16-year period. Semen and endocrine profiles were analyzed to evaluate the influence on the outcomes of orchiectomy versus testicular salvage. MATERIALS AND METHODS: From 1979 to 1995, 67 patients were identified who sustained unilateral testicular trauma. Of these patients 12 were located and 10 agreed to be evaluated. Injuries included gunshot wounds, stab wounds and blunt trauma, and treatment consisted of unilateral orchiectomy or testicular repair. The study protocol comprised a history and physical examination, routine semen analysis, determination of semen and serum antisperm antibody titers (Immunobead* assay) and a modified gonadotropin stimulation test. Results were compared with a group of semen donors with proved fertility. RESULTS: In the 7 patients who underwent unilateral orchiectomy mean sperm density was normal but significantly decreased compared with that of the fertile controls (81.6 versus 132.6 x 10(6)/ml., p = 0.04). Sperm motility was not significantly affected. Baseline follicle-stimulating hormone (FSH) and luteinizing hormone (LH), and post-stimulation LH were significantly increased in this group compared with controls (p < 0.01). In the group that underwent testicular repair sperm density, motility, and baseline and post-stimulation FSH and LH levels were not significantly different from controls. In all patients in both groups testosterone levels and contralateral testicular size were normal. Only 1 patient in the repair group had an elevated serum and semen antisperm titer. CONCLUSIONS: While the testicular salvage group had no significant seminal or endocrine abnormality, the orchiectomy group had a significant decrease in sperm density and elevation of baseline FSH and LH. These preliminary data suggest that testicular salvage is more protective of overall testicular function than orchiectomy.

Adult↗

PubMed1998

Alternative splicing of insulin-like growth factor I mRNA is developmentally regulated in the rat and mouse with preferential exon 2 usage in the mouse.

The synthesis of an IGF-I complementary RNA (cRNA) standard containing the primer sequences for each of the four IGF-I mRNA alternative transcript forms (class 1Ea, 1Eb, 2Ea and 2Eb) and a polyA+ tail allowed the determination of the absolute abundance of each form in growing rats and mice by quantitative RT-PCR. In rat liver, class 1Ea mRNA was the most abundant form representing 90% of the total IGF-I mRNA. Though the relative proportion was maintained, the absolute abundance was maximal at 4 weeks of age and had declined by 6 weeks. In contrast, class 2Ea mRNA was the predominant transcript in mouse liver (70% of total IGF-I mRNA) and the 2Eb mRNA form was also 10-fold higher than that detected in rat liver. These results suggest that rats and mice differ both in their transcription initiation at two leader exons and in their alternative splicing activities for exon 5.

Alternative Splicing↗

PubMed1998

Overexpression of growth hormone affects alternatively spliced IGF-I mRNA expression in oMt1a-oGH transgenic mice.

Restorative growth hormone (GH) treatment of hypophysectomized rats differentially enhances the transcription of alternative IGF-I mRNA classes in liver. The goal of the present study was to determine the effects of GH overexpression on various classes of hepatic IGF-I mRNA in GH transgenic mice. Unstimulated oMt1a-oGH transgenic mice had low levels of transgene expression, and therefore were used to determine the effects of long-term, slightly elevated GH levels on the abundance on each alternative IGF-I mRNA class. The acute effects of high GH levels on the expression of alternative IGF-I mRNA were studied by gavaging transgenic mice with 25 mM zinc sulfate to activate oMt1a-oGH transgene expression. Long-term, low levels of oGH transgene expression in unstimulated transgenic mice resulted in a 73% down regulation of IGF-I 2Ea mRNA but not 1Ea and 2Eb mRNA. Acute stimulation of transgene expression triggered a rapid, 240% increase in 1Ea mRNA levels within 4 hours of transgene expression while 2Ea mRNA was down regulated to nearly non-detectable levels by 6 hours. IGF-I 2Eb mRNA was not affected by the short-term GH elevation. Our results showed that IGF-I 1Ea and 2Ea mRNA were differentially regulated by chronic low or acute high levels of GH. These results suggest that the regulation of IGF-I 1Ea and 2Ea mRNA transcription involve different postreceptor molecules and/or feedback mechanisms.

Alternative Splicing↗

PubMed1998

Pharmacological comparison of UTP- and thapsigargin-induced arachidonic acid release in mouse RAW 264.7 macrophages.

