bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a
Vibrio cholerae
RTX toxin gene cluster that is tightly linked to the cholera toxin prophage
Authors: Lin Wei , Fullner Karla Jean , Clayton Rebecca , Sexton Jessica A. , Rogers Marc B. , Calia Kerstin E. , Calderwood Stephen B. , Fraser Claire , Mekalanos John J.
Published: July 2002

We identify and characterize a gene cluster in El Tor
Vibrio cholerae
that encodes a cytotoxic activity for HEp-2 cells
in vitro
. This gene cluster contains four genes and is physically linked to the cholera toxin (CTX) element in the
V. cholerae
genome. We demonstrate by using insertional mutagenesis that this gene cluster is required for the cytotoxic activity. The toxin, RtxA, resembles members of the RTX (repeats in toxin) toxin family in that it contains a GD-rich repeated motif. Like other RTX toxins, its activity depends on an activator, RtxC, and an associated ABC transporter system, RtxB and RtxD. In
V. cholerae
strains of the classical biotype, a deletion within the gene cluster removes
rtxC
and eliminates cytotoxic activity. Other strains, including those of the current cholera pandemic, contain a functional gene cluster and display cytotoxic activity. Thus, the RTX gene cluster in El Tor O1 and O139 strains might have contributed significantly to their emergence. Furthermore, the RTX toxin of
V. cholerae
may be associated with residual adverse properties displayed by certain live, attenuated cholera vaccines.
A gene therapy for cancer using intramuscular injection of plasmid DNA encoding interferon α
Authors: Horton Holly M. , Anderson Deborah , Hernandez Pepe , Barnhart Kerry M. , Norman Jon A. , Parker Suezanne E.
Published: July 2002

A cancer treatment is described in which i.m. injection of plasmid DNA (pDNA) encoding murine interferon α (mIFN-α) leads to potent antitumor effects on primary and metastatic tumors in mice. Mice bearing s.c. B16F10 melanoma, Cloudman melanoma, or glioma 261 tumors were injected i.m. with mIFN-α pDNA. In all three tumor models, a significant reduction in tumor volume and enhancement of survival was found after IFN pDNA therapy. The mIFN-α pDNA could be injected as infrequently as once every other week and still produce a significant antitumor effect, and, in a metastatic tumor model, the therapy markedly reduced the number of lung tumor metastases. Depletion of immune cell subsets indicated that CD8+T cells were required for the antitumor response. These studies demonstrate that primary and metastatic tumors can be treated systemically by i.m. injection of a plasmid encoding a cytokine gene.
Direct interaction of lignin and lignin peroxidase from
Phanerochaete chrysosporium
Authors: Johjima Toru , Itoh Noriyuki , Kabuto Mari , Tokimura Fusayo , Nakagawa Tamon , Wariishi Hiroyuki , Tanaka Hiroo
Published: July 2002

Binding properties of lignin peroxidase (LiP) from the basidiomycete
Phanerochaete chrysosporium
against a synthetic lignin (dehydrogenated polymerizate, DHP) were studied with a resonant mirror biosensor. Among several ligninolytic enzymes, only LiP specifically binds to DHP. Kinetic analysis revealed that the binding was reversible, and that the dissociation equilibrium constant was 330 μM. The LiP–DHP interaction was controlled by the ionization group with a pK
a
of 5.3, strongly suggesting that a specific amino acid residue plays a role in lignin binding. A one-electron transfer from DHP to oxidized intermediates LiP compounds I and II (LiPI and LiPII) was characterized by using a stopped-flow technique, showing that binding interactions of DHP with LiPI and LiPII led to saturation kinetics. The dissociation equilibrium constants for LiPI–DHP and LiPII–DHP interactions were calculated to be 350 and 250 μM, and the first-order rate constants for electron transfer from DHP to LiPI and to LiPII were calculated to be 46 and 16 s
−1
, respectively. These kinetic and spectral studies strongly suggest that LiP is capable of oxidizing lignin directly at the protein surface by a long-range electron transfer process. A close look at the crystal structure suggested that LiP possesses His-239 as a possible lignin-binding site on the surface, which is linked to Asp-238. This Asp residue is hydrogen-bonded to the proximal His-176. This His–Asp⋅⋅⋅proximal-His motif would be a possible electron transfer route to oxidize polymeric lignin.
Imaging adenoviral-directed reporter gene expression in living animals with positron emission tomography
Authors: Gambhir Sanjiv S. , Barrio Jorge R. , Phelps Michael E. , Iyer Meera , Namavari Mohammad , Satyamurthy Nagichettiar , Wu Lily , Green Leeta A. , Bauer Eileen , MacLaren Duncan C. , Nguyen Khoi , Berk Arnold J. , Cherry Simon R. , Herschman Harvey R.
Published: July 2002

We are developing quantitative assays to repeatedly and noninvasively image expression of reporter genes in living animals, using positron emission tomography (PET). We synthesized positron-emitting 8-[18F]fluoroganciclovir (FGCV) and demonstrated that this compound is a substrate for the herpes simplex virus 1 thymidine kinase enzyme (HSV1-TK). Using positron-emitting FGCV as a PET reporter probe, we imaged adenovirus-directed hepatic expression of the HSV1-tk reporter gene in living mice. There is a significant positive correlation between the percent injected dose of FGCV retained per gram of liver and the levels of hepatic HSV1-tk reporter gene expression (
r
2
> 0.80). Over a similar range of HSV1-tk expression
in vivo
, the percent injected dose retained per gram of liver was 0–23% for ganciclovir and 0–3% for FGCV. Repeated, noninvasive, and quantitative imaging of PET reporter gene expression should be a valuable tool for studies of human gene therapy, of organ/cell transplantation, and of both environmental and behavioral modulation of gene expression in transgenic mice.
The chemokine monocyte chemoattractant protein-1 induces functional responses through dimerization of its receptor CCR2
Authors: Rodríguez-Frade José Miguel , Vila-Coro Antonio J. , Martín de Ana Ana , Albar Juan Pablo , Martínez-A. Carlos , Mellado Mario
Published: July 2002

