bluuebunny/crossref_metadata_2025 huggingface dataset:
Proceedings of the Australian Physiological and Pharmacological Society Symposium: The Hypothalamus DISTRIBUTION, REGULATION AND ROLE OF HYPOTHALAMIC GALANIN SYSTEMS: RENEWED INTEREST IN A PLEIOTROPIC PEPTIDE FAMILY
Authors: Gundlach Andrew L , Burazin Tanya Cd , Larm Jari A
Published: March 2003

1. Galanin peptide and galanin receptor‐binding sites are known to be widely distributed within the central nervous system, particularly in the hypothalamus in the preoptic area, the paraventricular (PVN) and supraoptic (SON) nuclei and the arcuate nucleus/median eminence.

2. The present brief review focuses on some recent studies of the regional and cellular localization of mRNA encoding galanin and two galanin receptor subtypes (GalR1 and GalR2) in the hypothalamus, regulation of galanin and/or galanin receptor expression in various nuclei by physiological stimuli, electrophysiological effects of galanin on hypothalamic neurons and the isolation and cloning of galanin‐like peptide (GALP), a putative endogenous ligand for GalR2.

3. In situ hybridization studies in rat brain have demonstrated an abundance of GalR1 mRNA in SON, magnocellular (m) and parvocellular (p) PVN and dorsomedial, ventromedial and arcuate nuclei. In contrast, GalR2 mRNA is enriched in pPVN, but not mPVN, and is not detected in SON. In addition, GalR2 mRNA is present in the dorsomedial nucleus and is enriched in the arcuate nucleus compared with GalR1 transcripts, with numerous labelled cells in all subdivisions.

4. Neurons of the SON and PVN contain vasopressin and/or oxytocin, along with several other peptides, and the production and release of these hormones and peptides are modulated by various physiological stimuli. In relation to galanin systems, GalR1 and galanin expression is increased in magnocellular neurons by salt loading and is downregulated by lactation, consistent with an increased inhibition by galanin of vasopressin release following osmotic stimulation and a decreased inhibition of oxytocin release during lactation.

5. Powerful inhibitory effects of galanin on the electrical (and secretory) activity of magnocellular neurons and complex presynaptic actions of galanin on the synaptic release of glutamate in the arcuate nucleus in vitro suggest an active role for multiple galanin receptor subtypes in the regulation of these hypothalamic systems in vivo.

6. The recent isolation of a peptide from porcine hypothalamus (GALP‐1–60) that is structurally related to galanin and appears to be selective for GalR2 over GalR1 and the subsequent cloning of GALP cDNA from pig, rat and humans should allow studies to help reveal the physiological role played by galanin receptor subtypes (especially GalR2) and their multiple ligands in the hypothalamus and other brain areas.
Changes In Endogenous Monoamines In Aged Rats
Authors: Lee Jj , Chang Ck , Liu Im , Chi Tc , Yu Hj , Cheng Jt
Published: March 2003

1. It has been documented that ageing may alter endogenous neurotransmitters. However, these results are controversial. Thus, in the present study, cerebral cortex and plasma from male Wistar rats aged 8 weeks and 6, 12 or 24 months were used to investigate the changes in monoamines using electrochemical detection.

2. A marked decrease in L‐dihydroxyphenylalanine (L‐DOPA) was observed in aged rats. Like the decrease in dopamine (DA), levels of 5‐hydroxytryptamine (5‐HT) and 5‐hydroxyindolacetic acid (5‐HIAA), the major metabolite of 5‐HT, in aged rats were decreased in the cerebral cortex and plasma. Plasma levels of noradrenaline and levels of adrenaline in the cerebral cortex were also decreased in aged rats. Moreover, levels of 3,4‐dihydroxyphenylacetic acid (DOPAC), a metabolite of DA, in the cerebral cortex and plasma were reduced by ageing. The level of homovanillic acid (HVA) in all samples was markedly increased with ageing.

3. The ratio of DOPAC/DA and 5‐HIAA/5‐HT, being closely linked with the activity of monoamine oxidase, was increased in the cerebral cortex and plasma with ageing. The ratio of HVA/DOPAC, an index of the activity of catechol‐O‐methyltransferase, was also higher in the cerebral cortex and plasma of aged rats.

4. These data suggest that ageing may alter endogenous monoamines in both the brain and peripheral tissues.
Role Of Calcitonin Gene‐Related Peptide In Ischaemic Preconditioning In Diabetic Rat Hearts
Authors: Lu Rong , Hu Chang‐Ping , Peng Jun , Deng Han‐Wu , Li Yuan‐Jian
Published: March 2003

1. It has been suggested that calcitonin gene‐related peptide (CGRP) is involved in the protection provided by ischaemic preconditioning in rat hearts and that ischaemic preconditioning is absent in diabetic rat hearts.

2. In the present study, we tested the relationship between sensory nerve function and ischaemic preconditioning in diabetic rats.

3. In 4‐ and 8‐week diabetic rats and age‐matched non‐ diabetic controls, 30 min global ischaemia and 40 min reperfusion caused a significant decrease in cardiac function and a marked increase in creatine kinase (CK) release. Ischaemic preconditioning, by three cycles of 5 min ischaemia and 5 min reperfusion, improved the recovery of cardiac function and decreased CK release during reperfusion in 4‐week diabetic rat hearts. However, the cardioprotection afforded by ischaemic preconditioning was lost in 8‐week diabetic rat hearts. Pretreatment with CGRP for 5 min also significantly improved the recovery of cardiac function and decreased CK release in rats subjected to 4 or 8 weeks of diabetes.

