Stimulation of Protein Synthesis in Pachytene Primary Spermatocytes from the Human Testis by Pyruvate

A sequential enzymatic incubation in collagenase and trypsin was carried out to yield a suspension of viable single cells from the seminiferous epithelium of adult human testis. The cell suspension predominantly consisted of pachytene primary spermatocytes (15%), round spermatids (32%), and condensing spermatids and residual bodies (21%). Human pachytene spermatocytes were isolated by unit gravity sedimentation using the methods originally developed for murine tissue. The spermatocyte-enriched fraction was 79% pure. When the effect of energy sources on protein synthesis by spermatocytes was examined, the highest rate of protein synthesis with pyruvate was found among four kinds of substrates added at a concentration of 10 mM. As shown with murine spermatocytes, the rate of protein synthesis by the human spermatocytes is probably regulated by pyruvate.     

Effect of Hypophysectomy on Ribonucleic Acid Synthesis in Primary Spermatocytes Isolated from Rat Testes

RNA synthesis was examined in pachytene spermatocytes isolated from intact immature rats and from hypophysectomized immature rats (2–4 days after hypophysectomy). Hypophysectomy did not result in changes in the synthesis and turnover of "heterogeneous" RNA as well as the synthesis and processing of rRNA from spermatocytes. The uptake and incorporation of ³H-uridine in the spermatocyte population isolated from hypophysectomized rats, however, was decreased. These contradictory results were explained by radioautographic experiments which showed that 25 ± 6% of the pachytene spermatocytes isolated from hypophysectomized rats were unlabelled after incubation with ³H-uridine, whereas only 3 ± 2% of the pachytene spermatocytes from intact rats were unlabelled. It appeared therefore that in the absence of gonadotropic hormones from the pituitary gland, RNA synthesis in many spermatocytes remained unchanged for at least several days, although an increased number of the spermatocytes became inactive.     

Unanticipated Changes in Steady-state mRNA Levels for Glyceraldehyde-3-Phosphate Dehydrogenase in Rat Tibiae

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels are commonly used as an internal control to normalize gene expression data based on the belief that this gene is constitutively expressed. However, GAPDH mRNA levels increased by more than 2.5-fold in tibiae of hindlimb unloaded female rats compared to L32 mRNA levels. Similarly, GAPDH mRNA levels increased compared to 18S ribosomal RNA. Treatment with growth hormone and alcohol show no disparity in GAPDH mRNA levels whereas in some experiments, parathyroid hormone and 17-estradiol increased GAPDH mRNA levels. Taken together, these findings indicate that it is essential to demonstrate that GAPDH expression is not altered prior to using the gene as an internal control.     

Histopathological Changes in Testes of Adult Inbred Rats Immunized Against Pachytene Spermatocytes or Sertoli Cells

Adult inbred Lewis/Wistar rats, immunized against pachytene spermatocytes or Sertoli cells prepared from maturing rats of the same strain, develop histopathological changes in the testes comparable in several respects to those reported by others in classical experimental allergic orchitis. Early changes in the structure of pachytene spermatocytes are followed by germ cell degeneration, and defoliation of germinal cells into the lumen. Concomitantly, abnormalities develop in Sertoli cell structure, and the lymph-testis barrier becomes increasingly permeable. Sertoli cells are the only testicular somatic cells which appear to be directly sensitive during the immunologic response. Disturbances in Sertoli cell functions are postulated to be crucial in the etiology of aspermatogenesis. The data are discussed in relation to the possible roles in spermatogenesis of specific antigenic determinants present on the surfaces of mature Sertoli cells and advanced germinal cells located within the adluminal compartment of the seminiferous tubule.     

Thymidine Phosphorylase and Dihydropyrimidine Dehydrogenase Expression Levels in Tumor and Normal Tissue Specimens of T3 Human Colorectal Carcinoma