1. Although stimulation of mouse RAW 264.7 macrophages by UTP elicits a rapid increase in intracellular free Ca2+ ([Ca2+]i), phosphoinositide (PI) turnover, and arachidonic acid (AA) release, the causal relationship between these signalling pathways is still unclear. In the present study, we investigated the involvement of phosphoinositide-dependent phospholipase C (PI-PLC) activation, Ca2+ increase and protein kinase activation in UTP-induced AA release. The effects of stimulating RAW 264.7 cells with thapsigargin, which cannot activate the inositol phosphate (IP) cascade, but results in the release of sequestered Ca2+ and an influx of extracellular Ca2+, was compared with the effects of UTP stimulation to elucidate the multiple regulatory pathways for cPLA2 activation. 2. In RAW 264.7 cells UTP (100 microM) and thapsigargin (1 microM) caused 2 and 1.2 fold increases, respectively, in [3H]-AA release. The release of [3H]-AA following treatment with UTP and thapsigargin were non-additive, totally abolished in the Ca2+-free buffer, BAPTA (30 microM)-containing buffer or in the presence of the cPLA2 inhibitor MAFP (50 microM), and inhibited by pretreatment of cells with pertussis toxin (100 ng ml(-1)) or 4-bromophenacyl bromide (100 microM). By contrast, aristolochic acid (an inhibitor of sPLA2) had no effect on UTP and thapsigargin responses. 3. U73122 (10 microM) and neomycin (3 mM), inhibitors of PI-PLC, inhibited UTP-induced IP formation (88% and 83% inhibition, respectively) and AA release (76% and 58%, respectively), accompanied by a decrease in the [Ca2+]i rise. 4. Wortmannin attenuated the IP response of UTP in a concentration-dependent manner (over the range 10 nM-3 microM), and reduced the UTP-induced AA release in parallel. RHC 80267 (30 microM), a specific diacylglycerol lipase inhibitor, had no effect on UTP-induced AA release. 5. Short-term treatment with PMA (1 microM) inhibited the UTP-stimulated accumulation of IP and increase in [Ca2+]i, but had no effect on the release of AA. In contrast, the AA release caused by thapsigargin was increased by PMA. 6. The role of PKC in UTP- and thapsigargin-mediated AA release was shown by the blockade of these effects by staurosporine (1 microM), Ro 31-8220 (10 microM), Go 6976 (1 microM) and the down-regulation of PKC. 7. Following treatment of cells with SK&F 96365 (30 microM), thapsigargin-, but not UTP-, induced Ca2+ influx, and the accompanying AA release, were down-regulated. 8. Neither PD 98059 (100 microM), MEK a inhibitor, nor genistein (100 microM), a tyrosine kinase inhibitor, had any effect on the AA responses induced by UTP and thapsigargin. 9. We conclude that UTP-induced cPLA2 activity depends on the activation of PI-PLC and the sustained elevation of intracellular Ca2+, which is essential for the activation of cPLA2 by UTP and thapsigargin. The [Ca2+]i-dependent AA release that follows treatment with both stimuli was potentiated by the activity of protein kinase C (PKC). A pertussis toxin-sensitive pathway downstream of the increase in [Ca2+]i was also shown to be involved in AA release.

Animals↗

PubMed1998

Distinct PKC isoforms mediate the activation of cPLA2 and adenylyl cyclase by phorbol ester in RAW264.7 macrophages.

