bluuebunny/crossref_metadata_2025 huggingface dataset:
Ecology of stream fish: insights gained from an individual‐based approach to juvenile Atlantic salmon
Authors: Juanes F. , Letcher B. H. , Gries G.
Published: November 2003

Abstract – Using data from an ongoing study of juvenile Atlantic salmon growth and survival in tributaries of the Connecticut River, USA, we compare standard population‐level approaches to those focusing on individuals. We highlight the potential benefits of resampling individually tagged stream fish as compared to standard approaches. Specifically we focus on growth, survival, movements and population estimation. The advantages of estimating sizes and growth rates from individual size trajectories include obtaining growth histories and the ability to perform retrospective analysis of the consequences of different life‐history strategies. An example might be the patterns of growth leading to either early maturity or migration. Resampling known individuals is the only way we know to chart both short‐term and long‐term movements and to assign growth and mortality consequences to such movements. Finally, individual‐level data permit robust estimation of survival and density/abundance using methods such as Cormack/Jolly‐Seber. The results indicate that population estimates were about 10% lower using individual data than using population data, that survival from sample to sample was typically >90%, that the majority of recaptured fish did not move during the summer, that growth was rapid during spring and most fish lost mass during the summer and that growth trajectories for maturing and non‐maturing fish showed substantially different patterns. An individual‐based approach to stream fish ecology provides the opportunity to explore the mechanisms responsible for population‐level patterns but comes at the cost of significant field effort. Tradeoffs between increased data resolution and the effort required to obtain the data must be considered before undertaking individual‐based field studies of stream fishes.NOTE
The potential of flat‐bed passive integrated transponder antennae for studying habitat use by stream fishes
Authors: Greenberg L. A. , Giller P. S.
Published: November 2003

Abstract – Describing habitat use by stream fishes is important from both basic ecological and fisheries management points of view. The most widely used methods of measuring habitat use vary in degree of effort required, level of intrusiveness and in the level of spatial and temporal resolution. In this paper, we describe a remote monitoring technique that can provide detailed and continuous data on habitat use of individual fish in the field. The technique is based on the passive integrated transponder (PIT) system, in which a newly developed flat‐bed antenna is placed on the stream bottom and simply requires a PIT‐tagged fish to swim over it. We present data obtained from work using this new technology on brown trout (Salmo trutta) in stream enclosures, in which we describe habitat use and temporal patterns of movement by individuals and relate such data to growth rate and sex of the individual fish as well as to pool depth and time of day. In addition, we describe the range of applications of the flat‐bed PIT‐antenna as well as the advantages and disadvantages of using the system.NOTE
Living with failure: the prey capture success of young brook charr in streams
Authors: McLaughlin R. L. , Grant J. W. A. , Noakes D. L. G.
Published: November 2003

Abstract – We examined the prey capture success of recently emerged brook charr (Salvelinus fontinalis) foraging in shallow, clear, still‐water pools along the edges of streams. Fewer than 42% of attacks ended with ingestion either because of difficulty distinguishing suitable prey from unsuitable items or because of difficulty capturing evasive prey. Probabilities of capture upon attack and ingestion upon capture depended upon where attacks were directed in the water column, the fish's level of activity at the time of attack and its fork length, and the sampling date. In general, success was higher for larger, sedentary fish attacking prey in the lower portion of the water column than for smaller, active fish attacking prey at the water surface. The frequency of items attacked was only a moderate predictor of the frequency of prey ingested. Poor capture success is an important aspect of the early life history of brook charr in particular and probably of young salmonines in general.NOTE
Variation in rates of food consumption and evidence for compensatory responses in the three‐spined stickleback, Gasterosteus aculeatus L. in relation to growth and reproduction
Authors: Ali M. , Wootton R. J.
Published: November 2003

Abstract – Evidence that rates of food consumption differ between individuals in a population is noted. Preliminary estimates of individual variation in a population of three‐spined sticklebacks (Gasterosteus aculeatus L.) are provided and compared with similar estimates for a population of Phoxinus. Experimental studies are reviewed to demonstrate the capacity of sticklebacks to compensate for variations in food availability by compensatory changes in food consumption. These changes allowed sticklebacks on fluctuating rations to maintain growth and reproductive performances comparable to sticklebacks receiving constant, daily rations. Such studies start to define the scope for compensation that buffers the effect of environmental variability in food supply.NOTE
Evaluation of the effects of catch‐and‐release angling on the Atlantic salmon (Salmo salar) of the Ponoi River, Kola Peninsula, Russian Federation
Authors: Whoriskey F. G. , Prusov S. , Crabbe S.
Published: November 2003

Abstract – We studied the effects of catch‐and‐release fishing upon the Ponoi River's Atlantic salmon populations. The Ponoi River is located on the Kola Peninsula of the Russian Federation, and has recently been developed for sports fishing. Angler exploitation rates are estimated to range from 10.4% to 19% of the river's salmon, thus the possibility of significant levels of post‐release mortality is of concern. We radio‐tracked fish caught and released by anglers in 1995 and 1996. Despite our simple equipment and the large size of the river, we were able to relocate most fish. These fish had high rates of survival, and anglers recaptured about 11% of them per year a second time. This is very similar to the recapture rates observed for Floy‐tagged fish released in an angler‐based mark‐recapture assessment. We also held 62 angled fish for 24 hours in a live cage to evaluate rates of delayed mortality. Only one of the 62 fish died, and it was heavily scarred with gillnet marks. Most fish that are fatally stressed by angling die within 24 h (e.g., Booth et al. 1995). In 1996, up to 10% of our Floy‐tagged fish were angled and released twice, and about 0.5% were angled and released three times. No significant biases were detected in the post‐angling movement patterns of these fish. The multiple captures and lack of movement bias suggest that fish behavior was little altered by the angling experience. Nine fish Floy tagged prior to spawning have been recovered as typical emaciated kelts. Three were killed, and a post mortem exam showed all had spawned. Parr numbers at all monitored sites have been steadily increasing since the advent of catch‐and‐release fishing. By contrast, parr growth rates are generally unchanged or significantly better.NOTE
Enhanced replication of HIV‐1 in vivo in pigtailed macaques (Macaca nemestrina)
Authors: Bosch Marnix L. , Schmidt Ann , Chen Jiangli , Florey Mary‐Jo , Agy Michael , Morton William R.
Published: November 2003

