bluuebunny/crossref_metadata_2025 huggingface dataset:
Sugar–starch combinations in food and the relationship to dental caries in low‐risk adolescents
Authors: Campain Adele Christine , Morgan Michael Vivian , Evans Robin Wendell , Ugoni Antony , Adams Geoffrey Grant , Conn Jennifer Ann , Watson Maxwell John
Published: July 2003

The aim of this 2‐year prospective cohort study was to determine whether food‐level sugar–starch combinations are predictors of dental caries in a low‐risk adolescent population. A total of 645 subjects, aged 12–13 yr at baseline, were recruited from 25 secondary colleges in the north‐west region of metropolitan Melbourne, Australia. Examinations to record dental caries status were conducted annually. Dental caries was diagnosed according to the criteria of the World Health Organization. Dietary information was collected by four, continuous 4‐d records. Demographic data was collected by parental self‐administered questionnaire. A total of 504 subjects provided complete information for analysis. Approximately 37% of subjects experienced an increment in caries. In the multivariate model, only the low sugar–high starch food group was a significant predictor of caries increment on all surfaces and pit and fissure surfaces. For both these surfaces, significant interactions with starch at low sugar and across those clusters with a maximum proportion of sugar and/or starch (that is, high sugar–low starch, medium sugar‐medium starch and high sugar–low starch) were found. Sugar–starch interactions may be predictive of caries risk in a low‐risk adolescent population. Changing patterns of food consumption and the widespread exposure to various fluoride vehicles are possibly altering the diet–dental caries dynamic that once existed.
The impact of stomatological disease on oral health‐related quality of life
Authors: Llewellyn Carrie Diane , Warnakulasuriya Saman
Published: July 2003

The clinical diagnosis of stomatological disease may indicate its cause and prognosis; however, it says little about the resulting level of impairment from the patient's perspective. The primary objective of this study was to test whether patients attending an outpatient oral medicine clinic would have worse oral health related quality of life (OHR‐QoL) compared with the general population. In addition, we aimed to assess whether anxiety or depression could be predicted by OHR‐QoL and to explore the relationship between clinical diagnoses, OHR‐QoL and anxiety/depression. Data were collected from patients (n = 97) through face‐to‐face interviews using the Oral Health Impact Profile Short form (OHIP‐14) to measure OHR‐QoL, the Hospital Anxiety and Depression Scale (HADS) for psychiatric morbidity, and a visual analogue scale for self‐rated general health. Age‐ and sex‐matched controls (n = 388) were provided from a normative data set collected in a UK national survey in 1998. Participants had significantly lower OHR‐QoL scores than the general population on all domains and overall OHR‐QoL scores. Of the variance in anxiety, 55% was predicted by general health ratings and OHR‐QoL domains of ‘psychological discomfort’ and ‘psychological disability’. Of the variance in depression, 54% was predicted by general health ratings and OHR‐QoL domains of ‘functional limitation’ and ‘social disability’. Patient centred, routine assessment of OHR‐QoL provides an additional dimension that may help to improve awareness of the impact of disease on the individual's life and enhance the clinical decision‐making process.
Protective effects of melatonin against myocardial injury induced by isoproterenol in rats
Authors: Acikel Mahmut , Buyukokuroglu Mehmet Emin , Aksoy Hulya , Erdogan Fazli , Erol Mustafa Kemal
Published: July 2003

Abstract: This study was performed to determine whether melatonin could have a protective effect against myocardial injury (MI) induced by isoproterenol (ISO) in rats. Twenty‐four rats were divided into three treatment groups: (1) control (n = 8): saline solution. (2) ISO (n = 8): ISO only. (3) melatonin + ISO (n = 8). Melatonin (10 mg/kg/day, i.p.) was administered 30 min before the initiation of ISO (150 mg/kg/day, s.c.). Drugs and saline were given at 14:00 hr for two consecutive days. At the end of the second day, blood samples were taken from the abdominal aorta shortly after the rats were anesthetized for the purpose of measuring cardiac troponins T (cTnT) and I (cTnI); hearts were removed, preserved and examined microscopically. Additionally, based on the histological changes in myocardial tissue, the rats were divided into three groups: no change, mild changes and moderate and/or marked changes. The mean cTnT and cTnI values were significantly increased in ISO group compared with control group [(1.29 ± 0.22 ng/mL versus 0.46 ± 0.07 ng/mL, P < 0.0001) and (0.56 ± 0.11 ng/mL versus 0.21 ± 0.01 ng/mL, P < 0.001)], respectively, and were significantly reduced in the ISO + melatonin group (0.65 ± 0.06 ng/mL for cTnT and 0.25 ± 0.01 ng/mL for cTnI) compared with the ISO only group (P < 0.01), respectively. cTnT and cTnI values were significantly increased in rats with moderate and/or marked cardiac changes compared with hearts where there were mild changes and no change (P < 0.05). ISO + melatonin group showed less histological changes than the ISO group (P < 0.01). In conclusion, this study revealed a protective effect of melatonin against ISO‐induced MI in rats, and its potential clinical application in the treatment of MI.
Orally administered melatonin reduces oxidative stress and proinflammatory cytokines induced by amyloid‐β peptide in rat brain: a comparative, in vivo study versus vitamin C and E
Authors: Rosales‐Corral Sergio , Tan Dun‐Xian , Reiter Russel J. , Valdivia‐Velázquez Miguel , Martínez‐Barboza Gabriela , Pablo Acosta‐Martínez J. , Ortiz Genaro G.
Published: July 2003