The modulatory effects of protein kinase C (PKC) on the activation of cytosolic phospholipase A2 (cPLA2) and adenylyl cyclase (AC) have recently been described. Since the signalling cascades associated with these events play critical roles in various functions of macrophages, we set out to investigate the crosstalk between PKC and the cPLA2 and AC pathways in mouse RAW 264.7 macrophages and to determine the involvement of individual PKC isoforms. The cPLA2 and AC pathways were studied by measuring the potentiation by the phorbol ester PMA of ionomycin-induced arachidonic acid (AA) release and prostagladin E1 (PGE1)-stimulated cyclic AMP production, respectively. PMA at 1 microM caused a significant increase in AA release both in the presence (371%) and absence (67%) of ionomycin induction, while exposure of RAW 264.7 cells to PMA increased PGE1 stimulation of cyclic AMP levels by 208%. Treatment of cells with staurosporine and Ro 31-8220 inhibited the PMA-induced potentiation of both AA release and cyclic AMP accumulation, while Go 6976 (an inhibitor of classical PKC isoforms) and LY 379196 (a specific inhibitor of PKCbeta) inhibited the AA response but failed to affect the enhancement of the cyclic AMP response by PMA. Long term pretreatment of cells with PMA abolished the subsequent effect of PMA in potentiating AA release, but only inhibited the cyclic AMP response by 42%. Neither PD 98059, an inhibitor of MEK, nor genistein, an inhibitor of tyrosine kinases, had any effect on the ability of PMA to potentiate AA or cyclic AMP production. The potentiation of AA release, but not of cyclic AMP formation, by PMA was sensitive to inhibition by wortmannin. This effect was unrelated to the inhibition of PKC activation as deduced from the translocation of PKC activity to the cell membrane. Western blot analysis revealed the presence of eight PKC isoforms (alpha, betaI, betaII, delta, epsilon, mu, lambda and xi) in RAW 264.7 cells and PMA was shown to induce the translocation of the alpha, betaI, betaII, delta, epsilon and mu isoforms from the cytosol to the cell membrane within 2 min. Pretreatment of cells with PMA for 2-24 h resulted in a time-dependent down-regulation of PKCalpha, betaI, betaII, and delta expression, while the levels of the other four PKC isozymes were unchanged after PMA treatment for 24 h. A decrease in the potentiation of AA release by PMA was observed, concomitant with the time-dependent down-regulation of PKC. These results indicate that PKCbeta has a crucial role in the mediation of cPLA2 activation by the phorbol ester PMA, whereas PMA utilizes PKC epsilon and/or mu to up-regulate AC activity.

Adenylyl Cyclases↗

PubMed1998

Mutations in the Delta1-pyrroline 5-carboxylate dehydrogenase gene cause type II hyperprolinemia.

We surveyed Delta1-pyrroline 5-carboxylate dehydrogenase genes from four patients with hyperprolinemia type II using RT-PCR amplification, genomic PCR amplification and direct sequencing. We found four mutant alleles, two with frameshift mutations [A7fs(-1) and G521fs(+1)] and two with missense mutations (S352L and P16L). To test the functional consequences of three of these, we expressed them in a P5CDh-deficient strain of Saccharomyces cerevisiae . In contrast to wild-type human P5CDh, yeast expressing S352L and G521fs(+1) failed to grow on proline and had no detectable P5CDh activity. The P16L allele, however, produced fully functional P5CDh and subsequent analysis suggests that it is polymorphic in the relevant (Spanish) population. Interestingly, the G521fs(+1) allele segregates in the large Irish Traveller pedigree used to define the HPII phenotype. To our knowledge, this is the first description of the molecular basis for this inborn error.

1-Pyrroline-5-Carboxylate Dehydrogenase↗

PubMed1998

Lipoxygenase metabolites as mediators of UTP-induced intracellular acidification in mouse RAW 264.7 macrophages.

In previous studies, we have shown that mouse RAW 264.7 macrophages possess pyrimidinoceptors, coupled to a phosphoinositide-specific phospholipase C, with a higher specificity for UTP than for ATP. In the current study, we explored the mechanism involved in the UTP-induced intracellular acidification seen in this cell line. UTP (30 microM) caused a reversible pHi decrease of 0.16 +/- 0.01 unit; this effect was not influenced by the removal of extracellular Cl- or Na+ ions or by pretreatment with 5-(N-ethyl-N-isopropyl)-amiloride (10 microM), 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 microM), staurosporine (1 microM), or Ro 31-8220 (1 microM) but was completely abolished by the removal of extracellular Ca2+. UTP (30 microM), thapsigargin (1 microM), and ionomycin (1 microM) each induced a similar extent of external Ca2+-dependent acidification with a similar time-dependency, but the effects were nonadditive. To further investigate the Ca2+-dependent mechanism, we studied the involvement of arachidonic acid (AA) and eicosanoid metabolites. The addition of AA (10 microM) but not arachidic acid (100 microM) produced a reduction in pHi. UTP, thapsigargin, and ionomycin induced Ca2+-dependent AA release. Furthermore, 4-bromo-phenacyl bromide [30 microM, a phospholipase A2 (PLA2) inhibitor-, nordihydroguaiaretic acid (50 microM, a lipoxygenase inhibitor), and MK-886 (10 microM, a 5-lipoxygenase-activating protein inhibitor) abolished the UTP- or ionomycin-induced responses, whereas indomethacin (30 microM, a cyclooxygenase inhibitor) and baicalein (10 microM, a selective 12-lipoxygenase inhibitor) had no effect. MAFP (a cPLA2 inhibitor) and REV 5901 (a 5-lipoxygenase inhibitor as well as a competitive antagonist of peptide leukotrienes), but not RHC 80267 (a diacylglycerol lipase inhibitor), also inhibited the UTP-induced response. In contrast, the pHi response to AA was unaffected by the presence of 4-bromo-phenacyl bromide or the removal of extracellular Ca2+ ions but abolished by addition of NDGA. Exogenous 5-hydroperoxyeicosatetraenoic acid (2 microM) also produced marked acidification, and UTP and ionomycin both induced peptide leukotriene formation. In conclusion, this is the first report indicating that lipoxygenase metabolites act as mediators of the Ca2+-dependent acidification seen in macrophages in response to UTP or ionomycin via activation of cPLA2 and AA release.