Cytokines interact with hematopoietin superfamily receptors and stimulate receptor dimerization. We demonstrate that chemoattractant cytokines (chemokines) also trigger biological responses through receptor dimerization. Functional responses are induced after pairwise crosslinking of chemokine receptors by bivalent agonistic antichemokine receptor mAb, but not by their Fab fragments. Monocyte chemoattractant protein (MCP)-1-triggered receptor dimerization was studied in human embryonic kidney (HEK)-293 cells cotransfected with genes coding for the CCR2b receptor tagged with YSK or Myc sequences. After MCP-1 stimulation, immunoprecipitation with Myc-specific antibodies revealed YSK-tagged receptors in immunoblotting. Receptor dimerization also was validated by chemical crosslinking in both HEK-293 cells and the human monocytic cell line Mono Mac 1. Finally, we constructed a loss-of-function CCR2bY139F mutant that acted as a dominant negative, blocking signaling through the CCR2 wild-type receptor. This study provides functional support for a model in which the MCP-1 receptor is activated by ligand-induced homodimerization, allowing discussion of the similarities between bacterial and leukocyte chemotaxis.
Abnormal photoresponses and light-induced apoptosis in rods lacking rhodopsin kinase
Authors: Chen Ching-Kang , Burns Marie E. , Spencer Maribeth , Niemi Gregory A. , Chen Jeannie , Hurley James B. , Baylor Denis A. , Simon Melvin I.
Published: July 2002

Phosphorylation is thought to be an essential first step in the prompt deactivation of photoexcited rhodopsin.In vitro, the phosphorylation can be catalyzed either by rhodopsin kinase (RK) or by protein kinase C (PKC). To investigate the specific role of RK, we inactivated both alleles of the RK gene in mice. This eliminated the light-dependent phosphorylation of rhodopsin and caused the single-photon response to become larger and longer lasting than normal. These results demonstrate that RK is required for normal rhodopsin deactivation. When the photon responses of RK−/− rods did finally turn off, they did so abruptly and stochastically, revealing a first-order backup mechanism for rhodopsin deactivation. The rod outer segments of RK−/− mice raised in 12-hr cyclic illumination were 50% shorter than those of normal (RK+/+) rods or rods from RK−/− mice raised in constant darkness. One day of constant light caused the rods in the RK−/− mouse retina to undergo apoptotic degeneration. Mice lacking RK provide a valuable model for the study of Oguchi disease, a human RK deficiency that causes congenital stationary night blindness.
Solid-state synthesis and mechanical unfolding of polymers of T4 lysozyme
Authors: Yang Guoliang , Cecconi Ciro , Baase Walter A. , Vetter Ingrid R. , Breyer Wendy A. , Haack Julie A. , Matthews Brian W. , Dahlquist Frederick W. , Bustamante Carlos
Published: July 2002

Recent advances in single molecule manipulation methods offer a novel approach to investigating the protein folding problem. These studies usually are done on molecules that are naturally organized as linear arrays of globular domains. To extend these techniques to study proteins that normally exist as monomers, we have developed a method of synthesizing polymers of protein molecules in the solid state. By introducing cysteines at locations where bacteriophage T4 lysozyme molecules contact each other in a crystal and taking advantage of the alignment provided by the lattice, we have obtained polymers of defined polarity up to 25 molecules long that retain enzymatic activity. These polymers then were manipulated mechanically by using a modified scanning force microscope to characterize the force-induced reversible unfolding of the individual lysozyme molecules. This approach should be general and adaptable to many other proteins with known crystal structures. For T4 lysozyme, the force required to unfold the monomers was 64 ± 16 pN at the pulling speed used. Refolding occurred within 1 sec of relaxation with an efficiency close to 100%. Analysis of the force versus extension curves suggests that the mechanical unfolding transition follows a two-state model. The unfolding forces determined in 1 M guanidine hydrochloride indicate that in these conditions the activation barrier for unfolding is reduced by 2 kcal/mol.
Detection and selective dissociation of intact ribosomes in a mass spectrometer
Authors: Rostom Adam A. , Fucini Paola , Benjamin Dennis R. , Juenemann Ralf , Nierhaus Knud H. , Hartl F. Ulrich , Dobson Christopher M. , Robinson Carol V.
Published: July 2002

Intact
Escherichia coli
ribosomes have been projected into the gas phase of a mass spectrometer by means of nanoflow electrospray techniques. Species with mass/charge ratios in excess of 20,000 were detected at the level of individual ions by using time-of-flight analysis. Once in the gas phase the stability of intact ribosomes was investigated and found to increase as a result of cross-linking ribosomal proteins to the rRNA. By lowering the Mg
2+
concentration in solutions containing ribosomes the particles were found to dissociate into 30S and 50S subunits. The resolution of the charge states in the spectrum of the 30S subunit enabled its mass to be determined as 852,187 ± 3,918 Da, a value within 0.6% of that calculated from the individual proteins and the 16S RNA. Further dissociation into smaller macromolecular complexes and then individual proteins could be induced by subjecting the particles to increasingly energetic gas phase collisions. The ease with which proteins dissociated from the intact species was found to be related to their known interactions in the ribosome particle. The results show that emerging mass spectrometric techniques can be used to characterize a fully functional biological assembly as well as its isolated components.
Overexpression of the human VPAC
2
receptor in the suprachiasmatic nucleus alters the circadian phenotype of mice
Authors: Shen Sanbing , Spratt Christopher , Sheward W. John , Kallo Imre , West Katrine , Morrison Christine F. , Coen Clive W. , Marston Hugh M. , Harmar Anthony J.
Published: July 2002