4. The content of CGRP in the coronary effluent during ischaemic preconditioning was significantly increased in 4‐week diabetic rat hearts (P < 0.05). However, only a slight increase in the release of CGRP was shown in 8‐week diabetic rat hearts (P > 0.05).

5. In summary, the present results suggest that the protection afforded by ischaemic preconditioning is attenuated in diabetic rats and that the change may be related to the reduction in CGRP release in diabetic rat hearts.
John Ludbrook APPS Symposium Neural Mechanisms In The Cardiovascular Responses To Acute Central Hypovolaemia
Authors: Evans Roger G , Ventura Sabatino , Dampney Roger Al , Ludbrook John
Published: March 2003

1. The haemodynamic response to acute central hypovolaemia consists of two phases. During phase I, arterial pressure is well maintained in the face of falling cardiac output (CO) by baroreceptor‐mediated reflex vasoconstriction and cardio‐acceleration. Phase II commences once CO has fallen to a critical level of 50–60% of its resting value, equivalent to loss of approximately 30% of blood volume.

2. During phase II, sympathetic vasoconstrictor and cardiac drive fall abruptly and cardiac vagal drive increases. In humans, this response is invariably associated with fainting and has been termed vasovagal syncope.

3. In both experimental animals and in humans, the responses to acute central hypovolaemia are greatly affected by anaesthetic agents, in that the compensatory responses during phase I (e.g. halothane) or their failure during phase II (e.g. alfentanil) are blunted or abolished.

4. Therefore, our present knowledge of the neurochemical basis of the response to hypovolaemia depends chiefly on the results of experiments in conscious animals. Use of techniques for simulating haemorrhage has greatly enhanced this research effort, by allowing the effects of multiple treatments on the response to acute central hypovolaemia to be tested in the same animal.

5. The results of such experiments indicate that phase II of the response to hypovolaemia is triggered, at least in part, by a signal from cardiac vagal afferents. There is also strong evidence that phase II depends on brainstem δ1‐opioid receptor and nitrergic mechanisms and can potentially be modulated by circulating or neuronally released adrenocorticotropic hormone, brainstem serotonergic pathways operating through 5‐HT1A receptors and opioids acting through μ‐ and κ‐opioid receptors in the brainstem.

6. Phase II also appears to require input from supramedullary brain centres. Future studies should determine how these neurotransmitter systems interact and their precise neuroanatomical arrangements.
Organs/Systems Potentially Involved In One Model Of Programmed Hypertension In Sheep
Authors: Dodic Miodrag , Baird Rebecca , Hantzis Vicky , Koukoulas Irene , Moritz Karen , Peers Arianne , Wintour E Marelyn
Published: March 2003

1. When pregnant ewes and their fetuses are exposed to the synthetic glucocorticoid dexamethasone for 2 days early in pregnancy (days 26–28; term 145–150 days), female offspring have increased blood pressure relative to a control group. In one series, this was shown to be due to increased cardiac output, concomitant with a reset mean arterial pressure/heart rate reflex. The first group of such animals had, by the age of 7 years, left ventricular hypertrophy and reduced cardiac functional capacity.

2. The elevation in blood pressure is not maintained by any change in the peripheral renin–angiotensin system (RAS).

3. There is, however, preliminary evidence that some aspects of local RAS (particularly in the kidney and brain) could have participated in the ‘programming’ event. The levels of mRNA for angiotensin II receptors (AT1, AT2) and angiotensinogen are increased in the kidney of such dexamethasone‐treated fetuses in late gestation (130 days), some 100 days after steroid treatment. Similar increases in AT1 mRNA in the medulla oblongata of the fetal brain and large increases of mRNA for angiotensinogen occur in the hypothalamus.

4. These findings, together with evidence from the literature, suggest that both the kidney and parts of the brain are affected by events that also ‘program’ high blood pressure in the offspring of animals in which the intra‐uterine environment has been perturbed at some stage.
Fetal Origins of Adult Disease Fetal Origins Of Adult Disease?
Authors: Morley Ruth , Dwyer Terence
Published: March 2003

1. Associations between lower birthweight and higher blood pressure, increased risk of type 2 diabetes and coronary heart disease (CHD) have been observed in a number of different populations worldwide.

2. The reason for this is still debated. Some believe that the observed associations can be explained on the basis of differences in postnatal growth, socioeconomic confounding or genetic factors. Two published studies of birthweight and CHD, with information on later size, suggest that both gestational and postnatal exposures are important. Associations between birthweight and blood pressure, seen in cohorts of twins treated as individuals, have generally remained when data are analysed within twin pairs. Furthermore, similar associations are seen in studies of animals with relative genetic homogeneity kept in standard conditions. These findings suggest that neither socioeconomic nor genetic factors are wholly responsible for the observed associations.

3. If then, there is an underlying causal association, two issues are of fundamental importance. First, is fetal growth (for which birthweight is a summary measure) involved in the causal pathway or is the causal factor a fetal exposure independently associated with fetal growth and increased risk of adult cardiovascular disease? The answer is important in terms of our understanding, the potential for intervention and estimation of the public health implications. Second, are the classic risk factors for CHD in the causal chain between fetal exposures or growth and adult CHD? Most prospective studies measure these factors, but their role as intermediates is unproven.

