bluuebunny/crossref_metadata_2025 huggingface dataset:
INDIVIDUALISM, COLLECTIVISM AND THE MARKETIZATION OF SOCIAL SECURITY: Chile and China Compared
Authors: Midgley James , Tang Kwong‐leung
Published: June 2005

Ideological beliefs are of central importance in current debates about the marketization of social security. However, developments in Chile and China suggest that the conventional individualist‐collectivist dichotomy, which has framed ideological debates on social security, fails to capture the complexities of ideological differences as well as the imperative of economic development which appears to be driving social security policy in both countries. Examining events in these countries, it is suggested that the individualist‐collectivist dichotomy needs to be reconsidered. It is also argued at a normative level that these ideologies no longer provide a viable basis for social security policy, and that the “developmentalist” approach emerging in China merits further scrutiny.
TILTING TOWARD MARKETIZATION: Reform of the Canadian Pension Plan
Authors: Drover Glenn
Published: June 2005

Public concern about the earnings‐related Canada Pension Plan has forced the Canadian government to move toward fuller funding, a new investment policy, and changes to benefits and administration. Together, the three initiatives, and particularly the first two, amount to a modest degree of marketization. The assumption behind the reform is that the Canada Pension Plan will remain public and mandatory but the change will create a board and an investment policy which are more sensitive to market pressures and less amendable to government interference. The Canada Pension Plan is one of three tiers of support for the elderly in retirement. It is a contributory social insurance scheme which protects against loss of income. The other two are a social allowance (Old Age Security) as well as an income tested benefit (Guaranteed Income Supplement) which provide basic income support and employment based retirement pension plans as well as group and individual registered retirement savings plans which supplement the other two tiers on a voluntary basis. This article examines the reform of the Canada Pension Plan. After a brief examination of Canada's retirement benefits in an international context, the article summarizes recent debates in Canada, government policies to develop fuller funding and new investment strategies, and reflections on possible developments in the future.
HEALTH CARE POLICY IN THE GERMAN SOCIAL INSURANCE STATE: From Solidarity to Privatization?
Authors: Hinrichs Karl
Published: June 2005

Statutory health insurance is a centerpiece of the German welfare state, which considers itself to be a “social insurance state.” At the same time, due to a large volume of interpersonal redistributions that occur in health insurance, it is the most ambitious branch of the country's social insurance system. The stability of the health scheme thus depends on a “culture of solidarity” to maintain the legitimacy of these redistributions. This article analyzes recent changes in the legislative framework of the statutory health insurance. It asks whether these changes which predominantly aim to contain employers’ nonwage labor costs by making the insured bear a larger share of total health care spending, are possibly weakening the moral infrastructure of the welfare state. To this end, findings from qualitative interviews with insured persons are evaluated in view of recently approved and currently proposed legislative changes to the health scheme. The analytical focus is the question whether the two equity principles of this scheme, delivering health care according to medical need and financing it according to the “ability‐to‐pay,” are becoming endangered.
PRIVATIZATION OF SOCIAL SECURITY IN SOUTHEAST ASIA
Authors: Ramesh M
Published: June 2005

This article examines the main social security programs, narrowly defined to include income maintenance for the aged and health care, in Indonesia, Malaysia, Singapore and Thailand, and reveals the large degree to which the statutory programs depend on private provision, particularly, private financing. It further shows that efforts are underway to reduce, or at least to arrest the expansion of role of the state by a corresponding expansion in the role of private provision and financing. The expansion of social security for the aged in Thailand is an exception in this regard. I conclude that both inefficiency and inequity have been promoted.
MEGA‐EVENTS, URBAN DEVELOPMENT, AND PUBLIC POLICY
Authors: Burbank Matthew J. , Andranovich Greg , Heying Charles H.
Published: June 2005

As cities compete for jobs and capital in the context of limited federal aid and increasing global economic competition, a new and potentially high‐risk public policy strategy for stimulating local economic growth has emerged. This megaevent strategy entails the quest for a high‐profile event to serve as a stimulus to, and justification for, local development. How and why do American cities commit their resources to seeking a mega‐event? And, if a city lands a megaevent, how does that event affect local development policy? To address these questions, we examine the experiences of three American cities which have bid for and organized the Olympics in the contemporary era: Los Angeles, Atlanta, and Salt Lake City.
THE IDEOLOGICAL ROOTS OF DEFICIT REDUCTION POLICY
Authors: Taylor Andrew J.
Published: June 2005

This article identifies those members of Congress who have most often supported deficit reduction on floor roll‐call votes since 1980. An examination of the reciprocal relationship between fiscal policy preference and the more holistic concept of ideology reveals that at an abstract level conservatives and Republicans continue to support fiscal restraint. When we examine specific issues, however, we find that more moderate legislators are generally the most supportive of deficit reduction in the contemporary policy process. This, in turn, suggests a bifurcation of fiscal policy as it relates to ideology. It seems that even though legislators do sometimes think of fiscal policy along the traditional lines of budgetary balance or deficits, the issue is now more often recognized as consisting of the two distinct and separate sub‐issues of government expenditures and revenue policy.
THE SUCCESS of the 1993 BUDGET RECONCILIATION BILL at REDUCING the FEDERAL BUDGET DEFICIT
Authors: Fisher Patrick
Published: June 2005

This article discusses the process by which President Clinton achieved a budgetdeficit reduction package in Congress and the implications of this process. It concludes that future administrations will have more flexibility in debating whether the federal government should increase its scope or reduce taxes.
MEETING THE MAASTRICHT TARGETS
Authors: Kahrs Karl H.
Published: June 2005