The neuropeptides vasoactive intestinal peptide (VIP) and pituitary
adenylate cyclase-activating polypeptide (PACAP) belong to a
superfamily of structurally related peptide hormones that includes
glucagon, glucagon-like peptides, secretin, and growth
hormone-releasing hormone. Microinjection of VIP or PACAP into the
rodent suprachiasmatic nucleus (SCN) phase shifts the circadian
pacemaker and VIP antagonists, and antisense oligodeoxynucleotides have
been shown to disrupt circadian function. VIP and PACAP have equal
potency as agonists of the VPAC
2
receptor
(VPAC
2
R), which is expressed abundantly in the SCN, in a
circadian manner. To determine whether manipulating the level of
expression of the VPAC
2
R can influence the control of the
circadian clock, we have created transgenic mice overexpressing the
human VPAC
2
R gene from a yeast artificial chromosome (YAC)
construct. The YAC was modified by a strategy using homologous
recombination to introduce (
i
) the HA epitope tag
sequence (from influenza virus hemagglutinin) at the carboxyl terminus
of the VPAC
2
R protein, (
ii
) the
lacZ
reporter gene, and (
iii
) a
conditional centromere, enabling YAC DNA to be amplified in culture in
the presence of galactose. High levels of
lacZ
expression were detected in the SCN, habenula, pancreas, and testis of
the transgenic mice, with lower levels in the olfactory bulb and
various hypothalamic areas. Transgenic mice resynchronized more quickly
than wild-type controls to an advance of 8 h in the light-dark
(LD) cycle and exhibited a significantly shorter circadian period in
constant darkness (DD). These data suggest that the VPAC
2
R
can influence the rhythmicity and photic entrainment of the circadian
clock.
N-terminal RAG1 frameshift mutations in Omenn's syndrome: Internal methionine usage leads to partial V(D)J recombination activity and reveals a fundamental role
in vivo
for the N-terminal domains
Authors: Santagata Sandro , Gomez Carlos A. , Sobacchi Cristina , Bozzi Fabio , Abinun Mario , Pasic Srdjan , Cortes Patricia , Vezzoni Paolo , Villa Anna
Published: July 2002

Omenn's syndrome is an autosomal recessive primary
immunodeficiency characterized by variable numbers of T lymphocytes of
limited clonality, hypereosinophilia, and high IgE levels with a
paradoxical absence of circulating B lymphocytes. We have previously
attributed this disorder to missense mutations that render the
RAG1/RAG2 recombinase only partially active. Here we report seven
Omenn's patients with a novel class of genetic lesions: frameshift
mutations within the 5′ coding region of RAG1. Interestingly, we
demonstrate in transient expression experiments that these frameshift
deletion alleles remain partially functional for both deletional and
inversional recombination and can hence explain the partial
rearrangement phenotype observed in these patients. The rearrangement
activity is mediated by truncated RAG1 proteins that are generated by
alternative ATG usage 3′ to the frameshift deletion and that
demonstrate improper cellular localization. Taken together, our results
suggest a novel mechanism for the development of immunodeficiency in a
subset of Omenn's syndrome patients.
Targeted inhibition of calcineurin prevents agonist-induced cardiomyocyte hypertrophy
Authors: Taigen Tyler , De Windt Leon J. , Lim Hae W. , Molkentin Jeffery D.
Published: July 2002

Cardiac hypertrophy is a major predictor of future morbidity and mortality. Recent investigation has centered around identifying the molecular signaling pathways that regulate cardiac myocyte reactivity with the goal of modulating pathologic hypertrophic programs. One potential regulator of cardiomyocyte hypertrophy is the calcium-sensitive phosphatase calcineurin. We show here that calcineurin enzymatic activity, mRNA, and protein levels are increased in cultured neonatal rat cardiomyocytes by hypertrophic agonists such as angiotensin II, phenylephrine, and 1% fetal bovine serum. This induction of calcineurin activity was associated with an increase in calcineurin Aβ (CnAβ) mRNA and protein, but not in CnAα or CnAγ. Agonist-dependent increases in calcineurin enzymatic activity were specifically inhibited with an adenovirus expressing a noncompetitive peptide inhibitor of calcineurin known as cain [Lai, M. M., Burnett, P. E., Wolosker, H., Blackshaw, S. & Snyder, S. H. (1998)
J. Biol. Chem.
273, 18325–18331]. Targeted inhibition of calcineurin with cain or an adenovirus expressing only the calcineurin inhibitory domain of AKAP79 attenuated cardiomyocyte hypertrophy and atrial natriuretic factor expression in response to angiotensin II, phenylephrine, and 1% fetal bovine serum. These data demonstrate that calcineurin is an important regulator of cardiomyocyte hypertrophy in response to certain agonists and suggest that cyclosporin A and FK506 function to attenuate cardiac hypertrophy by specifically inhibiting calcineurin.
Effects of heat shock, heat shock protein 40 (HDJ-2), and proteasome inhibition on protein aggregation in cellular models of Huntington's disease
Authors: Wyttenbach Andreas , Carmichael Jenny , Swartz Jina , Furlong Robert A. , Narain Yolanda , Rankin Julia , Rubinsztein David C.
Published: July 2002