4. Intervention studies are the best way to test causal hypotheses, but our level of understanding is insufficient to justify such studies in humans, so we rely on animal studies to formally test causal hypotheses. In the present paper, we discuss design and statistical issues in relation to animal studies. The challenge in this field is to devise ways to identify and test potential causal hypotheses in humans.
The Kallikrein–Kinin System In Humans
Authors: Campbell Duncan J
Published: March 2003

1. Kinin peptides are implicated in many physiological and pathological processes, including the regulation of blood pressure and sodium homeostasis, inflammation and the cardioprotective effects of preconditioning. In humans, the plasma and tissue kallikrein–kinin systems (KKS) generate bradykinin and kallidin peptides, respectively.

2. We established methodology for the measurement of bradykinin and kallidin peptides and their metabolites in order to study the function of the plasma and tissue KKS in humans.

3. Bradykinin peptides were more abundant than kallidin peptides in blood and cardiac atrial tissue, whereas kallidin peptides were predominant in urine. The levels of kinin peptides in tissue were higher than in blood, confirming the primary tissue localization of the KKS.

4. Angiotensin‐converting enzyme inhibition increased blood levels of bradykinin and kallidin peptides.

5. Blood levels of kallidin peptides were suppressed in patients with severe cardiac failure, indicating that the activity of the tissue KKS is suppressed in this condition.

6. Bradykinin peptide levels were increased in the urine of patients with interstitial cystitis, suggesting a role for these peptides in the pathogenesis and/or symptomatology of this condition.

7. Cardiopulmonary bypass, a model of activation of the contact system, activated both the plasma and tissue KKS.

8. Measurement of individual bradykinin and kallidin peptides and their metabolites gives important information about the operation of the plasma and tissue KKS and their role in physiology and disease states.
Is Primary Aldosteronism Underdiagnosed In Clinical Practice?
Authors: Fiquet‐Kempf Béatrice , Launay‐Mignot Pauline , Bobrie Guillaume , Plouin Pierre‐François
Published: March 2003

1. Primary aldosteronism is a syndrome consisting of hypertension, suppressed renin activity or concentration and high aldosterone levels in plasma or urine. The main steps in diagnosis are the determination of renin and aldosterone levels, the demonstration of renin–aldosterone dissociation and discrimination between idiopathic hyperplasia and Conn’s adenoma, with only Conn’s adenoma amenable to surgery.

2. Patients with resistant hypertension and/or hypokalaemia should be screened for primary aldosteronism with simple, redundant hormonal tests. The aldosterone to renin ratio is a logical initial screening test, a high ratio demonstrating renin–aldosterone dissociation. Criteria for a high ratio should be determined in each laboratory.

3. In patients with documented primary aldosteronism, computed tomography scan and adrenal vein sampling help to distinguish between idiopathic hyperplasia and Conn’s adenoma.

4. Patients with low renin hypertension, idiopathic hyperplasia and Conn’s adenoma have overlapping values for plasma concentrations of potassium, renin and aldosterone and the aldosterone to renin ratio. Because primary aldosteronism subtypes are quantitative diseases, the true prevalence of primary aldosteronism cannot be defined.

5. The use of sensitive screening tests (e.g. aldosterone to renin ratio) gives a higher prevalence of diagnosed cases of primary aldosteronism, but not of surgically correctable forms. Therefore, there is no clinical evidence that primary aldosteronism is underdiagnosed.

6. There is a need for tests to predict the postoperative blood pressure outcome of surgery in subjects with Conn’s adenoma.
MINERALOCORTICOIDS AND CARDIAC FIBROSIS: THE DECADE IN REVIEW
Authors: Funder John
Published: March 2003

1. Over the past decade, aldosterone has been shown to have direct extra‐epithelial actions and substantial (patho)physiological roles in the cardiovascular system in the context of inappropriate salt status. In experimental studies on uninephrectomized rats given 0.9% NaCl solution to drink, these include blood pressure elevation via activation of circumventricular mineralocorticoid receptors in the central nervous system and production of pressure‐independent cardiac hypertrophy and fibrosis by a direct effect on the heart.

2. In the Randomized Aldactone Evaluation Study (RALES) trial, patients with severe congestive heart failure (CHF) were continued on their current therapy (angiotensin‐converting enzyme inhibitor, diuretic etc.) and given either placebo or spironolactone at an average dose of 26 mg/day. Mineralocorticoid receptor inhibition was accompanied by a 30% improvement in mortality and 35% less hospitalization, striking confirmation of a pathophysiological role for aldosterone in CHF.

3. Although the current basic and clinical studies are conflicting, there is evidence both for aldosterone synthesis by the failing human heart and for substantial cardiac metabolism of aldosterone. The extent to which this potential paracrine source for aldosterone may be involved in cardiac hypertrophy and cardiac fibrosis remains to be established.

4. Belatedly, aldosterone‐induced proteins (e.g. serum and glucocorticoid‐regulated kinase (SGK)) have been identified in epithelial mineralocorticoid target tissue. Studies are currently in progress on the cellular and molecular mechanisms involved in the coronary vasculitis provoked early in the mineralocorticoid/salt model, which, in turn, appears to trigger the subsequent perivascular and interstitial fibrotic response.
Angiotensin At1Receptor Signalling Pathways In Neurons
Authors: Sumners Colin , Fleegal Melissa A , Zhu Mingyan
Published: March 2003

1. The aim of the present article is to review the intracellular signal transduction pathways that are influenced by the peptide angiotensin (Ang) II, acting via its type 1 (AT1) receptor, in neurons.

2. The AT1receptors couple to a wide variety of signalling pathways in peripheral tissues, such as kidney, heart and vascular smooth muscle. A similar diversity of signalling mechanisms exists for AT1receptors in neurons.