This article documents the role of debt management and budgetary targets as emerging factors in economic integration. The author examines the negotiations behind the Maastrict Treaty with a particular focus on Germany and France. This study also examines the long and short‐term outlook for economic success by analyzing the domestic and international economic and political forces at work in the process of economic integration. Such an understanding is critical as regional trading blocs spread and resistance to these trends becomes organized.
THE IMPACT MAXIMUM BENEFIT AMOUNTS HAVE ON THE LENGTH OF TIME FAMILIES RECEIVE AFDC‐BASIC BENEFITS
Authors: Barton Thomas R. , Pillai Vijayan K. , Dietz Tracy J.
Published: June 2005

Analysts and program evaluators have rightly focused on the impact policies have on participants achieving programs’ goals and objectives. Yet a potentially neglected aspect of these analyses is the impact policies themselves have on who is initially eligible to participate in the programs and the impact policy parameters have on the length of time people participate in the program. This article reports the findings of a study on the length of time families receive AFDC (now TANF) benefits in two counties in a high benefit state, Wisconsin, between 1987 and 1989 and a low benefit state, Texas, from mid‐1989 through 1992 and the influence the states’ maximum benefits have on who is eligible to receive benefits and how families lose eligibility. Results of a life table analysis indicate that families in Texas receive benefits over significantly less time. However, results of a multivariate analysis indicate that when selected personal and familial attributes are held constant, the difference disappears.
THE SOCIAL CONSTRUCTION OF MEXICAN AND CUBAN IMMIGRANTS BY POLITICIANS
Authors: Magaña Lisa , Short Robert
Published: June 2005

Schneider and Ingram (1993) propose that the amount and quality of service that target groups receive from their government is related to two factors: political power (strong or weak); and the target groups’ image in the public eye (positive or negative), also known as “social constructions.” This research examines political candidates’ social constructions of Mexican and Cuban immigrants in major newspapers. Using newspaper articles from Lexis/Nexus (an electronic newspaper database), we content analyzed 495 major US newspapers over a six‐year period. Our results indicate that Latin American immigration, particularly Mexican immigration, is championed as a political platform in ways consistent with Schneider and Ingram's model.
EXPANDING THE LIMITS OF POWER: The Federal Reserve and the Implementation of Functional Regulation in the Gramm‐Leach‐Bliley Era
Authors: Holyoke Thomas T.
Published: June 2005

Research on bureaucratic behavior suggests that agencies are more likely to use the implementation process to extend their power and influence under particular circumstances. I argue that when an agency has been delegated considerable power by Congress, but provided only vague guidance on how to implement this authority, an atmosphere of uncertainty and competition is created. Under such a circumstance the agency will feel pressured to further extend its power in order to defend its regulatory turf against competitors and protect the authority it was delegated. I test this proposition by examining the behavior of the Federal Reserve as it implements the functional regulation provisions of the Gramm‐Leach‐Bliley Act. Evidence from the Federal Reserve's dealings with the Securities and Exchange Commission during the approval of the Schwab – U. S. Trust merger provides evidence that the Fed is indeed acting to extend its power and influence.
THE USE OF BARGAINING GAMES IN LOCAL DEVELOPMENT POLICY
Authors: Steinacker Annette
Published: June 2005

Granting location incentives is a common city policy to attract new businesses, despite the frequent belief that the benefits are too generous. Can cities do a better job determining when to agree to these concessions and when to refuse? I argue that analyzing the concession decision with a simple bargaining game illustrates: (1) that the negotiation strategy a city uses should be based on the characteristics of the firm in each case and (2) that even poor cities with few attractive location features can avoid making large concessions. Development experiences in Miami‐Dade are used to illustrate the model.
Antiproliferative activity of melatonin by transcriptional inhibition of cyclin D1 expression: a molecular basis for melatonin‐induced oncostatic effects
Authors: Cini Grazia , Neri Bruno , Pacini Alessandra , Cesati Valentina , Sassoli Chiara , Quattrone Silvia , D'Apolito Maria , Fazio Antonina , Scapagnini Giovanni , Provenzani Alessandro , Quattrone Alessandro
Published: June 2005

Abstract:  Melatonin is endowed with a growth inhibitory effect in MCF‐7 breast cancer cells whose mechanism has been related to an antiestrogenic activity exerted by inhibition of binding of the estradiol–estrogen receptor complex to its DNA responsive element. Looking for downstream gene determinants of this effect, we performed a transcriptome profiling by high‐density microarrays of estrogen‐treated MCF‐7 cells exposed or not to melatonin. We found that cyclin D1 was one of the main downregulated genes by melatonin. Validation experiments clearly confirm that in MCF‐7 cells the estrogen‐induced growth inhibitory activity of melatonin is consistently associated with inhibition of estrogen‐elicited cyclin D1 induction. This effect is almost purely transcriptional. Reporter gene assays indicate that the same portion of the cyclin D1 promoter which confers estrogen sensitivity, encompassing a potential cAMP responsive element binding site, is repressed by melatonin. Transcriptional downregulation of cyclin D1 is the key molecular event for melatonin's antiproliferative activity, as this activity can be completely and selectively rescued by transient cyclin D1 overexpression. Finally, we provide indirect evidence that the effect of melatonin on the cyclin D1 promoter is mediated by the c‐jun and ATF‐2 proteins, known to bind the minimal estrogen‐sensitive cyclin D1 promoter element. These findings establish for the first time a molecular link between melatonin and its effects on the cell cycle, providing at the same time a rationale for its use in adjuvant chemotherapy.
Antioxidant activity of melatonin and a pinoline derivative on linoleate model system
Authors: Mekhloufi J. , Bonnefont‐Rousselot D. , Yous S. , Lesieur D. , Couturier M. , Thérond P. , Legrand A. , Jore D. , Gardès‐Albert M.
Published: June 2005

