bluuebunny/crossref_metadata_2025 huggingface dataset:
Fluoreszenzdiagnostik mit 5-ALA-Thermogel bei zervikaler intraepithelialer Neoplasie
Authors: Andikyan Vaagn , Kronschnabl Manuela , Hillemanns Marija , Wang Xiuli , Stepp Herbert , Hillemanns Peter
Published: December 2003

Fragestellung: Die lokale Applikation von flüssigen Pharmaka vor die Portio uteri ist ein bisher ungelöstes Problem. Deshalb wurde die Pharmakokinetik und Selektivität von 5-Aminolävulinsäure (5-ALA) aus 5-ALA-Thermogel bei Patientinnen mit zervikaler intraepithelialer Neoplasie (CIN) evaluiert. Das neuartige Thermogel Pluronic F127, welches im kalten Zustand in flüssiger Form vorliegt, geht bei über 31°C in einen gelartigen Zustand über und gewährleistet so eine verbesserte Anhaftung von 5-ALA an die Portio. Methoden: Bei 27 Patientinnen mit CIN 1–3 wurden 0,5–12 h vor Konisation 10 ml eines 4-, 10- oder 20%igen 5-ALA-Thermogels auf die Portio uteri aufgetragen. Danach wurden Biopsien von CIN-Läsionen und normalem Gewebe entnommen, die histologisch, fluoreszenzmikroskopisch und fluoreszenzspektrometrisch untersucht wurden. Ergebnisse: Das Thermogel Pluronic F127 erwies sich einfach in der klinischen Handhabung und sehr geeignet als Medium für die lokale Applikation. Die Porphyrinfluoreszenz erreichte ihr Maximum bei der Verwendung von 10%igem 5-ALA-Thermogel und einem Inkubationsintervall von über 2 h. Es wurde eine statistisch signifikante höhere Porphyrin-Fluoreszenzintensität in den CIN-Arealen (1116 ± 241 WE) festgestellt als im normalen umgebenden Plattenepithel (704 ± 166 WE, p < 0,05). Nach 4–6 h war eine maximale Porphyrin-Fluoreszenzintensität im dysplastischen Epithel zu sehen. Die Tumorselektivität erreicht nach über 2 h eine Tumor-zu-Normalgewebe-Ratio von 3,5 bei CIN 3. Schlussfolgerungen: Aufgrund dieser Ergebnisse eignet sich das neuartige Wirkprinzip des Thermogels Pluronic F127 als neue Form der lokalen präzervikalen Applikation von Pharmaka wie Photosensibilisatoren. Für die photodynamische Therapie ist eine Verwendung von 10%igem 5-ALA-Thermogel mit einem Zeitintervall von 4–6 h zu empfehlen.
Compositional Lipoprotein Changes and Low-Density Lipoprotein Susceptibility to Oxidation in Chronic Renal Failure Patients with Heavy Proteinuria
Authors: Karabina Sonia Athena , Pappas Haralampos , Miltiadous George , Bairaktari Eleni , Christides Dimitris , Tselepis Alexandros , Elisaf Moses , Siamopoulos Konstantinos
Published: December 2003

Background: There are limited data regarding qualitative lipoprotein abnormalities in undialysed uremic patients without proteinuria. In this report, we focused on lipoprotein changes observed in uremic patients with proteinuria as well as on the susceptibility of low-density lipoprotein (LDL) of these patients to oxidative modification in vitro. Methods: 20 patients with chronic renal failure [serum creatinine >1.6 mg/dl (141.4 µmol/l)], but not yet on renal replacement therapy, and with heavy proteinuria (>2 g/24 h), and 18 age- and sex-matched healthy individuals participated in the study. In both patients and controls, venous blood was collected for determination of serum lipid and lipoprotein levels, lipoprotein subfraction profile and chemical composition, as well as the susceptibility of LDL subfractions to oxidation. Results: Patients exhibited a more atherogenic lipid profile compared with the control population. Furthermore, the total very LDL + intermediate-density lipoprotein mass was increased in patients compared with controls, while this subfraction was triglyceride enriched in uremic patients. The total LDL concentration was significantly higher in patients compared with controls due mainly to an increase in the mass of all lipoprotein subfractions. It is noteworthy that the mass of small dense LDL was significantly elevated in patients compared with controls (135 ± 12 vs. 115 ± 11 mg/dl, p = 0.01), an increase which was more pronounced in hypertriglyceridemic patients. Furthermore, the subfraction high-density lipoprotein-2 mass was significantly lower in uremic patients compared with controls. Finally, no significant differences in the lag time, the rate of oxidation and the relative electrophoretic mobility values in each LDL subfraction between the two groups were observed. Conclusion: We conclude that uremic patients with heavy proteinuria exhibit compositional lipoprotein changes that are less marked than those observed in nonuremic patients with nephrotic syndrome. However, there is no evidence that circulating LDL isolated from these patients is more susceptible to oxidation in vitro than lipoprotein isolated from age- and gender-matched controls.
Urinary Sediment Podocalyxin in Children with Glomerular Diseases
Authors: Kanno Katsue , Kawachi Hiroshi , Uchida Yoshiaki , Hara Masanori , Shimizu Fujio , Uchiyama Makoto
Published: December 2003

