bluuebunny/crossref_metadata_2025 huggingface dataset:
Influences of dietary adaptation and source of resistant starch on short-chain fatty acids in the hindgut of rats
Authors: Henningsson Åsa M. , Margareta E. , Nyman G. L. , Björck Inger M. E.
Published: April 2003

The effect of adaptation time on the concentration and pattern of short-chain fatty acids (SCFA) formed in the hindgut of rats given resistant starch (RS) in the form of raw potato starch (RPS) or high-amylose maize starch (HAS) was evaluated. Each starchy material was tested in diets containing 100 g indigestible carbohydrate/g DM, and fed for 13, 28 and 42 d. At the end of each period, the content of SCFA was determined in caecum, distal colon and faeces. The caecal concentration of total and individual SCFA increased for both diets with increasing adaptation time. The concentration of butyric acid was higher in the group fed RPS than in that fed HAS at all adaptation times. The caecal proportion of butyric acid was low both in rats fed RPS and HAS (6 and 4 %, respectively) following 13 d of adaptation. However, after 28 d of adaptation, the proportion of butyric acid had increased to 19 % in rats given RPS. A longer adaptation period (42 d) did not increase the proportion of butyric acid further. With HAS, there was also a significant (P<0·01) increase in the proportion of butyric acid with longer adaptation time. However, the increase was much slower and the proportion of butyric acid reached 6 and 8 % after 28 and 42 d respectively. It is concluded that the pattern of SCFA formed from RS in rats is dependent on adaptation time. It cannot be excluded that the different patterns of SCFA reported in the literature for RS may be due to the time of adaptation.
Effects of altitude, ambient temperature and solar radiation on fasting heat production in yellow cattle (Bos taurus)
Authors: Han Xing-Tai , Xie Ao-Yun , Bi Xi-Chao , Liu Shu-Jie , Hu Ling-Hao
Published: April 2003

Growing yellow cattle (Bos taurus, n 30, 1·0–3·5 years old and 75–240 kg) from their native altitude (2000–2800 m) were used to evaluate the effects of altitude, ambient temperature (Ta) and solar radiation on the basal energy metabolism in this large mammal. Fasting heat production (FHP) was measured at altitudes of 2260, 3250 and 4270 m on the Tibetan plateau both in the summer and winter respectively, after a 90 d adaptation period at each experimental site. The gas exchanges of the whole animal were determined continuously for 3 (2260 and 3250 m) or 2 (4270 m) d after a 96 (2260 and 3250 m) or 48 (4270 m) h starvation period, using closed-circuit respiratory masks. Increasing altitude from 2260 to 3250 m at similar Ta in the summer significantly elevated FHP for all animals (P<0·01), and from 3250 to 4270 m for young cattle (P<0·05); increasing altitude from 2260 to 3250 m in the winter also significantly elevated FHP (P<0·05), but the increase was mainly due to the decrease of Ta and the increase in wind speed. No results were obtained at 4270 m in the winter, due to the problems of the animals, adaptating to the altitude. The magnitude of FHP elevation caused by increasing altitude was greater with summer sunshine or winter wind than without them. Increase of Ta from 10·0 to 22·0°C, in the presence of solar radiation, slightly (2260 m) or significantly (3250 and 4270 m, P<0·01) elevated FHP, but slightly reduced it in the absence of solar radiation; decrease of Ta from 0·0 to −30·0°C linearly increased FHP. At 3250 and 4270 m, FHP at the same Ta was higher with summer sunshine or winter wind (3250 m) than without them, but this did not occur at 2260 m. In conclusion, high altitude elevates FHP in yellow cattle in the warm season, and the summer solar radiation and winter wind at high altitude significantly increase metabolic rate. It may be also concluded that the effects of solar radiation on metabolic rate depend on the altitude and the environmental temperature.
Population dynamics of Lymnaea columella and its natural infection by Fasciola hepatica in the State of Minas Gerais, Brazil
Authors: Coelho L.H.L. , Lima W.S.
Published: April 2003

Studies on the population dynamics and natural infection of Lymnaea columella by Fasciola hepatica were carried out from September 1999 to December 2000 in a low-lying area near Itajubá in the Brazilian State of Minas Gerais. A total of 626 snails were collected monthly at nine different sites, counted, and dissected to search for larvae of F. hepatica. The highest populations of L. columella were reached in October of 1999 and August of 2000, and the highest natural infection rates of snails by F. hepatica were reached in September 1999 (5.2%) and July 2000 (3.9%). The removal by farmers of aquatic plants from the drainage furrows caused a drastic reduction in this snail population.
Distribution of intestinal parasitoses in relation to environmental and sociocultural parameters in La Plata, Argentina
Authors: Gamboa M.I. , Basualdo J.A. , Córdoba M.A. , Pezzani B.C. , Minvielle M.C. , Lahitte H.B.
Published: April 2003