Animals↗

PubMed1998

Image analysis assessment of testicular touch preparation cytologies effectively quantifies human spermatogenesis.

PURPOSE: We have recently demonstrated that computer assisted image analysis of paraffin embedded testicular tissue based on deoxyribonucleic acid content and morphology characteristics is an effective method for the quantitative assessment of spermatogenesis. We assess the use of testicular touch preparation image analysis as a technique for quantification of spermatogenesis. MATERIALS AND METHODS: Air dried, touch imprints of testicular tissue from obstructed azoospermic and severely oligozoospermic patients were obtained at the time of biopsy. Image analysis using a filter based on deoxyribonucleic acid content and cellular morphological characteristics was performed on Feulgen stained touch preparation imprints as well as paraffin embedded sections. RESULTS: Image analysis of 52 testicular touch preparations from 48 azoospermic or severely oligozoospermic men revealed significant differences (p < 0.05) in the percentages of spermatid and spermatozoa, and 2N and 4N cells among seminiferous tubules exhibiting the 5 diagnostic categories of obstruction with normal spermatogenesis, maturation arrest at the spermatocyte stage, maturation arrest at spermatid stage, hypospermatogenesis and Sertoli cell only. Similar differences were observed in the image analysis data of the corresponding paraffin embedded testicular sections. CONCLUSIONS: Computer assisted image analysis of testicular touch preparation is an effective quantitative method of spermatogenesis evaluation.

Adult↗

PubMed1997

Serine/threonine kinase activity associated with the cytoplasmic domain of the lymphotoxin-beta receptor in HepG2 cells.

The lymphotoxin-beta receptor (LT-betaR) has been shown to be the receptor for the membrane-bound lymphotoxin heterotrimers LTalpha1/beta2 and LTalpha2/beta1. The extracellular domain of LT-betaR shows extensive similarity with members of the tumor necrosis factor receptor family, while its cytoplasmic domain is distinct and lacks any inherent enzymatic activity. This suggests that the interaction of LT-betaR with other molecules might be important for signal transduction. Here we demonstrate the association of a fusion protein, comprising glutathione S-transferase and the cytoplasmic domain of LT-betaR (GST-LT-betaR(CD)), with several proteins in the size range 29-80 kDa from HepG2 cell lysates. We present evidence that two of these proteins are serine/threonine kinases, which associate with amino acids 324-377 of the cytoplasmic domain of LT-betaR and phosphorylate this receptor. The characteristics of these novel kinases indicate that they are distinct from the previously described tumor necrosis factor receptor-associated kinases. This suggests the presence of novel signal transduction pathway(s) for LT-betaR.

Amino Acids↗

PubMed1997

Inhibition of ecto-ATPase by the P2 purinoceptor agonists, ATPgammaS, alpha,beta-methylene-ATP, and AMP-PNP, in endothelial cells.