Huntington's disease (HD), spinocerebellar ataxias types 1 and 3 (SCA1, SCA3), and spinobulbar muscular atrophy (SBMA) are caused by CAG/polyglutamine expansion mutations. A feature of these diseases is ubiquitinated intraneuronal inclusions derived from the mutant proteins, which colocalize with heat shock proteins (HSPs) in SCA1 and SBMA and proteasomal components in SCA1, SCA3, and SBMA. Previous studies suggested that HSPs might protect against inclusion formation, because overexpression of HDJ-2/HSDJ (a human HSP40 homologue) reduced ataxin-1 (SCA1) and androgen receptor (SBMA) aggregate formation in HeLa cells. We investigated these phenomena by transiently transfecting part of huntingtin exon 1 in COS-7, PC12, and SH-SY5Y cells. Inclusion formation was not seen with constructs expressing 23 glutamines but was repeat length and time dependent for mutant constructs with 43–74 repeats. HSP70, HSP40, the 20S proteasome and ubiquitin colocalized with inclusions. Treatment with heat shock and lactacystin, a proteasome inhibitor, increased the proportion of mutant huntingtin exon 1-expressing cells with inclusions. Thus, inclusion formation may be enhanced in polyglutamine diseases, if the pathological process results in proteasome inhibition or a heat-shock response. Overexpression of HDJ-2/HSDJ did not modify inclusion formation in PC12 and SH-SY5Y cells but increased inclusion formation in COS-7 cells. To our knowledge, this is the first report of an HSP increasing aggregation of an abnormally folded protein in mammalian cells and expands the current understanding of the roles of HDJ-2/HSDJ in protein folding.
MIR16, a putative membrane glycerophosphodiester phosphodiesterase, interacts with RGS16
Authors: Zheng Bin , Chen Dan , Farquhar Marilyn Gist
Published: July 2002

We have identified the protein MIR16 (for
M
embrane
I
nteracting protein of
R
GS16) from a yeast two-hybrid screen by using RGS16 as bait. MIR16 shares strong homology with bacterial glycerophosphodiester phosphodiesterases. It interacts with RGS16 and, more weakly, with several other selected RGS proteins. Analysis of deletion mutants showed that the N-terminal region of the RGS domain in RGS16 is required for its interaction with MIR16. MIR16 is an integral membrane glycoprotein, because it remained associated with membrane fractions after alkaline treatment and because, in some cells, it is sensitive to digestion with endoglycosidase H. By immunofluorescence and immunoelectron microscopy, MIR16 was localized on the plasma membrane in liver and kidney and on intracellular membranes in rat pituitary and cultured pituitary cells. MIR16 represents the only integral membrane protein identified thus far to interact with an RGS domain and, to our knowledge, is the only mammalian glycerophosphodiester phosphodiesterase that has been cloned. The putative enzymatic activity of MIR16 and its interaction with RGS16 suggest that it may play important roles in lipid metabolism and in G protein signaling.
School Improvement: The need for vision and reprofessionalisation
Authors: Halsall Rob
Published: July 2002

The New Structure of School Improvement: inquiring schools and achieving students Bruce Joyce, Emily Calhoun & David Hopkins

Schools Making a Difference: let's be realistic! Martin Thrupp

Learning Beyond the Classroom Tom Bentley
The millennial challenge: Modelling the agentic self in context
Authors: Little Todd D.
Published: July 2002

Behind every challenge is an opportunity. As a milestone for both societal and scientific development, the new millennium highlights the growing challenge of change and diversity. Modelling the agentic self in context is the opportunity it reveals. Developmentalists can seize this opportunity by increasing our emphasis on three aspects of developmental research. First, developmental research can examine the influence of context on development more aggressively to test the generality of developmental mechanisms. Second, developmental research can integrate an organismic perspective on human development more explicitly to account for the role of human agency in development. Third, developmental research can apply advanced statistical and analysis methods more sophisticatedly to effectively model the agentic self as it evolves across time and context.
Multiple dimension processing by newborns
Authors: Laplante David P. , Vorkapich L. , Orr R. Robert , Neville K. E.
Published: July 2002

Newborn looking toward novel stimuli containing a single or two novel dimensions was assessed in two studies. In Study 1, newborns were habituated to a visual stimulus containing stimulus movement and line orientation information. Following habituation, stimuli containing two novel dimensions were attended to more than stimuli containing a single novel dimension. It was also demonstrated that newborns detect stimulus movement changes. In Study 2, stimuli containing novel colour and line orientation information, following habituation, were attended to across three test trials more than stimuli containing a novel colour only. Colour discrimination was also demonstrated. It was concluded that newborns appear to attend to stimuli containing two novel dimensions differently than those containing a single novel dimension. It was suggested that newborns employ an analytic mode of processing to detect novel dimensions. Further research is required to verify this conclusion and to determine what component is first attended to.
Girls’ Expression of Their Own Perspectives in the Presence of Varying Numbers of Boys
Authors: Benenson Joyce F. , Del Bianco Rejeanne , Philippoussis Maria , Apostoleris Nicholas H.
Published: July 2002

