bluuebunny/crossref_metadata_2025 huggingface dataset:
Fluoxetine versus Vitex agnus castus extract in the treatment of premenstrual dysphoric disorder
Authors: Atmaca Murad , Kumru Selahattin , Tezcan Ertan
Published: March 2003

Clinical trials have demonstrated that serotonin reuptake inhibitors (SRIs) and the extract of Vitex agnus castus are effective for the treatment of premenstrual dysphoric disorder (PMDD). However, to the best of our knowledge, there has been no study comparing the efficacy of the SRIs with Vitex agnus castus (AC) extract. Therefore, the aim of the present study was to compare the efficacy of fluoxetine, a selective serotonin reuptake inhibitor (SSRI), with that of the AC extract, a natural choice. After a period of 2 screening months to screen the patients for suitability, 41 patients with PMDD according to DSM‐IV were recruited into the study. The patients were randomized to fluoxetine or AC for 2 months of single‐blind, rater‐ blinded and prospective treatment period. The outcome measures included the Penn daily symptom report (DSR), the Hamilton depression rating scale (HAM‐D), and the clinical global impression‐severity of illness (CGI‐SI) and ‐improvement (CGI‐I) scales. At endpoint, using the clinical criterion for improvement, a similar percentage of patients responded to fluoxetine (68.4%, n = 13) and AC (57.9%, n = 11). There was no statistically significant difference between the groups with respect to the rate of responders. This preliminary study suggests that patients with PMDD respond well to treatment with both fluoxetine and AC. However, fluoxetine was more effective for psychological symptoms while the extract diminished the physical symptoms. Copyright © 2002 John Wiley & Sons, Ltd.
Differential effects of alcohol and alcohol expectancy on risk‐taking during simulated driving
Authors: Burian Scott E. , Hensberry Rebecca , Liguori Anthony
Published: March 2003

This study examined the separate and combined effects of alcohol (0.0 or 0.5 g/kg) and alcohol expectancies (none or 2–3 standard drinks) on risk‐taking using a simulated‐driving lane choice task. In this task, risk‐taking was operationalized as choosing a cone‐defined lane with a higher relative probability of hitting a cone. When alcohol was received but not expected, the probability of a risky lane choice increased compared with when alcohol was neither expected nor received. However, when subjects both expected and received alcohol, the probability of a risky lane choice was significantly decreased compared with when alcohol was neither expected nor received. These findings suggest that the knowledge of dose received can differentially influence the pharmacological effect of alcohol on decision‐making. Copyright © 2003 John Wiley & Sons, Ltd.
Childhood cancer registries in Ontario, Canada: Lessons learned from a comparison of two registries
Authors: Greenberg Mark L. , Barr Ronald D. , DiMonte Bruna , McLaughlin Eva , Greenberg Corin
Published: March 2003

Two childhood cancer registries exist in Ontario. One (POGO) accrues by active registration by pediatric cancer centers, utilizing a histologically based classification system. The other accrues by passive linkage within a larger adult oriented cancer registry (OCR) using a topographically based classification. A reportedly high incidence of childhood cancer based on the latter registry prompted a comparison of the content of the two registries over a 2‐year period with the hypothesis that there would be systematic accrual errors. All registrations in both registries for the specified period were reviewed systematically and validated by pathology reports. A small number (2.6%) of registrations in the passive registry were not incident cases, while 2 particular pathologic diagnoses were included in the histologically based registry and not the topographically based registry. These were low grade gliomas and Langerhans cell histiocytosis (LCH). The validated annual incident rate (15.6 per 100,000 children 0–14 years of age, excluding LCH) is slightly higher than that reported in other industrialized countries. Ninety‐six percent of children aged 0–14 were treated in pediatric oncology centers, while only 46% of adolescents aged 15–17 were treated in such centers. Of the remaining adolescents, more than one‐third had lymphomas. This maldistribution of care provided for the young adolescent population may compromise their survival prospects. The results of this study should prompt revision of health care policy and patterns of service delivery. © 2003 Wiley‐Liss, Inc.
Vascular targeting effect of combretastatin A‐4 phosphate dominates the inherent angiogenesis inhibitory activity
Authors: Ahmed Bisan , van Eijk Loes I. , Bouma‐ter Steege Jessica C.A. , van der Schaft Daisy W.J. , van Esch Anita M. , Joosten‐Achjanie Susan R. , Lambin Philippe , Landuyt Willy , Griffioen Arjan W.
Published: March 2003

The current research aimed to define hypothesis‐based anti‐angiogenic properties of the vascular targeting agent combretastatin A‐4 phosphate (combreAp). The in vitro wound assay indicated that combreAp potently inhibited migration of endothelial cells (EC). A significant inhibition of migration could already be measured after 2 hr of treatment. In a three‐dimensional (3D) tube formation assay, combreAp inhibited sprout formation at concentrations that did not inhibit the proliferation of EC. At sub‐ng concentrations the half‐maximal response was reached. Interestingly, although combreAp is considered a vascular targeting agent, the human tumor cell lines tested were found to be 20–30 times more sensitive for combreAp than the human umbilical vein endothelial cells (HUVEC). A similar response difference between rat EC and R1 rat rhabdomyosarcoma tumor cells was observed. The growth inhibition in EC was only in part mediated by induction of apoptosis. The growth delay results obtained with the in vivo rodent tumor models involving repeat dosing of combreAp can partly be explained by anti‐angiogenic activity of the compound. The results obtained with the various in vitro and in vivo assays substantiate an anti‐angiogenic profile of combreAp, largely at the level of EC migration. This mechanism may operate to a different extent in different tumor types. © 2003 Wiley‐Liss, Inc.
Restricted expression of membrane type 1‐matrix metalloproteinase by myofibroblasts adjacent to human breast cancer cells
Authors: Bisson Christèle , Blacher Silvia , Polette Myriam , Blanc Jean‐Frédéric , Kebers Florence , Desreux Joëlle , Tetu Bernard , Rosenbaum Jean , Foidart Jean‐Michel , Birembaut Philippe , Noel Agnès
Published: March 2003