3. We outline the known neuronal AT1receptor signalling pathways as they relate to function. Pathways that couple activation of AT1receptors to short‐term changes in neuronal membrane ionic currents and firing rate will be reviewed. These are different from the pathways that elicit longer‐term changes in enzyme activity and gene expression and, ultimately, increases in noradrenaline synthesis.

4. Novel AT1receptor signalling pathways discovered through gene expression profiling and their potential functional significance have been discussed.
Selective Peripheral Regulation Of Noradrenaline And Adrenaline Release By Nitric Oxide
Authors: Elayan Hamzeh H , Kennedy Brian P , Ziegler Michael G
Published: March 2003

1. Nitric oxide (NO) has complex effects on the sympathoadrenal and cardiovascular systems and may act at both central and peripheral loci. Nitric oxide appears to act directly on blood vessels and indirectly by modulating the sympathoadrenal system. In the present study, we investigated the contribution of catecholamine release from peripheral vascular and adrenal sympathetic nerves to the cardiovascular effects of the NO synthesis inhibitor NG‐nitro‐L‐arginine methyl ester (L‐NAME; 10 mg/kg). Our experiments were perfomed in pithed vagotomized rats to remove the influence of central and baroreflex pathways.

2. Spinal cord stimulations for 30 s periods at 1, 2, 5 and 10 Hz using pulses of 1 msec at 10 V caused marked increases in plasma adrenaline and noradrenaline. NG‐Nitro‐L‐arginine methyl ester did not alter resting plasma catecholamine concentrations. However, L‐NAME generally more than doubled stimulation‐evoked release of adrenaline while reducing the extent of noradrenaline release relative to vehicle (saline)‐treated controls.

3. NG‐Nitro‐L‐arginine methyl ester significantly enhanced the vasopressor responses to spinal cord stimulation. The α1‐adrenoceptor antagonist prazosin (0.2 mg/kg) reduced the pressor responses of electrically stimulated L‐NAME‐treated rats to levels below those of vehicle‐treated control rats.

4. In the absence of electrical stimulation, L‐NAME raised the blood pressure of pithed rats without altering plasma catecholamines and the pressor effect was briefly attenuated by L‐arginine, but was unaffected by prazosin.

5. We conclude that the augmented pressor response to sympathetic stimulation in L‐NAME‐treated pithed rats is due largely to enhanced adrenal adrenaline release mediated by a peripheral mechanism. Stimulation of α1‐adrenoceptors plays a major role in the pressor response to electrical stimulation of L‐NAME‐treated rats, but this is not due to L‐NAME augmentation of noradrenaline release from vascular sympathetic nerves.
Different Effects Of β‐Adrenoceptor Desensitization On Inhibitory Actions In Guinea‐Pig Trachealis
Authors: Mizutani Hiroshi , Kume Hiroaki , Ito Yasushi , Takagi Kenzo , Yamaki Kenichi
Published: March 2003

1. To determine the inhibitory effects of agents that pass through and bypass β‐adrenoceptors under conditions of tolerance to β‐adrenoceptor agonists, we examined the inhibition by the β‐adrenoceptor agonists forskolin and theophylline against contraction induced by methacholine (MCh) after exposure to higher concentrations of a β‐adrenoceptor agonist for a long time in guinea‐pig tracheal smooth muscle, using isometric tension records.

2. After exposure to procaterol (0.0003–3 μmol/L) for 45 min, the inhibitory effect of 0.03 μmol/L procaterol on 1 μmol/L MCh‐induced contraction was attenuated in a concentration‐dependent manner, whereas after exposure to isoprenaline (0.0003–3 μmol/L) for an equivalent time, the inhibitory effect of isoprenaline was markedly attenuated at each concentration. However, after exposure to 3 μmol/L procaterol for 45 min, the inhibitory actions of forskolin and theophylline were, conversely, augmented.

3. These phenomena were observed under conditions whereby the response to MCh returned to control levels 6 h after removal of 3 μmol/L procaterol. The percentage inhibition produced by 0.1 μmol/L forskolin against 1 μmol/L MCh after exposure to normal bathing solution or 3 μmol/L procaterol for 45 min was 9.8 ± 5.5 and 82.8 ± 6.5%, respectively (P < 0.001). These values for 100 μmol/L (18 μg/mL) theophylline on MCh resposnes were 9.9 ± 8.5 and 88.0 ± 4.4% (n = 6 for both), respectively (P < 0.001).

4. The inhibitory action of agents that bypass β‐adrenoceptors was markedly augmented under conditions of β‐adrenoceptor desensitization in airway smooth muscle.

5. In conclusion, procaterol is less potent in causing desensitization of β‐adrenoceptors than isoprenaline. The activity of adenylyl cyclase may be enhanced after exposure to a high concentration of β‐adrenoceptor agonists.
Why do neuroprotective drugs that are so promising in animals fail in the clinic? An industry perspective
Authors: Green A Richard
Published: March 2003

1. No neuroprotective drug has yet been shown to be effective in treating acute ischaemic stroke in the clinic, despite evidence of efficacy in animal models.

2. An academic/industry round‐table group recently published guidelines to be met if a drug was to be progressed to clinical trial.

3. Major points included obtaining full dose–response evaluation and measurement of the therapeutic time window for efficacy, functional behavioural testing in addition to measurement of infarct volume, measurement of physiological parameters, use of appropriate models (transient and permanent focal ischaemia) and reproducibility of data by external laboratories.