PURPOSE: Thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) are important enzymes related to the metabolism of 5-fluorouracil and its derivatives. We evaluated the association between the clinicopathological factors and these enzymes in patients with T3 colorectal carcinoma. METHODS: The TP and DPD expression levels in 15 patients with T3 colorectal carcinomas were measured in tumor and adjacent normal tissue specimens by enzyme-linked immunosorbent assay. Correlations between each enzyme and clinicopathological factors were also statistically evaluated. RESULTS: The TP levels in tumor and normal tissue specimens were 77.9 +/- 33.6 and 24.7 +/- 10.3, respectively (P < 0.001). The DPD levels in tumor and normal tissue specimens were 44.1 +/- 18.2 and 53.1 +/- 24.1, respectively (P = 0.46). The TP/DPD ratios in tumor and normal tissue specimens were 1.84 +/- 0.52 and 0.53 +/- 0.26, respectively (P < 0.001). The tumor/normal ratios of TP level in patients with and without liver metastasis were 1.79 +/- 0.91 and 4.67 +/- 2.51, respectively (P = 0.024). CONCLUSION: The measurement of the enzyme expression levels of TP and DPD is considered to be useful for better understanding the conditions of tumor progression. The mechanisms of regulation of these enzymes thus require further evaluation.     

IGFBP 3在白藜芦醇抑制胃癌细胞增殖过程中的作用

目的研究在白藜芦醇（Res）抑制胃癌细胞增殖过程中,胰岛素样生长因子结合蛋白 3（IGFBP 3）表达的变化。方法噻唑蓝（MTT）法测定Res对BGC 823细胞增殖的抑制程度,实时荧光定量聚合酶链反应（qRT PCR）和Western blot技术检测Res处理后BGC 823细胞中IGFBP 3表达变化,siRNA技术沉默IGFBP 3基因表达,MTT法观察Res对BGC 823细胞增殖的影响,流式细胞技术测定各组细胞凋亡率。结果Res能够抑制BGC 823生长,经20,40,80,160 μmol·L－1Res处理后,细胞存活率分别为（82.35±10.65）%,（74.30±12.36）%,（62.80±14.66）%,（50.75±11.14）%。Res刺激后,IGFBP 3表达水平升高,与对照组比较,IGFBP 3 mRNA水平增高2.96 倍（P＜0.05）, IGFBP 3蛋白水平变化与其mRNA一致。siRNA技术沉默IGFBP 3表达后,160 μmol·L－1Res刺激BGC 823细胞,细胞存活率下降（78.5±9.86）%,但高于同浓度下Res刺激的未经IGFBP 3沉默的BGC 823细胞（P＜0.05）。IGFBP 3沉默后,160 μmol·L－1Res处理后BGC 823细胞凋亡率（20.13±9.12）%,明显低于未经IGFBP 3沉默的BGC 823细胞［（35.48±11.12）%］,且差异有统计学意义（P＜0.05）。结论Res可以抑制BGC 823细胞增殖,促进其凋亡。该机制涉及IGFBP 3高表达。     

Overexpression of Human Hydroxysteroid (17β) Dehydrogenase 2 Induces Disturbance in Skeletal Development in Young Male Mice

To understand the function of human hydroxysteroid (17β) dehydrogenase 2 (HSD17B2) in the peripheral tissues in vivo, we studied the bone development in transgenic male mice ubiquitously expressing human HSD17B2. Bones of HSD17B2TG and WT males (26 days and 2 and 6 mo old) were analyzed by pQCT and histomorphometry, and data were correlated with serum testosterone (T), IGF‐I, and osteocalcin concentrations. At the age of 26 days, the body weight of HSD17B2TG males was significantly lower, and the lengths of the tibia and femur of the HSD17B2TG males were significantly shorter. Histomorphometric and pQCT analyses showed lower trabecular and cortical BMD, a markedly smaller area of cortical bone at both of the diaphyses, and a smaller percentage of trabecular bone volume and thickness in the HSD17B2TG males. The data suggested slower osteoblast differentiation and a slower bone formation rate of femoral diaphysis on the periosteum but faster on the endocortical surface in HSD17B2TG males. The altered bone parameters were correlated with low serum T, IGF‐I, and osteocalcin concentrations at the prepubertal age. Interestingly, after puberty, the bone parameters analyzed in the adult HSD17B2TG males were mostly normal, consistent with the normal body weight and normalized serum concentrations of IGF‐I and T. In conclusion, HSD17B2TG males presented with growth retardation and a decreased bone formation rate at prepubertal age. These changes were associated with lower serum IGF‐I, osteocalcin, and T concentrations. It is concluded that the enforced constitutive expression of HSD17B2 disturbs the coordinated action of IGF‐I and sex steroids essential for pubertal bone growth.     