Abstract:  This study aimed at investigating the in vitro protective effects of GWC22, a novel pinoline derivative [6‐ethyl‐1‐(3‐methoxyphenyl)‐2‐propyl‐1,2,3,4‐tetrahydro‐beta‐carboline] chlorhydrate, against radiation‐induced oxidation of linoleate initiated by hydroxyl radicals (•OH). Using linoleate micelles (10−2 m) as lipid model, two indexes of peroxidation have been measured, i.e. conjugated dienes and hydroperoxides. Similar determinations were performed with melatonin in order to compare the protective effects of the two compounds. It was observed that, the higher the concentration of GWC22 (or melatonin) (3 × 10−5 to 10−4 m), the stronger the antioxidant ability. In these in vitro assays, GWC22 showed a better antioxidant effect than melatonin for a given antioxidant concentration. A reaction scheme has been proposed to explain the inhibitory effect of an antioxidant via the propagating steps of the lipid peroxidation. Indeed, we have suggested that melatonin and GWC22 may compete with the fatty acid to scavenge lipid peroxyl radicals (LOO•). We have estimated a lower limit for the LOO• rate constant for GWC22 (≥1.4 × 105/m/s) and for melatonin (≥2.8 × 104/m/s) assuming that the k‐value of the propagating step in linoleate (LOO• + linoleate) was 1.4 × 103/m/s. The difference of reactivity between melatonin and GWC22 in this model system is assumed to be related to their relative lipophilicity.
Melatonin protects against MPTP/MPP+‐induced mitochondrial DNA oxidative damage in vivo and in vitro
Authors: Chen Liu‐Ji , Gao Yan‐Qin , Li Xue‐Jun , Shen Di‐Han , Sun Feng‐Yan
Published: June 2005

Abstract:  The effects of melatonin on the mitochondrial DNA (mtDNA) damage induced by 1‐methyl‐4‐phenyl‐1, 2, 3, 6‐tetrahydropyridine (MPTP) and 1‐methyl‐4‐phenylpyridine ion (MPP+) were investigated both in vivo and in vitro. MPTP (24 mg/kg, s.c.) induced a rapid increase in the immunoreactivity of 8‐hydroxyguanine (8‐oxoG), a common biomarker of DNA oxidative damage, in the cytoplasm of neurons in the Substantia Nigra Compact of mouse brain. Melatonin preinjection (7.5, 15 or 30 mg/kg, i.p.) dose‐dependently prevented MPTP‐induced DNA oxidative damage. In SH‐SY5Y cells, MPP+ (1 mm) increased the immunoreactivity of 8‐oxoG in the mitochondria at 1 hr and in the nucleus at 3 hr after treatment. Melatonin (200 μm) preincubation significantly attenuated MPP+‐induced mtDNA oxidative damage. Furthermore, MPP+ time‐dependently increased the accumulation of mitochondrial oxygen free radicals (mtOFR) from 1 to 24 hr and gradually decreased the mitochondrial membrane potential (Ψm) from 18 to 36 hr after incubation. At 72 hr after incubation, MPP+ caused cell death in 49% of the control. However, melatonin prevented MPP+‐induced mtOFR generation and Ψm collapse, and later cell death. The present results suggest that cytoprotection of melatonin against MPTP/MPP+‐induced cell death may be associated with the attenuation of mtDNA oxidative damage via inhibition of mtOFR generation and the prevention of Ψm collapse.
Coexpression of MT1 and RORα1 melatonin receptors in the Syrian hamster Harderian gland
Authors: Tomás‐Zapico Cristina , Antonio Boga José , Caballero Beatriz , Vega‐Naredo Ignacio , Sierra Verónica , Álvarez‐García Óscar , Tolivia Delio , Josefa Rodríguez‐Colunga María , Coto‐Montes Ana
Published: June 2005

Abstract:  Melatonin acts through several specific receptors, including membrane receptors (MT1 and MT2) and members of the RZR/ROR nuclear receptors family, which have been identified in a large variety of mammalian and nonmammalian cells types. Both membrane and nuclear melatonin receptors have been partially characterized in Harderian gland of the Syrian hamster. Nevertheless, the identities of these receptors were unknown until this study, where the coexistence of MT1 and RORα1 in this gland was determined by nested RT‐PCR followed by amplicon sequencing and Western‐blot. Furthermore, the cellular localization of both receptors was determined by immunohistochemistry. Thus, MT1 receptor was localized exclusively at the basal side of the cell acini, supporting the hypothesis that this receptor is activated by the pineal‐synthesized melatonin. On the contrary, although a RORα1‐immunoreactivity was observed in nuclei of epithelial cells of both sexes, an extranuclear specific staining, which was more frequently among those cells of males, was also seen. The implication of this possible nuclear exclusion of RORα1 on the role of this indoleamine against oxidative stress is discussed.
Learning and memory deficits in rats induced by chronic thinner exposure are reversed by melatonin
Authors: Baydas Giyasettin , Ozveren Faik , Akdemir Ismail , Tuzcu Mehmet , Yasar Abdullah
Published: June 2005