Non‐human primate models for acquired immunodeficiency syndrome (AIDS) are important for studies of prevention and intervention strategies. Ideally, such models would make use of human immunodeficiency virus type 1 (HIV‐1) and animals that are readily available for research. HIV‐1 was obtained from an infected macaque, and passaged sequentially in three groups of two Macaca nemestrina neonates each. Evidence for
enhanced viral replication was first found in one of the group 2 animals,
and in both group 3 animals. Observations that underlie this conclusion
are sustained viral recovery from peripheral blood mononuclear cells
(PBMCs), increased and accelerated production of antiviral antibodies,
and the ability to detect plasma viral ribonucleic acid (RNA) months
after infection. There was no evidence of CD4 depletion in any of the
animals during the follow‐up period. These data suggest that a useful
non‐human primate model for AIDS can be attained in pigtailed macaques
(M. nemestrina).
Derivation and characterization of a highly pathogenic isolate of human immunodeficiency virus type 2 that causes rapid CD4+ cell depletion in Macaca nemestrina
Authors: McClure Janela , Schmidt Ann M. , Rey‐Cuille Marie‐Anne , Bannink Jeannette , Misher Lynda , Tsai Che‐Chung , Anderson David M. , Morton William R. , Hu Shiu‐Lok
Published: November 2003

With few exceptions, humans are the only species known to develop acquired immunodeficiency syndrome (AIDS) after human immunodeficiency virus (HIV) infection. We report here that an isolate of HIV type 2, EHO, readily established persistent infection in 100% of Macaca
nemestrina in three consecutive transmission studies. Of the eight
infected animals, five showed persistently high virus load and six
developed AIDS‐like diseases or CD4+ cell depletion within 4
years of infection. The pathology and clinical signs closely parallel
those of HIV‐1 infection of humans, including lymphadenopathy, anemia,
CD4+ cell depletion, and opportunistic infections. A cell‐free
virus stock was established from the lymph nodes of an animal that
developed AIDS‐like diseases. This virus, HIV‐2/287, was highly
pathogenic in M. nemestrina, causing CD4+ cell
depletion within 2–8 weeks post‐infection. While both HIV‐2 EHO and
HIV‐2/287 use predominantly CXCR4, the latter shows greatly enhanced
replicative capacity in macaque peripheral blood mononuclear cells
(PBMCs). The establishment of a human immunodeficiency virus that causes
rapid and reproducible CD4+ cell depletion in macaques could facilitate the study of HIV pathogenesis and the development of effective vaccines and therapy against AIDS.
Total numbers of lymphocyte subsets in different lymph node regions of uninfected and SIV‐infected macaques
Authors: Halbach Astrid , Nierwetberg* Dagmar , Müller Justus G. , Sauer Ursula , Kerkau Thomas , Stolte Nicole , Hofmann Petra , Czub Stephanie , Ter Meulen Volker , Sopper Sieghart
Published: November 2003

Human immunodeficiency virus (HIV) infection leads to a decline of CD4+ T‐cells in blood. Because blood represents only a small proportion of the total lymphocyte pool, it is important to investigate other lymphoid organs. So far, only relative proportions of lymphocyte subsets in single peripheral lymph node (LN) regions of HIV‐infected patients and simian immunodeficiency virus (SIV)‐infected macaques have been documented. We have therefore quantified the absolute numbers of lymphocyte subsets in blood and six different LN regions of 10 uninfected and 26 SIV‐infected macaques. In addition, we have determined the expression of markers of activation and differentiation. Already, in uninfected monkeys, there were significant differences in the cellular composition of different LN regions. Infection with SIV resulted in drastic changes in the proportion as well as absolute numbers of different lymphocyte subsets. Moreover, the relative contribution of the single LN regions to the total lymphocyte pool was also altered.
SIVcpz from a naturally infected Cameroonian chimpanzee: Biological and genetic comparison with HIV‐1 N
Authors: Müller‐Trutwin M.C. , Corbet S. , Souquière S. , Roques P. , Versmisse P. , Ayouba A. , Delarue S. , Nerrienet E. , Lewis J. , Martin P. , Simon F. , Barré‐Sinoussi F. , Mauclère P.
Published: November 2003

Thus far, simian immunodeficiency virus from chimpanzees (SIVcpz) genomes have been characterized as Pan troglodytes troglodytes and
show a strong relation with human immunodeficiency virus (HIV)‐1 N in
their env genes. We fully characterized another SIVcpz from P.
t. troglodytes. This chimpanzee (Cam5) was, as was also the host of
SIVcpz‐cam3, wild born in Cameroon, a region where all three groups of
HIV‐1 (M, N and O) co‐occur. In contrast to other SIVcpz, SIVcpz‐cam5 was
isolated immediately after the rescue of the animal. Our data demonstrate
that SIVcpz‐cam5, like SIVcpz‐cam3, grows easily on human peripheral
blood mononuclear cells (PBMCs) and uses CCR5 as a co‐receptor similar to
HIV‐1 N YBF30. Phylogenetic analysis based on the entire env gene
shows that SIVcpz‐cam5 falls into the same unique subcluster as HIV‐1 N
YBF30, SIVcpz‐cam3 and SIVcpz‐US. A phylogenetic relationship was also
found with the vif gene of HIV‐1 N. This study provides proof that
HIV‐1 N related viruses circulate in wild P. t. troglodytes.
Identification of SIV env‐specific CTL in the jejunal mucosa in vaginally exposed, seronegative rhesus macaques (Macaca mulatta)
Authors: Wilson Lawrence A. , Murphey‐Corb Michael , Martin Louis N. , Harrison Richard M. , Ratterree Marion S. , Bohm Rudolf P.
Published: November 2003