Environmental parameters influencing the distribution of parasite species in three neighbourhoods of differing socioeconomic conditions in La Plata, Argentina were analysed. Coproparasitological screenings were performed in children up to 14 years old from a marginal zone (100), a suburban neighbourhood (101), and an urban area (91) in 1999--2000. The presence of parasite species in environmental samples (water and soil) and the degree of association among parasite communities was documented and evaluated. The prevalence of infection in each population was 73.0%, 54.4% and 35.2%, respectively. The frequencies of helminths and pathogenic protozoa were both higher in the marginal zone, where sanitary and environmental conditions were significantly inferior compared with the other zones. The high prevalence of intestinal parasites in this infantile population was related to parasitic contamination of the soil and water sources in addition to deficient sanitary and sociocultural conditions. Calculation of an equitability index revealed that the specific richness was less equitable once socioeconomic conditions and hygienic practices were improved. This study demonstrates the need to implement management practices for the control of intestinal parasitoses in accordance with the environmental and sociocultural characteristics of a given ecosystem.
Involvement of cysteine proteinases in excystment ofParagonimus ohiraimetacercariae induced by sodium cholate and A23187
Authors: Ikeda T.
Published: April 2003

The involvement of intrinsic proteinases in the excystment ofParagonimus ohiraimetacercariae was studied inin vitroexcystment induced by sodium (Na) cholate, a bile salt and A23187, a Ca2+ionophore. The effects of various proteinase inhibitors on thein vitroexcystment were examined and similar inhibitory profiles were obtained. Benzyloxycarbonyl-L-leucyl-L-leucinal (Z-Leu-Leu-H), a cysteine proteinase inhibitor and 4-(2-aminoethyl)-benzenesulfonyl fluoride (Pefabloc SC), a serine proteinase inhibitor completely inhibited excystment, while L-3-carboxy-2,3-trans-epoxypropionyl-leucylamido (4-guanidino)-butane (E-64), a cysteine proteinase inhibitor and leupeptin, a cysteine/serine proteinase inhibitor permitted partial excystment at a lower rate, but inhibited it from proceeding from the partial excystment stage. In secretions released from metacercariae during excystment, proteinase activities detected towards various fluorogenic peptidyl substrates were almost completely inhibited by Z-Leu-Leu-H and E-64, but not by Pefabloc SC. Sodium cholate induced a higher secretion of cysteine proteinases and a higher rate of excystment than A23187. Profiles of cysteine proteinase activities towards five peptidyl substrates detected were markedly different among the two secretions and the lysate of newly excysted juveniles. Newly excysted juveniles released cysteine proteinases with similar activity profiles and levels to metacercariae induced by Na cholate-incubation, whereas the release of cysteine proteinases was reduced compared with metacercariae induced by A23187-incubation. These results provide valuable information about the involvement of intrinsic proteinases in metacercarial excystment.
Fasciola gigantica: larval productivity of three different miracidial isolates in the snail Lymnaea truncatula
Authors: Dar Y. , Vignoles P. , Rondelaud D. , Dreyfuss G.
Published: April 2003

Bimiracidial infections of Lymnaea truncatula with three isolates of Fasciola gigantica, originating from China, Egypt and Madagascar, were carried out to determine the effect of geographic origin of the parasite on the larval productivity of redial generations. The prevalences of experimental infections in snails exposed to strains from Madagascar, China and Egypt were 20.8%, 60.0% and 80.0%, respectively. At day 49 post-exposure (p.e.), the total number of free rediae in snails infected with the Egyptian isolate was significantly higher than that recorded in the Madagascan group. On the other hand, at day 49 p.e., the majority of cercariae in the Chinese and Egyptian groups were produced by R2a rediae (70.6% and 66.6% of cercariae produced by all live rediae), while, in the Madagascan group, the cercariae were produced mainly by the first redial generation. Snails infected with the Egyptian isolate of miracidia developed more live rediae and, consequently, could produce a higher number of cercariae. As a result, L. truncatula snails were highly adapted to infections with the Egyptian and Chinese isolates of F. gigantica.
Taenia solium taeniasis/cysticercosis in Papua, Indonesia in 2001: detection of human worm carriers
Authors: Margono S.S. , Ito A. , Sato M.O. , Okamoto M. , Subahar R. , Yamasaki H. , Hamid A. , Wandra T. , Purba W.H. , Nakaya K. , Ito M. , Craig P.S. , Suroso T.
Published: April 2003

A preliminary study to detect human worm carriers of Taenia solium in Papua (Irian Jaya), Indonesia was carried out using stool examinations for the detection of copro-antigens and adult proglottids after chemotherapy, and confirmation by mitochondrial DNA analysis using expelled proglottids and metacestodes developed in NOD/Shi-scid mice from eggs of expelled proglottids. Approximately 8.6% of the local population in Kama (5/58), 1 km from the local capital city centre, Wamena, were confirmed to harbour adult T. solium using these techniques.
A molecular phylogeographic study based on DNA sequences from individual metacercariae of Paragonimus mexicanus from Guatemala and Ecuador
Authors: Iwagami M. , Monroy C. , Rosas M.A. , Pinto M.R. , Guevara A.G. , Vieira J.C. , Agatsuma Y. , Agatsuma T.
Published: April 2003