Background: In an immunofluorescence study using antibody to podocalyxin, we reported that urinary excretion of podocytes reflected podocyte injury in glomeruli. However, this method has some problems, since it is basically urine cytology. To overcome problems with this test, we measured whole podocalyxin content in urine sediment by enzyme-linked immunosorbent assay (ELISA). Methods: Urinary sediment podocalyxin (u-sed-PCX) content of the first morning urine was quantified by ELISA after solubilization by detergent. We measured urine samples from children with various glomerular diseases and from healthy volunteers as controls. The glomerular diseases were classified into two categories: group I (inflammatory glomerular, 5 diseases) and group II (non-inflammatory glomerular, 3 diseases). Results: (1) The level of u-sed-PCX was significantly higher in the urine from patients with glomerular diseases (groups I and II, median (interquartile range (IQR)): 2 (0.6–18.5), n = 111) compared with controls (0 (0–0.4), n = 135), and the level of u-sed-PCX in group I diseases (3.4 (0.6–27.2), n = 90) was significantly higher than those in group II diseases (0.9 (0.1–2.5), n = 21). (2) The presence of PCX in urine sediment was confirmed by Western blot analysis. (3) The degree of proteinuria was significantly correlated with the level of u-sed-PCX in group I (rs = 0.539, p < 0.001), but not in group II. (4) In group I, the level of u-sed-PCX was significantly higher in the acute phase than in the chronic phase (p < 0.01). (5) Comparison of histological findings of renal biopsies with u-sed-PCX showed a significant correlation in acute extracapillary lesions (p < 0.05). (6) Persistent high level of u-sed-PCX paralleled good histological progression in renal biopsies. Conclusion: Quantification of urinary sediment podocalyxin by ELISA is a reliable and useful laboratory marker for the estimation of the severity of active glomerular injury and a urinary index of acute extracapillary changes.
Matrix Metalloproteinase-9 Expression in Renal Biopsies of Patients with HIV-Associated Nephropathy
Authors: Ahuja Tejinder S. , Gopalani Anisa , Davies Peter , Ahuja Harleen
Published: December 2003

Background/Aims: The normal architecture and function of the kidney are maintained by a critical turnover of the extracellular matrix (ECM). Although increased synthesis of ECM leads to progression of renal failure, the role of degradation of ECM by metalloproteinases in progressive nephropathies is unclear. Recently, in an animal model of membranous glomerulonephritis (Heymann nephritis), visceral epithelial cell (VEC) injury has been found to rapidly increase matrix metalloproteinase-9 (MMP-9) synthesis by these cells during the period of maximal proteinuria. Because injury to glomerular VECs may be an important initiating factor in the pathogenesis of focal segmental glomerulosclerosis (FSGS), especially in HIV-associated nephropathy (HIVAN), we determined the expression of MMP-9 and its inhibitors in renal biopsies of patients with HIVAN. Methods: RNA was isolated from cortical renal tissue of 6 patients with HIVAN. Only those biopsies which displayed characteristic findings of HIVAN including collapsing FSGS, cystic dilatation of the tubules, and proliferation of VECs were used. As controls, we obtained renal cortical tissue from normal areas of kidneys removed due to malignancies. MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 mRNA expressions were determined using real-time quantitative reverse transcription-polymerase chain reaction. In all 6 patients with HIVAN, MMP-9 and TIMP-1 were overexpressed. We localized MMP-9 protein to the glomeruli with immunohistochemical detection. Conclusion: MMP-9 is overexpressed in the glomeruli of patients with HIVAN and may be involved in the pathogenesis of the disease.
Biology of the Human Podocyte
Authors: Saleem Moin A.
Published: December 2003

The glomerulus is a complex physiological structure that, as well as maintaining a selective filtration barrier, participates in the control of renal blood flow and blood pressure. Recently there has been a surge of interest in the podocyte, as the main player in this functional unit, driven by key discoveries of genes mutated in hereditary conditions, that result in breakdown of barrier functions. This has been accompanied by the development of powerful new molecular, cellular and animal tools to enable study at a level of detail not previously possible. How this emerging information is shaping our understanding of glomerular biology, and ultimately our understanding of the pathophysiology of glomerular diseases, is outlined here.
Effect of Mycophenolate Mofetil on Glomerulosclerosis and Renal Oxidative Stress in Rats
Authors: van den Branden Christiane , Ceyssens Bart , Pauwels Marina , van Wichelen Greet , Heirman Ingeborg , Jie Ni , Verbeelen Dierik
Published: December 2003

Background/Aims: Mycophenolate mofetil (MMF) is known to attenuate glomerulosclerosis in experimental models of renal failure. We investigated whether this is mediated by reduction of oxidative stress. Methods: Effects of MMF on oxidative stress are studied in an experimental rat model (NA model) involving unilateral nephrectomy and two intravenous injections with adriamycin (2 mg/kg). Rats are sacrificed after 2 and 6 weeks. Glomerulosclerosis and tubulointerstitial lesions are demonstrated by histological techniques. Presence of macrophages/monocytes (ED1) and myofibroblasts (α-SMA) is demonstrated by immunohistochemistry. Oxidative stress is evaluated by enzymatic measurements (AOE), spectrofluorometry (TBARS), immunohistochemistry (MDA and HNE) and histology (ferric iron deposition). Results: The NA model shows proteinuria, hypercholesterolemia, beginning glomerulosclerosis, tubulointerstitial sclerosis and tubular dilatation, glomerular, periglomerular and interstitial presence of α-SMA and increased presence of macrophages/monocytes after 6 weeks. Oxidative stress in renal cortex is apparent (increased cortex TBARS concentration, increased glomerular presence of MDA and HNE, decreased activity of antioxidant enzymes, ferric iron deposition in proximal tubules) after 6 weeks. MMF administration results in a decrease of glomerulosclerosis, interstitial sclerosis, glomerular and periglomerular expression of α-SMA and the number of ED1-positive cells in tubulointerstitium and glomeruli. Proteinuria and cholesterolemia are not decreased. TBARS level, and activities of catalase, Mn and Cu/Zn superoxide dismutase as well as the presence of ferric iron in the proximal tubules are not changed by MMF treatment. Cortex activity of glutathione peroxidase returns to normal. Conclusion: MMF has a favorable effect on glomerular and interstitial fibrosis in the NA model of kidney disease, but not on proteinuria and cholesterolemia. Improvement of fibrosis cannot be explained by major changes in oxidative stress or antioxidant defense.
Blocking VLA-4 Prevents Progression of Experimental Crescentic Glomerulonephritis
Authors: Khan Sarah B. , Allen Andrew R. , Bhangal Gurjeet , Smith Jennifer , Lobb Roy R. , Cook H. Terence , Pusey Charles D.
Published: December 2003