We previously reported major histocompatibility complex Class I‐restricted cytotoxic T lymphocytes (CTL) in jejunal lamina propria (LP) of monkeys following colonic exposure to subinfectious SIV doses. Those monkeys with strong mucosal CTL responses specific for simian immunodeficiency virus (SIV) envelope (env) were protected from later colonic challenge with a heterologous pathogenic virus dose. Here, env‐specific CTL were similarly induced in jejunal LP in five of eight non‐progesterone treated macaques that were vaginally exposed to SIV, but not infected. Subsequent vaginal challenge following progesterone treatment produced systemic infection. The only two monkeys that had jejunal env‐specific CTL detectable post‐challenge developed significantly lower plasma virus loads, and had delayed disease progression. Either vaginal or colonic exposure to subinfectious SIV doses can induce CTL detectable in jejunal LP. The association of such CTL with protection or delayed disease upon challenge suggests that successful vaccine protection against SIV/HIV may require CTL responses in the mucosa.
Macaque blood‐derived antigen‐presenting cells elicit SIV‐specific immune responses
Authors: Zhu Yong‐de , Koo Kevin , Bradshaw Jeffrey D. , Sutton William F. , Kuller LaRene , Bucala Richard , Anderson David , Mossman Sally P. , Villinger François , Haigwood Nancy L.
Published: November 2003

Natural blood‐borne antigen‐presenting cells (APCs) were tested for their ability to augment antigen presentation for SIV vaccines. Fibrocytes and monocyte‐derived dendritic cells (DCs) were isolated from multiple Macaca fascicularis. Macaque fibrocytes displayed the
characteristic cellular morphology and stained positive for CD34 and
collagen, as observed in human and murine fibrocytes. Macaque DCs were
generated from monocytes by culturing in granulocyte–macrophage
colony stimulating factor and interleukin‐4 (IL‐4). Two days after
maturation, cells were enriched for the DC marker CD83. Fibrocytes and
DCs were each transfected with green fluorescence protein expression
plasmids or DNA expression vectors encoding all of the SIVmne structural
and regulatory genes. Autologous DCs were re‐infused into macaques
subcutaneously (sc) following transfection; mixing with recombinant SIV
antigens or inactivated whole SIV in vitro; or mock‐treatment.
Autologous monocyte‐derived DCs pulsed with whole inactivated SIV were
re‐infused and elicited cellular and/or humoral responses in vivo in eight of ten vaccinated macaques.
SHIV89.6P pathogenicity in cynomolgus monkeys and control of viral replication and disease onset by human immunodeficiency virus type 1 Tat vaccine
Authors: Cafaro Aurelio , Caputo Antonella , Maggiorella Maria Teresa , Baroncelli Silvia , Fracasso Claudio , Pace Monica , Borsetti Alessandra , Sernicola Leonardo , Negri Donatella R.M. , Ten Haaft Peter , Betti Monica , Michelini Zuleika , Macchia Iole , Fanales‐Belasio Emanuele , Belli Roberto , Corrias Franco , Buttò Stefano , Verani Paola , Titti Fausto , Ensoli Barbara
Published: November 2003

The Tat protein of human immunodeficiency virus (HIV) is produced very early after infection, plays a key role in the virus life cycle and in acquired immunodeficiency syndrome (AIDS) pathogenesis, is immunogenic and well conserved among all virus clades. Notably, a Tat‐specific immune response correlates with non‐progression to AIDS. Here, we show that a vaccine based on the Tat protein of HIV blocks primary infection with the simian/human immunodeficiency virus (SHIV)89.6P and prevents the CD4 T cell decline and disease onset in cynomolgus monkeys. No signs of virus replication were found in five out of seven vaccinated macaques for almost 1 year of follow‐up. Since the inoculated virus (derived from rhesus or from cynomolgus macaques) is shown to be highly pathogenic in cynomolgus macaques, the results indicate efficacy of Tat vaccination in protection against highly pathogenic virus challenge. Finally, the studies of the Tat‐specific immunological responses indicate a correlation of protection with a cytotoxic T cell response. Thus, a Tat‐based vaccine is a promising candidate for preventive and therapeutic vaccination in humans.
Mucosal challenge of Macaca nemestrina with simian immunodeficiency virus (SIV) following SIV nucleocapsid mutant DNA vaccination*
Authors: Gorelick Robert J. , Lifson Jeffrey D. , Yovandich Jason L. , Rossio Jeffrey L. , Piatak Jr Michael , Scarzello Anthony J. , Knott Walter B. , Bess Jr Julian W. , Fisher Bradley A. , Flynn Bernard M. , Henderson Louis E. , Arthur Larry O. , Benveniste Raoul E.
Published: November 2003

A simian immunodeficiency virus (SIV)(Mne) DNA clone was constructed that produces viruses containing a four amino acid deletion in the second zinc finger of the nucleocapsid (NC) domain of the Gag polyprotein. Viruses produced from this clone, although non‐infectious both in
vitro and in vivo, complete a majority of the steps in a
single retroviral infection cycle. Eight pig‐tailed macaques (Macaca
nemestrina) were inoculated intramuscularly and subcutaneously three times over the course of 24 weeks with the NC mutant expressing DNA. These macaques, and four controls, were then challenged mucosally (intrarectally) with the homologous virus (SIV Mne CL E11S) and monitored for evidence of infection and clinical disease. Prior to challenge, a measurable humoral immune response was noted in four of eight immunized macaques. After challenge, all 12 macaques became infected, although four immunized animals greatly restricted their viral replication, and one immunized animal that controlled replication remains antibody negative. No disease has been evidence during the 46‐week period of monitoring after challenge.
Enhanced safety and efficacy of live attenuated SIV vaccines by prevaccination with recombinant vaccines
Authors: Jones Leslie , Ahmad Shabbir , Chan Kenneth , Verardi Paulo , Morton William R. , Grant Richard , Yilma Tilahun
Published: November 2003