4. The present paper examines both failed compounds and disodium 4‐[(tert‐butylimino) methyl] benzene‐1, 3‐disulphonate N‐oxide (NXY‐059), a nitrone radical‐trapping agent currently in clinical development. It aims to determine whether these guidelines were met by compounds that have failed and, thus, determine whether following the guidelines, as is being done with NXY‐059, will increase the chances of developing efficacious drugs for treating acute ischaemic stroke.

5. It is concluded that we will only achieve the goal of producing a clinically effective neuroprotective agent if the guidelines have been met by the novel compound under investigation.
Gender differences in the regulation of vascular tone
Authors: Thompson Janell , Khalil Raouf A
Published: March 2003

1. The greater incidence of hypertension and coronary artery disease in men and post‐menopausal women compared with premenopausal women has suggested vascular protective effects of the female sex hormone oestrogen. However, vascular effects of the female sex hormone progesterone and the male sex hormone testosterone have also been suggested.

2. Oestrogen, progesterone and testosterone receptors have been identified in the plasmalemma, cytosol and nuclear compartments of vascular cells. The interaction of sex hormones with their specific receptors triggers not only long‐term genomic vascular effects, but also acute non‐genomic vascular responses.

3. Sex hormones may activate endothelium‐dependent vascular relaxation pathways, including the nitric oxide–cGMP and prostacyclin–cAMP pathways and a hyperpolarizing factor pathway.

4. Sex hormones may also inhibit the mechanisms of vascular smooth muscle contraction, such as [Ca2+]i, protein kinase C and other protein kinases.

5. The sex hormone‐induced stimulation of endothelium‐dependent vascular relaxation and inhibition of vascular smooth muscle contraction may contribute to the gender differences in vascular tone and may represent potential beneficial vascular effects of hormone‐replacement therapy during natural and surgically induced deficiencies of gonadal hormones.
Green tea catechins evoke a phasic contraction in rat aorta via H2O2‐mediated multiple‐signalling pathways
Authors: Shen Jian‐Zhong , Zheng Xiu‐Feng , Wei Er‐Qing , Kwan Chiu‐Yin
Published: March 2003

1. The contractile effects of tea polyphenols (TP) and its four principle catechins, namely (–)‐epicatechin (EC), (–)‐epicatechin‐3‐gallate (ECG), (–)‐epigallocatechin (EGC) and (–)‐epigallocatechin‐3‐gallate (EGCG), on rat aorta contractility were investigated using the isometric tension recording technique.

2. At concentrations of 5–100 mg/L, TP evoked phasic contraction of rat aorta in a concentration‐dependent but endothelium‐independent manner. Of the four catechins tested, EGCG and EGC (3–300 µmol/L), but not EC and ECG, mimicked the contractile response to TP, suggesting that the epigallol moiety in the B ring may be associated with the contractile effect.

3. Contractions in response to EGCG and EGC were not affected by several endogenous vasoconstrictor receptor antagonists, but could be abolished by 10 µmol/L BAPTA‐AM, a membrane‐permeable Ca2+ chelator, or attenuated by removal of extracellular Ca2+, suggesting the involvement of both intracellular and extracellular Ca2+ in evoking the contraction.

4. Pretreatment with non‐selective Ca2+ channel antagonists mefenamic acid (10 µmol/L), tetrandrine (30 µmol/L) and SKF 96365 (30 µmol/L), but not nifedipine (1 µmol/L), the selective inhibitor of voltage‐dependent Ca2+ channels, inhibited the contractile responses to EGC and EGCG, indicating the involvement of Ca2+ influx via non‐voltage dependent Ca2+ channels.

5. Several intracellular Ca2+ channel modulators, including procaine (5 mmol/L), dantrolene (30 µmol/L) and 2‐amino ethoxydiphenyl borate (50 µmol/L; an inositol 1,4,5‐trisphosphate receptor inhibitor), also inhibited EGCG‐ and EGC‐induced contractions, thus suggesting a role of intracellular Ca2+ release in these contractions.

6. Both EGCG‐ and EGC‐induced contractions were depressed, to different degrees, by inhibitors of several receptor‐coupled enzymes, including phospholipase C, protein kinase C, phospholipase A2 and tyrosine kinase. Furthermore, both EGCG‐ and EGC‐induced contractions were completely abolished by catalase, but not by superoxide dismutase or mannitol/dimethyl sulphoxide.

7. Taken together, these data show, for the first time, that TP and its related catechins that contain an epigallol structure in the B ring, as in EGCG and EGC, exert direct contractile effects on rat aortic smooth muscle via a H2O2‐mediated pathway.
Cardiac mitochondrial complex activity is enhanced by heat shock proteins
Authors: Sammut Ivan A , Harrison Joanne C
Published: March 2003

1. Prolonged ischaemia and reperfusion in heart transplantation results in mitochondrial dysfunction and loss of cardio‐energetics. Improved myocardial tolerance to ischaemia–reperfusion can be increased by de novo synthesis of heat shock protein (Hsp) groups, transiently expressed following mild hyperthermic or oxidative stress. Consideration of the roles of various Hsp in ischaemic–reperfused myocardium can provide new insights into potential therapeutic adjuncts to cardiac surgery.

2. Several Hsp classes have been located within or in association with mitochondrial elements. Cardiac Hsp research has focused primarily on the 70 kDa group, involved in protein folding functions within the cytosol and matrix. Similarly, Hsp 60 and 10 have been shown to form a mitochondrial chaperonin complex conferring protection to ischaemia‐challenged myocytes. Equally pertinent is Hsp 32, an isoform of the haem‐metabolizing enzyme heme oxygenase.