Background/Aims: Integrins are adhesion molecules of fundamental importance to the recruitment of leucocytes in inflammation. The α4β1 integrin (VLA-4) is a leucocyte ligand for endothelial vascular cell adhesion molecule-1 (VCAM-1), fibronectin and osteopontin. We addressed the role of VLA-4 in mediating progressive renal injury in vivo using a blocking monoclonal antibody (mAb) in a rat model of crescentic glomerulonephritis. Methods: WKY rats with nephrotoxic nephritis were given anti-VLA-4 or control mAb at 2.5 mg/kg by i.p. injection on alternate days. In separate experiments, antibodies were given from days 5–13, from days 13–21 or from days 14–28. Results: Early treatment with anti-VLA-4 mAb from days 5–13 showed a significant effect on renal function, with a reduction in albuminuria (p < 0.01) and a higher creatinine clearance (p < 0.05). Delayed treatment from days 13–21 also showed a reduction in albuminuria (p < 0.05) and serum creatinine (p < 0.05). However, there was no significant effect on glomerular or interstitial scarring in these two experiments. In the late treatment study, in which anti-VLA-4 mAb was administered from days 14–28, serum creatinine was reduced (p < 0.05), creatinine clearance was improved (p < 0.05), and renal survival was significantly prolonged (p < 0.05). Interstitial scarring was significantly less in treated rats (p < 0.05). Glomerular macrophage and CD8+ cell counts were higher in anti-VLA-4 mAb treated rats (p < 0.05), possibly reflecting greater glomerular scarring in control animals. Conclusion: Leucocyte VLA-4 mediates pro-inflammatory and pro-fibrotic effects within the kidney, independent of any role in recruitment of leucocytes into the kidney. Blocking VLA-4 is a promising therapeutic approach in human glomerulonephritis.
Uremic Toxins Overload Accelerates Renal Damage in a Rat Model of Chronic Renal Failure
Authors: Satoh Masami , Hayashi Hideo , Watanabe Maki , Ueda Kyoko , Yamato Hideyuki , Yoshioka Toshimasa , Motojima Masaru
Published: December 2003

Uremic toxins have been suggested to promote progression of chronic renal failure by damaging tubular cells. Previous in vitro studies have indicated that some uremic toxins induce oxidative stress and activate NF-ĸB to upregulate plasminogen activator inhibitor-1 in tubular cells. These mechanisms may promote tubulointerstitial fibrosis. The present study examined whether uremic toxins induce glomerular and tubulointerstitial damage in vivo. Two uremic toxins, hippuric acid (HA) or indoleacetic acid (IAA), were tested in two independent experiments (HA-treated rats vs. non-HA-treated controls, IAA-treated rats vs. non-IAA-treated controls). The uremic toxins were administered to subtotally nephrectomized rats. Renal functions were measured periodically and glomerular sclerosis and interstitial fibrosis were examined at the end of the experimental period (18 and 24 weeks, respectively, after subtotal nephrectomy for HA and IAA treatments). Glomerular filtration rate (inulin clearance) at the end of the study period was significantly lower in uremic toxin-treated rats than in control rats (HA-treated rats: 0.090 ± 0.004 ml/min/100 g body weight vs. non-HA-treated controls: 0.125 ± 0.013, IAA-treated rats: 0.068 ± 0.006 versus non-IAA-treated controls: 0.100 ± 0.013; both p < 0.05). Beta-N-acetyl-glucoseamidase excretion was significantly higher in uremic toxin-treated rats than in control rats (HA-treated: 0.55 ± 0.05 U/day vs. control: 0.39 ± 0.04 at week 18, IAA-treated: 0.35 ± 0.02 vs. control: 0.26 ± 0.07 at week 16; both p < 0.05). Glomerular sclerosis index was significantly higher in uremic toxin-treated rats than in control rats (HA-treated: 0.85 ± 0.16 versus control: 0.48 ± 0.10, IAA-treated: 1.13 ± 0.25 vs. control: 0.57 ± 0.10; both p < 0.05). Significant enlargement of interstitial fibrosis was observed in indoleacetic acid-treated rats. These results indicate that overload of uremic toxins accelerates the loss of kidney function, glomerular sclerosis and tubulointerstitial injury in a rat model of chronic renal failure. The present study suggests the potential benefit of early intervention to remove various uremic toxins in delaying the onset of end-stage renal failure in patients with progressive renal disease.
AT1 Receptor Expression in Glomeruli from NO-Deficient Rats
Authors: Vandermeersch Sophie , Stefanovic Vadislav , Hus-Citharel Annette , Ardaillou Raymond , Dussaule Jean-Claude , Chansel Dominique
Published: December 2003