Ecto-ATPase is a plasma membrane-bound enzyme that sequentially dephosphorylates extracellular nucleotides such as ATP. This breakdown of ATP and other nucleotides makes it difficult to characterize and classify P2 purinoceptors. We have previously shown that the P2 purinergic antagonists, PPADS, suramin and reactive blue, act as ecto-ATPase inhibitors in various cell lines. Here, we show that the P2 purinergic agonists, ATPgammaS, alpha,beta-methylene ATP (alpha,beta-MeATP) and AMP-PNP, inhibit the ecto-ATPase of bovine pulmonary artery endothelial cells (CPAE), with pIC50 values of 5.2, 4.5 and 4.0, respectively. In CPAE, FPL67156, a selective ecto-ATPase inhibitor, also inhibits ecto-ATPase activity, with a pIC50 value of 4.0. In addition, alpha,beta-MeATP (3-100 microM), which itself does not induce phosphoinositide (PI) turnover, left-shifted the agonist-concentration effect (E/[A]) curves for ATP, 2MeS-ATP and UTP by approximate 100-300 fold, while those for ATPgammaS and AMP-PNP were only shifted approximately 2-3 fold. Moreover, in the presence of alpha,beta-MeATP, not only was the potentiation effect of PPADS on the UTP response lost, but a slight inhibition of the UTP response by PPADS was also seen. Thus, we conclude that the action of ATPgammaS, alpha,beta-MeATP and AMP-PNP as ecto-ATPase inhibitors account for their high agonist potency, and also provide information for the development of ecto-ATPase inhibitors of high selectivity and potency.

Adenosine Triphosphatases↗

PubMed1997

Priming effects of lipopolysaccharide on UTP-induced arachidonic acid release in RAW 264.7 macrophages.

Stimulation of mouse RAW 264.7 macrophages with UTP activates both the inositol phosphate signal transduction pathway and the phospholipase A2 pathway. In the present study, we investigated the interactions between bacterial lipopolysaccharide and UTP in these two systems and the underlying mechanisms involved. While the UTP-induced release of arachidonic acid was only 2.9-fold that in controls, priming the cells with 1 microgram/ml lipopolysaccharide for 1 h before UTP treatment resulted in 9.2-fold arachidonic acid release upon stimulation with UTP. Lipopolysaccharide priming was both concentration- and time-dependent with a peak effect after 1 h treatment at a concentration of 1 microgram/ml. Lipopolysaccharide treatment affect neither the basal nor the UTP-stimulated inositol phosphate formation and [Ca2+]i rise. Pretreatment of the cells with staurosporine, calphostin, N-(2-aminoethyl)-5-isoquinolinesulfonamide H-7), genistein or K-252a led marked inhibition of the priming effect, suggesting that both protein kinase C and tyrosine kinase are involved in the lipopolysaccharide effect. Buffering intracellular Ca2+ levels using [1,2-bis-(o-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester] (BAPTA/AM) or pretreatment with either N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H-89), 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD098059) or {1-N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl] -4-phenyl-piperazine (KN-62) did not affect the lipopolysaccharide-induced priming effect. Primed UTP stimulation was inhibited by actinomycin D and cycloheximide, indicating a requirement for both gene expression and protein translation. To further examine whether the stimulatory effects of lipopolysaccharide on phospholipase A2 activity were independent of [Ca2+]i levels but dependent on protein phosphorylation, a fixed Ca2+ concentration and inhibitors of protein phosphatases were used in primed permeabilized cells. Arachidonic acid release from permeabilized cells containing 100 nM Ca2+ was high in lipopolysaccharide-primed cells and potentiated by addition of microcystin, orthovanadate or FK 506. These results that the Ser/Thr and tyrosine phosphorylation cascades induced by protein kinase C and tyrosine kinase, respectively, are required for the arachidonic acid potentiation effect of lipopolysaccharide, which was independent of modulation of the upper stream signaling pathways of UTP.

Animals↗

PubMed1997

In situ end-labeling of human testicular tissue demonstrates increased apoptosis in conditions of abnormal spermatogenesis.