The only vaccines shown to be protective against intravenous challenge with virulent virus in the simian immunodeficiency virus (SIV)/macaque model are attenuated live SIVs. However, these vaccines have several disadvantages: 1) they persist indefinitely in vaccinated macaques; 2) they are pathogenic to neonatal macaques; and 3) they are lethal in some adult macaques. To enhance the safety and efficacy of these vaccines, we immunized macaques first with recombinant vaccines and then inoculated the animals with SIVΔnef. In the first experiment,
preimmunized macaques advanced to disease slower than controls after
challenge with virulent SIV; five animals survived for 3 years without
disease and only the vaccine virus (SIVΔnef) could be isolated at this time. In the second experiment, preimmunized animals had lower virus loads and no disease compared to controls.
Induction of HIV‐1‐specific T‐helper responses and type 1 cytokine secretion following therapeutic vaccination of macaques with a recombinant fowlpoxvirus co‐expressing interferon‐gamma
Authors: Dale C. Jane , Anne  , Zhao  , Jones Stephen L. , Boyle David B. , Ramshaw Ian A. , Kent Stephen J.
Published: November 2003

Preventive and/or therapeutic vaccines against Human Immunodeficiency Virus (HIV‐1) are urgently required. Induction of cellular immunity is favoured since these responses correlate with control of HIV‐1. Recombinant fowlpoxvirus (FPV) vaccines encoding both HIV‐1 gag/pol and interferon‐gamma (FPV gag/pol‐IFNΓ) were hypothesised to enhance HIV‐specific cellular immunity and were further evaluated in macaques previously infected with HIV‐1. A novel assay to detect IFNΓ secretion following HIV antigen stimulation of whole blood was developed to further assess the safety and immunogenicity of the FPV gag/pol‐IFNΓ vaccine. Immunisation with FPV gag/pol‐IFNΓ safely enhanced HIV‐specific IFNΓ secretion following ex vivo
stimulation of whole blood, greater than that observed following FPV
gag/pol vaccination not co‐expressing IFNΓ. Both HIV‐specific
IFNΓ‐spot‐forming cells by ELISPOT and CD69 expression by
CD4+ lymphocytes were also enhanced following FPV gag/pol‐IFNΓ vaccination. Hence, the FPV‐HIV vaccine co‐expressing IFNΓ stimulated HIV‐specific T cell responses in macaques, and should be further evaluated as a therapeutic or preventive HIV vaccine.
A vaccine strategy utilizing a combination of three different chimeric vectors which share specific vaccine antigens
Authors: Heeney Jonathan L. , Koopman Gerrit , Rosenwirth Brigitte , Bogers Willy , Van Dijk Jeanette , Nieuwenhuis Ivonne , Niphuis Henk , Ten Haaft Peter , Hanke Thomas , Rhodes Gary , Berglund Peter , Burny Arsene , Bex Francoise , Sutter Gerd , Liljeström Peter
Published: November 2003

A large number of recombinant of viral and bacterial systems have been engineered as vectors to express foreign genes for vaccination and/or gene therapy. A common problem is the immune response to the vector itself. The presence of anti‐vector immune responses may preclude sufficient ‘priming’ or immunogenicity if pre‐existing immune responses are present, or they may impair optimal ‘boosting’ upon repeated immunization or delivery with the same vector. To circumvent this problem we developed a strategy using different chimeric vectors which share only the expression of common specific antigens desired for immunization. This approach not only has the advantage of avoiding increased anti‐vector responses, but allows the use of combinations of vectors which could subsequently present the same or related antigen differently to the immune system as well as at alternative sites to induce the optimal type of immunity against the pathogen of interest.
Extended pharmacological profiles of rat P2Y2 and rat P2Y4 receptors and their sensitivity to extracellular H+ and Zn2+ ions
Authors: Wildman Scott S , Unwin Robert J , King Brian F
Published: November 2003

Two molecularly distinct rat P2Y receptors activated equally by adenosine‐5′‐triphosphate (ATP) and uridine‐5′‐triphosphate (UTP) (rP2Y2 and rP2Y4 receptors) were expressed in Xenopus oocytes and studied extensively to find ways to pharmacologically distinguish one from the other.

Both P2Y subtypes were activated fully by a number of nucleotides. Tested nucleotides were equipotent at rP2Y4 (ATP=UTP=CTP=GTP=ITP), but not at rP2Y2 (ATP=UTP>CTP>GTP>ITP). For dinucleotides (ApnA, n=2–6), rP2Y4 was only fully activated by Ap4A, which was as potent as ATP. All tested dinucleotides, except for Ap2A, fully activated rP2Y2, but none were as potent as ATP. ATPγS and BzATP fully activated rP2Y2, whereas ATPγS was a weak agonist and BzATP was inactive (as an agonist) at rP2Y4 receptors.

Each P2Y subtype showed different sensitivities to known P2 receptor antagonists. For rP2Y2, the potency order was suramin>>PPADS= RB‐2>TNP‐ATP and suramin was a competitive antagonist (pA2, 5.40). For rP2Y4, the order was RB‐2>>suramin>PPADS> TNP‐ATP and RB‐2 was a competitive antagonist (pA2, 6.43). Also, BzATP was an antagonist at rP2Y4 receptors.

Extracellular acidification (from pH 8.0 to pH 5.5) enhanced the potency of ATP and UTP by 8–10‐fold at rP2Y4 but did not affect agonist responses at rP2Y2 receptors.