Chronic inhibition of nitric oxide synthase promotes renin-dependent hypertension and renal injury. The present study examines how renal angiotensin II receptors are expressed in this model. NG-nitro-L-arginine methyl ester (L-NAME) was given orally to rats for 1 month and was associated or not with captopril during the 4 last days of the administration. 125I-[Sar1, Ile8]-Ang II binding, AT1 mRNA and cytosolic calcium were studied in isolated glomeruli from L-NAME and control rats and in cultured mesangial cells from normal rats. Renal injury was marked in rats receiving L-NAME. Type I angiotensin II (AT1) receptor number and mRNA expression were decreased (p < 0.05) in glomeruli isolated from L-NAME-treated rats compared with controls, unless they received captopril in combination. The low level of AT1 receptor expression was associated with an attenuated rise of cytosolic calcium in response to angiotensin II. Angiotensin-converting enzyme activity in glomeruli and angiotensin II concentration in renal cortex were increased (p < 0.05) in rats receiving L-NAME alone, whereas aminopeptidase A activity was not modified. To better discriminate between the direct and indirect effects of nitric oxide deficiency, rat mesangial cells were exposed or not for 24 h to S-nitroso-N-acetyl penicillamine, a nitric oxide donor. Angiotensin II binding, AT1 mRNA expression and calcium response to angiotensin II were decreased in presence of the nitric oxide donor (p < 0.01). These results suggest that the decrease of AT1 receptor expression after 1 month of L-NAME treatment does not depend on a direct effect of nitric oxide deficiency but results from the high local angiotensin II concentration due to the stimulated angiotensin-converting enzyme activity. They also show that the renin-angiotensin dependence of this model of hypertension does not result from the overexpression of AT1 receptors.
Influence of Murine Renal Sexual Dimorphism on Amiloride-Induced Hyperkalemia
Authors: Cremades Asunción , Vicente-Ortega Vicente , Peñafiel Rafael
Published: December 2003

The present study investigated the influence of androgens in potassium homeostasis both under normal conditions and when potassium balance was affected by the mild diuretic amiloride. Our results indicate the existence of a clear sexual dimorphism in renal and plasma potassium content in CD1 mice, adult males having a higher plasma potassium concentration than females or 40-day-old male mice (4.6 ± 1.0 vs. 3.9 ± 0.9 mEq/l). Plasma K+ concentration was increased by testosterone treatment and decreased after orchiectomy, while the opposite occurred in the case of renal potassium concentration. Amiloride was well tolerated by CD1 female mice, but induced severe hyperkalemia in male mice, where it was also associated with a high rate of mortality. Testosterone treatment increased the toxicity of the diuretic in both CD1 female and male mice. Histopathological analysis of kidneys from CD1 male mice treated with amiloride revealed alterations at the proximal and distal tubule level. These results strongly suggest that androgens may produce adverse effects in renal function.
Turning on the male – SRY, SOX9 and sex determination in mammals
Authors: Knower K.C. , Kelly S. , Harley V.R.
Published: December 2003

The decision of the bi-potential gonad to develop into either a testis or ovary is determined by the presence or absence of the Sex-determining Region gene on the Y chromosome (SRY). Since its discovery, almost 13 years ago, the molecular role that SRY plays in initiating the male sexual development cascade has proven difficult to ascertain. While biochemical studies of clinical mutants and mouse genetic models have helped in our understanding of SRY function, no direct downstream targets of SRY have yet been identified. There are, however, a number of other genes of equal importance in determining sexual phenotype, expressed before and after expression of SRY. Of these, one has proven of central importance to mammals and vertebrates, SOX9. This review describes our current knowledge of SRY and SOX9 structure and function in the light of recent key developments.
Expression studies of the PIS-regulated genes suggest different mechanisms of sex determination within mammals
Authors: Pannetier M. , Servel N. , Cocquet J. , Besnard N. , Cotinot C. , Pailhoux E.
Published: December 2003

In mammals, the Y-located SRY gene is known to induce testis formation from the indifferent gonad. A related gene, SOX9, also plays a critical role in testis differentiation in mammals, in birds and reptiles. It is now assumed that SRY acts upstream of SOX9 in the sex determination cascade, but the regulatory link which should exist between these two genes remains unknown. Studies on XX sex reversal in polled goats (PIS mutation: Polled Intersex Syndrome) have led to the discovery of a female-specific locus crucial for ovarian differentiation. This genomic region is composed of at least two genes, FOXL2 and PISRT1, which share a common transcriptional regulatory region, PIS. In this review, we present the expression pattern of these PIS-regulated genes in mice. The FOXL2 expression profile of mice is similar to that described in goats in accordance with a conserved role of this ovarian differentiating gene in mammals. On the contrary, the PISRT1 expression profile is different between mice and goats, suggesting different mechanisms of the primary switch in the testis determination process within mammals. A model based on two different modes of SOX9 regulation in mice and other mammals is proposed in order to integrate our results into the current scheme of gonad differentiation.
Structure, evolution and expression of the FOXL2 transcription unit
Authors: Cocquet J. , De Baere E. , Gareil M. , Pannetier M. , Xia X. , Fellous M. , Veitia R.A.
Published: December 2003