The membrane type‐1 matrix metalloproteinase (MT1‐MMP), a protease originally identified in breast carcinoma, is characterized by its capacity to activate other MMPs (MMP‐2 and MMP‐13) and to degrade extracellular matrix. Our study was undertaken to localize and identify the MT1‐MMP expressing cells in human breast adenocarcinomas. A textural analysis of images obtained by immunohistochemistry and in situ hybridization showed precisely the co‐expression of alpha smooth muscle actin (αSM actin) and MT1‐MMP in myofibroblasts. MT1‐MMP expression is confined to myofibroblasts in close contact with tumor cells. In sharp contrast, the expression of MMP‐2 was more widely distributed in both αSM actin positive and negative cells close to and at distance from cancer cell clusters. Our in vitro observations are consistent with the higher level of MT1‐MMP expression and of MMP‐2 activation observed in αSM actin positive fibroblasts derived from breast tumors, as compared to normal breast fibroblasts. Collectively, these results implicate myofibroblasts as major producer of MT1‐MMP in breast cancer and emphasize the importance of stromal‐epithelial cell interactions in their progression. © 2003 Wiley‐Liss, Inc.
Mutation analysis of DMBT1 in glioblastoma, medulloblastoma and oligodendroglial tumors
Authors: Pang Jesse Chung‐sean , Dong Zhiqian , Zhang Rong , Liu Yanhui , Zhou Liang‐fu , Chan Bik Wan , Poon Wai Sang , Ng Ho‐keung
Published: March 2003

DMBT1 has been implicated as a candidate tumor suppressor gene on chromosome 10q for brain, gastrointestinal and lung cancer. Homozygous deletion and lack of expression are 2 known mechanisms for inactivating DMBT1. We evaluated whether somatic mutation, which represents a major inactivation mechanism for most tumor suppressor genes, occurs in the DMBT1 gene. A total of 102 primary brain tumors, consisting of 25 glioblastoma multiforme, 24 medulloblastoma and 53 oligodendroglial tumors, were analyzed by conformation‐sensitive gel electrophoresis in all 54 coding exons of DMBT1. Twelve different base substitutions were detected in 26 (25%) tumors. Eight base substitutions resulted in amino acid changes and 4 were silent. These base changes were also detected in tumor‐matched blood samples, however, indicating that the base variations represent genetic polymorphisms. We also assessed homozygous deletions of the DMBT1 gene in the series and found that 16 of 95 (5 glioblastomas, 5 medulloblastomas, 6 oligodendroglial tumors; total 17%) tumors harbor such alteration. High‐quality blood DNA samples were available in 5 tumors carrying homozygous deletion and, using long‐range PCR, 3 of these blood samples showed germline hemizygous deletions in a region between introns 10 and 26 of DMBT1. Our results showed that mutation does not play a role in inactivation of DMBT1 in brain tumors. Intragenic homozygous deletion of DMBT1 is common in brain tumors and is likely a result of a germline deletion of 1 allele followed by loss of the second allele during tumor development. © 2003 Wiley‐Liss, Inc.
Imatinib inhibits various types of activating mutant kit found in gastrointestinal stromal tumors
Authors: Chen Hui , Isozaki Koji , Kinoshita Kazuo , Ohashi Akiko , Shinomura Yasuhisa , Matsuzawa Yuji , Kitamura Yukihiko , Hirota Seiichi
Published: March 2003

Mutations of proto‐oncogene c‐KIT in gastrointestinal stromal tumors (GISTs) are considered to cause a constitutive activation of KIT responsible for their oncogenesis. Imatinib has therapeutic potential for GISTs because of its inhibitory effect on KIT kinase activity. To investigate the effect of Imatinib on various c‐KIT mutations found in GISTs, we examined kinase activity of KIT, cell proliferation and tumorigenicity of transfectants with various c‐KIT mutations. Murine lymphoid Ba/F3 cells transfected with one of the three types of mutants (KITdel559–560, KIT642Glu, and KIT820Tyr) or wild‐type KIT were used for the experiments. Phosphorylation of KIT, mitogen‐activated protein (MAP) and Akt was studied by immunoblotting with or without immunoprecipitation. In vitro studies on cell proliferation using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenylcetrazolium bromide colorimetric assay and in vivo tumorigenicity assay using nude mice were also carried out. Imatinib could inhibit the KIT, MAP and Akt phosphorylation of all the transfectants but had a weaker effect on KIT820Tyr. Imatinib potently inhibited the proliferation of cells transfected with KIT820Tyr at the concentration of 10 μM whereas it inhibited the other 3 types at 1 μM. Moreover, Imatinib could inhibit the tumor formation in nude mice transplanted with transfectants. In various types of activating mutant KIT, Imatinib could inhibit the constitutive activation of KIT signal transduction and cell proliferation both in vitro and in vivo although the effect of Imatinib on KIT820Tyr was weaker than that on KITdel559–560 or KIT642Glu. © 2003 Wiley‐Liss, Inc.
Promoter hypermethylation profile of tumor‐associated genes p16, p15, hMLH1, MGMT and E‐cadherin in oral squamous cell carcinoma
Authors: Viswanathan Muthusamy , Tsuchida Nobuo , Shanmugam Govindaswamy
Published: March 2003