Abstract:  Thinner is a neurotoxic mixture which is widely used as an aromatic industrial solvent. This product has been shown to cause functional and structural changes in the central nervous system. We investigated the effect of exposure to high concentrations (3000 p.p.m.) of thinner for 45 days (1 hr/day) on cognitive functions and the levels of neural cell adhesion molecules (NCAM) and lipid peroxidation products (LPO) in the hippocampus, cortex and cerebellum of rats. The actions of melatonin on the effects produced by thinner exposure were also tested. Thinner exposure caused a significant increase in LPO (malondialdehyde and 4‐hydroxyalkenals) in all brain regions. Melatonin administration significantly reduced LPO and elevated glutathione levels in the brain regions. NCAM (180 kDa) was significantly decreased in hippocampus and cortex of thinner‐exposed rats. Furthermore, thinner‐exposed rats showed cognitive deficits in passive avoidance and Morris water maze tasks, whereas in the rats chronically treated with melatonin these effects were reversed. This study indicates that treatment with melatonin prevents learning and memory deficits caused by thinner exposure possibly by reducing oxidative stress and regulating neural plasticity.
Melatonin levels decrease in type 2 diabetic patients with cardiac autonomic neuropathy
Authors: Tutuncu Neslihan B. , Batur Mustafa K. , Yildirir Aylin , Tutuncu Tanju , Deger Ahmet , Koray Zehra , Erbas Belkis , Kabakci Giray , Aksoyek Serdar , Erbas Tomris
Published: June 2005

Abstract:  The present study has been designed to determine melatonin levels in type 2 diabetic patients and test the relationship between the autonomic nervous system and melatonin dynamics. Thirty‐six type 2 diabetic patients and 13 age‐matched healthy subjects were recruited for the study. Circadian rhythm of melatonin secretion was assessed by measuring serum melatonin concentrations between 02:00–04:00 and 16:00–18:00 hr. Melatonin dynamics were re‐evaluated with respect to autonomic nervous system in diabetic patients with autonomic neuropathy who were diagnosed by the cardiovascular reflex tests, heart rate variability (HRV), and 24‐hr blood pressure monitoring. Nocturnal melatonin levels and the nocturnal melatonin surge were low in the diabetic group (P = 0.027 and 0.008 respectively). Patients with autonomic neuropathy revealed decreased melatonin levels both at night and during day when compared with healthy controls (P < 0.001 and 0.004 respectively) while the melatonin dynamics were similar to controls in patients without autonomic neuropathy. Nocturnal melatonin level was positively correlated with nocturnal high and low frequency components of HRV (P = 0.005 and 0.011 respectively) and systolic and diastolic blood pressures at night (P = 0.002 and 0.004 respectively) in patients with autonomic neuropathy. We found a negative correlation between nocturnal melatonin levels and the degree of systolic blood pressure decrease at night (r = −0.478, P = 0.045). As a conclusion this study has shown that circadian rhythm of melatonin secretion is blunted in type 2 diabetic patients and there is a complex relationship between various components of autonomic nervous system and melatonin secretion at night. Among the patients with autonomic neuropathy those with more preserved HRV and the systolic nondippers (<10% reduction in blood pressure during the night relative to daytime values) have more pronounced melatonin surge at night.
Seasonality of pineal gland activity and immune functions in chickens
Authors: Majewski Paweł , Adamska Iwona , Pawlak Joanna , Barańska Anna , Skwarło‐Sońta Krystyna
Published: June 2005

Abstract:  The immunomodulatory action of melatonin in different animal species is already well known, although the mechanism(s) by which the indoleamine influences the immune system have yet to be fully elucidated. Previously, we have shown both anti‐inflammatory and opioid‐mediated influence of exogenous melatonin on thioglycollate‐induced peritonitis in young chickens. In the present study, the kinetics of peritonitis and splenocyte proliferation were compared in chickens reared in both seasons under the same L:D 12:12 conditions. These two aspects of the immune response were correlated with the diurnal rhythm of pineal gland function, measured by the activity of N‐acetyltransferase (NAT), a key enzyme in melatonin biosynthesis. The results revealed seasonal changes in the circadian rhythm of pineal NAT activity occurring in parallel to the natural local geophysical seasons. These changes appeared to influence the development of peritonitis and splenocyte responsiveness to mitogenic stimulation in vitro. Moreover, the existence of bidirectional communication between the pineal gland and the activated immune system was supported by the decreased activity of pineal NAT in chickens with peritonitis compared with control birds.
Microcirculatory effects of melatonin in rat skeletal muscle after prolonged ischemia
Authors: Wang Wei Z. , Fang Xin‐Hua , Stephenson Linda L. , Baynosa Richard C. , Khiabani Kayvan T. , Zamboni William A.
Published: June 2005