FOXL2 is a putative transcription factor involved in ovarian development and function. Its mutations in humans are responsible for the blepharophimosis syndrome, characterized by eyelid malformations and premature ovarian failure (POF). Here we have performed a comparative sequence analysis of FOXL2 sequences of ten vertebrate species. We demonstrate that the entire open reading frame (ORF) is under purifying selection leading to strong protein conservation. We also review recent data on FOXL2 transcript and protein expression. FOXL2 has been shown 1) to be the earliest known sex dimorphic marker of ovarian determination/differentiation in vertebrates, 2) to have, at least in mammals, an ovarian expression persisting until adulthood. The conservation of its sequence and pattern of expression suggests that FOXL2 might be a key factor in the early development of the vertebrate female gonad and involved later in adult ovarian function. Finally, we provide arguments for the existence of an alternative transcript in rodents, that may arise from a differential polyadenylation. Although it has only been demonstrated in rodents, its presence/absence in other species deserves further investigation.
Origin and possible roles of the Sox8 transcription factor gene during sexual development
Authors: Takada S. , Koopman P.
Published: December 2003

Sox8 is a member of the Sox family of developmental transcription factor genes and is closely related to Sox9, a critical gene involved in mammalian sex determination and differentiation. Both genes encode proteins with the ability to bind similar DNA target sequences, and to activate transcription in in vitro assays. Expression studies indicate that the two genes have largely overlapping patterns of activity during mammalian embryonic development. A knockout of Sox8 in mice has no obvious developmental phenotype, suggesting that the two genes are able to act redundantly in a variety of developmental contexts. In particular, both genes are expressed in the developing Sertoli cell lineage of the developing testes in mice, and both proteins are able to activate transcription of the gene encoding anti-Müllerian hormone (AMH), through synergistic action with steroidogenic factor 1 (SF1). We have hypothesized that Sox8 may substitute for Sox9 in species where Sox9 is expressed too late to be involved in sex determination or regulation of Amh expression. However, our studies involving the red-eared slider turtle indicate that Sox8 is expressed at similar levels in males and females throughout the sex-determining period, suggesting that Sox8 is neither a transcriptional regulator for Amh, nor responsible for sex determination or gonad differentiation in that species. Similarly, Sox8 is not expressed in a sexually dimorphic pattern during gonadogenesis in the chicken. Since a functional role(s) for Sox8 is implied by its conservation during evolution, the significance of Sox8 for sexual and other aspects of development will need to be uncovered through more directed lines of experimentation.
Cell aggregation precedes the onset of Sox9-expressing preSertoli cells in the genital ridge of mouse
Authors: Moreno-Mendoza N. , Harley V. , Merchant-Larios H.
Published: December 2003

Sox9 is expressed at the onset of the genital ridge formation in both sexes. It is assumed that Sry, the testis determining gene, turns Sox9 on in male embryos because it is turned off in female embryos. Spatial expression of Sry follows a cranio-caudal pattern. Here, we asked if Sox9 is expressed in the same cell lineage and with a similar pattern as Sry. A correlative study between the structural changes in the genital ridge and the immunocytochemical localization of Sox9-positive cells was undertaken. We used a transgenic strain expressing the green fluorescent protein (GFP) that considerably enhanced the cell context where the first Sox9-positive cells appear. Although Sox9-positive cells are located among loose mesenchymal cells by stages of 8–14 tail somites (ts) in both sexes, they are absent in the thickening coelomic epithelium of females. At 15 ts the first Sox9-positive cells appear within the core of the condensed cells only in male genital ridges. At 17 ts, a gradient of Sox9-positive cells in males is apparent, closely following the cranio-caudal pattern of cell aggregation seen in genital ridges of both sexes. Hence, our results suggest that Sox9 is expressed only in loose mesenchymal cells in both sexes and that expression of Sox9 in males requires the prior aggregation of cells in the genital ridges. The correspondence of Sox9 and Sry pattern of expression supports that both genes are expressed in the preSertoli cell lineage in the core of the genital ridges.
Expression of DMRT1 in the mammalian ovary and testis – from marsupials to mice
Authors: Pask A.J. , Behringer R.R. , Renfree M.B.
Published: December 2003

Doublesex and mab3 related transcript (DMRT1) was identified as a candidate gene for human 9p24.3 associated sex reversal. DMRT1 orthologues have highly conserved roles in sexual differentiation from flies and worms to humans. A DMRT1 orthologue was isolated from a marsupial, the tammar wallaby Macropus eugenii. The wallaby gene is highly conserved with other vertebrate DMRT1 genes, especially within the P/S and DM domains. It is expressed in the differentiating testis from the late fetus, during pouch life and in the adult. As in eutherian mammals, DMRT1 protein was localized in the germ cells and the Sertoli cells of the testis, but in addition it was detected in the Leydig cells, peri-tubular myoid cells and within the acrosome of the sperm heads. DMRT1 protein was also detected in the fetal and adult ovary pre-granulosa, granulosa and germ cells. Similarly, we also detected DMRT1 in the granulosa cells of all developing follicles in the adult mouse ovary. This is the first report of DMRT1 expression in the adult mammalian ovary, and suggests a wider role for this gene in mammals, in both the testis and ovarian function.
Molecular characterization of three gonad cell lines
Authors: Beverdam A. , Wilhelm D. , Koopman P.
Published: December 2003