Inhibition of Proliferation by Omega-3 Fatty Acids in Chemoresistant Pancreatic Cancer Cells

Background Pancreatic cancer-gemcitabine (GEM) chemoresistance has been demonstrated to be associated with enhanced NF-kB activation and antiapoptotic protein synthesis. The well-known capacity of omega-3 fatty acids (n-3 FAs) to inhibit NF-kB activation and promote cellular apoptosis has the potential to restore or facilitate gemcitabine chemosensitivity. Methods Four pancreatic cancer cell lines (MIA PaCa-2, BxPC-3, PANC-1, and L3.6), each with distinct basal NF-kB and differing GEM sensitivity profiles, were administered: 100 uM of (1) n-3FA, (2) n-6FA, (3) GEM, (4) n-3FA + GEM, or (5) n-6FA + GEM for 24 and 48 hours. Proliferation was assessed using the WST-1 assay. To define the mechanism(s) of altered proliferation, electron mobility shift assay for NF-kB activity, western blots of phoshoStat3, phosphoIκB, and poly(ADP-ribose) polymerase (PARP) cleavage were performed in the MIA PaCa-2 cell line. Results All cell lines demonstrated a time/dose-dependent inhibition of proliferation in response to n-3FA. For MIA PaCa-2 cells, n-3FA and n-3FA + GEM treatment resulted in reduction of I-kB phosphorylation and NF-kB activation when compared with n-6FA control. n-3FA and combination treatment also significantly decreased Stat3 phosphorylation, whereas GEM alone had no effect. n-3FAs and n-3FA + GEM groups demonstrated increased PARP cleavage, mirroring NF-kB activity and Stat3 phosphorylation. Conclusions n-3 FA treatment is specifically associated with inhibition of proliferation in these four pancreatic cell lines irrespective of varied gemcitabine resistance. An experimental paradigm to screen for potential contributory mechanism(s) in altered pancreatic cancer cellular proliferation was defined, and using this approach the co-administration of n-3 FA with GEM inhibited GEM-induced NF-kB activation and restored apoptosis in the MIA PaCa-2 cell-line. Supported by: NIDDK K08 DK DK60778 (Espat)     

In vitro Studies on Histochemical Localization of Testicular δ5 -3β-Hydroxysteroid Dehydrogenase Activity from Indomethacin Pretreated Rats — Effect of Prostaglandins and Luteinizing Hormone

In-vitro-Untersuchungen uber die histochemische Lokalisation der δ⁵ -3β-Hydroxysteroid Dehydrogenase Activity from Indomethacin Pretreated Rats — Effect of Prostaglandins and Luteinizing Hormone
Bei der Verwendung von mit Indomethacin vorbehandelten Rattenhoden als experi-mentelle Grundlage für in vitro Untersuchungen über den Effekt von LH und Prostaglandinen auf die δ⁵-3β-hydroxysteroiddehydrogenase stellte sich eine stärkere Stimulation der Enzymaktivität heraus, wenn das Inkubationsmedium LH und PGE2 in einer Konzentration von jeweils 1 μg/ml enthielt. Der Effekt von 1 μg/ml LH allein war geringer, aber der Verstärkungseffekt von PGE2 auf die testikuläre Antwort der LH-Zu-fuhr konnte nicht mehr beobachtet werden, wenn das Verhältnis von LH zu PGE2 bei 1: 10 lag.
Weder PGE2 noch PGF2A führten zu einer Stimulation der Enzymaktivitäten bei Konzentrationen von 1, 10 oder 20 μg/ml im Inkubationsmedium. PGF2A scheint bei Konzentrationen von 1 oder 10 μg/ml nicht mit der LH-Wirkung auf diese steroidgene-tischen Enzyme zu interferieren. Im Gegensatz dazu wirkten die beiden Prostaglandine PGE2 und PGF2A bei Konzentrationen von 20 μg/ml antagonistisch auf die Wirkung von LH auf die testikuläre δ⁵-3β-hydroxysteroid-Dehydrogenaseaktivität.
Auf der Grundlage dieser Untersuchungsergebnisse scheint das Prostaglandin E2 in gewisser Weise für das Zustandekommen der steroidogenetischen Wirkung von LH in den Hoden notwendig zu sein.     

Inhibition of 3 alpha-hydroxysteroid oxidoreductase and 5 alpha-reductase activity by antiandrogens and indomethacin in the rat prostate.