Abstract: To determine the efficacy of antioxidants in reducing amyloid‐β‐induced oxidative stress, and the neuroinflammatory response in the central nervous system (CNS) in vivo, three injections of fibrillar amyloid‐β (fAβ) or artificial cerebrospinal fluid (aCSF) into the CA1 region of the hippocampus of the rat were made. Concomitantly, one of the three free radical scavengers, i.e. melatonin, vitamin C, or vitamin E was also administered. Besides being a free radical scavenger, melatonin also has immunomodulatory functions. Antioxidant treatment reduced significantly oxidative stress and pro‐inflammatory cytokines. There were no marked differences between melatonin, vitamin C, and vitamin E regarding their capacity to reduce nitrites and lipoperoxides. However, melatonin exhibited a superior capacity to reduce the pro‐inflammatory response induced by fAβ.
Melatonin prevents free radical formation due to the interaction between β‐amyloid peptides and metal ions [Al(III), Zn(II), Cu(II), Mn(II), Fe(II)]
Authors: Zatta Paolo , Tognon Giuseppe , Carampin Paolo
Published: July 2003

Abstract: Alzheimer's disease, among other pathological features, is characterized by an over‐accumulation of amyloid‐β peptide, metal ions, and oxidative stress proteins in the brain. Amyloid‐β aggregated peptides with bound metal ions may initiate free radical generation with consequent protein and lipid oxidation, reactive oxygen species formation and eventually neuronal death. Melatonin is able to dramatically reduce the free radical formation which follows the interaction between transition metal ions and amyloid‐β. This paper reports the scavenging effect of melatonin of reactants generated by amyloid peptides in combination with some metal ions.
Amikacin‐induced acute renal injury in rats: protective role of melatonin
Authors: Parlakpinar Hakan , Ozer M. K. , Sahna Engin , Vardi Nigar , Cigremis Yilmaz , Acet Ahmet
Published: July 2003

Abstract: It is well established that some agents such as aminoglycosides generate free oxygen radicals, leading to an increased oxireductase production, which in turn increases tissue toxicity. The aim of this study is to test whether melatonin, the chief secretory product of the pineal gland and a highly effective antioxidant and free radical scavenger, reduces the nephrotoxicity caused by amikacin (AK). Herein, we investigated the physiologic and pharmacological role of melatonin in influencing AK‐induced nephrotoxicity. For this, pinealectomized (Px) and sham operated (non‐Px) rats were used. Both AK and melatonin were administered to all groups. We investigated the effects of melatonin on AK‐induced changes in levels of malondialdehyde (MDA), a lipid peroxidation product, glutathione (GSH), an antioxidant whose levels are influenced by oxidative stress, and blood urea nitrogen (BUN) and serum creatine (Cr) levels. Morphologic changes in the kidney were also examined by using light microscopy. MDA levels were found to be higher in Px than in non‐Px AK‐treated animals. Melatonin administration to Px rats reduced MDA levels. In relative to non‐Px rats, Px animals treated with AK had significantly lower GSH concentrations while melatonin administration elevated GSH levels in the kidney; however, this stimulatory effect of melatonin was not observed in non‐Px AK‐treated rats. Treatment with AK alone resulted in significantly higher plasma Cr and BUN levels. Repeated administration of melatonin prevented the AK‐induced elevation of plasma Cr and BUN levels. Morphologic damage to renal tubules as a result of AK was more severe in the renal cortex than in the medulla. The damage to the kidney induced by AK was reversed by melatonin in the Px rats. In conclusion, these results show that physiologic melatonin concentrations are important in reducing AK‐induced renal damage, while pharmacologic concentrations of melatonin did not add to the beneficial effect.
Temporal integration of melatonin infusion duration: signal averaging versus frequency dependence
Authors: Piatkowska Jolanta M. , Prendergast Brian J. , Gorman Michael R.
Published: July 2003

Abstract: Day length affects somatic and reproductive physiology of Siberian hamsters via regulation of the duration of nocturnal pineal melatonin secretion. Nightly ‘long’ (e.g. 12 hr) or ‘short’ (e.g. 6 hr) melatonin signals inhibit or stimulate gonadal growth, respectively. When long and short signals are presented in combination, however, neuroendocrine mechanisms exhibit a frequency‐dependent response, stimulating gonadal growth only if short signals are presented every second night or more frequently. The present experiments further assessed formal models for the temporal integration of melatonin signals changing abruptly in duration from night to night. Photo‐inhibited Siberian hamsters were housed in constant light and infused subcutaneously with various combinations of nightly short or long melatonin signals according to one of the several regimes that varied the frequency of short melatonin signal occurrence, average duration of the nightly melatonin signal, or both. Six weeks of nightly alternating short and long signals yielded different gonadal responses depending on the average melatonin signal duration. Moreover, when average melatonin signal duration was held constant between groups, gonadal stimulation was independent of the frequency of the constituent melatonin signals except when the duration of the short signal was reduced to 3 hr. Thus, neuroendocrine mechanisms do not solely categorize melatonin signals as either long or short but attend also to the duration of each component signal. In the majority (six of seven) of infusion regimes, reproductive responses to chimeric patterns of long and short melatonin signals were compatible with a simple signal‐averaging mechanism.
Melatonin protects against lipid peroxidation and membrane rigidity in erythrocytes from patients undergoing cardiopulmonary bypass surgery
Authors: Ochoa Julio J. , Vílchez María J. , Palacios Miguel A. , García Joaquín J. , Reiter Russel J. , Muñoz‐Hoyos Antonio
Published: July 2003