Extracellular Zn2+ ions (0.1–300 μM) coapplied with ATP inhibited agonist responses at rP2Y4 but not at rP2Y2 receptors.

These two P2Y receptors differ significantly in terms of agonist and antagonist profiles, and the modulatory activities of extracellular H+ and Zn2+ ions. These pharmacological differences will help to distinguish between rP2Y2 and rP2Y4 receptors, in vivo.

British Journal of Pharmacology (2003) 140, 1177–1186. doi:10.1038/sj.bjp.0705544
Catalase has negligible inhibitory effects on endothelium‐dependent relaxations in mouse isolated aorta and small mesenteric artery
Authors: Ellis Anthie , Pannirselvam Malarvannan , Anderson Todd J , Triggle Chris R
Published: November 2003

The current study examined the hypothesis that endothelial production of hydrogen peroxide (H2O2) mediates relaxations to acetylcholine (ACh) in aorta and small mesenteric arteries (SMA) from mice.

Relaxations to ACh (0.01–10 μM) and H2O2 (0.1–1000 μM) were produced in aorta and SMA isolated from wild‐type C57BL/6 mice and type II diabetic mice (db/db). In SMA, relaxations to ACh were produced in the presence of Nω‐nitro‐L‐arginine methyl ester (100 μM) and indomethacin (Indo, 10 μM).

1‐H[1,2,4]oxadiazolo[4,3‐]quinoxalin‐1‐one (10 μM) significantly reduced ACh‐induced relaxations in SMA, abolished responses in aorta, but had no effect on relaxations induced by H2O2. Catalase (2500 U ml−1) abolished responses to H2O2, but did not alter relaxations to ACh in the SMA and only caused a small rightward shift in responses to ACh in the aorta.

ACh‐, but not H2O2‐, mediated relaxations were significantly reduced by tetraethylammonium (10 mM), the combination of apamin (1 μM) and charybdotoxin (100 nM), and 25 mM potassium chloride (KCl). Higher KCl (60 mM) abolished relaxations to both ACh and H2O2. Polyethylene glycolated superoxide dismutase (100 U ml−1), the catalase inhibitor 3‐amino‐1,2,4‐triazole (3‐AT, 50 mM) and treatment with the copper chelator diethyldithiolcarbamate (3 mM) did not affect relaxations to ACh.

H2O2‐induced relaxations were endothelium‐independent and were not affected by ethylene diamine tetraacetic acid (EDTA 0.067 mM), 4‐aminopyridine (1 mM), ouabain (100 μM) and barium (30 μM), 3‐AT or Indo.

Although the data from this study show that H2O2 dilates vessels, they do not support the notion that H2O2 mediates endothelium‐dependent relaxations to ACh in either aorta or SMA from mice.

British Journal of Pharmacology (2003) 140, 1193–1200. doi:10.1038/sj.bjp.0705549
Decreased expression of glial cell line‐derived neurotrophic factor signaling in rat models of neuropathic pain
Authors: Nagano Masatoshi , Sakai Atsushi , Takahashi Naoki , Umino Masahiro , Yoshioka Koichi , Suzuki Hidenori
Published: November 2003

In an attempt to clarify whether glial cell line‐derived neurotrophic factor (GDNF), a survival factor for subpopulations of primary afferent neurons, is involved in the states of neuropathic pain, we observed changes in the expressions of GDNF and its signal‐transducing receptor Ret after nerve injury in two rat models of neuropathic pain.

In the rats treated with sciatic nerve ligation (chronic constrictive injury (CCI) model) or spinal nerve ligation at L5 (SNL model), the thresholds of paw withdrawal in response to mechanical or heat stimuli began to decrease on the injured side within the first week after the operation and the decreases in the thresholds persisted for more than 2 weeks.

In CCI‐treated rats, the GDNF contents in L4 and L5 dorsal root ganglia (DRGs) on the injured side were markedly decreased at day 7 after the operation and stayed at low levels at day 14. In SNL‐treated rats, comparable reductions of GDNF levels in L4 and L5 DRGs on the injured side were observed at 14 postoperative days.

Significant decreases of the percentages of DRG neurons expressing Ret were also observed at L4 DRGs in CCI‐treated rats at 7 and 14 postoperative days and in SNL‐treated rats at 14 days.

In CCI‐ or SNL‐treated rats, continuous intrathecal administration of GDNF (12 μg day−1) using an osmotic pump suppressed the increased sensitivities to nociceptive stimuli to control levels.

The present results suggested that the dysfunction of GDNF signaling in the nociceptive afferent system may contribute to the development and/or maintenance of neuropathic pain states.

British Journal of Pharmacology (2003) 140, 1252–1260. doi:10.1038/sj.bjp.0705550
Inhibition of human α7 nicotinic acetylcholine receptors by open channel blockers of N‐methyl‐D‐aspartate receptors
Authors: Maskell Peter D , Speder Pauline , Newberry Nigel R , Bermudez Isabel
Published: November 2003

Human α7 nicotinic acetylcholine (ACh) receptors were expressed in Xenopus oocytes and the effects of the N‐methyl‐D‐aspartate (NMDA) receptor open channel blockers memantine and cerestat on this receptor were examined using two‐electrode voltage‐clamp recordings and 125I‐α‐bungarotoxin (125I‐α‐bgtx) binding.

Memantine and cerestat produced complete inhibition of ACh‐induced inward currents with affinities similar to that reported for native NMDA receptors. Cerestat, IC50 1.7 (−1; +2) μM, was more potent than memantine, IC50 5 (−3;+8) μM, and the effects of both drugs were fully and rapidly reversible.

Inhibition of α7 receptor function was voltage‐independent, and it occurred at concentrations far lower than those needed to inhibit (never completely) binding of 125I‐α‐bgtx to α7 receptors, suggesting that the effects of memantine or cerestat are noncompetitive.