Aberrant promoter hypermethylation of tumor‐associated genes leading to their inactivation is a common event in many cancer types. Using a sensitive restriction‐multiplex PCR method, we studied the promoter hypermethylation profile of the p16, p15, hMLH1, MGMT and E‐cad genes in oral squamous cell carcinoma (OSCC) of Indians. We analyzed a total of 51 samples for the p15 tumor‐suppressor gene and 99 samples for each of the remaining genes. Our studies indicate an incidence of promoter hypermethylation of 23% each for p16 and p15, 8% for hMLH1, 41% for MGMT and 35% for E‐cad. We observed aberrant hypermethylation of the promoter region of at least 1 of these genes in 74.5% of cases (n = 51) for which all the 5 genes were studied. Abnormal methylation was detected in tumors irrespective of stage and location in the oral cavity, whereas no abnormal methylation was detectable in normal oral squamous tissues obtained from 25 OSCC patients. Detection of aberrant hypermethylation patterns of cancer‐associated genes listed above is therefore suitable for diagnosis of OSCC in individuals at high risk for this disease. © 2003 Wiley‐Liss, Inc.
Expression of the human cachexia‐associated protein (HCAP) in prostate cancer and in a prostate cancer animal model of cachexia
Authors: Wang Zejing , Corey Eva , Hass G. Michael , Higano Celestia S. , True Lawrence D. , Wallace David , Tisdale Michael J. , Vessella Robert L.
Published: March 2003

Prostate cancer (CaP) patients with disseminated disease often suffer from severe cachexia, which contributes to mortality in advanced cancer. Human cachexia‐associated protein (HCAP) was recently identified from a breast cancer library based on the available 20‐amino acid sequence of proteolysis‐inducing factor (PIF), which is a highly active cachectic factor isolated from mouse colon adenocarcinoma MAC16. Herein, we investigated the expression of HCAP in CaP and its potential involvement in CaP‐associated cachexia. HCAP mRNA was detected in CaP cell lines, in primary CaP tissues and in its osseous metastases. In situ hybridization showed HCAP mRNA to be localized only in the epithelial cells in CaP tissues, in the metastatic foci in bone, liver and lymph node, but not in the stromal cells or in normal prostate tissues. HCAP protein was detected in 9 of 14 CaP metastases but not in normal prostate tissues from cadaveric donors or patients with organ‐confined tumors. Our Western blot analysis revealed that HCAP was present in 9 of 19 urine specimens from cachectic CaP patients but not in 19 urine samples of noncachectic patients. HCAP mRNA and protein were also detected in LuCaP 35 and PC‐3M xenografts from our cachectic animal models. Our results demonstrated that human CaP cells express HCAP and the expression of HCAP is associated with the progression of CaP and the development of CaP cachexia. © 2003 Wiley‐Liss, Inc.
Tenascin‐C upregulates matrix metalloproteinase‐9 in breast cancer cells: Direct and synergistic effects with transforming growth factor β1
Authors: Kalembeyi Ilunga , Inada Hiroyasu , Nishiura Rika , Imanaka‐Yoshida Kyoko , Sakakura Teruyo , Yoshida Toshimichi
Published: March 2003

Tenascin‐C (TN‐C) and matrix metalloproteinases (MMPs) are highly expressed in cancer tissues and probably promote cell migration during cancer progression. TN‐C and MMPs are often co‐localized in areas of active tissue remodeling in pathologic conditions, suggesting reciprocal regulation. To investigate whether TN‐C regulates MMPs expression in cancer cells, we first exposed mammary cancer cells derived from TN‐C‐deficient mice to TN‐C and examined MMPs expression. TN‐C was then compared with fibronectin (FN), laminin (LN), basic fibroblast growth factor (b‐FGF) and transforming growth factor‐beta1 (TGF‐β1). Results of endpoint RT‐PCR, quantitative real‐time RT‐PCR and gelatin zymography demonstrated that TN‐C, strongly and dose dependently, upregulates MMP‐9 expression in murine mammary cancer cells. TN‐C weakly induced MMP‐2, MMP‐3 and MMP‐13. FN and LN induced MMP‐9 to lesser extents compared with TN‐C. b‐FGF had no effect on MMP‐9 expression. TGF‐β1 induced MMP‐9 expression in a dose‐dependent manner, and this induction was significantly enhanced by addition of TN‐C. TN‐C and TGF‐β1 also upregulated MMP‐9 expression in the human breast cancer cell line MDA‐MB‐231. Neutralization with specific anti‐TGF‐β1 antibody showed decreased expression of MMP‐9, indicating that TGF‐β controls the baseline MMP‐9 expression by a direct autocrine mechanism. Under neutralization of TGF‐β, addition of TN‐C still upregulated MMP‐9. Conversely, neutralization of endogenous TN‐C (in a TN‐C‐positive mammary cancer cell line) downregulated TGF‐β‐induced MMP‐9 expression. Thus, TN‐C induces MMP‐9 expression directly and by collaboration with TGF‐β. These findings reveal a novel role of TN‐C in breast cancer progression. © 2003 Wiley‐Liss, Inc.
The p75NTR tumor suppressor induces caspase‐mediated apoptosis in bladder tumor cells
Authors: Tabassum Arshia , Khwaja Fatima , Djakiew Daniel
Published: March 2003