Abstract: The first aim of this study was to test whether there are changes in erythrocyte lipid peroxidation products and membrane fluidity during cardiac surgery involving cardiopulmonary bypass. Secondly, in vitro tests were performed to examine whether melatonin alters induced lipid peroxidation and reduced membrane fluidity in erythrocytes from these patients. Fifteen patients undergoing cardiac surgery involving cardiopulmonary bypass (CPB) were selected. Five blood samples were taken at different times during surgery for analysis of thiobarbituric acid reactive substances (TBARS) content and membrane fluidity of the erythrocytes. TBARS are an index of the level of lipid peroxidation. The results revealed an increase in TBARS levels and a parallel decrease in erythrocyte membrane fluidity (increased membrane rigidity) after the onset of CPB with respect to the reference sample. Likewise, in vitro induction of lipid peroxidation in the erythrocyte samples from CPB patients followed a similar pattern. The changes in TBARS levels and membrane fluidity were suppressed by pre‐incubation of erythrocytes with melatonin prior to the induction (by 70 μm Fe2+ + 250 μm ascorbate) of lipid peroxidation in a concentration‐dependent manner. The results constitute a persuasive argument for the use of melatonin for preventive and therapeutic purposes during CPB.
Oxidative injury to the locus coeruleus of rat brain: neuroprotection by melatonin
Authors: Chen Kuen‐Bor , Lin Anya Maan‐Yuh , Chiu Tsai‐Hsien
Published: July 2003

Abstract: Neurodegeneration in the locus coeruleus (LC) has been documented in several central nervous system (CNS) neurodegenerative diseases. In the present study, iron‐induced oxidative injury in the LC was investigated in chloral‐hydrate anesthetized rats. Three days after bilateral infusion of iron in the LC, both vertical and horizontal locomotor activities were decreased. Seven days after unilateral infusion of iron, lipid peroxidation was elevated in the infused LC, and the norepinephrine content was depleted in the ipsilateral hippocampus of the brain. Furthermore, the immunohistochemical study demonstrated a reduction in tyrosine hydroxylase‐positive neurons in the infused LC. The involvement of programmed cell death (apoptosis) in iron‐induced oxidative injury in the LC was investigated. Forty‐eight hours after iron infusion, cytosolic cytochrome c was elevated in the infused LC. Moreover, terminal deoxytransferase‐mediated dUTP‐nick end labeling (TUNEL)‐positive cells, an indicative of apoptosis, were detected in the infused LC. In an attempt to prevent oxidative injury in the LC, melatonin was systemically administered. Intraperitoneal injection of melatonin attenuated iron‐induced behavioral changes in locomotor activity as well as iron‐induced increases in cytosolic cytochrome c and TUNEL‐positive cells. Moreover, melatonin diminished iron‐induced oxidative injury. At the same time, the level of glial derived neurotrophic factor (GDNF) was elevated in the LC of melatonin‐treated rats. Our data suggests that oxidative stress because of iron results in apoptosis in the infused LC and causes degeneration of the coeruleohippocampal noradrenergic system in the rat brain. Furthermore, melatonin, among other mechanisms, may exert its neuroprotection via up‐regulation of GDNF levels in CNS.
Long‐term in vivo pineal microdialysis
Authors: Sun Xing , Liu Tiecheng , Deng Jie , Borjigin Jimo
Published: July 2003

Abstract:  This study describes the development of a new technique for long‐term measurement of daily 5‐hydroxytryptamine (5‐HT) and melatonin contents in the pineal gland of freely moving rats. The technique features a number of novel improvements over previous protocols. It allows visualization of the pineal gland for accurate targeting of the guide cannula, which minimizes bleeding; incurs no direct injury to the surrounding brain tissues; and causes no interference with the sympathetic innervation from the superior cervical ganglia. Robust releases of melatonin and indole precursors were continuously monitored quantitatively and reproducibly for more than 2 wk in the same animal. In addition, effects of pharmacological agents on in vivo pineal circadian rhythms can be studied reproducibly over time, and gene expression profiles can be correlated with physiological consequences in single animals. Using these approaches, it is found that β‐adrenergic activation leads to decreased release of 5‐HT, and that increased cAMP signaling in vivo results in activation of N‐acetyltransferase gene induction and melatonin production. These studies will enhance the understanding of signaling pathways that regulate pineal 5‐HT and melatonin synthesis and secretion.
Early neuropathological Alzheimer's changes in aged individuals are accompanied by decreased cerebrospinal fluid melatonin levels
Authors: Zhou Jiang‐Ning , Liu Rong‐Yu , Kamphorst Wouter , Hofman Michel A. , Swaab Dick F.
Published: July 2003

Abstract: Neuropathology is the most reliable criterion for diagnosing Alzheimer's disease (AD). A well‐established system for staging the spread of neuropathological changes in AD is available. The clinical use of a biomarker that reflects the neuropathological change occurring in brain tissue has not yet been established. Melatonin is a product that plays not only a major role in the regulation of the circadian rhythms but may also exert neuroprotective effects in AD. Melatonin levels were determined in ventricular postmortem cerebrospinal fluid (CSF) of 121 subjects. Braak staging and a modified Braak staging for cortex (MBSC) were used to evaluate the severity of AD neuropathology. The present study revealed that not only the Braak stages of AD, but also the MBSC were negatively correlated with CSF melatonin levels. By using MBSC, we now demonstrate for the first time that CSF melatonin levels were significantly decreased in the aged individuals with early neuropathological changes in the temporal cortex, where the AD process starts. Those individuals that did not have any neurofibrillary tangle (NFT) or neuritic plaque (NP) in the temporal cortex, had much higher melatonin levels (287 ± 68 and 280 ± 64 pg/mL, respectively) than those individuals that had a few NFTs and NPs (82 ± 4 and 39 ± 8 pg/mL, respectively) in the temporal cortex. These results suggest that the decrease in CSF melatonin levels may be an early event in the development of AD possibly occurring even before the clinical symptoms.
Oxidation of melatonin by singlet molecular oxygen (O2(1Δg)) produces N1‐acetyl‐N2‐formyl‐5‐methoxykynurenine
Authors: de Almeida Eduardo A. , Martinez Glaucia R. , Klitzke Clécio F. , de Medeiros Marisa H. G. , Mascio Paolo Di
Published: July 2003