OBJECTIVE: To determine, using an in situ end-labeling technique, whether the frequency of apoptosis is increased in testis biopsy specimens that demonstrate abnormal spermatogenesis. DESIGN: Immunohistochemical analysis was performed on archived paraffin-embedded testis biopsy specimens. Apoptotic indices, defined as the number of apoptotic bodies per the total number of cells or the number of Sertoli cells, were calculated after counting all the intratubular spermatogenic cells and Sertoli cells in 20 tubules. SETTING: Major academic male factor infertility clinic. PATIENT(S): Forty-eight testis biopsy specimens were obtained for routine clinical purposes from 38 men with azoospermia or severe oligozoospermia. INTERVENTION(S): In situ end-labeling was performed on archived paraffin-embedded testis biopsy specimens using terminal deoxynucleotidyl transferase. MAIN OUTCOME MEASURE(S): Apoptotic indices. RESULT(S): Significantly increased apoptotic indices were observed in patients with spermatocyte arrest, spermatid arrest, and hypospermatogenesis compared with patients with normal spermatogenesis and the Sertoli cell-only pattern. CONCLUSION(S): In situ end-labeling of testis biopsy specimens from infertile men demonstrates increased apoptosis in maturation arrest and hypospermatogenesis states compared with normal spermatogenesis and the Sertoli cell-only pattern. This unique observation implicates a prominent role for this form of programmed cell death in the pathophysiology of maturation arrest and hypospermatogenesis states.

Adult↗

PubMed1997

Apoptotic frequency is increased in spermatogenic maturation arrest and hypospermatogenic states.

PURPOSE: Increased testicular apoptosis has been observed in maturation arrest and hyposper-matogenesis states in rodent models, but this process has not yet been characterized in humans. We hypothesized that increased cell death present with accelerated apoptosis is significant in pathophysiology of many male infertility states associated with abnormal spermatogenesis. We examined frequency of apoptotic bodies in human testis biopsy specimens from infertile men using morphometric analysis of hematoxylin and eosin stained paraffin sections. MATERIALS AND METHODS: Testis biopsy specimens were obtained for routine clinical purposes from azoospermic and severely oligozoospermic men and were stained with hematoxylin and eosin. Apoptotic bodies were identified using established morphometric criteria. Apoptotic indexes, defined as apoptotic bodies per total number of cells and per Sertoli cells, were calculated after counting all intratubular spermatogenic cells and Sertoli cells in 20 tubules. RESULTS: A total of 51 biopsies was performed in 50 men. Significantly increased apoptotic body per total cell and apoptotic body per Sertoli cell ratios were observed in maturation arrest and hypospermatogenesis states in comparison to Sertoli cell only and normal spermatogenesis (p < 0.05, Mann-Whitney test). CONCLUSIONS: Increased apoptosis in maturation arrest and hypospermatogenesis states compared to normal but obstructed spermatogenesis and Sertoli cell only were observed, indicating a prominent role for this form of programmed cell death in human male infertility.

Apoptosis↗

PubMed1997

Involvement of protein kinase C in the UTP-mediated potentiation of cyclic AMP accumulation in mouse J774 macrophages.

1. We have investigated the effects of nucleotide analogues on cyclic AMP formation in mouse J774 macrophages and the mechanisms involved. 2. UTP, in the concentration range 0.1-100 microM, induced concentration-dependent potentiation of prostaglandin E1 (PGE1)-induced cyclic AMP formation, but had no effect on basal cyclic AMP formation. UDP showed an equal potency, while 2-methylthio ATP, alpha, beta-methylene ATP and beta,gamma-methylene ATP gave either a slight increase or had no effect at concentrations up to 100 microM. ATP, although 100 fold less effective than UTP, also caused cyclic AMP potentiation, but had no effect on agonist-stimulated or basal cyclic AMP levels. 3. The cyclic AMP potentiation effect of UTP correlated with increased [Ca2+]i and inositol phosphate (IP) formation over the same concentration range. 4. Ionomycin, which evokes an increase in [Ca2+]i without affecting IP formation, did not cause an increase in cyclic AMP content, indicating that UTP-induced cyclic AMP regulation is not due to activation of Ca(2+)-sensitive adenylyl cyclase isoforms. 5. Although reduced, UTP potentiation was seen in cells incubated in a Ca(2+)-free and/or BAPTA-containing medium. Under these conditions, the UTP-increased IP accumulation was similarly reduced. 6. Exposure of cells to phorbol 12-myristate 13-acetate (PMA) also increased PGE1 stimulation of cyclic AMP levels, and the UTP-induced potentiation of cyclic AMP formation was inhibited by either staurosporine or Ro 31-8220. Pretreatment of cells with PMA for 4-24 h resulted in marked attenuation of UTP-stimulated cyclic AMP potentiation. 7. Pretreatment with pertussis toxin (24 h, 100 ng ml-1) did not significantly affect UTP-induced cyclic AMP potentiation and IP formation, although it increased the cyclic AMP response to PGE1. 8. Analysis of J774 cells by Western blotting with antibodies specific for different protein kinase C (PKC) isoforms shows the presence of the beta I, beta II, delta, epsilon, eta, mu, lambda and zeta isoforms. Moreover, UTP significantly increased the level of PKC beta I, beta II, delta, epsilon, mu, lambda and zeta immunoreactivity in the membrane fraction and decreased the cytosolic reactivity of PKC beta II, delta, epsilon and zeta. 9. Immunoblot studies also indicate the presence of type II adenylyl cyclase. 10. These results indicate that PKC is required for the potentiation of adenylyl cyclase activity by macrophage pyrimidinoceptors, which exhibit a higher specificity for UTP and UDP than for ATP.