This study was designed to examine the hypothesis that girls’ expression of their own perspectives would decrease as the ratio of girls to boys decreased. In each of 12 classes, two target girls were observed in three contexts: with four other girls (all-girls group), with two other girls and two boys (girls-in-majority group), and with four boys (girls-in-minority group). The number and duration of episodes in which the target girls clearly took a differing perspective from another child were analysed across the three contexts. Results showed a significant decrease in duration of episodes of firmly taking a different perspective from the all-girls to the girls-in-majority contexts. However, there was no difference in duration of episodes of firmly taking a different perspective between the all-girls and the girls-in-minority contexts. In contrast to previous literature, findings strongly suggest that girls’ expression of their own perspectives is influenced by several mechanisms and not simply the ratio of girls to boys.
Intergenerational Roots of Finnish Women’s Sex-atypical Careers
Authors: Nummenmaa Anna Raija , Nummenmaa Tapio
Published: July 2002

The study investigates background factors influencing the pursuit of sex-atypical careers by Finnish women ( N = 235). Adolescents were investigated three times during an 8-year follow-up; the first time when the girls were in the ”nal grade of high school and planning their careers, the second time when they had gained a place either in professional education or at university, and ”nally when they had taken their place in working life. Background information was obtained about parents and grandparents. Recursive models with latent generational family variables were ”tted. Nonagricultural background of the grandparents predicted a somewhat higher education for the parents, which in turn predicted higher academic success in high school leading to an interest in, and the realisation of, sex-atypical careers.
Maternal Transmission of Nicotine Dependence: Psychiatric, Neurocognitive and Prenatal Factors
Authors: Niaura Raymond , Bock Beth , Lloyd Elizabeth E. , Brown Richard , Lipsitt Lewis P. , Buka Stephen
Published: July 2002

This paper reviews the literature on maternal influences on smoking behaviors of offspring from the perspective of neuropsychiatric deficits that may be transmitted from mother to child. In particular, we review what is known regarding associations between: (1) in‐utero exposure to smoking, (2) adolescent neurocognitive functioning and psychiatric comorbidity, and (3) the patterns of smoking and progression of nicotine dependence. Furthering our knowledge of these differences in susceptibility to nicotine dependence among youth will provide additional avenues for prevention and intervention efforts targeted toward those at high risk for dependence.
Oral (Drinking Water) Two-Generation Reproductive Toxicity Study of Bromodichloromethane (BDCM) in Rats
Authors: Christian M. S. , York R. G. , Hoberman A. M. , Fisher L. C. , Brown W. Ray
Published: July 2002

Bromodichloromethane (BDCM) was tested for reproductive toxicity in a two-generation study in CRL SD rats. Thirty rats/sex/group/generation were continuously provided BDCM in drinking water at 0 (control carrier, reverse osmosis membrane-processed water), 50, 150, and 450 ppm (0,4.1 to 12.6, 11.6 to 40.2, and 29.5 to 109.0 mg/kg/day, respectively). Adult human intake approximates 0.8 μg/kg/day (0.0008 mg/kg/day). P and F1 rats were observed for general toxicity (viability, clinical signs, water and feed consumption, body weights, organ weights [also three weanling F1 and F2 pups/sex/litter], histopathology [10/sex, 0-and 450-ppm exposure groups]) and reproduction (mating, fertility, abortions, premature deliveries, durations of gestation, litter sizes, sex ratios, viabilities, maternal behaviors, reproductive organ weights [also three weanling F1 and F2 pups/sex/litter], sperm parameters, and implantations. F1 rats were evaluated for age at vaginal patency or preputial separation. Ten P and F1 rats/sex from the 0-and 450-ppm exposure groups and rats at 50 and 150 ppm with reduced fertility were evaluated for histopathology (gross lesions, testes, intact epididymis, all F1 dams for number of primordial follicles). Developmental parameters in offspring included implantation and pup numbers, sexes, viabilities, body weights, gross external alterations, and reproductive parameters (F1 adults). Toxicologically important, statistically significant effects at 150 and/or 450 ppm included mortality and clinical signs associated with reduced absolute and relative water consumption, reduced body weights and weight gains, and reduced absolute and relative feed consumption (P and F1 rats). Significantly reduced body weights at 150 and 450 ppm were associated with reduced organ weights and increased organ weight ratios (% body and/or brain weight). Histopathology did not identify abnormalities. Small delays in sexual maturation (preputial separation, vaginal patency) and more F1 rats with prolonged diestrus were also attributable to severely reduced pup body weights. Mating, fertility, sperm parameters, and primordial ovarian follicular counts were unaffected. The no-observable-adverse-effect level (NOAEL) and the reproductive and developmental NOAELs for BDCM were at least 50 ppm (4.1 to 12.6 mg/kg/day), 5125 to 15,750 times the human adult exposure level, if delayed sexual maturational associated with severely reduced body weights is considered reproductive toxicity. If considered general toxicity, reproductive and developmental NOAELs for BDCM are greater than 450 ppm (29.5 to 109.0 mg/kg/day), or 36,875 to 136,250 times the human adult exposure level. Regardless, these data indicate that BDCM should not be identified as a risk to human reproductive performance or development of human conceptuses.
Enhanced Serine Protease Activities in the Sulfur Mustard-Exposed Homogenates of Hairless Guinea Pig Skin
Authors: Kam Chih-Min , Selzler Joe , Schulz Susan M. , Bongiovanni Rudolfo , Powers James C.
Published: July 2002