To facilitate the study of the regulation and downstream interactions of genes involved in gonad development it is important to have a suitable cell culture model. We therefore aimed to characterize molecularly three different mouse gonad cell lines. TM3 and TM4 cells were originally isolated from prepubertal mouse gonads and were tentatively identified as being of Leydig cell and Sertoli cell origin, respectively, based upon their morphology and hormonal responses. The third line is a conditionally immortalized cell line, derived from 10.5–11.5 days post-coitum (dpc) male gonads of transgenic embryos carrying a temperature-sensitive SV40 large T-antigen. We studied by reverse transcription-polymerase chain reaction (RT-PCR) the expression profiles of a number of genes known to be important for early gonad development. Moreover, we assessed these cell lines for their capacity to induce Sox9 transcription upon expression of Sry, a key molecular event occurring during sex determination. We found that all three cell lines were unable to upregulate Sox9 expression upon transfection of Sry-expression constructs, even though these cells express many of the studied embryonic gonad genes. These observations point to a requirement for SRY cofactors for direct or indirect upregulation of Sox9 expression during testis determination.
Expression pattern of a gonadoblastoma candidate gene suggests a role of the Y chromosome in prostate cancer
Authors: Lau Y.-F.C. , Lau H.W. , Kömüves L.G.
Published: December 2003

The contribution of specific genes on the Y chromosome in the etiology of prostate cancer has been undefined. Genetic mapping studies have identified a gonadoblastoma locus on the human Y chromosome (GBY) that predisposes the dysgenetic gonads of XY sex-reversed patients to tumorigenesis. Recently a candidate gene, the testis-specific protein Y-encoded (TSPY) that resides on the GBY critical region, has been demonstrated to express preferentially in tumor cells in gonadoblastoma and testicular germ cell tumors. TSPY shares high homology to a family of cyclin B binding proteins and has been considered to possibly play a role in cell cycle regulation or cell division. To address the possible involvement of the TSPY gene in prostate cancer, both in situ mRNA hybridization and immunohistochemistry techniques were used to study the expression of this putative GBY gene in prostate specimens. Our results demonstrated that TSPY was expressed at low levels in normal epithelial cells and benign prostatic hyperplasia (BPH), but at elevated levels in tumor cells of prostate cancers at various degrees of malignancy. Sequence analysis of RT-PCR products obtained from both prostatic and testicular tissues using specific primers flanking the open reading frame of the TSPY mRNA revealed a complex pattern of RNA processing of the TSPY transcripts involving cryptic intron splicing and/or intron skipping. The variant transcripts encode a variety of polymorphic isoforms or shortened versions of the TSPY protein, some of which might possess different biochemical and/or functional properties. The abbreviated transcripts were more abundant in prostatic cancer tissues than the testicular ones. Although the exact nature of such variant TSPY transcripts and proteins is still unclear, their differential expression suggests that the TSPY gene may also be involved in the multi-step prostatic oncogenesis besides its putative role in gonadoblastoma and testicular seminoma.
Dppa3 is a marker of pluripotency and has a human homologue that is expressed in germ cell tumours
Authors: Bowles J. , Teasdale R.P. , James K. , Koopman P.
Published: December 2003

We identified a transcript named 11M2 on the basis of its strong male-specific expression pattern in the developing mouse gonad. 11M2 was found to be expressed by gonad primordial germ cells (PGCs) of both sexes and down-regulated in female PGCs as they enter prophase I of the first meiotic division, similar to the expression of Oct4. Mouse EST analysis revealed expression only in early-stage embryos, embryonic stem cells and pre-meiotic germ cells. 11M2 corresponds to a recently reported gene variously known as PGC7, stella or Dppa3. We have identified the human orthologue of Dppa3 and find by human EST analysis that it is expressed in human testicular germ cell tumours but not in normal human somatic tissues. The expression patterns of mouse and human DPPA3, in undifferentiated embryonic cells, embryonic germ cells and adult germ cell tumours, together suggest a role for this gene in maintaining cell pluripotentiality.
Sex chromosomes, sex determination, and sex-linked sequences in Microtidae
Authors: Marchal J.A. , Acosta M.J. , Bullejos M. , Díaz de la Guardia R. , Sánchez A.
Published: December 2003

The Arvicolidae is a widely distributed rodent group with several interesting characteristics in their sex chromosomes. Here, we summarize the actual knowledge of some of these characteristics. This mammalian group has species with abnormal sex determination systems. In fact, some species present the same karyotype in both males and females, with total absence of a Y chromosome, and hence of SRY and ZFY genes. Other species present fertile, sex-reversed XY females, generally due to mutations affecting X chromosomes. Furthermore, in Microtus oregoni males and females are gonosomic mosaic (the females are XO in the soma and XX in the germ cells, while the males are XY in the soma and OY in the germ cells). Regarding sex chromosomes, some species present enlarged (giant) sex chromosomes because of the presence of large blocks of constitutive heterochromatin, which have been demonstrated to be highly heterogeneous. Furthermore, we also consider the alterations affecting composition and localization of sex-linked genes or repeated sequences. Finally, this rodent group includes species with synaptic and asynaptic sex chromosomes. In fact, several species with asynaptic sex chromosomes have been described. It is interesting to note that within the genus Microtus both types of sex chromosomes are present.
The rice field eel as a model system for vertebrate sexual development
Authors: Cheng H. , Guo Y. , Yu Q. , Zhou R.
Published: December 2003