3. Our studies have shown that mitochondrial respiratory enzyme activity can be protected by Hsp, affording protection to cardiac energetics during preservation for transplantation. Upregulation of Hsp 32, 60 and 72 in rats, achieved by mild hyperthermic stress, improved cardiac function, ultrastructure and mitochondrial respiratory and complex activities in ex vivo perfused hearts subjected to cold cardioplegic arrest and ischaemia–reperfusion. Pre‐ischaemic mitochondrial complex activities were increased in heat stress versus sham‐treated groups for complex I, IV and V.

4. Investigation of the direct effect of upregulation of Hsp 72 by gene transfection resulted in a similar pattern of response, with increased complex I activity and improved ventricular function.

5. These studies provide the first evidence of Hsp‐mediated enhancement of mitochondrial energetic capacity. Enhanced protection of mitochondrial energetics, as a result of increased Hsp expression, contributes to the recovery of myocardial function in ischaemia–reperfusion.
Fragmenting oligochaete Enchytraeus japonensis: A new material for regeneration study
Authors: Myohara Maroko , Yoshida‐Noro Chikako , Kobari Fumiko , Tochinai Shin
Published: March 2003

Enchytraeus japonensis, a recently described terrestrial oligochaete, reproduces asexually by fragmentation and subsequent regeneration. Taking notice of its high potential as a new material for regeneration study, detailed studies were undertaken on the regeneration and reproduction of E. japonensis. The full‐grown body divided into 6–13 fragments that regenerated into complete individuals in 4 days, grew to full length in 10 days, and then fragmented again. Regeneration of the head and tail was epimorphic, involving blastema formation, while old segments in the regenerating fragment morphallactically transformed into the appropriate segments to retain the proper body proportions, which could be visualized by histochemistry for alkaline phosphatase. Artificially cut fragments regenerated either normally or into dicephalic monsters with biaxial heads depending on the conditions. Fragmentation could be induced by decapitation, and sexual reproduction was also found inducible in the laboratory. These findings, together with its simple metameric morphology and ease of culture and handling, suggest that E. japonensis is an excellent material for studying animal regeneration.
Temporal expression patterns of 39 Brachyury‐downstream genes associated with notochord formation in the Ciona intestinalis embryo
Authors: Hotta Kohji , Takahashi Hiroki , Erives Albert , Levine Michael , Satoh Nori
Published: March 2003

Expression of the Brachyury (Ci‐Bra) gene of the ascidian Ciona intestinalis is initiated at the 64‐cell stage. Gene expression is restricted to notochord precursor cells, and Ci‐Bra plays a key role in notochord differentiation. In a previous study, nearly 50 cDNA clones for potential Ci‐Bra‐downstream genes that are expressed in notochord cells were isolated. The present determination, by whole‐mount in situ hybridization, of the temporal expression patterns of 19 notochord‐specific and 20 notochord‐predominant genes demonstrated that the timings of initiation of the expression of various genes was not identical. The expression of several genes was initiated as early as the gastrula stage. However, the expression of most of the notochord‐specific genes commenced at the neural plate stage. Partial nucleotide sequence data of these clones suggest that genes expressed earlier encode potential transcriptional factors and/or nuclear proteins, while those expressed later encode proteins implicated in cell adhesion, signal transduction, regulation of the cytoskeleton, and components of the extracellular matrix. These gene activities may be associated with changes in cell shape and adhesion during the intercalation and extension of the notochord cells.
Pax‐6 expression during retinal regeneration in the adult newt
Authors: Kaneko Yuko , Matsumoto Gen , Hanyu Yoshiro
Published: March 2003

The present study examined the expression of Pax‐6 during retinal regeneration in adult newts using in situ hybridization. In a normal retina, Pax‐6 is expressed in the ciliary marginal zone, the inner part of the inner nuclear layer, and the ganglion cell layer. After surgical removal of the neural retina, retinal pigment epithelial cells proliferate into retinal precursor cells and regenerate a fully functional retina. At the beginning of retinal regeneration, Pax‐6 was expressed in all retinal precursor cells. As regeneration proceeded, differentiating cells appeared at the scleral and vitreal margins of the regenerating retina, which had no distinct plexiform layers. In this stage, the expression of Pax‐6 was localized in a strip of cells along the vitreal margin of the regenerating retina. In the late stage of regeneration, when the layer structure was completed, the expression pattern of Pax‐6 became similar to that of a normal retina. It was found that Pax‐6 is expressed in the retinal precursor cells in the early regenerating retina and that the expression pattern of Pax‐6 changed as cell differentiation proceeded during retinal regeneration.
Fate mapping study of the endoderm in the posterior part of the 1.5‐day‐old chick embryo
Authors: Matsushita Susumu
Published: March 2003

Various endodermal sites posterior to the caudal‐most somite were marked in ovo with the vital dye DiI, and the fate of marked endoderm was analyzed after 2 or 3 days’ reincubation. The endoderm in this area became gut epithelium posterior to the caudal jejunum and yolk sac. The area occupied by the cells that were to contribute to the dorsal part of the digestive tube lay centrally around the area overlaid by axial and paraxial mesoderm, with the preventral digestive area lying outside with considerable overlapping, which was surrounded by the preyolk sac area. During the formation of the posterior digestive tube, the endoderm was elongated anteroposteriorly to a considerable degree. Cells that contributed to the cloaca and those that produced descendants in the large intestine occupied similar areas posterior to the center of the sinus rhomboidalis, which were included in the pre‐ileal area extending more anteriorly. Prejejunal cells generally localized in a more anterior position than pre‐ileal cells. Pre‐allantoic cells were located in a rather small area around the posterior primitive streak.
Efficient targeting of gene expression in chick embryos by microelectroporation
Authors: Momose Tsuyoshi , Tonegawa † Akane , Takeuchi Jun , Ogawa Hidesato , Umesono Kazuhiko , Yasuda Kunio
Published: March 2003