p75NTR was identified as a tumor and metastasis suppressor that functions in part via induction of apoptosis in tumor cells. To examine p75NTR‐dependent apoptosis in tumor cells, we demonstrated that a dose‐dependent increase in p75NTR expression was associated with a concomitant increase in the mitochondrial proapoptotic effector proteins Bad, Bax and Bik and a decrease in the mitochondrial prosurvival effector proteins phospho‐Bad, Bcl‐2 and Bcl‐xL. Significantly, p75NTR‐dependent induction of cytochrome c release from the mitochondria occurred during CHX potentiation of apoptosis. Furthermore, p75NTR expression largely suppressed expression of IAP‐1 and induced cleavage of procaspase‐9 and procaspase‐7 but not of procaspases 2, 3, 6, 8 and 10. A specific peptide inhibitor of procaspase‐9 cleavage also inhibited cleavage of procaspase‐7, indicating that caspase‐7 is downstream of caspase‐9. As end points of apoptosis, we observed p75NTR‐dependent annexin V binding to the plasma membrane, an indicator of early apoptotic events, and Hoechst staining of DNA nuclear fragmentation, an indicator of late apoptotic events, whereas control tumor cells that lack expression of the p75NTR protein did not exhibit either of these apoptotic markers. Together, these results delineate the mitochondria‐mediated apoptotic pathway of the p75NTR tumor‐suppressor gene product. © 2003 Wiley‐Liss, Inc.
High delta‐like 1 expression in a subset of neuroblastoma cell lines corresponds to a differentiated chromaffin cell type
Authors: van Limpt Vera A.E. , Chan Alvin J. , van Sluis Peter G. , Caron Huib N. , van Noesel Carel J.M. , Versteeg Rogier
Published: March 2003

The childhood tumour neuroblastoma originates from neural crest‐derived progenitors of the sympathetic nervous system. By Serial Analysis of Gene Expression (SAGE), we previously identified the Drosophila Delta homologue Dlk1 as one of the genes most highly expressed in the neuroblastoma cell line SK‐N‐FI. The Delta‐Notch pathway controls many differentiation steps in Drosophila and man. We analysed expression of 21 genes of this pathway in 21 neuroblastoma cell lines. Dlk1 expression was very high in 5 cell lines, while another subset expressed Notch3. The imprinting of Dlk1 was faithfully preserved in neuroblastomas. The single paternal allele can therefore produce over 0.5% of all cellular mRNAs. Dlk1 maps to 14q32, a region that exhibited LOH in 31/170 (18%) tumours. The random parental origin of the deleted alleles excluded Dlk1 as target of the LOH. In addition, Dlk1 was not amplified, rearranged or mutated in neuroblastoma cell lines and tumours. We therefore analysed whether high Dlk1 expression marks a specific differentiation stage of the sympatho‐adrenal lineage. Many neuroblastomas arise in the adrenal medulla, which predominantly consists of chromaffin cells. Normal adrenal medulla exhibited equally high Dlk1 levels as the SK‐N‐FI cell line. Chromaffin cells in young children are marked by noradrenalin production, which is mediated by dopamine‐beta‐hydroxylase (DBH). DBH expression in the neuroblastoma cell lines almost perfectly corresponded to Dlk1 expression. Neuroblastoma cell lines with high Dlk1 expression are therefore arrested in a relatively late stage of chromaffin lineage differentiation, while Notch3 expression might correspond to earlier precursor stages or to an alternative developmental fate. © 2003 Wiley‐Liss, Inc.
Incidence of ocular melanoma in Australia from 1990 to 1998
Authors: Vajdic Claire M. , Kricker Anne , Giblin Michael , McKenzie John , Aitken Joanne , Giles Graham G. , Armstrong Bruce K.
Published: March 2003

Routinely collected incidence data have often lacked specific identification of ocular melanoma in the past and with increasing diagnosis and management of this disease by noninvasive techniques may now underestimate the true incidence. We attempted to accurately measure the incidence of ocular melanoma in Australia from 1990 to 1998 using 2 population‐based sources, cancer registries and ophthalmologists. We examined the distribution across the continent, by latitude and in subpopulations, and evaluated the extent of nonnotification to cancer registries. One‐half (51%) of the incident cases from 1996 to 1998 were diagnosed clinically and had no tissue diagnosis. An estimated 20% of melanomas, mainly those lacking a tissue diagnosis, were not notified to Australian cancer registries, but only 1.3% were not notified by ophthalmologists. Expert reviewers agreed that a high proportion (95%) of clinically diagnosed lesions were probable or possible melanomas. Incidence was significantly higher in men than in women, especially at older ages, relatively uniform across the Australian states with only weak evidence of a latitude gradient, and higher in rural than in urban areas. The incidence of ocular melanoma in people born in Southern Europe was half and in those born in Asia only 20% of the rate in people born in Australia and New Zealand. We concluded that it was possible to identify a high proportion of cases by surveying relevant sources additional to cancer registries and to be reasonably confident of the accuracy of the clinical diagnoses. The higher incidence in older men, who probably have higher sun exposure, than in older women and in residents of rural areas, where outdoor work is more prevalent, than in urban areas; the low incidence in dark‐eyed populations, who may have a lower sensitivity to or less transmission of solar radiation to the choroid; and the preferential location of ocular melanomas in ocular sites known to receive the highest exposure to solar radiation all support a role for solar radiation in the aetiology of ocular melanoma. The absence of a strong latitude gradient in incidence does not argue against such a role because exposure of the eye to solar UV is probably determined most by the horizon sky, where ambient solar UV is less affected by latitude. © 2003 Wiley‐Liss, Inc.
Biology of spermatozoa maturation: An overview with an introduction to this issue
Authors: Toshimori Kiyotaka
Published: March 2003