We studied the effects of antiandrogens in vitro on inhibition of 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) and 5 alpha-reductase activities in the rat prostate. Kinetic and inhibition experiments were analyzed by thin layer chromatography. Cyproterone acetate (CA), chlormadinone acetate (CMA), and TZP-4238 (TZP), which is more potent than other antiandrogens, were used as inhibitors and were compared with indomethacin IND, which is a recognized 3 alpha-HSOR inhibitor. The IC50S of CA, CMA, IND, and TZP for 3 alpha-HSOR reductase in cytosol were about 5, 10, 10, and 100 microM, respectively, and inhibition was competitive. The IC50 of IND for 3 alpha-HSOR reductase in microsomes was 20 microM. The IC50S of other inhibitors were > 100 microM, and inhibition was noncompetitive. The IC50S of CA, CMA, IND, and TZP for 3 alpha-HSOR oxidase in cytosol were > 100 microM, and inhibition was competitive or noncompetitive. Inhibition of 3 alpha-HSOR oxidation was not observed in microsomes. The difference between these inhibition patterns suggests that there may be 4 isoenzymes in rat prostatic tissue. The IC50S of MK-906, CMA, and TZP for 5 alpha-reductase in prostate homogenate were about 0.01, 200, and 200 microM, respectively.     

The Effects of Bone Remodeling Inhibition by Alendronate on Three-Dimensional Microarchitecture of Subchondral Bone Tissues in Guinea Pig Primary Osteoarthrosis

We assessed whether increase of subchondral bone density enhances cartilage stress during impact loading, leading to progressive cartilage degeneration and accelerated osteoarthrosis (OA) progression. Sixty-six male guinea pigs were randomly divided into six groups. During a 9-week treatment period, four groups received twice-weekly subcutaneous injections of alendronate (ALN) in two doses: two groups received 10 μg/kg and two groups received 50 μg/kg. The two control groups received vehicle. After 9 weeks, one 10 μg/kg ALN group, one 50 μg/kg ALN group, and one control group were killed. The remaining three groups (17-week groups) were left for an additional 8 weeks, receiving the same treatment regimen before death. The left proximal tibiae were scanned by micro-computed tomography to quantify the microarchitecture of subchondral bone, followed by mechanical testing and determination of collagen and mineral. The control groups had typical OA-related cartilage degeneration at 9 and 17 weeks, whereas the 50 μg/kg ALN group had even worse degeneration in the medial condyle. It is unclear whether there is a direct or a secondary effect of ALN on the cartilage. The 9-week ALN group had significantly greater subchondral plate thickness. The 9- and 17-week groups had similar changes of cancellous bone microarchitecture, with greater volume fraction and connectivity and an extremely plate-like structure. The 9-week ALN group had greater bone mineral concentration, and the 17-week ALN group had reduced collagen concentration and greater mineral concentration. Treatment with ALN did not significantly change the mechanical properties of the cancellous bone.     

Restoration of Mineral Depositions by Dexamethasone in the Matrix of Nonmineralizing Osteoblastic Cells Subcloned from MC3T3-E1 Cells

The original osteoblastic cell line, MC3T3-E1, was derived from normal mouse bone tissue and mineralized without any specific factors in vitro. This cell line may be slightly unstable because of high differentiation, and some of these cells sometimes lost the ability for mineral deposition. In this study, a new cell line was cloned which lost the ability for mineral deposition from MC3T3-E1 cells. This cell line, termed MC3T3-NM4, was not observed to undergo mineral deposition for up to at least 36 days even in media containing beta-glycerophosphate. The alkaline phosphatase (ALP) activity was also not increased. The lack of calcifying ability was found to be restored by the addition of dexamethasone in the media. This restoration was accompanied by an increase in ALP activity and osteocalcin level. It was suggested that this restoration was not due to artificial mineralization resulting from cell death. Received: 3 March 1999 / Accepted: 25 May 2000 / Online publication: 22 September 2000     

Inhibition of TGF‐β signaling by 1D11 antibody treatment increases bone mass and quality in vivo