Adenylate Cyclase Toxin↗

PubMed1997

Effects of depolarization and NMDA antagonists on the role survival of cerebellar granule cells: a pivotal role for protein kinase C isoforms.

Primary cultures of cerebellar granule cells (CGCs) grown in high-K+ (25 mM; K25) medium progressively differentiate in vitro. Differentiation is noticeable after 3-4 days in vitro (DIV) and reach a mature stage after 8 DIV. Longer cultivation of CGCs (>13 DIV) triggers the processes of spontaneous cell death. However, if cultured in normal physiological K concentration (5 mM; K5), a significant proportion of the cells dies by the end of the first week in culture. To address the role of protein kinase C (PKC) in the development of CGCs, we measured the kinase activity as well as the protein level of the kinase isoforms. As the K25 CGC culture proceeded, the PKC activity time-dependently increased by 3.2-fold, reaching a steady state at 8 DIV. Western blot analysis using PKC isoform-specific antibodies revealed an increase in levels of PKC alpha, gamma, mu, lambda, and iota from 2 to 8 DIV. A slight increase or decrease at 4 DIV was observed for PKC epsilon and betaII, respectively, whereas no significant change was observed for betaI. The isoforms of delta, theta, eta, and zeta were not detected. Comparing the 14 DIV cultures with the 10 DIV cultures, the immunoreactivities of PKC iota and epsilon were decreased, those of PKC alpha, betaI, betaII, gamma, and lambda were unchanged, whereas that of PKC mu was still increased. In K5 cultures, the immunoreactivity of each PKC isoform at 2-4 DIV was similar to that observed in K25 cells, although no remarkable differentiation features were observed. Coordinated with the appearance of cell death at 8 DIV in low-K+ cultures, levels of PKC alpha, mu, lambda, and iota, but not the others, were markedly decreased. The NMDA receptor antagonists MK-801 and 2-amino-5-phosphopentanoic acid markedly prevented the age-induced apoptosis of CGCs, and the cells survived >18 DIV under these conditions. The cytoprotective effect of MK-801 was concomitant with the increases in levels of PKC gamma, lambda, iota, and mu at 10 and 14 DIV. In addition, the PKC epsilon level was increased at 14 DIV but decreased at early stages, whereas PKC alpha, betaI, and betaII levels were unchanged, as compared with K25 culture alone. Taken together, induction and up-regulation of PKC isoforms may play an important role in the maintenance of CGC survival by depolarization and MK-801.

2-Amino-5-phosphonovalerate↗

PubMed1997

Image analysis assessment of the abnormal testis biopsy in male infertility.

PURPOSE: We previously demonstrated that testis biopsy image analysis is an effective method for quantifying intratubular spermatogenic cells in the obstructed testis with normal spermatogenesis. As an extension of the initial report, we describe using the quantitative ploidy and morphological characteristics of cells counted with image analysis in abnormal testis biopsies obtained for a male infertility evaluation. MATERIALS AND METHODS: Image analysis using a specifically designed filter was performed on Feulgen stained 5 microns, sections of paraffin embedded testicular tissue. Archival testicular tissue had been obtained using standard biopsy techniques from patients with azoospermia or severe oligospermia. Qualitative classification was based on standard evaluation of hematoxylin and eosin processed tissue. RESULTS: There were 62 biopsies performed in 58 men. Significant differences in the intratubular content of haploid (spermatozoa and spermatids), diploid and tetraploid cells were found among the 5 categories of abnormalities: the Sertoli-cell-only syndrome, spermatocyte arrest, spermatid arrest, hypospermatogenesis and normal spermatogenesis. Moderate variability was found in the proportion of cell types in spermatid arrest and hypospermatogenesis. CONCLUSIONS: Testis biopsy image analysis provides a quantitative method for categorizing abnormalities of intratubular cell content present in male infertility states by using deoxyribonucleic acid content and morphology characteristics. The limitations of the present qualitative analysis system are emphasized by the moderate variability evident within the current categories of spermatid arrest and hypo-spermatogenesis states.