Mammalian spermatozoa undergo morphological, biochemical, and physiological modifications initially in the testis (testicular maturation) and later in the epididymis (epididymal maturation). These maturational changes are commensurate with the functional events that occur in developing germ cells and maturing spermatozoa. This special issue reviews the recent, relevant topics dealing with spermatozoa maturation and focuses on the events that occur in internal components such as the nucleus, the acrosome, the perinuclear theca, the fibrous sheath, and the cytoplasmic droplet as well as the plasma membrane. These structures/elements and the constituent proteins of which they are comprised undergo a variety of sequential modifications starting from their origination in developing germ cells up to epididymal maturation. Several steps of the maturation processes on the sperm plasma membrane are mediated by external enzymes and secretions derived from the epithelium lining of the genital tract. Degradation of some of the constituent proteins and the elimination of defective spermatozoa are controlled by the degradation/recycling system, the ubiquitin system. These maturational modifications are necessary for spermatozoa to become fertilization‐competent cells and to be stored safely in the male. Microsc. Res. Tech. 61:1–6, 2003. © 2003 Wiley‐Liss, Inc.
Contribution of epididymal secretory proteins for spermatozoa maturation
Authors: Dacheux Jean‐Louis , Gatti Jean Luc , Dacheux Françoise
Published: March 2003

The final stages of sperm differentiation occur outside the gonad and are not under the genomic control of germ cells. Only sequential interactions with the medium surrounding the sperm are believed to induce the final steps of spermatogenesis. The epididymis, a long tubule with very active secretory and reabsorption functions, is able to create sequential changes in the composition of luminal fluid throughout its length. The chronologies of the changes, which occur on/in the sperm with those in their surrounding environment, are described. Correlations between the highly regionalized epididymal activities and sperm characteristics linked to their survival and fertility potential are presented in this review. Microsc. Res. Tech. 61:7–17, 2003. © 2003 Wiley‐Liss, Inc.
Glycan modifying enzymes in luminal fluid of rat epididymis: Are they involved in altering sperm surface glycoproteins during maturation?
Authors: Tulsiani Daulat R.P.
Published: March 2003

Testicular spermatozoa undergo morphological and biochemical alterations, collectively termed epididymal maturation, in the intraluminal environment of epididymis. As a result of these modifications, the spermatozoon becomes a motile and functionally competent cell capable of undergoing capacitation and binding to the zona pellucida, the extracellular coat that surrounds the mammalian oocyte. Although details of all the changes are not fully known, several studies provide evidence suggesting that sperm plasma membrane undergoes extensive biochemical changes, including organization and modification of surface glycoproteins as spermatozoa transit from the proximal to the distal epididymis. In this article, I have attempted to summarize results with two sets of glycoprotein (glycan)‐modifying enzymes, namely, glycohydrolases (hydrolytic enzymes) and glycosyltransferases (synthetic enzymes) present in the epididymal luminal fluid (LF). The in vitro experimental approaches described in this report demonstrate that: 1) a PNA‐positive glycoprotein(s) (containing O‐linked glycan) of 135–150 kDa subunit molecular mass which is present on the surface of caput (but not the cauda) spermatozoa can be degalactosylated by the enzymatic digestion with LF β‐D‐galactosidase; and 2) an N‐linked glycan chain(s) which is present on a sperm surface glycoprotein (apparent subunit molecular mass of 86 kDa) can be fucosylated in vitro when distal caput sperm (or sperm plasma membrane‐rich fractions) are incubated in the presence of a nucleotide sugar (GDP[14C]fucose). Combined, these results strongly suggest a role for the glycan‐modifying enzymes in degalactosylation and fucosylation of sperm surface glycoproteins during epididymal transit. Microsc. Res. Tech. 61:18–27, 2003. © 2003 Wiley‐Liss, Inc.
Acquisition of volume regulatory response of sperm upon maturation in the epididymis and the role of the cytoplasmic droplet
Authors: Cooper Trevor G. , Yeung Ching‐Hei
Published: March 2003

In certain cases of infertility in domestic species, and in the homozygous c‐ros tyrosine kinase knockout mouse, males are infertile as a result of unopposed sperm swelling. This induces flagellar angulation, preventing their normal migration in the female tract. Angulation always occurs at the site of the cytoplasmic droplet located in mature sperm at the annulus (midpiece – principal piece junction). This article reviews the literature on the fate of the sperm's cytoplasmic droplet and suggests a role for it in volume regulation. A hypothesis is presented that during epididymal transit sperm acquire osmolytes accumulated by the epididymal epithelium. This is possibly driven by a mechanism of regulatory volume increase invoked by the hypertonicity of epididymal luminal fluid. These osmolytes are subsequently used for regulatory volume decrease after ejaculation into the relatively hypotonic female tract. Study of the mechanisms of sperm volume regulation may highlight new contraceptive leads. Microsc. Res. Tech. 61:28–38, 2003. © 2003 Wiley‐Liss, Inc.
Structural modification of the hamster sperm acrosome during posttesticular development in the epididymis
Authors: Olson Gary E. , Winfrey Virginia P. , Nagdas Subir K.
Published: March 2003