Skin homogenates of hairless guinea pigs exposed percutaneously to sulfur mustard (SM) were investigated by measuring four serine protease activities (elastase, tryptase, chymase, and Asp-ase) using sensitive chromogenic substrates. The homogenate samples from skin area directly exposed to S M showed enhanced elastase activities. The tryptase (trypsin-like enzyme) activity also increased slightly; however, the chymase (chymotrypsin-like enzyme) and Aspase (cleave after aspartic acid) activities did not show any increase. The enhanced elastase activities after SM exposure indicate that inflammation is present in the SM lesions. The inhibitory potency of MeO-Suc-Ala-Ala-Pro-Val-CH2Cl (an elastase inhibitor) and two amidine derivatives (inhibitors of trypsin-like enzyme) was tested against the activities present in samples from both exposed and control tissues. The elastase inhibitor decreased the hydrolysis of elastase substrate in the samples from SM-exposed skin to a greater extent than the samples from control tissues. The two trypsin inhibitors decreased the activity in samples from exposed and control tissues equally well. These substrate and inhibitor studies facilitate the characterization of various proteases affected by SM and may be useful for elucidating the mechanism of SM-induced vesication.
Historical Aspects of Phototoxicology
Authors: Urbach Frederick
Published: July 2002

Phototoxicology deals with the injurious effects of ultraviolet and visible radiation. The principal phototoxiceffectis the erythema, edema, and pain (“sunburn”) produced in unacclimatized skin by exposure to solar radiation. Similarly, pho-tokeratitis (“snow blindness”) is a phototoxic phenomenon which was described as early as 410 BC by Xenophon. That it was ultraviolet radiation that was the proximate cause of these photo-toxic effects was discovered by Charcot (1858) in experiments with electric arcs. Dubreuilh (1896) related outdoor solar exposure to incidence of skin cancers; and in 1899, Raab noticed that fluorescent chemicals could be activated by visible light to produce killing of microorganisms. By the middle of the twentieth century, phototoxic effects, primarily on skin, were discovered to be due to in trinsic and to topically applied materials (e.g., porphyrins, coal tar). The phototoxic effects of medicinal compounds (e.g., fluoroquinolones), have recently become important. Photoactive agents and their phototoxic effects have long been used in the therapy of such skin diseases as vitiligo and psoriasis, and more recently for the treatment of malignancies.
Assays for Photocarcinogenesis: Relevance of Animal Models
Authors: Forbes P. Donald , Sambuco Christopher P.
Published: July 2002

In the three and a half decades since the first published reports of squamous cell carcinomas in the skin of hairless mice exposed to ultraviolet radiation, this animal model has been developed and utilized extensively in basic and applied photobiology. Quantitative aspects of photocarcinogenesis had been investigated initially in haired mice; subsequently, the discipline of photoimmunology has developed largely on the basis of information derived from haired animals. Classical selective breeding methods, a few fortuitous mutations along the way, and the advent of genetic engineering have all enhanced the utility of the laboratory animal models. The benefits have included advances in our understanding of many influences on photocarcinogenesis such as genetic diversity, animal age, epidermal DNA damage and repair, wavelength dependency (action spectrum), ultraviolet radiation dose and its delivery, interactions with chemicals, and nutrition. As in other fields of basic and biomedical research, photobiological data have not been considered journalistically newsworthy or particularly contentious until they acquired potential economic impact. Thus, photocarcinogenesis became a more familiar word (at least along the Washington Beltway) when stratospheric ozone first appeared to be threatened by chlorofluorocarbons, and trends in photocarcinogenesis seemed less arcane when it appeared that the trends might be modified by consumer products and pharmaceuticals. The greatest challenge has not been in finding imaginative ways to exploit the models and to push the frontiers of science, but rather has been the uncertainty about quantitatively extrapolating the findings to humans. Research models would hardly be useful if they were not different from people (i.e., smaller, quicker to respond, shorter lived), and that fact encompasses differences in anatomy, physiology, metabolism, surface-to-weight ratios, etc. Selected examples from photomedicine (e.g., chronic solar damage, occupational exposure to tar, psoriasis phototherapy) tend to confirm that several basic principles about photocarcinogenesis are shared by man and mouse; other risk assessments await development and refinement, or remain to be corrected by experience.
Symposium VIII: Regulatory Update: Redbook Presentations from the 17th Annual Meeting of the American College of Toxicology, 11-13 November 1996
Authors: Collins Thomas F. X.
Published: July 2002

Thomas F. X. Collins
Use of Pharmacokinetic Data Under the FDA's Redbook II Guidelines for Direct Food Additives
Authors: Roth William L. , Young John F.
Published: July 2002

Experience with food additive petitions submited after publication of the Food and Drug Administration's Redbook I (U. S. FDA, 1982) guide lines indicated a number of areas in which improvements were needed, and advances in toxicol-ogy testing during the last decade required additional rev is ions. In March 1993, the FDA's Center for Food Safety and Applied Nutrition (CFSAN) distributed copies of a draft of Redbook II for public comment. Since that time, revisions have been made based on comments received on the initial draft. This article describes the rationale for Redbook II guidance on the design of pharm acoki-netic studies and discusses some common problems the FDA has encountered in reviewing pharmacokine tic data submitted as part of food additive petitions. Points emphasized are that (1) pharmaco kinetic information is needed for the interpretation of toxicity studies and is most use ful when conducted before major toxicity studies, (2) the use of whole-body autoradiography is encouraged as a means to select tissues of interest, and as a substitute for dissection and tis-sue sampling, (3) kinetic and mechanistic studies conducted with blood compo-nents, tissue slices, hepatocytes, and othercell types in vitro ofien provide more useful information on the fate of chemicals in specific tissues than information extracted from whole-animal studies. The intention of th e new guide lines for pharmaco kinetic studies is to increase the information content of data gathered and to encourage the use of pharmaco kinetic models and results in the selection of doses for subchronic, chronic, and developmental toxicity studies.
High Glucose Concentration Alters Cell Proliferation Dynamics in Human Hepatoma Cells
Authors: Rao Prathibha S. , Lyn-Cook Beverly D. , Littlefield Neil A. , Mehendale Harihara M.
Published: July 2002