Abstract: It has been shown that melatonin exhibits antioxidant properties. Chemical structures of some of the products formed by the interaction of melatonin with reactive oxygen and nitrogen species have been elucidated. Despite some evidence that the reaction of melatonin with singlet molecular oxygen (O2(1Δg)) produces N1‐acetyl‐N2‐formyl‐5‐methoxykynurenine (AFMK), it has not been fully documented. In this investigation, melatonin was oxidized by photosensitization with methylene blue or by a clean chemical source of O2(1Δg), the thermodecomposition of N,N′‐di(2,3‐dihydroxypropyl)‐1,4‐naphtalenedipropanamide (DHPNO2). The resulting product was characterized by high performance liquid chromatography, coupled to electrospray ionization mass spectrometry and also by 1H, 13C and dept135 nuclear magnetic resonance spectroscopy. An isotopically labeled DHPN18O2 was also prepared and used as a chemical source of labeled 18[O2(1Δg)] to unequivocally characterize the end product. The results uncovered by this work confirm the hypothesis that oxidation of melatonin by O2(1Δg) produces AFMK.
The Wieliczka Salt Mine
Authors: Hallett Don
Published: July 2003

Just a few kilometres south of Cracow, in southern Poland, the scenery changes abruptly from the fluvial plain of the Vistula river, mantled by tills and loess deposits of the Pleistocene glaciation, to a boldly undulating topography representing the outermost thrust sheet of the Carpathian mountains. These northern foothills of the Carpathians are known in Poland as the Beskid Mountains. The Carpathian thrust front can be traced from Upper Silesia through Cracow and Przemysl in southern Poland as far as the Ukraine and Romania. At several places along the thrust front, salt deposits have been found, which have been mined over many centuries. One of the best known mines is located at Wieliczka, 15 km south‐east of Cracow. The mine is a popular tourist location, but it also illustrates some spectacular geological features.
Multicentric adenomatoid tumors involving uterus, ovary, and appendix
Authors: Hanada Seiji , Okumura Yutaka , Kaida Kiyotaka
Published: July 2003

A case of large, and multicentric adenomatoid tumors involving the uterus, right ovary and appendix is described. The uterine tumor was exceptionally large, about 15 cm in maximal diameter, and located in the right fundal region of the uterus with a short, thick pedicle as in a subserous pedunculated uterine fibroid.
Risk of postnatal depression after emergency delivery
Authors: Koo Vincent , Lynch Janine , Cooper Stephen
Published: July 2003

Aim:  To identify whether women having emergency delivery are at increased risk of developing postnatal depression (PND).

Methods:  This is a retrospective comparative cohort study design. Two hundred and fifty Malaysian women were part of a previous study examining the prevalence of PND in a multiracial country and the effects of postnatal rituals. All women were at least 6 weeks post‐partum when asked to complete the Edinburgh Postnatal Depression Scale (EPDS). Sociodemographic and birth data were obtained.

Results:  Data collected were divided into two groups: 55 emergency delivery and 191 non‐emergency delivery. There were four missing data. There was no significant difference in the mean age, parity, gestational period, baby birthweight, 5 min baby Apgar score and EPDS scores of the two groups. However, the analysis of PND indicated that women with emergency delivery had a relative risk of 1.81 compared with women with non‐emergency delivery. The comparison of the two groups using χ2 indicated a significant (χ2= 3.94, d.f. = 1, P= 0.04) increase in the presence of PND in the emergency delivery.

Conclusion:  When compared with women having non‐emergency delivery, women having emergency delivery had about twice the risk of developing PND. Special attention to this group appears warranted.
The social impact of livestock: A developing country perspective
Authors: RIETHMULLER Paul
Published: July 2003

Agriculture in developing countries is influenced not only by available agricultural resources but also by a complex web of interactions based on social, economic, cultural and political factors. The relationship is two way – what happens in agricultural industries influences each of the factors just mentioned. This paper focuses on the social factors in relation to the livestock industries. Livestock are an increasingly important part of the agricultural sector of developing countries and social and cultural factors may be more important for livestock than for cropping industries.
Twenty‐eight new microsatellite loci in chicken and their cross‐species amplification in Japanese quail and helmeted guinea fowl
Authors: KAYANG Boniface Baboreka , INOUE‐MURAYAMA Miho , TAKAHASHI Hideaki , MINEZAWA Mitsuru , TSUDZUKI Masaoki , MIZUTANI Makoto , ITO Shin’ichi
Published: July 2003

Twenty‐eight original chicken microsatellite markers were isolated and characterized to determine their utility as cross‐reactive markers for comparative genetic mapping in the order Galliformes. Primer pairs were typed in 12 unrelated chickens and also tested on Japanese quail and helmeted guinea fowl deoxyribonucleic acid (DNA). Polymorphism was observed in 23 (82.1%) of the markers and the average number of alleles per locus was 2.9 while the mean heterozygosity was 0.19. Eleven (39.3%) of the chicken markers cross‐reacted with Japanese quail DNA and 2 (7.1%) with helmeted guinea fowl DNA. The cross‐reactive markers described would serve as useful resources for comparative genetic mapping in poultry species belonging to the order Galliformes.
Development of transferred xenogeneic vole embryos in mouse uteri
Authors: WIDAYATI Diah Tri , OHMORI Yasushige , WAKITA Tsuyoshi , FUKUTA Katsuhiro
Published: July 2003