During vertebrate embryonic development, a key to unraveling specific functions of gene products is the capability to manipulate expression of the gene of interest at the desired time and place. For this, we developed a ‘microelectroporation’ technique by which DNA can be locally introduced into a targeted site of avian embryos, restricting spatial expression of the protein products during development. This technique involved injection of DNA solution in ovo around the target tissue and pinpoint application of an electric field by tungsten electrodes, allowing efficient and reproducible targeted gene transfer, for example, into an optic vesicle, somites, cranial mesoderm and limb mesenchyme. Because of the locality of gene introduction and its expression, survival rates of the embryos were high: approximately 90% of the embryos injected in optic vesicles were alive for at least 1 day after microelectroporation. The instantaneous gene transfer into embryonic cells allowed rapid expression of protein products such as green fluorescence protein within 2.5 h with fluorescence maintained for 3 days of incubation. This improved technique provides a convenient and efficient way to express transgenes in a spatially and temporally restricted manner in chicken embryos.
Nuage constituents arising from mitochondria: Is it possible?
Authors: Reunov Arkadiy , Isaeva Valeria , Au Doris , Wu Rudolf
Published: March 2003

An ultrastructural study of nuage–mitochondria complexes in spermatogonia of the sea urchin, Anthocidaris crassispina, was carried out. Release of mitochondrial contents into the cytoplasm was observed. The mitochondrial derivatives persisted as cristae‐containing globules of friable material that subsequently contacted and integrated with nuage. The present ultrastructural findings agree with the results of other researchers who proposed that germ plasm substance probably produced by the nucleus is supplemented by the mitochondrial genome.
Involvement of BMP‐4/msx‐1 and FGF pathways in neural induction in the Xenopus embryo
Authors: Ishimura Akihiko , Maeda Ryu , Takeda Masatoshi , Kikkawa Mika , Daar Ira Owen , Maéno Mitsugu
Published: March 2003

The msx homeodomain protein is a downstream transcription factor of the bone morphogenetic protein (BMP)‐4 signal and a key regulator for neural tissue differentiation. Xmsx‐1 antagonizes the dorsal expression of noggin and cerberus, as revealed by in situ hybridization and reverse transcription–polymerase chain reaction assays. In animal cap explants, Xmsx‐1 and BMP‐4 inhibit the neural tissue differentiation induced by noggin or cerberus. A loss‐of‐function study using the Xmsx‐1/VP‐16 fusion construct indicated that neural tissue formation was directly induced by the injection of fusion ribonucleic acid, although the expression of neural cell adhesion molecule (N‐CAM) in the cap was less than that in the cap injected with tBR or noggin. In contrast to the single cap assay, unexpectedly, both BMP‐4 and Xmsx‐1 failed to inhibit neurulation in the ectodermal explants to which the organizer mesoderm was attached. The results of cell‐lineage tracing experiments indicated that the neural cells were differentiated from the animal pole tissue where the excess RNA of either BMP‐4 or Xmsx‐1 was injected, whereas notochord was differentiated from the organizer mesoderm. Neural tissue differentiated from BMP‐4‐injected ectodermal cells strongly expressed posterior neural markers, such as hoxB9 and krox20, suggesting that the posterior neural cells differentiated regardless of the existence of the BMP signal. The introduction of a dominant‐negative form of the fibroblast growth factor (FGF) receptor (XFD) into the ectodermal cells drastically reduced the expression of pan and posterior neural markers (N‐CAM and hoxB‐9) if co‐injected with BMP‐4 RNA, although XFD alone at the same dose did not shut down the expression of N‐CAM in the combination explants. Therefore, it is proposed that an FGF‐related molecule was involved in the direct induction of posterior neural tissue in the inducing signals from the organizer mesoderm in vivo.
The maternal Xenopusβ‐catenin signaling pathway, activated by frizzled homologs, induces goosecoid in a cell non‐autonomous manner
Authors: Brown Jeffrey D. , Hallagan Sarah E. , McGrew L. Lynn , Miller Jeffrey R. , Moon Randall T.
Published: March 2003

In spite of abundant evidence that Wnts play essential roles in embryonic induction and patterning, little is known about the expression or activities of Wnt receptors during embryogenesis. The isolation and expression of two maternal Xenopus frizzled genes, Xfrizzled‐1 and Xfrizzled‐7, is described. It is also demonstrated that both can activate the Wnt/β‐catenin signaling pathway as monitored by the induction of specific target genes. Activation of the β‐Catenin pathway has previously been shown to be necessary and sufficient for specifying the dorsal axis of Xenopus. β‐Catenin is thought to work through the cell‐autonomous induction of the homeobox genes siamois and twin, that in turn bind to and activate the promoter of another homeobox gene, goosecoid. However, it was found that the β‐catenin pathway regulated the expression of both endogenous goosecoid, and a goosecoid promoter construct, in a cell non‐autonomous manner. These data demonstrate that maternal Frizzleds can activate the Wnt/β‐catenin pathway in Xenopus embryos, and that induction of a known downstream gene can occur in a cell non‐autonomous manner.
Xoom is maternally stored and functions as a transmembrane protein for gastrulation movement in Xenopus embryos
Authors: Hasegawa Kouichi , Sakurai Nobuko , Kinoshita Tsutomu
Published: March 2003