Complex developmental mechanisms of vertebrates are unraveled using comparative genomic approaches. Several teleosts, such as zebrafish, medaka and pufferfish, are used as genetic model systems because they are amenable to studies of gene function. The rice field eel, a freshwater fish, is emerging as a specific model system for studies of vertebrate sexual development because of its small genome size and naturally occurring sex reversal. Data presented here support the use of the rice field eel as another important fish model for comparative genome studies, especially in vertebrate sexual development. This model system is complementary rather than redundant.
Sex and death in birds: A model of dosage compensation that predicts lethality of sex chromosome aneuploids
Authors: Graves J.A.M.
Published: December 2003

Birds show female heterogamety, with ZZ males and ZW females. It is still not clear whether the W is female-determining, or whether two doses of the Z chromosomes are male-determining, or both. This question could easily be settled by the sexual phenotypes of ZZW and ZO birds, in the same way that the sexual phenotypes of XXY and XO showed that the Y is male determining in humans, but that the dosage of an X-borne gene determines sex in Drosophila. However, despite extensive searches, no ZZW or ZO diploid birds have been satisfactorily documented, so we must assume that these genotypes are embryonic lethals. Given that ZW and ZZ are viable and the W contains few genes it is not clear why this should be so. Here I propose that sex chromosome aneuploids are lethal in chicken because, to achieve dosage compensation, a locus on the W chromosome controls the upregulation of genes on the Z in ZW females. ZO birds would therefore have only half the normal dose of Z-linked gene product and ZZW would have twice the amount, both of which would undoubtedly be incompatible with life. Reports of other aneuploids and triploids are also consistent with this hypothesis.
Steroids, aromatase and sex differentiation of the newt Pleurodeles waltl
Authors: Kuntz S. , Chardard D. , Chesnel A. , Grillier-Vuissoz I. , Flament S.
Published: December 2003

In the newt Pleurodeles waltl, genetic sex determination obeys female heterogamety (female ZW, male ZZ). In this species as in most of non-mammalian vertebrates, steroid hormones play a key role in sexual differentiation of gonads. In that context, male to female sex reversal can be obtained by treatment of ZZ larvae with estradiol. Male to female sex reversal has also been observed following treatment of ZZ larvae with testosterone, a phenomenon that was called the “paradoxical effect”. Female to male sex reversal occurs when ZW larvae are reared at 32°C during a thermosensitive period (TSP) that takes place from stage 42 to stage 54 of development. Since steroids play an important part in sex differentiation, we focussed our studies on the estrogen-producing enzyme aromatase during normal sex differentiation as well as in experimentally induced sex reversal situations. Our results based on treatment with non-aromatizable androgens, aromatase activity measurements and aromatase expression studies demonstrate that aromatase (i) is differentially active in ZZ and ZW larvae, (ii) is involved in the paradoxical effect and (iii) might be a target of temperature. Thus, the gene encoding aromatase might be one of the master genes in the process leading to the differentiation of the gonad in Pleurodeles waltl.
Expression of Dmrt1 protein in developing and in sex-reversed gonads of amphibians
Authors: Aoyama S. , Shibata K. , Tokunaga S. , Takase M. , Matsui K. , Nakamura M.
Published: December 2003

Many genes are known to be involved in gonadal differentiation in vertebrates. Dmrt1, a gene that encodes a transcription factor with a DM-domain, is considered to be one of the essential genes controlling testicular differentiation in mammals, birds, reptiles, amphibians and fish. However, it still remains unknown which testicular cells of animals other than mice and chicks express Dmrt1 protein. For an explanation of its role(s) in testicular differentiation in vertebrates, the expression of the Dmrt1 protein needs to be studied. For this purpose, we conducted an immunohistochemical study of this protein in an amphibian by using an antibody specific for Dmrt1. No positive signal was found in the indifferent gonad of tadpoles of Rana rugosa at early stages. However, in the testis of tadpoles at later stages (XV–XXV) and in frogs one month after metamorphosis, this protein was expressed in interstitial cells and Sertoli cells. In the testis of adult frogs, germ cells also expressed Dmrt1 protein. RT-PCR analysis revealed that the gene for this protein was not transcribed at any time during ovarian development, but was expressed in the female to male sex-reversed gonad. This was true when immunohistological studies were performed. In addition, Southern blot analysis showed Dmrt1 to be an autosomal gene. Taken together, our findings indicate that Dmrt1 protein is expressed by interstitial cells, Seroli cells and germ cells in the testis of R. rugosa. Dmrt1 may thus be very involved in the testicular differentiation of amphibians.
Differential Dmrt1 transcripts in gonads of the protandrous black porgy, Acanthopagrus schlegeli
Authors: He C.-L. , Du J.-L. , Wu G.-C. , Lee Y.-H. , Sun L.-T. , Chang C.-F.
Published: December 2003

Black porgy, Acanthopagrus schlegeli Bleeker, is a marine protandrous hermaphrodite fish. A Dmrt1 cDNA was cloned and characterized and in order to study the process of sex change in this species, mRNA transcripts of Dmrt1 were monitored. Dmrt1 was specifically transcribed in testis and seminal vesicle in 2-year-old black porgy according to RT-PCR and Southern analysis. A real-time quantification PCR analysis was further developed for the measurement of Dmrt1 transcripts. Dmrt1 transcripts were at significantly higher levels in bisexual testis than bisexual ovary in 1+ and 2+ year-old fish. Dmrt1 transcripts decreased in the functional and bisexual testis of 3-year-old fish. Much higher levels of Dmrt1 transcripts in the bisexual ovary were detected in 1+ year-old fish than in 2+ and 3-year-old fish. No differences in Dmrt1 transcripts were found in bisexual ovaries of 2+ and 3-year-old fish and female ovaries of 3-year-old fish. The data suggest there is relationship of Dmrt1 to the sex change of protandrous black porgy.
Analysis of repetitive DNA sequences in the sex chromosomes of Oreochromis niloticus
Authors: Harvey S.C. , Boonphakdee C. , Campos-Ramos R. , Ezaz M.T. , Griffin D.K. , Bromage N.R. , Penman P
Published: December 2003