The acrosome of the mature spermatozoon functions as a regulated secretory vesicle which performs several critical functions in mammalian fertilization. Acrosome assembly occurs throughout spermiogenesis and continues during posttesticular sperm maturation in the epididymis, resulting in a structurally polarized membrane‐bounded organelle that contains an assortment of hydrolases and a stable infrastructure termed the acrosomal matrix. The role of stable acrosomal matrix assemblies in acrosomal biogenesis and function are poorly understood. This article presents ultrastructural, immunocytochemical, and biochemical data on the remodeling of the hamster acrosomal matrix during spermiogenesis and posttesticular sperm maturation in the epididymis. Specific posttranslational modifications of the major acrosomal matrix protein are evident in late, step 16, spermatids and matrix protein processing continues within specific acrosomal subdomains of caput epididymal spermatozoa. At the completion of sperm maturation, the acrosomal matrix consists of two structurally distinct domains which are adherent to the outer acrosomal membrane and exhibit a localized distribution pattern. Coincident with acrosomal matrix differentiation, a paracrystalline cytoskeletal complex is assembled onto the outer acrosomal membrane of epididymal spermatozoa. This cytoskeletal network appears to establish transmembrane structural interactions with the acrosomal matrix and may maintain attachment of the acrosomal cap to the sperm head during the early steps of the acrosome reaction. Microsc. Res. Tech. 61:46–55, 2003. © 2003 Wiley‐Liss, Inc.
Perinuclear theca during spermatozoa maturation leading to fertilization
Authors: Müjica Adela , Navarro‐García Fernando , Hernández‐González Enrique O. , de Lourdes Juárez‐Mosqueda María
Published: March 2003

Mammalian spermatozoa acquire the capacity to fertilize the ovum and display motility during their passage through the epididymis. At the same time, they undergo changes in metabolic patterns, enzymatic activities, ability to bind to zona pellucida surface, and electrophoretic properties and, furthermore, stabilization of some sperm structures by the establishment of disulphide linkages takes place in several sperm structures. The cytoplasmic perinuclear theca (PT) is a unique extranuclear cytoskeletal element that surrounds the nucleus, which is proposed to be a structural scaffold to the sperm nucleus. The purpose of this review is to describe PT changes related to epididymal sperm maturation. We will focus mainly on the protein components of the PT of eutherian mammalian spermatozoa and on quantitative protein changes during sperm maturation. The protein constituents of the PT have not been completely defined and most of them are different from the cytoskeletal proteins of somatic cells. However, they are proteins with cytoskeletal features. The morphologic changes reported for PT and the proposed functions of PT are discussed. Microsc. Res. Tech. 61:76–87, 2003. © 2003 Wiley‐Liss, Inc.
Ubiquitin‐dependent proteolysis in mammalian spermatogenesis, fertilization, and sperm quality control: Killing three birds with one stone
Authors: Sutovsky Peter
Published: March 2003

Ubiquitin and ubiquitin‐like proteins control the degradation of substrates as diverse as cyclins, viral envelope proteins, plasma membrane receptors, and mRNAs. The ubiquitinated substrates are targeted towards the lysosomal or proteasomal degradation sites. The number and position of ubiquitin molecules bound to substrates' lysine residues and the number and position of ubiquitin molecules in polyubiquitin chains determine the astonishing substrate specificity of ubiquitin‐mediated proteolysis. Ubiquitin is likely to be expressed in mammalian gametes and embryos at any given developmental step, but the information on ubiquitin dependence of gametogenesis and fertilization is sketchy. Ubiquitin ligases E1, E2, E3, and UBC4 are active in the testis. Ubiquitin and proteasomal subunits can be detected in the human sperm centrosome that undergoes dramatic reduction during spermatid elongation. Spermatid histones are ubiquitinated as they are being transiently replaced by transitional proteins and permanently by protamines. Ubiquitin tagging of the sperm mitochondrial membranes may serve as a death sentence for paternal mitochondria at fertilization, thus promoting the maternal inheritance of mitochondrial DNA (mtDNA) in mammals. The defective spermatozoa become surface‐ubiquitinated during sperm descent down the epididymis. Finally, new evidence suggests the involvement of ubiquitin‐proteasome pathway in the zona penetration by the acrosome‐reacted spermatozoon. Such differential patterns of ubiquitination in the testis and epididymis, and inside the egg, may be necessary for reproductive success in humans and animals. Deciphering and eventually manipulating the ubiquitin‐dependent proteolysis in the reproductive system could allow us to redirect the mode of mtDNA inheritance after cloning and ooplasmic transplantation, provide germ line therapy in some cases of male infertility, develop new contraceptives, manage polyspermia during in vitro fertilization, and establish objective markers for infertility diagnostics, semen evaluation, and prediction of future fertility. Microsc. Res. Tech. 61:88–102, 2003. © 2003 Wiley‐Liss, Inc.
Fibrous sheath of mammalian spermatozoa
Authors: Eddy Edward M. , Toshimori Kiyotaka , O'Brien Deborah A.
Published: March 2003

The fibrous sheath is a unique cytoskeletal structure surrounding the axoneme and outer dense fibers and defines the extent of the principal piece region of the sperm flagellum. It consists of two longitudinal columns connected by closely arrayed semicircular ribs that assemble from distal to proximal throughout spermiogenesis. The fibrous sheath is believed to influence the degree of flexibility, plane of flagellar motion, and the shape of the flagellar beat. Nearly half of the protein in fibrous sheaths isolated from mouse sperm is AKAP4. This protein and two others, AKAP3 and TAKAP‐80, have anchoring sites for cAMP‐dependent protein kinase. AKAP3 also anchors ropporin, a spermatogenic cell‐specific protein that is linked through rhophilin to the small GTPase Rho. Other proteins associated with the fibrous sheath include two enzymes in the glycolytic pathway. Glyceraldehyde 3‐phosphate dehydrogenase‐s (GAPDS) is the product of a gene expressed only in spermatogenic cells, while hexokinase type 1‐s (HK1‐S) is derived from alternative transcripts present only in spermatogenic cells. Most of the other glycolytic enzymes in sperm have unique structural or functional properties. The fibrous sheath also contains a spermatogenic cell‐specific member of the μ‐class glutathione S‐transferase family (GSTM5) and an intermediate filament‐like protein (FS39). These and other observations indicate that the fibrous sheath functions as a scaffold for proteins in signaling pathways that might be involved in regulating sperm maturation, motility, capacitation, hyperactivation, and/or acrosome reaction and for enzymes in the glycolytic pathway that provide energy for the hyperactivated motility of sperm that allows them to penetrate the zona pellucida. Microsc. Res. Tech. 61:103–115, 2003. Published 2003 Wiley‐Liss, Inc.
Ribavirin reduces clinical signs and pathological changes of experimental autoimmune encephalomyelitis in Dark Agouti rats
Authors: Milicevic Irena , Pekovic Sanja , Subasic Sanja , Mostarica‐Stojkovic Marija , Stosic‐Grujicic Stanislava , Medic‐Mijacevic Ljubica , Pejanovic Vjera , Rakic Ljubisav , Stojiljkovic Mirjana
Published: March 2003