These results provide evidence that human α7 receptors are inhibited by memantine and cerestat and suggest that caution should be applied when using these compounds to study systems in which NMDA and nACh receptors co‐exist.

British Journal of Pharmacology (2003) 140, 1313–1319. doi:10.1038/sj.bjp.0705559
Expression and effects of cardiotrophin‐1 (CT‐1) in human airway smooth muscle cells
Authors: Zhou Danyi , Zheng Xueyan , Wang Lu , Stelmack Gerald , Halayko Andrew J , Dorscheid Delbert , Bai Tony R
Published: November 2003

Cellular hypertrophy and/or a reduced rate of apoptosis could increase airway smooth muscle mass. As cardiotrophin‐1 (CT‐1) induces hypertrophy and inhibits apoptosis in cardiomyocytes, we tested for the expression and effects of CT‐1 in human bronchial smooth muscle cells (HBSMC).

CT‐1 was detected in abundance in normal adult human lung and was expressed in both fetal and adult HBSMC.

Following serum deprivation, CT‐1 was released by reintroduction of serum and by TGF‐β2/IL‐4 in fetal but not adult cells. TGF‐β2/IL‐4 triggered the release of CT‐1 in serum‐fed adult cells. Hypoxia and strain had no effect on the release of CT‐1.

CT‐1 reduced the apoptosis induced both by serum deprivation and by Fas antibody/TNF‐α treatment in adult cells, with greater efficacy than other members of the IL‐6 superfamily. The MAPK/ERK kinase inhibitor PD98059 (1–10 μM) reduced the effect of CT‐1. Fetal cells were more resistant to apoptosis.

CT‐1 (10 ng ml−1) induced a significant increase in cell size as judged by protein/DNA ratios and flow cytometry. No effects on smooth muscle α‐actin or vimentin proteins were noted, although CT‐1 qualitatively alters the cytostructural distribution of SM22, an actin filament‐associated protein, and increased SM22 protein abundance. No effect on proliferation or migration was evident.

These data suggest CT‐1 expression primarily in fetal and synthetic HBSMC phenotypes. By reducing the rates of apoptosis and inducing hypertrophy, CT‐1 may contribute to increased smooth muscle mass in airway disease.

British Journal of Pharmacology (2003) 140, 1237–1244. doi:10.1038/sj.bjp.0705562
Adenosine 3′, 5′ cyclic monophosphate attenuates the production of fibronectin in the glomeruli of anti‐glomerular basement membrane antibody‐associated nephritic rats
Authors: Nagamatsu Tadashi , Nishiyama Tsutomu , Goto Isamu , Nagao Toshiyuki , Suzuki Yoshio
Published: November 2003

Excessive production of extracellular matrix is thought to be involved in the progression of glomerulonephritis and glomerulosclerosis. In chronic glomerulonephritis, fibronectin has been shown to accumulate in the glomeruli, accompanied by cell proliferation.

Glomerulonephritis was induced in rats by the injection of anti‐glomerular basement membrane antibody. The rats showed proteinuria and histological alterations in the glomeruli. An increase in fibronectin levels in the culture medium of isolated nephritic glomeruli was confirmed, and was associated with the development of nephritis. Immunohistochemical staining demonstrated a marked accumulation of fibronectin in the glomeruli of nephritic rats.

Attenuated generation of cyclic AMP was also observed in the nephritic glomeruli treated with forskolin, prostaglandin E1 or adenosine.

Forskolin, prostaglandin E2 and 8‐bromo‐cyclic AMP markedly reduced the production of fibronectin by the nephritic glomeruli compared with controls in a dose‐dependent manner. 8‐Bromo‐cyclic AMP suppressed the production of fibronectin by cultured mesangial cells.

These findings suggest that the attenuated generation of cyclic AMP in response to ligands is connected to the augmented accumulation of fibronectin in nephritic glomeruli, and may facilitate the development of methods for treating glomerulonephritis and glomerulosclerosis.

British Journal of Pharmacology (2003) 140, 1245–1251. doi:10.1038/sj.bjp.0705564
Papain immobilization on a nitrilon fibre carrier containing primary amine groups
Authors: Li Yan Feng , Jia Fu Yun , Li Jun Rong , Liu Gang , Li Yao Zeng
Published: November 2003

A carrier containing primary amine groups was synthesized by the reaction between nitrilon fibre and diethylenetriamine, and papain was immobilized on this kind of carrier by using glutaraldehyde as a coupling agent. The factors involved with the activity recovery of the immobilized papain and the enzymic properties of the resulting immobilized papain were studied in comparison with free papain, for which casein was chosen as a substrate. The results show that the activity recovery of immobilized papain can reach 47·3%, and that the immobilized papain exhibits better environmental adaptability and reusability than free papain.
Selecting and expressing protective single‐chain Fv fragment to stabilize L‐asparaginase against inactivation by trypsin
Authors: Guo Li , Yan Xiyun , Qian Shijun , Meng Guangzhen
Published: November 2003

Four non‐inhibitory specific single‐chain Fv (sc Fv) fragments directed against L‐asparaginase (ASNase) of Escherichia coli were selected from a synthetic phage‐display scFv library. The scFv46 fragment could enhance the resistance of ASNase to trypsin proteolysis, with 70% of the initial ASNase activity present after the ASNase–scFv46 complex had been treated with trypsin for 30 min at 37 °C, whereas little residual activity was detected without the scFv46 fragment. The scFv46 gene was cloned to an expression vector pET‐21a and expressed at high levels (about 45% of total cell protein) in E. coli BL21 (DE3) as inclusion bodies. The refolded and purified scFv46 fragment was proved to protect ASNase, and the protective effect was further confirmed by SDS/PAGE. It was found that under optimum conditions of molar ratio of scFv to ASNase, incubation time and temperature, the residual activity of the ASNase–scFv46 complex could reach about 78% after treatment with trypsin for 30 min at 37 °C. The results demonstrated that scFv fragments prepared by phage‐antibody library technology could be used to protect target proteins.
Yeast protease B‐digested skimmed milk inhibits angiotensin‐I‐converting‐enzyme activity
Authors: Roy Molay Kumar , Watanabe Yasuo , Tamai Youichi
Published: November 2003