An experimental model to study interspecific pregnancy using voles,Microtus arvalis, and green fluorescent protein gene‐induced transgenic mice is presented. Xenogeneic blastocysts from the vole were transferred into the uteri of pseudopregnant mice along with allogeneic blastocysts from green fluorescent protein gene‐induced transgenic mice. The uteri containing xeno‐allo combined transfers were examined from day 6 to 13 of gestation. Although the vole embryos implanted, the uteri containing vole embryos were smaller compared with those having allogeneic mouse embryos. On day 8, the uteri containing vole embryos hemorrhaged internally and no vole embryo was found in the pregnant uterus after day 11. Allogeneic mouse embryos developed normally despite the presence and abortion of the vole embryos. In uteri implanted with vole embryos, decidua were formed and numerous blood vessels were distributed around the embryo. Maternal blood cells infiltrated into the celomic cavity of the vole embryo through the discontinuous region of trophoblast. Periodic acid‐Schiff‐positive granulated metrial gland cells were remarkably increased in the decidual sites. These findings suggest that a disorder of embryo–maternal interaction might induce the appearance of numerous granulated metrial gland cells and rejection of the embryos.
Caprine somatic cell nuclear transfer using in vivo matured oocytes collected by laparoscopic follicular aspiration
Authors: OHKOSHI Katsuhiro , TAKAHASHI Seiya , KOYAMA Shin‐ichiro , AKAGI Satoshi , ADACHI Noritaka , FURUSAWA Tadashi , FUJIMOTO Jun‐ichiro , IZAIKE Yoshiaki , TOKUNAGA Tomoyuki
Published: July 2003

In vivo matured oocytes collected by laparoscopic follicular aspiration (LFA) from hormone treated female goats were used as recipient ooplasts for somatic cell nuclear transfer (SCNT). Japanese native (Shiba) goats were used as donor females and some donor females were used repeatedly (two or three times) at intervals of a few months. To induce synchronization of estrus, a sponge containing 0.5 g of progesterone was inserted into the vagina of each goat for 14 days. These animals were also treated with follicle stimulating hormone (FSH) in a series of 8 injections over 4 days. The first FSH injection was administered on the morning of day 9 of sponge insertion. On the morning of day 13, 50 µg of gonadotropin‐releasing hormone (GnRH) was injected into each animal. Twenty‐nine hours after GnRH injection, LFA was performed. After removal of cumulus cells, collected oocytes with the first polar body were selected and enucleated for nuclear transfer. Anterior pituitary cells isolated from an adult male Shiba goat were transfected with a DNA fragment containing the enhanced green flourescent protein gene and the puromycin resistance gene. A single donor cell was inserted into the perivitelline space of each enucleated oocyte and fusion was induced with one electric pulse of 20 V for 10 µs. The SCNT goat eggs were cultured in chemically defined medium at 38.5°C in 5% CO2, 5% O2, 90% N2 for 9 days. By LFA, 396 oocytes were collected from a total of 30 females. After removal of cumulus cells, 64% of them extruded the first polar body. The percentage of SCNT goat eggs produced using in vivo matured oocytes which developed to the blastocyst stage (20–21%) was significantly higher (P < 0.05) than that produced with in vitro matured oocytes (3–8%).
Molecular cloning of insulin‐like growth factor‐I complimentary DNA and promoter region in the domestic duck (Anas platyrhynchos)*
Authors: KANSAKU Norio , YAGI Eriko , NAKADA Asako , OKABAYASHI Hisato , GUEMENE Daniel , ZADWORNY David
Published: July 2003

Complementary DNA (cDNA) and the flanking region of insulin‐like growth factor‐I (IGF‐I) of domesticated duck were cloned. The nucleotide sequence analysis of the cDNA showed seven and eight bases different, respectively, from chicken and turkey IGF‐I cDNA within the coding region. The amino acid sequence of prepro IGF‐I differed by one and two amino acids from those observed in chicken and turkey, respectively. However, no amino acid substitution was observed in the mature IGF‐I region. Sequence analysis of the promoter region and exon 1 of the duck IGF‐I revealed a high degree of similarity to that of the chicken IGF‐I gene. These results suggest that the mechanisms which regulate expression of the IGF‐I gene may be widely conserved in avian species.
Synthesis of citrulline from ornithine by the small intestinal mucosa of cattle
Authors: SULTANA Halima , KITANO Ayumi , WADUD Shaila , TAKAHASHI Toshihiro , MORITA Tetsuo , ONODERA Ryoji
Published: July 2003

Three segments of cattle small intestine (duodenum, upper jejuno‐ileum and lower jejuno‐ileum) were examined in an in vitro system for activity of ornithine carbamoyltransferase (OCT; EC 2.1.3.3) which is involved in the synthesis of citrulline (Cit) from ornithine (Orn). The mucosa of the three segments of small intestine was collected from Japanese black cattle, homogenized and then centrifuged. The supernatant fraction was used as the crude OCT enzyme solution. The OCT activity was assayed by the production of Cit from Orn determined directly by HPLC. The optimal pH and temperature for OCT activities in the duodenum, upper jejuno‐ileum and lower jejuno‐ileum of cattle small intestine were 7.47 and 39°C. Little difference was observed between the three segments. The OCT activity in cattle kidney was also examined for comparison, and almost no activity was found. The OCT activities in crude enzyme solutions of the three segments of small intestine were stable for up to one month of storage at −20°C in Tris HCl buffer solution. Finally, the role of the small intestine in supplying Cit as a precursor for arginine synthesis in cattle kidney was discussed.
A quantitative study on arginine synthesis from argininosuccinic acid and citrulline by crude enzymes of cattle kidney
Authors: SULTANA Halima , INADA Mikiko , WADUD Shaila , TAKAHASHI Toshihiro , MORITA Tetsuo , ONODERA Ryoji
Published: July 2003