Abstract:  The purpose of this study was to determine microcirculatory effects and response of nitric oxide synthase (NOS) to melatonin in skeletal muscle after prolonged ischemia. A vascular pedicle isolated rat cremaster muscle model was used. Each muscle underwent 4 hr of zero‐flow warm ischemia followed by 2 hr of reperfusion. Melatonin (10 mg/kg) or saline as a vehicle was given by intraperitoneal injection at 30 min prior to reperfusion and the same dose was given immediately after reperfusion. After reperfusion, microcirculation measurements including arteriole diameter, capillary perfusion and endothelial‐dependent and ‐independent vasodilatation were performed. The cremaster muscle was then harvested to measure endothelial NOS (eNOS) and inducible NOS (iNOS) gene expression and enzyme activity. Three groups of rats were used: sham‐ischemia/reperfusion (I/R), vehicle + I/R and melatonin + I/R. As compared with vehicle + I/R group, administration of melatonin significantly enhanced arteriole diameter, improved capillary perfusion, and attenuated endothelial dysfunction in the microcirculation of skeletal muscle after 4 hr warm ischemia. Prolonged warm ischemia followed by reperfusion significantly depressed eNOS gene expression and constitutive NOS activity and enhanced iNOS gene expression. Administration of melatonin did not significantly alter NOS gene expression or activity in skeletal muscle after prolonged ischemia and reperfusion. Melatonin provided a significant microvascular protection from reperfusion injury in skeletal muscle. This protection is probably attributable to the free radical scavenging effect of melatonin, but not to its anti‐inflammatory effect.
Melatonin synthesis and melatonin‐membrane receptor (MT1) expression during rat thymus development: role of the pineal gland
Authors: Jimenez‐Jorge Silvia , Jimenez‐Caliani Antonio J. , Guerrero Juan M. , Naranjo Maria C. , Lardone Patricia J. , Carrillo‐Vico Antonio , Osuna Carmen , Molinero Patrocinio
Published: June 2005

Abstract:  To gain insight into the relationship between thymus and pineal gland during rat development, the melatonin content as well as the activity and expression of the two key enzymes for melatonin biosynthesis, i.e. N‐acetyltransferase (NAT) and hydroxyindole‐O‐methyltransferase (HIOMT), were studied in the thymus at fetal and postnatal stages. Moreover, melatonin‐membrane receptor (MT1) expression was also analyzed. We found both the expression and activity of thymic NAT and HIOMT at 18 days of fetal life. Additionally, there is production of melatonin in the thymus as well as MT1 expression at this fetal age. These results show values higher in day‐time than at night‐time. The pineal gland begins to produce significant levels of melatonin around postnatal day 16, and this synthesis shows a circadian rhythm with high values during the dark period; therefore the nocturnal serum melatonin may inhibit thymic melatonin production. To document this, we report an increased melatonin content of the thymus in pinealectomized rats compared with sham‐pinealectomized. In conclusion, these results show, for the first time, the presence of the biosynthetic machinery of melatonin and melatonin production in developing rat thymus and that the pineal gland may regulate this process.
A novel H28Y mutation in LEC rats leads to decreased NAT protein stability in vivo and in vitro
Authors: Huang Zheping , Deng Jie , Borjigin Jimo
Published: June 2005

Abstract:  Nocturnal melatonin production is reportedly controlled by the rhythms of serotonin N‐acetyltransferase (NAT, or arylalkylamine N‐acetyltransferase). While analyzing the melatonin synthetic pathways of Long Evans cinnamon (LEC) rats mutant for PINA, a pineal night‐specific ATPase defective in Wilson disease, we discovered that NAT activity and protein levels are greatly reduced in LEC rats, and that the highly conserved histidine 28 is mutated to tyrosine. To study the effect of H28Y, we isolated a new strain of rat termed LPN that is mutant for NAT but wildtype for both PINA and coat color. Compared with control rats, the LPN rats displayed low NAT protein levels and enzyme activities. These results suggest that the H28Y mutation in NAT is the cause of reduced NAT levels in vivo. The identical H28Y mutation was also found in Sprague–Dawley rats from Zivic–Miller, suggesting it may be a common mutation in rodents. When analyzed in bacterial cells and HEK293 cells, the mutation resulted in reduction of both NAT protein stability and catalytic activity, confirming that the in vivo NAT phenotype in LPN rats was due to the H28Y mutation. Further analysis of the NAT‐H28Y will focus on the mechanisms of the increased degradation both in vitro and in vivo, which will facilitate our understanding of how melatonin synthesis is controlled at the molecular level.
N‐acetyltransferase is not the rate‐limiting enzyme of melatonin synthesis at night
Authors: Liu Tiecheng , Borjigin Jimo
Published: June 2005

Abstract:  Circadian melatonin production in the pineal gland and retina is under the control of serotonin N‐acetyltransferase (NAT) and hydroxyindole‐O‐methyltransferase. Because NAT activity varies diurnally, it has been considered both the melatonin rhythm‐generating enzyme and the rate‐limiting enzyme of melatonin synthesis. In rats with dramatically reduced NAT activity due to a H28Y mutation in NAT, melatonin levels remained the same as in wildtype controls, suggesting that NAT does not determine the rate of melatonin production at night. Using a combination of molecular approaches with a sensitive in vivo measurement of pineal diurnal melatonin production, we demonstrate that (i) N‐acetylserotonin (NAS), the enzymatic product of NAT, is present in vast excess in the night pineals compared with melatonin; (ii) the continuous increase in NAT protein levels at late night does not produce a proportional increase in melatonin; and (iii) an increase in NAS in the same animal over several circadian cycles do not result in corresponding increase in melatonin output. These results strongly suggest that NAT is not the rate‐limiting enzyme of melatonin formation at night.
Quantitative estimation of channeling from early glycolytic intermediates to CO2 in intact Escherichia coli
Authors: Shearer Georgia , Lee Jennifer C. , Koo Jia‐an , Kohl Daniel H.
Published: June 2005