Xoom has been identified as a novel gene that plays an important role in gastrulation of Xenopus laevis embryo. Although Xoom is actively transcribed during oogenesis, distribution and function of its translation product have not yet been clarified. In the present study, the polyclonal antibody raised against Xoom was generated to investigate a behavior of Xoom protein. Anti‐Xoom antibodies revealed that there are two forms of Xoom protein in Xenopus embryos: (i) a 45 kDa soluble cytoplasmic form; and (ii) a 44 kDa membrane‐associated form. Two forms of Xoom protein were ubiquitously detected from unfertilized egg to tadpole stage, with a qualitative peak during blastula and gastrula stages. Immunohistochemical examination showed that Xoom protein is maternally stored in the animal subcortical layer and divided into presumptive ectodermal cells during cleavage stages. Enzymatic digestion of membrane protein and immunologic detection of Xoom showed that Xoom exists as a membrane‐associated protein. To examine a function of Xoom protein, anti‐Xoom antibodies were injected into blastocoele of stage 7 blastula embryo. Anti‐Xoom antibodies caused gastrulation defect in a dose‐ dependent manner. These results suggest that maternally prepared Xoom protein is involved in gastrulation movement on ectodermal cells.
Position‐specific and non‐colinear expression of the planarian posterior (Abdominal‐B‐like) gene
Authors: Nogi Taisaku , Watanabe Kenji
Published: March 2003

Hox genes are pivotal molecules in the control of morphogenesis along the anterior‐posterior (AP) axis in various bilaterians. Planarians are key animals for understanding the evolution of the bilaterian body plan. Furthermore, they are also known for their strong regeneration ability and are thought to use the Hox genes in the process of reconstruction of the AP axis. In the present paper, the identification and analysis of expression of two posterior (Abdominal‐B‐like) genes, DjAbd‐Ba and DjAbd‐Bb, is reported in the planarian Dugesia japonica. DjAbd‐Ba is expressed in the entire tail region and its anterior boundary is the posterior pharyngeal region. In contrast, DjAbd‐Bb is expressed in several types of cells throughout the body. During regeneration, the expression of DjAbd‐Ba rapidly recovers a pattern similar to that in the normal worm. These findings suggest the possibility that DjAbd‐Ba is involved in the specification of the tail region. The anterior boundary of the expression domain of the posterior gene DjAbd‐Ba is anterior to the domains of the central genes Plox4‐Dj and Plox5‐Dj. These expression patterns of planarian Hox genes seem out of the rule of spatial colinearity and may reflect an ancestral feature of bilaterian Hox genes.
Nucleus : cell volume ratio directs the timing of the increase in blastomere adhesiveness in starfish embryos
Authors: Masui Mizuko , Yoneda Mitsuki , Kominami Tetsuya
Published: March 2003

Blastomeres of starfish embryos begin to increase in adhesiveness after the eighth cleavage and form a monolayered hollow blastula. To investigate factors that affect the timing of the adhesiveness increase, we changed the volume of the cytoplasm or the ploidy of embryos and examined the morphologic changes in the descendent blastomeres during early cleavage stages. In parthenogenetic embryos, in which the ploidy is doubled, the timing of the increase in adhesiveness was accelerated by one cell cycle. In contrast, the timing was delayed by approximately one cell cycle in a large‐sized embryo formed by the fusion of an egg and a non‐nucleate egg fragment. These two sets of observations are in accord with the expectation from the classical concept that the DNA : cytoplasmic ratio may direct the timing of events in early development. However, observations of small‐sized embryos with a reduced amount of cytoplasm were contradictory to the expectation based on the DNA : cytoplasmic ratio; the timing of the increase in adhesiveness in half‐sized embryos was almost the same as in control embryos and the timing was delayed by only one cell cycle in quarter‐sized embryos. Measurement of the diameters of nuclei showed that the size of nuclei was variable, depending on the stage of development, the volume of cytoplasm and ploidy. We calculated a volume ratio of nucleus to cytoplasm (N : C volume ratio) for tetraploid, large‐, half‐ and quarter‐sized embryos. We found that the embryonic cells begin to adhere always when their N : C volume ratio reaches 0.06. A plausible model for the cellular timing mechanism of cell contact is proposed..
A hatching enzyme substrate in the Xenopus laevis egg envelope is a high molecular weight ZPA homolog
Authors: Lindsay LeAnn L. , Wallace Matthew A. , Hedrick Jerry L.
Published: March 2003

The Xenopus laevis egg envelope is composed of six or more glycoproteins, three of which have been cloned and identified as the mammalian homologs ZPA (ZP2), ZPB (ZP1) and ZPC (ZP3). The remaining glycoproteins are a triplet of high molecular weight components that are selectively hydrolyzed by the hatching enzyme. We have isolated one of these proteins and cloned its cDNA. The mRNA for the protein was found to be expressed only in early stage oocytes, as are other envelope components. From the deduced amino acid sequence, it was indicated to be a secreted glycoprotein with a characteristic ZP domain in the C‐terminal half of the molecule. The N‐terminal half was unrelated to any known glycoprotein. Comparative sequence analysis of the ZP domain indicated that it was derived from an ancestor of ZPA and ZPB, with the greatest identity to ZPA. This envelope component has been designated ZPAX.