Adult↗

PubMed1997

Testis biopsy image analysis effectively quantifies spermatogenic cell types.

PURPOSE: Evaluation of the testis biopsy specimen in male infertility has been hampered by the qualitative rather than quantitative nature of routine histological techniques and interpretation. Although useful, flow cytometry has demonstrated significant limitations due to its inability to differentiate among haploid cell types, spermatozoa and spermatids. Thus, deoxyribonucleic acid image analysis was performed on testis biopsy specimens to combine the advantages of quantitative ploidy and morphological characteristics to develop a more effective quantitative system. MATERIALS AND METHODS: Image analysis using a specifically designed filter was performed on Feulgen stained 5 microns. sections of paraffin embedded testicular tissue. The archival testicular tissue had been obtained using standard biopsy techniques from patients with azoospermia and normal spermatogenesis on routine hematoxylin and eosin processed tissue. RESULTS: The overall findings (mean plus or minus standard deviation) in 20 biopsies from 18 men are haploid 54.2 +/- 5.6, diploid 31.2 +/- 5.2 and tetraploid 15.0 +/- 4.6%. Spermatozoa and spermatids contributed 23.2 +/- 5.7 and 30.9 +/- 7.6% to the total cell content, respectively. A mean of 2,462 +/- 254 cells were counted per case. CONCLUSIONS: We used image analysis to evaluate testicular biopsies. This technique can quantitatively determine the percentages of various cell types within the seminiferous tubules. Furthermore, it can differentiate between spermatozoa and spermatids based on morphological characteristics. This technique, which combines ploidy and cell morphology characteristics, represents a significant advancement in the interpretation of the testicular biopsy, and has implications for redefining the presently used system of qualitative interpretative assessment.

Adult↗

PubMed1996

Cloning, characterization, and expression of cDNAs encoding human delta 1-pyrroline-5-carboxylate dehydrogenase.

Delta 1-pyrroline-5-carboxylate dehydrogenase (P5CDh; EC 1.5.1.12), a mitochondrial matrix NAD(+)-dependent dehydrogenase, catalyzes the second step of the proline degradation pathway. Deficiency of this enzyme is associated with type II hyperprolinemia (HPII), an autosomal recessive disorder characterized by accumulation of delta 1-pyrroline-5-carboxylate (P5C) and proline. As an initial step in understanding the biochemistry of human P5CDh and molecular basis of HPII, we utilized published peptide sequence data and degenerate primer polymerase chain reaction to clone two full-length human P5CDh cDNAs, differing in length by 1 kilobase pair (kb). Both cDNAs have the identical 1689-base pair open reading frame encoding a protein of 563 residues with a predicted molecular mass of 62 kDa. The long cDNA contains an additional 1-kb insert in the 3'-untranslated region that appears to be an alternatively spliced intron. The conceptual translation of human P5CDh has 89% sequence identity with the published human P5CDh peptide sequences and 42 and 26% identity with Saccharomyces cerevisiae and Escherichia coli P5CDhs, respectively, as well as homology to several other aldehyde dehydrogenases. Both P5CDh cDNA clones detect a single 3.2-kb transcript on Northern blots of multiple human tissues, indicating the long cDNA containing the 3'-untranslated intron represents the predominant transcript. The P5CDh structural gene appears to be single copy with a size of about 20 kb localized to chromosome 1. To confirm the identity of the putative P5CDh cDNAs, we expressed them in a P5CDh-deficient strain of S. cerevisiae. Both conferred measurable P5CDh activity and the ability to grow on proline as a sole nitrogen source.

1-Pyrroline-5-Carboxylate Dehydrogenase↗