A pathway intermediate is said to be ‘channeled’ when an intermediate just made in a pathway has a higher probability of being a substrate for the next pathway enzyme compared with a molecule of the same species from the aqueous cytoplasm. Channeling is an important phenomenon because it might play a significant role in the regulation of metabolism. Whereas the usual mechanism proposed for channeling is the (often) transient interaction of sequential pathway enzymes, many of the supporting data come from results with pure enzymes and dilute cell extracts. Even when isotope dilution techniques have utilized whole‐cell systems, most often only a qualitative assessment of channeling has been reported. Here we develop a method for making a quantitative calculation of the fraction channeled in glycolysis from in vivo isotope dilution experiments. We show that fructose‐1,6‐bisphosphate, in whole cells of Escherichia coli, was strongly channeled all the way to CO2, whereas fructose‐6‐phosphate was not. Because the signature of channeling is lost if any downstream intermediate prior to CO2 equilibrates with molecules in the aqueous cytosol, it was not possible to evaluate whether glucose‐6‐phosphate was channeled in its transformation to fructose‐6‐phosphate. The data also suggest that, in addition to pathway enzymes being associated with one another, some are free in the aqueous cytosol. How sensitive the degree of channeling is to growth or experimental conditions remains to be determined.
Two types of replication protein A in seed plants
Authors: Ishibashi Toyotaka , Koga Asami , Yamamoto Taichi , Uchiyama Yukinobu , Mori Yoko , Hashimoto Junji , Kimura Seisuke , Sakaguchi Kengo
Published: June 2005

Replication protein A (RPA), a heterotrimeric protein composed of 70, 32 and 14‐kDa subunits, has been shown to be essential for DNA replication, repair, recombination, and transcription. Previously, we found that, in two seed plants, rice and Arabidopsis, there are two different types of RPA70‐kDa subunit. Substantial biochemical and genetic characterization of these two subunits, termed OsRPA70a and OsRPA70b or AtRPA70a and AtRPA70b, respectively, is described in this report. Inactivation of AtRPA70a by transfer DNA insertion or RNA interference is lethal, so the complex containing RPA70a may be essential for DNA replication. Transfer DNA insertion and RNAi lines for AtRPA70b are morphologically normal, albeit hypersensitive to certain mutagens, such as UV‐B and methyl methanesulfonate, suggesting that RPA70b functions mostly in DNA repair. In two‐hybrid, pull‐down and coexpression analysis, OsRPA70b was found to interact more selectively than OsRPA70a with OsRPA32. The data suggest that two different types of RPA heterotrimer are present in seed plants, and that there may be additional 32 and 14‐kDa subunit homologs that interact with OsRPA70a. Each of the two probable plant RPA complexes may have different roles in DNA metabolism.
Histone H2A phosphorylation in DNA double‐strand break repair
Authors: Foster Elinor R. , Downs Jessica A.
Published: June 2005

DNA repair must take place within the context of chromatin, and it is therefore not surprising that many aspects of both chromatin components and proteins that modify chromatin have been implicated in this process. One of the best‐characterized chromatin modification events in DNA‐damage responses is the phosphorylation of the SQ motif found in histone H2A or the H2AX histone variant in higher eukaryotes. This modification is an early response to the induction of DNA damage, and occurs in a wide range of eukaryotic organisms, suggesting an important conserved function. One function that histone modifications can have is to provide a unique binding site for interacting factors. Here, we review the proteins and protein complexes that have been identified as H2AS129ph (budding yeast) or H2AXS139ph (human) binding partners and discuss the implications of these interactions.
A novel carbonic anhydrase from the giant clam Tridacna gigas contains two carbonic anhydrase domains
Authors: Leggat William , Dixon Ross , Saleh Said , Yellowlees David
Published: June 2005

This report describes the presence of a unique dual domain carbonic anhydrase (CA) in the giant clam, Tridacna gigas. CA plays an important role in the movement of inorganic carbon (Ci) from the surrounding seawater to the symbiotic algae that are found within the clam's tissue. One of these isoforms is a glycoprotein which is significantly larger (70 kDa) than any previously reported from animals (generally between 28 and 52 kDa). This α‐family CA contains two complete carbonic anhydrase domains within the one protein, accounting for its large size; dual domain CAs have previously only been reported from two algal species. The protein contains a leader sequence, an N‐terminal CA domain and a C‐terminal CA domain. The two CA domains have relatively little identity at the amino acid level (29%). The genomic sequence spans in excess of 17 kb and contains at least 12 introns and 13 exons. A number of these introns are in positions that are only found in the membrane attached/secreted CAs. This fact, along with phylogenetic analysis, suggests that this protein represents the second example of a membrane attached invertebrate CA and it contains a dual domain structure unique amongst all animal CAs characterized to date.
Redox‐regulated affinity of the third PDZ domain in the phosphotyrosine phosphatase PTP‐BL for cysteine‐containing target peptides
Authors: van den Berk Lieke C. J. , Landi Elena , Harmsen Etelka , Dente Luciana , Hendriks Wiljan J. A. J.
Published: June 2005