In vitro experiments were conducted to examine the synthesis of arginine (Arg) from argininosuccinic acid (ASA) and citrulline (Cit) by crude enzymes of cattle kidney cortex. Kidney samples, collected from Japanese black cattle, were homogenized in KCl solution (ice‐cold), and centrifuged at 27 000 × g for 20 min at 4°C, and the supernatant fluid was used as a crude enzyme solution. The enzyme solution was incubated at 39°C in Tris HCl buffer with 15 mmol/L ASA or with 10 mmol/L Cit in the presence of 10 mmol/L aspartic acid (Asp), 10 mmol/L ATP and 5 mmol/L MgCl2 to examine the activities of two enzymes, argininosuccinate lyase and argininosuccinate synthetase, which work at the terminal steps of Arg biosynthesis. The production of Arg from ASA, or ASA and Arg from Cit by argininosuccinate lyase and argininosuccinate synthetase activities, respectively, were determined directly by the HPLC method. The optimum pH for argininosuccinate lyase activity was 7.85. Unfortunately, the optimum pH for argininosuccinate synthetase activity could not be determined because no inhibitor of argininosuccinate lyase was used in the Cit incubation, so the ASA produced from Cit spontaneously converted to Arg during incubation with Cit. The maximum production of ASA from Cit was found at pH 6.45 under these conditions. We observed the optimum pH for the synthesis of Arg from Cit at 7.7. The production of Arg from ASA or Cit was quantitatively determined as 14.4 or 8.83 µmol/g kidney tissue/h, respectively, at the optimal pH values. This suggests that the daily production of Arg from ASA or Cit by the kidney might be sufficient to cover the daily requirement of Arg in cattle.
In vitro metabolism of tyrosine by rumen bacteria, protozoa and their mixture
Authors: KHAN Rokibul Islam , ANDO Sada , TAKAHASHI Toshihiro , MORITA Tetsuo , ONODERA Ryoji
Published: July 2003

An anaerobic in vitro study of tyrosine (Tyr) metabolism by rumen bacteria (B), protozoa (P) and their mixture (BP) was conducted using an HPLC analytical method to obtain some basic and systematic information on aromatic amino acid metabolism in the rumen. Most of the Tyr (> 96%) disappeared after 12 h incubation in B and BP, but only 58% in P. Tyr was converted mainly to p‐hydroxyphenylacetic acid (HPA) in all microbial suspensions. About 45% of disappeared Tyr in B and P, and about 35% in BP were converted to HPA. An appreciable amount of phenylalanine (Phe), 13 and 3% of disappeared Tyr, and a small amount of tryptophan (Trp), 8 and 1% of disappeared Tyr, were also produced from Tyr by rumen bacteria and protozoa, respectively. Small amounts of p‐hydroxyphenylpyruvic acid (about 4 and 6% of disappeared Tyr) were produced from Tyr in B and P, respectively. A moderately large amount of phenylacetic acid (14% of disappeared Tyr) was produced from Tyr in P which was 1.9 times higher than that in B. Phenylpropionic acid and p‐hydroxybenzoic acid were produced only in B and BP. It was concluded that the Tyr degradation ability of rumen bacteria was about 1.5 times higher than that of rumen protozoa. Degraded Tyr mainly produces HPA and then two other aromatic amino acids, Phe and Trp, which are considered essential amino acids for ruminants. Therefore, it is speculated that the requirement for Phe and Trp in ruminants may be partially fulfilled if Tyr is sufficiently supplied in rations.
Effects of supplemental protein sources during the grazing period on performance, ruminal characteristics and blood constituents in early weaned growing Wagyu steer calves
Authors: SUGIMOTO Masahito , YAYOTA Cizuru , KUZUOKA Syuji , SATO Yukinobu
Published: July 2003

We investigated the effects of grazing and supplemental protein sources during the grazing period on growth performance, ruminal characteristics and blood constituents in 15 growing Wagyu steer calves weaned at 3 months of age. Each animal was allotted one of three treatments: (1) GSBM, soybean meal (SBM) supplemented as a protein source during a grazing period; (2) GCGM, corn gluten meal (CGM) supplemented as a protein source during a grazing period; or (3) NG, no grazing, in which steers were fed hay and GSBM concentrate only. The estimated rumen undegradable protein (RUP) concentration of CGM was higher than that of SBM. High RUP supplementation during the grazing period did not influence feed intake or growth performance. Compared with the NG treatment, the average daily gain in GSBM and GCGM calves was significantly depressed (P < 0.05). Bodyweight/withers height ratio in the NG calves tended to be greater than in the GSBM and GCGM calves. Final paunch girth/heart girth ratio in the GSBM and GCGM calves was significantly greater (P < 0.05) than in the NG calves. Supplementation with CGM did not influence ruminal ammonia nitrogen and blood constituents. These results indicate that CGM supplementation during the grazing period did not enhance the growth of Wagyu calves because the CGM diet probably did not elevate the metabolizable protein supply to the grazing calves above that of the SBM diet in our conditions.
Production of aromatic amino acids and related compounds from p‐hydroxyphenylacetic acid by rumen microorganisms in vitro
Authors: KHAN Rokibul Islam , ANDO Sada , TAKAHASHI Toshihiro , MORITA Tetsuo , ONODERA Ryoji
Published: July 2003