Angiotensin‐1‐converting‐enzyme (ACE) inhibitoryactivity was identified in skimmed milk digested with cell‐free extract of yeast Saccharomyces cerevisiae. Simultaneously, a protease enzyme involved in the production of ACE‐inhibition materials in digested skimmed milk was purified to homogeneity from the cell‐free extracts of S. cerevisiae by ammonium sulphate fractionation and chromatography in DEAE‐Sephacel, D‐tryptophan methyl ester‐Sepharose 4B, Hiload Superdex G‐200 and HPLC Mono‐Q chromatography. The purified enzyme was identified as protease B, based on the molecular mass on SDS/PAGE and the N‐terminal amino acid sequence of the enzyme. The optimum pH for digestion of skimmed milk and production of ACE‐inhibition materials was pH 4.8. The IC50 of the hydrolysate was 0.42 mg of protein/ml when skimmed milk was digested with yeast protease B
Purification and properties of a lipase from Cephaloleia presignis (Coleoptera, Chrysomelidae)
Authors: Arreguín‐Espinosa Roberto , Arreguín Barbarín , González Carolina
Published: November 2003

A novel lipase from the insect Cephaloleia presignis was purified by a procedure involving ammonium sulphate precipitation, and Phenyl Toyopearl 650M, DEAE‐5PW and hydrophobic‐interaction column chromatographies. The purified lipase was homogeneous with a molecular mass of 31000 Da by SDS/PAGE and of 29000 Da by gel filtration on a Superose 12 column. The enzyme was indentified as a glycoprotein with a pI of 6.9. The enzyme unspecifically liberated short‐chain to long‐chain fatty acids from p‐nitrophenyl esters, methyl esters and triglycerides. The N‐terminal 28 amino acid residues were determined as AGTLGYATRHVLPIFTLDDYTGSNEMWG, which showed no similarity with known proteins, suggesting that the purified lipase may belong to a novel class of hydrolases.
An evaluation of hybridization kinetics in biosensors using a single‐fractal analysis
Authors: Vontel Sunita , Ramakrishnan Anand , Sadana Ajit
Published: November 2003

The diffusion‐limited hybridization kinetics of analyte in solution to receptor immobilized on a biosensor or immunosensor surface is analysed within a fractal framework. The data may be analysed by a single‐fractal analysis. This was indicated by the regression analysis provided by Sigmaplot [Sigmaplot Users Manual (1993) Jandel Scientific, San Rafael, CA]. It is of interest to note that the binding‐rate coefficient and the fractal dimension both exhibit changes in the same and in the opposite directions for the single example presented in each case. The binding‐rate coefficient(s) expressions developed as a function of the analyte (DNA) concentration in solution and the fractal dimension are of particular value, since they provide a means to better control biosensor or immunosensor performance and provide physical insights into the hybridization process.
Inhibitory and lytic effects of phenothiazine derivatives and related tricyclic neuroleptic compounds, on Entamoeba histolytica HK9 and HM1 trophozoites
Authors: Ondarza Raúl N. , Hernández Eva , Iturbe Angélica , Hurtado Gerardo
Published: November 2003

It has been shown previously that tricyclic neuroleptics like clomipramine and chlorpromazine have lethal effects on Leishmania donovani and L. major, and other studies indicate that the phenothiazine inhibitors of trypanothione reductase are potential anti‐trypanosomal and anti‐leishmanial drugs. With this in mind and our original observation on the presence of trypanothione in Entamoeba histolytica HK9, we examined the possible inhibitory effects of various phenothiazine and tricyclic derivatives on this human parasite. We found that drugs like clomipramine (KD002), the most potent in vitro inhibitor of trypanothione reductase among 30 tricyclic compounds tested, at 25 μM after 24 h of culture under aerobic conditions, caused a substantial decrease in the number of E. histolytica HK9 trophozoites, from approx. 15×106 to 5·37×106 cells, and at 100 μM to 0·8×106 cells. A substantial inhibitory effect on cell proliferation could also be demonstrated with metronidazol (used clinically against amoebiasis). Under similar experimental conditions other tricyclic and phenothiazine derivatives (OFKs), designed originally to inhibit the trypanothione reductase of trypanosomatides, had an inhibitory effect of 16 to 95%. For comparison, similar results were obtained using clomipramine and a phenothiazine derivative (OFK006) with Trypanosoma cruzi and Crithidia luciliae, except that with the latter the inhibitory effect of clomipramine was less dramatic. Experiments comparing two E. histolytica strains showed that normal cell proliferation under anaerobiosis was higher in strain HK9 than in HM1, which is highly virulent, but that metronidazol and clomipramine were less effective against HM1. Two other drugs tested, diphenydramine (KD005) and a phenothiazine derivative (OFK008), also had significant but lower inhibitory effects on both strains. The inhibitory activity on cell proliferation and the lytic effects on this human parasite by the tricyclic compounds clomipramine, chlorpromazine and others, as well as by the phenothiazine derivatives, indicate that they can be considered potential anti‐amoebic agents.
A kinetic study of the synthesis of ascorbate fatty acid esters catalysed by immobilized lipase in organic media
Authors: Luhong Tang , Hao Zhang , Shehate Manal M. , Yunfei Sun
Published: November 2003