PDZ domains are protein–protein interaction modules that are crucial for the assembly of structural and signalling complexes. They specifically bind to short C‐terminal peptides and occasionally to internal sequences that structurally resemble such peptide termini. The binding of PDZ domains is dominated by the residues at the P0 and P−2 position within these C‐terminal targets, but other residues are also important in determining specificity. In this study, we analysed the binding specificity of the third PDZ domain of protein tyrosine phosphatase BAS‐like (PTP‐BL) using a C‐terminal combinatorial peptide phage library. Binding of PDZ3 to C‐termini is preferentially governed by two cysteine residues at the P−1 and P−4 position and a valine residue at the P0 position. Interestingly, we found that this binding is lost upon addition of the reducing agent dithiothrietol, indicating that the interaction is disulfide‐bridge‐dependent. Site‐directed mutagenesis of the single cysteine residue in PDZ3 revealed that this bridge formation does not occur intermolecularly, between peptide and PDZ3 domain, but rather is intramolecular. These data point to a preference of PTP‐BL PDZ3 for cyclic C‐terminal targets, which may suggest a redox state‐sensing role at the cell cortex.
Role of the surface charges D72 and K8 in the function and structural stability of the cytochrome c6 from Nostoc sp. PCC 7119
Authors: Lange Christian , Luque Irene , Hervás Manuel , Ruiz‐Sanz Javier , Mateo Pedro L. , De la Rosa Miguel A.
Published: June 2005

We investigated the role of electrostatic charges at positions D72 and K8 in the function and structural stability of cytochrome c6 from Nostoc sp. PCC 7119 (cyt c6). A series of mutant forms was generated to span the possible combinations of charge neutralization (by mutation to alanine) and charge inversion (by mutation to lysine and aspartate, respectively) in these positions. All forms of cyt c6 were functionally characterized by laser flash absorption spectroscopy, and their stability was probed by urea‐induced folding equilibrium relaxation experiments and differential scanning calorimetry. Neutralization or inversion of the positive charge at position K8 reduced the efficiency of electron transfer to photosystem I. This effect could not be reversed by compensating for the change in global charge that had been introduced by the mutation, indicating a specific role for K8 in the formation of the electron transfer complex between cyt c6 and photosystem I. Replacement of D72 by asparagine or lysine increased the efficiency of electron transfer to photosystem I, but destabilized the protein. D72 apparently participates in electrostatic interactions that stabilize the structure of cyt c6. The destabilizing effect was reduced when aspartate was replaced by the small amino acid alanine. Complementing the mutation D72A with a charge neutralization or inversion at position K8 led to mutant forms of cyt c6 that were more stable than the wild‐type under all tested conditions.
Detection of nucleolar organizer and mitochondrial DNA insertion regions based on the isochore map of Arabidopsis thaliana
Authors: Chen Ling‐Ling , Gao Feng
Published: June 2005

Eukaryotic genomes are composed of isochores, i.e. long sequences relatively homogeneous in GC content. In this paper, the isochore structure of Arabidopsis thaliana genome has been studied using a windowless technique based on the Z curve method and intuitive curves are drawn for all the five chromosomes. Using these curves, we can calculate the GC content at any resolution, even at the base level. It is observed that all the five chromosomes are composed of several GC‐rich and AT‐rich regions alternatively. Usually, these regions, named ‘isochore‐like regions’, have large fluctuations in the GC content. Five isochores with little fluctuations are also observed. Detailed analyses have been performed for these isochores. A GC‐rich ‘isochore‐like region’ and a GC‐isochore in chromosome II and IV, respectively, are the nucleolar organizer regions (NORs), and genes located in the two regions prefer to use GC‐ending codons. Another GC‐isochore located in chromosome II is a mitochondrial DNA insertion region, the position and size of this region is precisely predicted by the current method. The amino acid usage and codon preference of genes in this organellar‐to‐nuclear transfer region show significant difference from other regions. Moreover, the centromeres are located in GC‐rich ‘isochore‐like regions’ in all the five chromosomes. The current method can provide a useful tool for analyzing whole genomic sequences of eukaryotes.
Identification of ATP‐NADH kinase isozymes and their contribution to supply of NADP(H) in Saccharomyces cerevisiae
Authors: Shi Feng , Kawai Shigeyuki , Mori Shigetarou , Kono Emi , Murata Kousaku
Published: June 2005

ATP‐NAD kinase phosphorylates NAD to produce NADP by using ATP, whereas ATP‐NADH kinase phosphorylates both NAD and NADH. Three NAD kinase homologues, namely, ATP‐NAD kinase (Utr1p), ATP‐NADH kinase (Pos5p) and function‐unknown Yel041wp (Yef1p), are found in the yeast Saccharomyces cerevisiae. In this study, Yef1p was identified as an ATP‐NADH kinase. The ATP‐NADH kinase activity of Utr1p was also confirmed. Thus, the three NAD kinase homologues were biochemically identified as ATP‐NADH kinases. The phenotypic analysis of the single, double and triple mutants, which was unexpectedly found to be viable, for UTR1, YEF1 and POS5 demonstrated the critical contribution of Pos5p to mitochondrial function and survival at 37 °C and the critical contribution of Utr1p to growth in low iron medium. The contributions of the other two enzymes were also demonstrated; however, these were observed only in the absence of the critical contributor, which was supported by complementation for some pos5 phenotypes by the overexpression of UTR1 and YEF1. The viability of the triple mutant suggested that a ‘novel’ enzyme, whose primary structure is different from those of all known NAD and NADH kinases, probably catalyses the formation of cytosolic NADP in S. cerevisiae. Finally, we found that LEU2 of Candida glabrata, encoding β‐isopropylmalate dehydrogenase and being used to construct the triple mutant, complemented some pos5 phenotypes; however, overexpression of LEU2 of S. cerevisiae did not. The complementation was putatively attributed to an ability of Leu2p of C. glabrata to use NADP as a coenzyme and to supply NADPH.
Differentiation stage‐dependent preferred uptake of basolateral (systemic) glutamine into Caco‐2 cells results in its accumulation in proteins with a role in cell–cell interaction
Authors: Lenaerts Kaatje , Mariman Edwin , Bouwman Freek , Renes Johan
Published: June 2005