Suspensions of mixed rumen bacteria (B), protozoa (P), and mixed rumen microorganisms (BP) prepared from rumen contents of fistulated goats were anaerobically incubated with 1 mM p‐hydroxyphenylacetic acid (HPA) at 39°C for 24 h. Tyrosine (Tyr), phenylalanine (Phe), tryptophan (Trp) and other related compounds in both supernatants and hydrolyzates of microbial cells in all incubations were analyzed by HPLC. Large amounts of Tyr (32.1, 42.7 and 36.1% of disappeared HPA in B, P and BP, respectively) were produced from HPA during a 12 h incubation period. The formation of Tyr in P (178.6 µmol/g MN) was 1.5 and 2 times higher than in B and BP, respectively. Phe (7–11% of the disappeared HPA) and Trp (3–6% of the disappeared HPA) were also synthesized from HPA in B, P, and BP. Phe synthesis in P (46.3 µmol/g MN) was 1.7 times higher than in B but, in contrast, Trp synthesis in B, was 1.6 times higher than in P. The metabolites p‐hydroxyphenylpyruvic acid (in the range of 5–14% of disappeared HPA), phenylacetic acid (1–11%), p‐hydroxybenzoic acid (3–7%) and benzoic acid (1–6%) were produced from HPA in B, P and BP. Phenylpropionic acid (6% of the disappeared HPA) was produced only in B and BP.
Effect of α‐cyclodextrin‐allyl isothiocyanate on ruminal microbial methane production in vitro
Authors: LILA Zeenat Ara , MOHAMMED Nazimuddin , KANDA Shuhei , KAMADA Toshihiko , ITABASHI Hisao
Published: July 2003

The objective of the present study was to investigate the effects of α‐cyclodextrin‐allyl isothiocyanate (CD‐AI) on ruminal microbial methane production and rumen fermentation of corn starch, soluble potato starch or hay plus concentrate (1.5:1) by mixed rumen microorganisms. Diluted rumen fluid (30 mL) was incubated anaerobically at 38°C for 6 and 24 h with or without CD‐AI (0, 0.4, 0.8, 1.6 and 3.2 g/L). The pH of the medium was unchanged by CD‐AI in all substrates. The molar proportion of acetate was decreased and propionate was increased with a corresponding decrease in acetate : propionate ratio (P < 0.05). Total volatile fatty acids and butyrate were increased (P < 0.05). Ammonia‐N was decreased (P < 0.05). Except with soluble potato starch, numbers of protozoa were unchanged after 6 h. As concentration of CD‐AI increased from 0 to 3.2 g/L, fermentation of corn starch, soluble potato starch and hay plus concentrate resulted in decreased (P < 0.05) methane production of 49–100% (6 h) and 14–100% (24 h); 39–100% (6 h) and 16–100% (24 h); and 45–100% (6 h) and 17–100% (24 h), respectively. When hay plus concentrate was used as substrate, methanogenic bacteria were decreased (P < 0.05) with 0.8 g/L of CD‐AI after 6 h. Excluding the lower dose level (0.4 g/L) of CD‐AI, digestibility of neutral detergent fiber of hay plus concentrate was decreased (P < 0.05) after 24 h. A suitable level of CD‐AI could therefore be used as a supplement to inhibit methane production and improve rumen fermentation without detrimental effects on fiber digestion.
Once‐weekly epoetin beta is highly effective in treating anaemic patients with lymphoproliferative malignancy and defective endogenous erythropoietin production
Authors: Cazzola Mario , Beguin Yves , Kloczko Janusz , Spicka Ivan , Coiffier Bertrand
Published: July 2003

Summary. Epoetin beta, three‐times weekly (t.i.w.), is effective in reversing anaemia in lymphoproliferative disorders. The current study investigated whether an epoetin beta dose of 30 000 IU given subcutaneously once weekly (q.w.) was at least as effective as 10 000 t.i.w. administration in anaemic patients with lymphoproliferative malignancy and defective endogenous erythropoietin (Epo) production. Overall, 241 anaemic patients with multiple myeloma, low‐grade non‐Hodgkin's lymphoma or chronic lymphocytic leukaemia, all with serum Epo values ≤ 100 mU/ml, were randomized to receive the q.w. (n = 119) or t.i.w. (n = 122) regimen for 16 weeks. The primary efficacy criterion, i.e. the time‐adjusted area under the haemoglobin–time curve from weeks 5–16, was comparable between the q.w. and t.i.w. groups [difference = − 0·20 g/dl (90% confidence interval − 0·52–0·11)]. Moreover, response rates were high and similar in both arms (72%vs 75%, q.w. and t.i.w. groups respectively). Baseline serum Epo was predictive of response: the lower serum Epo, the higher the likelihood of response (P = 0·002). Thus, epoetin beta administered q.w. is an effective and convenient treatment for anaemia in patients with lymphoproliferative disorders. Tailoring this treatment modality to subjects with defective endogenous Epo production represents a rational use of epoetin from both a medical and a community perspective.
Fidelity and reproducibility of antisense RNA amplification for the study of gene expression in human CD34+ haemopoietic stem and progenitor cells
Authors: Attia Mohamed A. , Welsh Jonathan P. , Laing Kenneth , Butcher Philip D. , Gibson Frances M. , Rutherford Tim R.
Published: July 2003