The aim of this study was to develop an in vitro model to investigate the molecular mechanisms of glucose-induced inhibition of cell proliferation. HuH7 cells were grown in the presence or absence of glucose for 7 days and cell proliferation was stimulated by exposure to thioacetamide. Lactate dehydrogenase leakage and 3H-thymidine incorporation were used as indices of toxicity and DNA synthesis, respectively. Cell cycle progression and protooncogene expression was monitored by flow cytometry and slot-blot analyses. Toxicity caused by thioacetamide regressed with time in the presence of 11 mM glucose (control). However, in the presence of 28 mM glucose, sustained toxicity was evident as mirrored by lactate dehydrogenase leakage. Peak DNA synthesis noted at 48 hours in the thioacetamide-treated group (11 mM glucose) was significantly diminished in the presence of 28 mM glucose. Increased c-myc expression was observed as early as 30 minutes in the thioacetamide-treated group. When cells were exposed to 28 mM glucose, c-myc expression was delayed and diminished. Methylation profile studies revealed no appreciable changes, but c-myc was significantly amplified in the control, thioacetamide-, and in the presence of 28 mM of glucose-treated groups which correlated with mRNA changes in these groups. In the glucose-pretreated group (28 mM) significant amplification of the c-myc gene was observed at later time points but there was no change in the mRNA expression, indicating that the expression was delayed. This study shows that high glucose concentrations diminish DNA synthesis and cell cycle progression normally stimulated by thioacetamide. It is concluded that high glucose concentration causes cell cycle arrest via perturbation in protooncogene expression and hence the use of high glucose concentrations in therapy should be carefully examined in situations where postsurgical healing and healing after xenobiotic-induced injury are encountered.
Adaptative response of antioxidant enzymes in different areas of rat brain after repeated d‐amphetamine administration
Authors: Carvalho Félix , Fernandes Eduarda , RemiãO Fernando , Gomes‐Da‐Silva Joana , Tavares Maria Amélia , Bastos Maria De Lourdes
Published: July 2002

d‐Amphetamine has been shown to be a potential brain neurotoxic agent, particularly to dopaminergic neurones. Reactive oxygen species indirectly generated by this drug have been indicated as an important factor in the appearance of neuronal damage but little is known about the adaptations of brain antioxidant systems to its chronic administration. In this study, the activities of several antioxidant enzymes in different areas of rat brain were measured after repeated administration of d‐amphetamine sulphate (sc, 20 mg/kg/day, for 14 days), namely glutathione‐S‐transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GRed), catalase, and superoxide dismutase (SOD). When compared to a pair‐fed control group, d‐amphetamine treatment enhanced the activity of GST in hypothalamus to 167%, GPx in striatum to 127%, in nucleus accumbens to 192%, and in medial prefrontal cortex to 139%, GRed in hypothalamus to 139%, as well as catalase in medial prefrontal cortex to 153%. However, the same comparison revealed a decrease in the activity of GRed in medial pre‐frontal cortex by 35%. Food restriction itself reduced GRed activity by 49% and enhanced catalase activity to 271% in nucleus accumbens. The modifications observed for the measured antioxidant enzymes reveal that oxidative stress probably plays a role in the deleterious effects of this drug in CNS and that, in general, the brain areas studied underwent adaptations which provided protection against the continuous administration of the drug.
Metastatic Liposarcoma: A Cause of Symptomatic Acute Pericarditis
Authors: Hatton Matthew Q. F. , Reid Robin , Barrett Ann
Published: July 2002

We describe a patient presenting with a myxoid liposarcoma of the lower thigh in whom an episode of acute pericarditis
indicated the recurrence of widespread metastatic disease.
Assessing the comprehension and production of language in young children: an account of the Reynell Developmental Language Scales III
Authors: Edwards Susan , Garman Michael , Hughes Arthur , Letts Carolyn , Sinka Indra
Published: July 2002

The Reynell Developmental Language Scales were first made available in an experimental version in 1969, with revisions in 1977 and 1985 (Reynell 1977, Reynell and Huntley 1985). Throughout this period and beyond, the Scales were amongst the most popular child language assessment tools to be used in the UK. There are a number of reasons for this: they were considered to be clinically practical in that even very young children readily co‐operated with the test procedures, the Scales measured both comprehension and production of language, and they were standardized on a large sample of British children. In terms of standardized testing, there were no serious UK contenders. However, in recent years, it became clear that some revisions were needed, especially for the Expressive Scale, which had come to be used far less often than the Comprehension Scale. The Expressive Scale was difficult to administer objectively and yielded uninformative results. In a recent volume devoted to assessment procedures used by speech and language therapists (Beech et al. 1993), although the Comprehension Scale is referred to (Lees 1993), there is no mention of the Expressive Scale. In addition, some items and materials used in both scales had become dated and were no longer considered appropriate. A revision was clearly required. This paper describes the new scales, the Reynell Developmental Language Scales III: The University of Reading Edition (RDLS III: Edwards et al. 1997). The new Scales were constructed to reflect current knowledge of language impairment and to incorporate findings from up‐to‐date research on structural and lexical development in child language. As a result, the existing Expressive Scale has been completely revised and substantial changes have been made to the Comprehension Scale.
Ca2+ Homeostasis in the Endoplasmic Reticulum: Coexistence of High and Low [Ca2+] Subcompartments in Intact HeLa Cells
Authors: Montero Mayte , Alvarez Javier , Scheenen Wilhelm J.J. , Rizzuto Rosario , Meldolesi Jacopo , Pozzan Tullio
Published: July 2002