Glutamine is an essential amino acid for enterocytes, especially in states of critical illness and injury. In several studies it has been speculated that the beneficial effects of glutamine are dependent on the route of supply (luminal or systemic). The aim of this study was to investigate the relevance of both routes of glutamine delivery to in vitro intestinal cells and to explore the molecular basis for proposed beneficial glutamine effects: (a) by determining the relative uptake of radiolabelled glutamine in Caco‐2 cells; (b) by assessing the effect of glutamine on the proteome of Caco‐2 cells using a 2D gel electrophoresis approach; and (c) by examining glutamine incorporation into cellular proteins using a new mass spectrometry‐based method with stable isotope labelled glutamine. Results of this study show that exogenous glutamine is taken up by Caco‐2 cells from both the apical and the basolateral side. Basolateral uptake consistently exceeds apical uptake and this phenomenon is more pronounced in 5‐day‐differentiated cells than in 15‐day‐differentiated cells. No effect of exogenous glutamine supply on the proteome was detected. However, we demonstrated that exogenous glutamine is incorporated into newly synthesized proteins and this occurred at a faster rate from basolateral glutamine, which is in line with the uptake rates. Interestingly, a large number of rapidly labelled proteins is involved in establishing cell–cell interactions. In this respect, our data may point to a molecular basis for observed beneficial effects of glutamine on intestinal cells and support results from studies with critically ill patients where parenteral glutamine supplementation is preferred over luminal supplementation.
Characterization of electrogenic bromosulfophthalein transport in carnation petal microsomes and its inhibition by antibodies against bilitranslocase
Authors: Passamonti Sabina , Cocolo Alessandra , Braidot Enrico , Petrussa Elisa , Peresson Carlo , Medic Nevenka , Macri Francesco , Vianello Angelo
Published: June 2005

Bilitranslocase is a rat liver plasma membrane carrier, displaying a high‐affinity binding site for bilirubin. It is competitively inhibited by grape anthocyanins, including aglycones and their mono‐ and di‐glycosylated derivatives. In plant cells, anthocyanins are synthesized in the cytoplasm and then translocated into the central vacuole, by mechanisms yet to be fully characterized. The aim of this work was to determine whether a homologue of rat liver bilitranslocase is expressed in carnation petals, where it might play a role in the membrane transport of anthocyanins. The bromosulfophthalein‐based assay of rat liver bilitranslocase transport activity was implemented in subcellular membrane fractions, leading to the identification of a bromosulfophthalein carrier (KM = 5.3 µm), which is competitively inhibited by cyanidine 3‐glucoside (Ki = 51.6 µm) and mainly noncompetitively by cyanidin (Ki = 88.3 µm). Two antisequence antibodies against bilitranslocase inhibited this carrier. In analogy to liver bilitranslocase, one antibody identified a bilirubin‐binding site (Kd = 1.7 nm) in the carnation carrier. The other antibody identified a high‐affinity binding site for cyanidine 3‐glucoside (Kd = 1.7 µm) on the carnation carrier only, and a high‐affinity bilirubin‐binding site (Kd = 0.33 nm) on the liver carrier only. Immunoblots showed a putative homologue of rat liver bilitranslocase in both plasma membrane and tonoplast fractions, isolated from carnation petals. Furthermore, only epidermal cells were immunolabelled in petal sections examined by microscopy. In conclusion, carnation petals express a homologue of rat liver bilitranslocase, with a putative function in the membrane transport of secondary metabolites.
Enzymatic properties of wild‐type and active site mutants of chitinase A fromVibrio carchariae, as revealed by HPLC‐MS
Authors: Suginta Wipa , Vongsuwan Archara , Songsiriritthigul Chomphunuch , Svasti Jisnuson , Prinz Heino
Published: June 2005

The enzymatic properties of chitinase A fromVibrio carchariaehave been studied in detail by using combined HPLC and electrospray MS. This approach allowed the separation of α and β anomers and the simultaneous monitoring of chitooligosaccharide products down to picomole levels. Chitinase A primarily generated β‐anomeric products, indicating that it catalyzed hydrolysis through a retaining mechanism. The enzyme exhibited endo characteristics, requiring a minimum of two glycosidic bonds for hydrolysis. The kinetics of hydrolysis revealed that chitinase A had greater affinity towards higherMrchitooligomers, in the order of (GlcNAc)6 > (GlcNAc)4 > (GlcNAc)3, and showed no activity towards (GlcNAc)2and pNP‐GlcNAc. This suggested that the binding site of chitinase A was probably composed of an array of six binding subsites. Point mutations were introduced into two active site residues – Glu315 and Asp392 – by site‐directed mutagenesis. The D392N mutant retained significant chitinase activity in the gel activity assay and showed ≈ 20% residual activity towards chitooligosaccharides and colloidal chitin in HPLC‐MS measurements. The complete loss of substrate utilization with the E315M and E315Q mutants suggested that Glu315 is an essential residue in enzyme catalysis. The recombinant wild‐type enzyme acted on chitooligosaccharides, releasing higher quantities of small oligomers, while the D392N mutant favored the formation of transient intermediates. Under standard hydrolytic conditions, all chitinases also exhibited transglycosylation activity towards chitooligosaccharides and pNP‐glycosides, yielding picomole quantities of synthesized chitooligomers. The D392N mutant displayed strikingly greater efficiency in oligosaccharide synthesis than the wild‐type enzyme.