In the Nile tilapia, Oreochromis niloticus, sex determination is primarily genetic, with XX females and XY males. While the X and Y chromosomes (the largest pair) cannot be distinguished in mitotic chromosome spreads, analysis of comparative hybridization of X and Y chromosome derived probes (produced, by microdissection and DOP-PCR, from XX and YY genotypes, respectively) to different genotypes (XX, XY and YY) has demonstrated that sequence differences exist between the sex chromosomes. Here we report the characterization of these probes, showing that a significant proportion of the amplified sequences represent various transposable elements. We further demonstrate that concentrations of a number of these individual elements are found on the sex chromosomes and that the distribution of two such elements differs between the X and Y chromosomes. These findings are discussed in relation to sex chromosome differentiation in O. niloticus and to the changes expected during the early stages of sex chromosome evolution.
Dental Plaque as a Biofilm: A Pilot Study of the Effects of Nutrients on Plaque pH and Dentin Demineralization
Authors: Zaura E. , ten Cate J.M.
Published: December 2003

Direct or in situ methods (e.g., confocal microscopy, microsensors) are used to study non-oral biofilms for almost two decades, and they have recently been introduced in the research of dental plaque. We combined a pH microsensor technique and transversal microradiography (TMR) in a pilot study where the effects of nutrient availability on plaque acidogenicity and on the development of caries-like lesions were assessed. One volunteer accumulated dental plaque for 7 days in 0.2-mm-wide and 0.8-mm-deep dentin grooves at four conditions: (1) saliva only, (2) 8 × 5 min/day dipping in 10% sucrose solution, (3) 7 × 5 min/day consumption of sweet cookies plus one meal/day, and (4) subject’s regular diet. Plaque pH versus time and depth profiles in the grooves were recorded ex vivo before and after sugar challenge. ‘Saliva’ plaque responded to sugar with slow pH decrease – minimum pH 5.6–5.8 was reached after 30 min, while sugar dipping resulted in metabolically active plaque (minimum pH 5.3–5.5 within 4–8 min). TMR analysis revealed no demineralization after these two periods. Metabolically active plaque leading to distinct lesions resulted from frequent plaque exposure to diets rich in starch and carbohydrates (groups 3 and 4). These findings strengthen the view that the plaque acidogenicity does not necessarily reflect cariogenicity, and that retention of food components may account for increased cariogenicity at plaque retention sites in the mouth.
Intraindividual Variability, Change, and Aging: Conceptual and Analytical Issues
Authors: Martin Mike , Hofer Scott M.
Published: December 2003

Background: Developmental researchers use a variety of research designs to examine aging-related changes. Most longitudinal studies of aging are based on research designs that feature successive, widely spaced, assessments to estimate changes in cognitive performance. Such designs assume that short-term variations in cognitive performance are small relative to long-term changes or have modeled such phenomena as nuisance parameters. Objective: There is now sufficient empirical evidence to establish intraindividual cognitive variability as a systematic source of individual differences and of important predictive value for aging-relevant outcomes. Methods: After an overview of types of change, potential underlying processes, and adequate analytic designs, we discuss consequences for lifespan aging research. Results: We emphasize that interpretations of both cross-sectional and longitudinal results need to consider and specify theoretical assumptions about short-term and long-term changes. Conclusions: Above and beyond the analysis of long-term mean changes, short- term changes are an important aspect of aging-related change, and their analysis may help to explain psychological processes of adaptation.
Static and Dynamic Longitudinal Structural Analyses of Cognitive Changes in Old Age
Authors: Ghisletta Paolo , Lindenberger Ulman
Published: December 2003

Background: Among the main data-analytical advances of recent decades are Latent Growth Models (LGM) and Multilevel Models (MLM) for the analysis of longitudinal data. Objective: We discuss the relative advantages and disadvantages of the two analytical methods and offer some practical guidelines concerning the choice between LGM and MLM based on (a) completeness and balance of the data, (b) theoretical functional form of change examined, (c) examination of the error structure, (d) theoretical relations among differential level effects and differential change effects, and (e) role of time-invariant covariates. Methods: To discuss LGM and MLM, we provide illustrations from applications to the Berlin Aging Study (BASE) and the Swiss Interdisciplinary Longitudinal Study on the Oldest Old (SWILSO-O). Results: As predicted by two-component theories of lifespan cognition, performance on a vocabulary test (indicator of broad crystallized intelligence) did not decline over time, while scores on a digit letter test (indicator of broad fluid intelligence) decreased over 6 years. Differential level effects were obtained on both variables, while average and differential change effects were obtained only for the digit letter test. In a second set of analyses, we tested the error-free effect that a broad fluid intelligence indicator exerted on the latent yearly change in a broad crystallized intelligence indicator, and vice versa. In both data sets we obtained strong evidence for a more reliable effect of the fluid indicator on the change in the crystallized indicator. This evidence provided support for the dedifferentiation hypothesis of cognitive abilities in very old age. Conclusions: New insights into cognitive aging phenomena can be gained with proper applications of LGM and MLM. We posit that the choice between LGM and MLM and their specification rests on theoretical and empirical motives to be defined a priori.