The kinetics of the synthesis of l‐ascorbyl laurate and l‐ascorbyl palmitate catalysed by immobilized lipase from l‐ascorbic acid and an acyl donor (lauric acid, palmic acid and their methyl and ethyl esters) in 2‐methylbutan‐2‐ol have been investigated. The factors affecting the reaction rate (shaking speed, temperature, water activity, enzyme concentration as well as substrate concentration) are discussed. The reaction conditions have been optimized as follows: shaking speed 200 rev./min, temperature 55 °C, enzyme 17–20% (w/w of substrate) for these substrates. The most suitable substrate concentration for all of these substrates was 300 mmol/l. The reactions were modelled. For lauric acid, methyl laurate and ethyl laurate: Km=74.3, 48.97 and 55.8 mmol/l, respectively; and Vmax=0.010764, 0.0114, 0.01116 mmol·min−1·g−1, respectively. The most suitable substrate was methyl laurate. For palmic acid, methyl palmitate and ethyl palmitate: Km=102, 94.9 and 104 mmol/l, respectively; and Vmax=0.0417, 0.0424 and 0.0435 mmol·min−1·g−1, respectively. The most suitable substrate was methyl palmitate.
Differences in the post‐translational modifications of human papillomavirus type 6b major capsid protein expressed from a baculovirus system compared with a vaccinia virus system
Authors: Fang NingXia , Frazer Ian H. , Fernando Germain J. P.
Published: November 2003

Virus‐like particles (VLPs) are being currently investigated in vaccines against viral infections in humans. There are different recombinant‐protein‐expression systems available for obtaining the necessary VLP preparation for vaccination. However, the differences in post‐translational modifications of the recombinant proteins obtained and their differences in efficacy in eliciting an anti‐viral response in vaccines are not well established. In this study we have compared the post‐translational modifications of human papillomavirus type‐6b major capsid protein L1 (HPV 6bL1) expressed using recombinant baculovirus (rBV) in Sf9 (Spodoptera frugiperda) insect cells, with the protein expressed using recombinant vaccinia virus (rVV) in CV‐1 kidney epithelial cells. Two‐dimensional gel electrophoresis of biosynthetically labelled rBV‐expressed HPV 6bL1 showed several post‐translationally modified variants of the protein, whereas rVV‐expressed HPV 6bL1 showed only a few variants. Phosphorylations were detected at threonine and serine residues for the L1 expressed from rBV compared with phosphorylation at serine residues only for the L1 expressed from rVV. HPV 6bL1 expressed using rBV incorporated [3H]mannose and [3H]galactose, whereas HPV 6bL1 expressed using rVV incorporated only [3H]galactose. We conclude that post‐translational modification of recombinant HPV 6bL1 can differ according to the system used for its expression. Since recombinant L1 protein is a potential human‐vaccine candidate, the implication of the observed differences in post‐translational modifications on immunogenicity of L1 VLPs warrants investigation.
Effect of amino acid substitution at the P3 and P4 subsites of fusion proteins on Kex2 protease activity
Authors: Suzuki Yuji , Ikeda Nobue , Kataoka Eri , Ohsuye Kazuhiro
Published: November 2003

The effect of amino acid substitution at the P3 and P4 subsites on the cleavage activity of a recombinant secretory‐type Kex2 protease (Kex2‐660) was investigated using recombinant fusion proteins. They were constructed from a truncated Escherichia coli β‐galactosidase (βG‐117S) followed by human parathyroid hormone amino acids 1–34 [hPTH(1‐34)] with a junction designed to allow cleavage with Kex2‐660. Kex2‐660 preferentially cleaved the fusion protein with Arg‐His at the P3‐P4 subsite, whereas it poorly cleaved the fusion proteins with Val‐Asp, Gly‐His, Cys‐His and Leu‐His compared with the original sequence, Val‐Lys. As for Asp and Glu at P4, they precluded the cleavage almost completely. Some of the substitutions were investigated kinetically using the fusion proteins and peptide‐substrate mimics corresponding to the 15 amino acids contained in the junction. The substitution Val‐Lys to Arg‐His resulted in a 2‐fold increase in kcat and the introduction of Asp at P4 of the peptide substrate resulted in a large increase in Km and a change in the optimum pH. The ordered cleavage of the fusion protein was attained by introducing the two dibasic sites, Arg‐His and Asp‐His, at P4‐P3. The fusion protein was cleaved with Kex2 only at the former site in 3.0 M urea at pH 8.0 and the liberated peptides containing the latter site could be cleaved further with Kex2 at pH 6.5 once purified in urea‐free solution. Thus Kex2 exhibited extended substrate specificity beyond P2‐P1 in the cleavage of the fusion proteins, although it depended on the reaction conditions.
Expression and immunological evaluation of the Escherichia coli ‐derived hepatitis C virus envelope E1 protein
Authors: Lorenzo Lázaro J. , García Odalys , Acosta‐Rivero Nelson , Duen˜as‐Carrera Santiago , Martínez Gillian , Álvarez‐Obregón Julio , Pichardo Dagmara , Ramos Astrid , Guerra Ivis , Morales Juan
Published: November 2003

Immunological response against envelope protein E1 is very important in natural hepatitis C virus (HCV) infection, although it is insufficient to clear the viraemia. The HCV genomic region encoding the first 149 amino acids of the envelope E1 protein (E1340, amino acids 192–340) was expressed in Escherichia coli (to a level of 30% of the whole cellular proteins) and purified to 85%. We measured the immune response in rabbits and mice as well as the reactivity against 37 human sera raised against the whole recombinant protein and E1‐encoding peptides. From this, 51.1% of human sera were found to react with E1340. High‐level antibodies against E1340 were obtained in rabbits and mice when immunized. These antibodies had a similar peptide‐recognition pattern to that described previously for human sera. The most reactive region was located at the N‐terminus of the E1 protein. Cellular immunity in mice was evaluated by delayed‐type hypersensitivity assay. It revealed the induction of a CD4+ T‐cell‐mediated response by this protein. This E1340 protein and the animal‐derived anti‐E1 sera are immunological tools that could aid in the monitoring and development of anti‐HCV therapies.