Transforming growth factor β (TGF‐β) is an abundant bone matrix protein that influences osteoblast and osteoclast interactions to control bone remodeling. As such, TGF‐β represents an obvious pharmacologic target with the potential to regulate both bone formation and resorption to improve bone volume and strength. To investigate the skeletal effect of TGF‐β inhibition in vivo, we used an antibody (1D11) specifically directed at all three isoforms of TGF‐β. Normal mice were treated with 1D11 or control antibody (4 weeks), and cortical and trabecular bone was assessed by micro–computed tomographic (µCT) scanning. Bone volume and cellular distribution were determined by histomorphometric analysis of vertebrae and long bones. Also, whole‐bone strength was assessed biomechanically by three‐point bend testing, and tissue‐level modulus and composition were analyzed by nanoindentation and Raman microspectroscopy, respectively. TGF‐β blockade by 1D11 increased bone mineral density (BMD), trabecular thickness, and bone volume by up to 54%, accompanied by elevated osteoblast numbers and decreased osteoclasts. Biomechanical properties of bone also were enhanced significantly by 1D11 treatment, with increased bending strength and tissue‐level modulus. In addition, Raman microspectroscopy demonstrated that 1D11‐mediated TGF‐β inhibition in the bone environment led to an 11% increase in the mineral‐to‐collagen ratio of trabecular bone. Together these studies demonstrate that neutralizing TGF‐β with 1D11 increases osteoblast numbers while simultaneously decreasing active osteoclasts in the marrow, resulting in a profound increase in bone volume and quality, similar to that seen in parathyroid hormone (PTH)–treated rodent studies. © 2010 American Society for Bone and Mineral Research.     

Inhibition by aminohydroxypropylidene bisphosphonate (AHPrBP) of 1,25(OH)2 vitamin D3-induced stimulated bone turnover in the mouse

Summary The purpose of this study was to evaluate whether the 1,25(OH)₂D₃-induced increased bone mineralization in the mouse occurs in response to stimulation of bone resorption. In order to inhibit bone resorption, 35-day-old mice were given 16 μmol/kg/day of (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (AHPrBP) for 10 days, the first injection occurring 3 days prior to the continuous infusion of 0.06, 0.13, or 0.20 μg/kg/day of 1,25(OH)₂D₃ for 7 days. Two groups of mice were treated with AHPrBP or 1,25(OH)₂D₃ alone. The skeletal changes were assessed by histomorphometric study of caudal vertebrae after double³H-proline and double tetracycline labelings for evaluation of the matrix apposition rate (MaAR) and mineral apposition rate (MiAR), respectively. Treatment with AHPrBP alone or combined to 1,25(OH)₂D₃ decreased the number of acid phosphatase-stained osteoclasts and reduced the endosteal MaAR and MiAR and the amount of osteoid. When given alone, 1,25(OH)₂D₃ increased serum calcium above normal, enhanced the number of histochemically active osteoclasts, and stimulated the endosteal MiAR. Pretreatment with AHPrBP blocked both the increase in serum calcium and the stimulation of the MiAR induced by 1,25(OH)₂D₃ infusion though serum 1,25(OH)₂D₃ levels rose according to the dose given. The results show that 1) the serum calcium and the bone resorbing responses to 1,25(OH)₂D₃ infusion are prevented by pretreatment with AHPrBP, and 2) the stimulatory effect of 1,25(OH)₂D₃ on the mineralization rate is blocked when bone resorption is inhibited. The data indicate that 1,25(OH)₂D₃ promotes bone mineralization in the mouse mainly in response to stimulation of bone resorption.     

Efficacy of PARP Inhibition in Metastatic Castration-resistant Prostate Cancer is Very Different with Non-BRCA DNA Repair Alterations: Reconstructing Prespecified Endpoints for Cohort B from the Phase 3 PROfound Trial of Olaparib

The PROfound trial evaluated the PARP inhibitor olaparib in metastatic castration-resistant prostate cancers harboring alterations in BRCA1/2 and ATM (cohort A) and in 12 other homologous recombination repair genes (cohort B). Olaparib led to more objective responses and longer radiographic progression-free survival than the control in cohort A and when cohorts A and B were combined. The efficacy of olaparib in cohort B was a secondary objective prespecified in the trial protocol but was not reported. Reconstructing patient-level data for cohort B, two of 54 patients (4%) in the olaparib arm and two of 24 patients (8%) in the control arm had a radiographic response, and there was no evidence that olaparib prolonged radiographic progression-free survival in cohort B (hazard ratio 0.88, 95% confidence interval 0.58–1.34). These results are in strong contrast to cohort A.Patient summaryA large clinical study concluded that treatment with the PARP inhibitor olaparib benefits men with metastatic castration-resistant prostate cancer whose tumors harbor alterations in 15 different DNA repair genes. In contrast to the group dominated by BRCA alterations, any potential benefit from olaparib was considerably less, if present at all, for men with prostate cancers harboring one of the 12 other, non-BRCA DNA repair alterations.