Two recombinant aequorin isoforms with different Ca2+ affinities, specifically targeted to the endoplasmic reticulum (ER), were used in parallel to investigate free Ca2+ homeostasis in the lumen of this organelle. Here we show that, although identically and homogeneously distributed in the ER system, as revealed by both immunocytochemical and functional evidence, the two aequorins measured apparently very different concentrations of divalent cations ([Ca2+]er or [Sr2+]er). Our data demonstrate that this contradiction is due to the heterogeneity of the [Ca2+] of the aequorin-enclosing endomembrane system. Because of the characteristics of the calibration procedure used to convert aequorin luminescence into Ca2+ concentration, the [Ca2+]er values obtained at steady state tend, in fact, to reflect not the average ER values, but those of one or more subcompartments with lower [Ca2+]. These subcompartments are not generated artefactually during the experiments, as revealed by the dynamic analysis of the ER structure in living cells carried out by means of an ER-targeted green fluorescent protein. When the problem of ER heterogeneity was taken into account (and when Sr2+ was used as a Ca2+ surrogate), the bulk of the organelle was shown to accumulate free [cation2+]er up to a steady state in the millimolar range. A theoretical model, based on the existence of multiple ER subcompartments of high and low [Ca2+], that closely mimics the experimental data obtained in HeLa cells during accumulation of either Ca2+ or Sr2+, is presented. Moreover, a few other key problems concerning the ER Ca2+ homeostasis have been addressed with the following conclusions: (a) the changes induced in the ER subcompartments by receptor generation of InsP3 vary depending on their initial [Ca2+]. In the bulk of the system there is a rapid release whereas in the small subcompartments with low [Ca2+] the cation is simultaneously accumulated; (b) stimulation of Ca2+ release by receptor-generated InsP3 is inhibited when the lumenal level is below a threshold, suggesting a regulation by [cation2+]er of the InsP3 receptor activity (such a phenomenon had already been reported, however, but only in subcellular fractions analyzed in vitro); and (c) the maintenance of a relatively constant level of cytosolic [Ca2+], observed when the cells are incubated in Ca2+-free medium, depends on the continuous release of the cation from the ER, with ensuing activation in the plasma membrane of the channels thereby regulated (capacitative influx).
Nucleocytoplasmic Shuttling Factors Including Ran and CRM1 Mediate Nuclear Export of NFAT In Vitro
Authors: Kehlenbach Ralph H. , Dickmanns Achim , Gerace Larry
Published: July 2002

We have developed a permeabilized cell assay to study the nuclear export of the shuttling transcription factor NFAT, which contains a leucine-rich export signal. The assay uses HeLa cells that are stably transfected with NFAT fused to the green fluorescent protein (GFP). Nuclear export of GFP–NFAT in digitonin-permeabilized cells occurs in a temperature- and ATP-dependent manner and can be quantified by flow cytometry. In vitro NFAT export requires the GTPase Ran, which is released from cells during the digitonin permeabilization. At least one additional rate-limiting export factor is depleted from permeabilized cells by a preincubation at 30°C in the absence of cytosol. This activity can be provided by cytosolic or nucleoplasmic extracts in a subsequent export step. Using this assay, we have purified a second major export activity from cytosol. We found that it corresponds to CRM1, a protein recently reported to be a receptor for certain leucine-rich export sequences. CRM1 appears to be imported into the nucleus by a Ran-dependent mechanism that is distinct from conventional signaling pathways. Considered together, our studies directly demonstrate by fractionation and reconstitution that nuclear export of NFAT is mediated by multiple nucleocytoplasmic shuttling factors, including Ran and CRM1.
Mutations in the Middle of the Transmembrane Domain Reverse the Polarity of Transport of the Influenza Virus Hemagglutinin in MDCK Epithelial Cells
Authors: Lin Sasa , Naim Hussein Y. , Chapin Rodriguez A. , Roth Michael G.
Published: July 2002

The composition of the plasma membrane domains of epithelial cells is maintained by biosynthetic pathways that can sort both proteins and lipids into transport vesicles destined for either the apical or basolateral surface. In MDCK cells, the influenza virus hemagglutinin is sorted in the trans-Golgi network into detergent-insoluble, glycosphingolipid-enriched membrane domains that are proposed to be necessary for sorting hemagglutinin to the apical cell surface. Site- directed mutagenesis of the hemagglutinin transmembrane domain was used to test this proposal. The region of the transmembrane domain required for apical transport included the residues most conserved among hemagglutinin subtypes. Several mutants were found to enter detergent-insoluble membranes but were not properly sorted. Replacement of transmembrane residues 520 and 521 with alanines converted the 2A520 mutant hemagglutinin into a basolateral protein. Depleting cell cholesterol reduced the ability of wild-type hemagglutinin to partition into detergent-insoluble membranes but had no effect on apical or basolateral sorting. In contrast, cholesterol depletion allowed random transport of the 2A520 mutant. The mutant appeared to lack sorting information but was prevented from reaching the apical surface when detergent-insoluble membranes were present. Apical sorting of hemagglutinin may require binding of either protein or lipids at the middle of the transmembrane domain and this normally occurs in detergent-insoluble membrane domains. Entry into these domains appears necessary, but not sufficient, for apical sorting.