Summary. Microarrays provide a powerful tool for the study of haemopoietic stem and progenitor cells (HSC). Because of the low frequency of HSC, it is rarely feasible to obtain enough mRNA for microarray hybridizations, and amplification will be necessary. Antisense RNA (aRNA) amplification is reported to give high‐fidelity amplification, but most studies have used only qualitative validation. Before applying aRNA amplification to the study of HSC, we wished to determine its fidelity and reproducibility, and whether statistically significant results can be obtained. We found that aRNA amplification introduced biases into relative RNA abundance. However, these biases were extremely consistent, and valid comparisons could be made, if amplified RNA was compared with amplified RNA. By applying this method to the effect of interferon‐γ and tumour necrosis factor‐α on normal primary CD34+ HSC, biologically significant differences could be detected, including potential mechanisms for resistance of CD34+ cells to CD95‐mediated apoptosis and evidence of the differentiating effects of the cytokines. Differences of twofold or less were detected, and most of these differences attained statistical significance after triplicate experiments. These data demonstrate that aRNA amplification can be used with microarray hybridization to study the transcriptional profiles of small numbers of primary CD34+ HSC.
Increased in vitro cellular drug resistance is related to poor outcome in high‐risk childhood acute lymphoblastic leukaemia
Authors: Frost Britt‐Marie , Nygren Peter , Gustafsson Göran , Forestier Erik , Jonsson Olafur G. , Kanerva Jukka , Nygaard Randi , Schmiegelow Kjeld , Larsson Rolf , Lönnerholm Gudmar ,   On behalf of the Nordic Society for Paediatric Haematology and Oncology
Published: July 2003

Summary. We determined the in vitro cellular drug resistance in 370 children with newly diagnosed acute lymphoblastic leukaemia (ALL). The resistance to each of 10 drugs was measured by the fluorometric microculture cytotoxicity assay (FMCA) and was related to clinical outcome. The median follow‐up time was 41 months. Risk‐group stratified analyses indicated that in vitro resistance to dexamethasone, doxorubicin and amsacrine were each significantly related to the probability of disease‐free survival. In the high‐risk (HR) group, increased in vitro resistance to dexamethasone (P = 0·014), etoposide (P = 0·025) and doxorubicin (P = 0·05) was associated with a worse clinical outcome. Combining the results for these drugs provided a drug resistance score with an independent prognostic significance superior to that of any other factor studied, with a relative risk of relapse in the most resistant group 9·8 times that in the most sensitive group (P = 0·007). The results in the intermediate‐risk (IR) and standard‐risk (SR) groups were less clear cut. In conclusion, our data indicate that in vitro testing of cellular drug resistance can be used to predict the clinical outcome in HR ALL, while the final evaluation of the results in IR and SR patients must await longer follow‐up.
Activated protein C resistance determined with a thrombin generation‐based test predicts for venous thrombosis in men and women
Authors: Tans Guido , Van Hylckama Vlieg Astrid , Thomassen M. Christella L. G. D. , Curvers Joyce , Bertina Rogier M. , Rosing Jan , Rosendaal Frits R.
Published: July 2003

Summary. Activated protein C (APC) resistance, determined with a thrombin‐generation‐based APC resistance test, may explain risk differences of venous thrombosis in users of second‐ and third‐generation oral contraceptives (OC). To clinically validate this test, we analysed the Leiden thrombophilia case–control study (474 patients with a first episode of deep vein thrombosis and 474 age‐ and sex‐matched control subjects). Data for men and women were analysed separately. As hormonal status in women is known to strongly influence the APC sensitivity ratio (APCsr), additional strata (OC use and menopausal state) were defined. The APCsr was higher in all patients than in control subjects. Odds ratios (OR), using the 90th percentile of all control subjects (APCsr > 4·5) as cut‐off, were: 7·5 [95% confidence interval (CI) 1·6–33·8] for men, 3·0 (95% CI 1·0–8·8) for premenopausal women not using OC, 4·8 (95% CI 1·6–14·7) for premenopausal women using OC and 4·7 (95% CI 1·4–15·6) for postmenopausal women. After excluding the carriers of factor V Leiden, the OR became infinite for men (no control had an APCsr > 4·5), 1·4 (95% CI 0·2–8·2) for premenopausal women not using OC, 3·4 (95% CI 1·1–10·8) for premenopausal women using OC and 3·6 (95% CI 0·6–20·5) for postmenopausal women. A high APCsr, determined with the thrombin‐generation‐based APC resistance test, predicts venous thrombotic risk, in populations with and without factor V Leiden. In addition, acquired APC resistance resulting from OC use predicts an increased risk for venous thrombosis independent of factor V Leiden.
Basic fibroblast growth factor‐stimulated ex vivo expansion of haematopoietic progenitor cells from human placental and umbilical cord blood
Authors: Kashiwakura Ikuo , Takahashi Tsuneo A.
Published: July 2003

Summary. We investigated whether basic fibroblast growth factor (bFGF) is effective in inducing ex vivo expansion of CD34+ haematopoietic progenitor cells derived from human placental and umbilical cord blood. bFGF significantly promoted the clonal growth of various haematopoietic progenitor cells, including granulocyte–macrophage colony‐forming units (CFU‐GM), mixed colony‐forming units (CFU‐Mix) and megakaryocyte colony‐forming units (CFU‐Meg) under semisolid culture conditions, with an optimal bFGF concentration of 30 ng/ml. CD34+ cells were then cultured in serum‐free liquid medium containing various combinations of early‐acting cytokines, including thrombopoietin (TPO), stem cell factor (SCF), interleukin 3 (IL‐3) and flt3‐ligand (FL), with or without bFGF, for 6 and 12 d. Without bFGF, TPO + IL‐3, TPO + SCF + FL and TPO +SCF + IL‐3 + FL dramatically increased the total numbers of erythroid progenitors, CFU‐GM and CFU‐Mix by d 12 of culture respectively. However, the addition of bFGF did not promote further proliferation of these progenitors, except for the erythroid progenitors, by d 6 when stimulated with all four cytokines. In contrast, total CFU‐Meg numbers were approximately doubled when these cultures were supplemented with bFGF, producing 100‐ to 120‐fold increases compared with the baseline control cultures. These results suggest that bFGF is effective in supporting the generation of megakaryocytic progenitor cells during ex vivo expansion.