The effect of ribavirin on development of experimental autoimmune encephalomyelitis (EAE) was investigated. The disease was induced in genetically susceptible Dark Agouti rats with syngeneic spinal cord homogenate in complete Freund's adjuvant (SCH‐CFA). Depending on the amount of mycobacteria in CFA, the animals developed either moderate or severe EAE. Ribavirin (1‐β‐D‐ribofuranosyl‐1,2,4‐triazole‐3‐carboxamide) was applied i.p. at a daily dosage of 30 mg/kg in two treatment protocols: from the start of immunization (preventive treatment) or from the onset of the first EAE signs after the induction (therapeutic treatment). Signs of EAE began between 7 and 9 days after induction and peaked at days 11–13. In moderate EAE (mean maximal severity score 3.33 ± 0.21), the recovery was completed by days 23–26, whereas, in severe EAE (mean maximal severity score 4.5 ± 0.23), obvious recovery was not detected. Preventive ribavirin treatment significantly decreased clinical signs after both moderate (score 1.75 ± 0.25, P < 0.05) and severe (score 3.62 ± 0.31, P < 0.015) immunization. Also, disease manifestations were reduced by therapeutic treatment of ribavirin (mean maximal severity score 2.5 ± 0.2 vs. 3.33 ± 0.21 in controls, P < 0.005) but less so in comparison with preventive treatment. Analysis of the effects of ribavirin on histopathologic changes in the spinal cord tissue revealed a reduction of mononuclear cell infiltrates, composed of T cells and macrophages/microglia, and the absence of demyelination, which were pronounced in control EAE animals. Beneficial effects of preventive and therapeutic treatment with ribavirin on development of EAE suggest this nucleoside analogue as a useful candidate for therapy in multiple sclerosis. © 2003 Wiley‐Liss, Inc.
Glutamate regulates caveolin expression in rat hippocampal neurons
Authors: Bu Jing , Bruckner Shane R. , Sengoku Tomoko , Geddes James W. , Estus Steven
Published: March 2003

Caveolae are cholesterol‐rich, membrane microdomains that appear critical to signaling between extracellular and intracellular macromolecules as well as cholesterol homeostasis. Caveolae formation is modulated by caveolin, a protein family that is the proteinaceous hallmark of caveolae. Very little is known regarding the events that modulate caveolin expression and regulation in neurons. To detect caveolin expression in neurons, primary rat hippocampal neurons were harvested at embryonic day 18, maintained for 7 days in vitro, and then analyzed for caveolin immunofluorescence. Caveolin‐1 immunoreactivity was detected in cells that were identified as neurons by morphology and concurrent microtubule‐associated protein (MAP2) staining. Changes in caveolin‐1 expression were evaluated by reverse transcriptase‐polymerase chain reaction (RT‐PCR) analyses of RNA isolated from hippocampal neurons treated with glutamate receptor agonists. Glutamate induced a concentration‐dependent increase in caveolin‐1 mRNA. The largest increases in caveolin‐1 mRNA were detected after 6 hours of treatment. Kainate and AMPA both mimicked glutamate effects on caveolin‐1 mRNA expression. Western blot analyses revealed that caveolin was induced at the protein level as well. Taken together, these data suggest that glutamate can regulate caveolin expression through kainate and AMPA ionotropic glutamate receptors. © 2003 Wiley‐Liss, Inc.
Minimal residues in linker domain of syntaxin 1A required for binding affinity to Ca2+/calmodulin‐dependent protein kinase II
Authors: Nomura Kazushige , Ohyama Akihiro , Komiya Yoshiaki , Igarashi Michihiro
Published: March 2003

The linker domain is important for the conformational change syntaxin 1A, which enables it to act as a SNARE for exocytosis. We found that when applied exogenously, the linker domain is a potent inhibitor of exocytosis through inhibiting interaction between autophosphorylated CaMKII and endogenous syntaxin 1A (Ohyama et al. [2002] J. Neurosci. 22:3342–3351). To identify the simplest and the most potent inhibitor for exocytosis, we further characterized the linker domain and determined the minimal number of residues required for CaMKII binding. The minimal length of the CaMKII‐binding site was 145–172 residues and a loss of G172 considerably weakened affinity for CaMKII. The basic amino acid clusters, R151 and K146, were indispensable for binding, whereas R148 was not. A comparison of the CaMKII‐binding in several syntaxin isoforms revealed that the substitution of S162 attenuated CaMKII‐binding activity. These results suggest that S162 is an important residue as well as the basic amino acid cluster within region 145–172 of the linker domain. © 2003 Wiley‐Liss, Inc.
Growth arrest of PC12 cells by nerve growth factor is dependent on the phosphatidylinositol 3‐kinase/Akt pathway via p75 neurotrophin receptor
Authors: Ito Hisanori , Nomoto Hiroshi , Furukawa Shoei
Published: March 2003

We recently isolated mutant PC12 cell clones (PC84 cells) by transfection of PC12 cells with nerve growth factor (NGF) cDNA. These cells secreted active NGF and extended short processes, but proliferated faster than the parental PC12 cells. Because the expression level of p75, a low‐affinity receptor for NGF, was significantly low, we suspected that NGF signaling via p75 was necessary for the growth arrest of the PC12 cells, and this was shown to be the case by repressing p75 function in PC12 cells. In this study, we examined the downstream signaling of p75, which would ultimately evoke the growth arrest. NGF is known to induce rapid phosphorylation of MAP kinase and Akt in PC12 cells, whereas in PC84 cells, MAP kinase was phosphorylated but the phosphorylation level of Akt was very low under the serum‐free condition. This finding suggested that the low expression level of p75 in PC84 cells was the reason for the low Akt activation. Because Akt is known to be activated via phosphatidylinositol (PI) 3‐kinase, we treated PC12 cells with a PI3‐kinase inhibitor, Wortmannin, and found these cells did not cease proliferation in the presence of NGF. Furthermore, anti‐p75 neutralizing antibody reduced NGF‐induced phosphorylation of Akt in PC12 cells under the serum‐free condition. Because we had already shown that PC12 cells treated with anti‐p75 neutralizing antibody did not cease proliferation in the presence of NGF, these results suggest that NGF activates Akt via p75, which is necessary for the NGF‐induced growth arrest of PC12 cells. © 2003 Wiley‐Liss, Inc.
Nerve growth factor regulates neuroectodermal tumor cell responses to mitogenic growth factors
Authors: Castellon Raquel , Mirkin Bernard L.
Published: March 2003

Previous studies showed that nerve growth factor (NGF) decreases the proliferation of neuroectodermal tumor (NET) cells (C‐1300 and Neuro2A murine neuroblastoma, PC12 rat pheochromocytoma) within 5–7 days in a dose‐dependent manner. This effect is regulated by the concentration of serum in the culture medium. Therefore, we hypothesized that NGF exerts its antimitogenic activities by interfering with the proliferative action of other growth factors. We studied the effects of short‐term vs. long‐term as well as endogenous vs. exogenous NGF on NET cell proliferation in response to various mitogenic growth factors. Retrovirus‐mediated transfer of the β‐NGF gene into NET cells activated TrkA and consistently decreased their proliferative responses to insulin‐like growth factor (IGF)‐I, IGF‐II, fibroblast growth factor‐2, and epidermal growth factor (EGF), down‐regulating EGF and IGF‐I binding sites. It also decreased tyrosine phosphorylation of ERK‐1, STAT3, and EGF or IGF receptors after treatment with IGF‐I or EGF. Long‐term incubation of NET cells with NGF mimicked the responses induced by β‐NGF gene transfer, albeit in a reversible manner. Short‐term NGF treatment augmented the proliferative responses to IGF‐I or EGF by enhancing cell survival. It also increased tyrosine phosphorylation of signal transducing proteins after exposure to IGF or EGF, an effect opposite to that of long‐term NGF treatments. Hence, long‐term NGF exposure in vitro might better reproduce the effects of NGF in vivo than short‐term treatments. Only long‐term exposure to NGF decreased the responses of NET cells to mitogenic growth factors by down‐regulating their receptors and attenuating signal transduction events required for cell proliferation. These results suggest that NGF could exert similar actions on cellular responses to growth factors in vivo. © 2003 Wiley‐Liss, Inc.
TorsinA in PC12 cells: Localization in the endoplasmic reticulum and response to stress
Authors: Hewett Jeffrey , Ziefer Philipp , Bergeron Daniele , Naismith Teri , Boston Heather , Slater Damien , Wilbur Jeremy , Schuback Deborah , Kamm Christoph , Smith Nicole , Camp Sara , Ozelius Laurie J. , Ramesh Vijaya , Hanson Phyllis I. , Breakefield Xandra O.
Published: March 2003

Most cases of early‐onset torsion dystonia are caused by deletion of GAG in the coding region of the DYT1 gene encoding torsinA. This autosomal dominant neurologic disorder is characterized by abnormal movements, believed to originate from neuronal dysfunction in the basal ganglia of the human brain. The torsins (torsinA and torsinB) are members of the “ATPases associated with a variety of cellular activities” (AAA+) superfamily of proteins that mediate chaperone and other functions involved in conformational modeling of proteins, protection from stress, and targeting of proteins to cellular organelles. In this study, the intracellular localization and levels of endogenous torsin were evaluated in rat pheochromocytoma PC12 cells following differentiation and stress. TorsinA, apparent MW 37 kDa, cofractionates with markers for the microsomal/endoplasmic reticulum (ER) compartment and appears to reside primarily within the ER lumen based on protease resistance. TorsinA immunoreactivity colocalizes with the lumenal ER protein protein disulfide isomerase (PDI) and extends throughout neurites. Levels of torsinA did not increase notably in response to nerve growth factor‐induced differentiation. None of the stress conditions tested, including heat shock and the unfolded protein response, affected torsinA, except for oxidative stress, which resulted in an increase in the apparent MW of torsinA and redistribution to protrusions from the cell surface. These findings are consistent with a relatively rapid covalent modification of torsinA in response to oxidative stress causing a change in state. Mutant torsinA may interfere with and/or compromise ER functions, especially in dopaminergic neurons, which have high levels of torsinA and are intrinsically vulnerable to oxidative stress. © 2003 Wiley‐Liss, Inc.