A molecular phylogeographic study of Paragonimus mexicanus collected from Guatemala and Ecuador was performed. Genomic DNA was extracted from individual metacercariae, and two gene regions (partial mitochondrial cytochrome c oxidase subunit 1 (CO1) and the second internal transcribed spacer of the nuclear ribosomal gene repeat (ITS2)) were amplified by the polymerase chain reaction (PCR). Sequences segregated in a phylogenetic tree according to their geographic origins. ITS2 sequences from Ecuador and Guatemala differed at only one site. Pairwise distances among CO1 sequences within a country were always lower than between countries. Nevertheless, genetic distances between countries were less than between geographical forms of P. westermani that have been suggested to be distinct species. This result suggests that populations from Guatemala and Ecuador are genetically differentiated perhaps at the level of subspecies.
Cadmium toxicity and snail–digenean interactions in a population of Lymnaea spp.
Authors: Morley N.J. , Crane M. , Lewis J.W.
Published: April 2003

The toxicity of cadmium to a population of Lymnaea peregra and L. stagnalis naturally infected with a range of digeneans and collected from a number of sites in the lower Thames Valley, UK was investigated. Lymnaeid snails were exposed to 100 μg l-1 cadmium and the effects on host survival and emergence of cercariae recorded. Overwintered L. peregra, but not L. stagnalis, showed significantly reduced survival compared to seasonally infected snails, i.e. snails which have acquired an infection during the spring or summer. A significant increase in survival with increasing snail size was demonstrated for L. stagnalis and for seasonally infected L. peregra only. Only L. stagnalis infected with Diplostomum spathaceum and L. peregra infected individually with D. spathaceum, Sanguinicola inermis, Echinoparyphium recurvatum and Notocotylus attenuatus demonstrated a significantly reduced survival compared to laboratory-bred controls. The exposure of L. stagnalis to cadmium resulted in a significant reduction in the emergence of D. spathaceum over a 5-day period but cadmium-exposed L. peregra showed no difference in the emergence of E. recurvatum cercariae over a 3-day exposure period. The mechanisms and importance of metal toxicity to snail–digenean interactions are discussed.
Prevalence of canine dirofilariasis in Taiwan
Authors: Wu Ching-Cheng , Fan Ping-Chin
Published: April 2003

Between 1993 and 1997, 837 stray dogs from North Taiwan were necropsied and examined for dog heartworm infections. A thick smear from 20 ml of peripheral blood from each dog was also prepared and examined for microfilariae (mf). The overall prevalence of adult worms in the dog population was 57%. The prevalence of mf in 1228 house dogs from different parts of Taiwan was also determined from 20 ml of peripheral blood in the same way. The overall prevalence of mf was 25%, with a value of 30% in the main island of Taiwan, this being 15 times higher than that in the offshore islands (2%). In Taiwan, the prevalence ranged from 4% in Hualien County, East Taiwan, to 41% in Nantou County, Central Taiwan. The mf prevalences on offshore islands were 1% on Liuchiu and 2% on Lanyu. The mf density per 20 ml blood in 82 house dogs was found to be 23 mf per dog, with a range of 3–97 mf per dog. A total of 477 stray dogs were found to be infected with adult worms of Dirofilaria immitis. The mean number of 7 worms per dog was obtained, with a range of 1–55 worms per dog. These results indicate that the prevalence of canine dirofilariasis has increased in Taiwan over the past 10 years. Moreover, the prevalence may be related to the wind speed, temperature, relative humidity, and altitude in the different areas surveyed.
On the longevity of Schistosoma curassoni
Authors: Vercruysse J. , Rollinson D. , van Heerden M. , Southgate V.R.
Published: April 2003

It is demonstrated that Schistosoma curassoni, a parasite of sheep, cattle and goats in parts of West Africa, will live for at least 8 years 5 months in a sheep. The sheep was exposed to 500 cercariae of S. curassoni liberated from infected Bulinus wrighti. The sheep died of natural causes, and at post-mortem 28 pairs of adult S. curassoni were removed from the mesenteric and rectal veins. All female worms were gravid, and eggs were hatched from faeces to produce miracidia. The development of immune responses of the host had apparently little or no effect on the viability of the eggs. Histological studies of the liver, small and large intestines revealed mild pathological symptoms. The longevity of S. curassoni is the first record of longevity of schistosomes to be based on worm counts.
In vitrometabolism of an insect neuropeptide by homogenates of the nematodeCaenorhabditis elegans
Authors: Masler E.P.
Published: April 2003

The cytosolic fraction of homogenates from the free-living soil nematodeCaenorhabditis elegansis capable of metabolizing the insect neuropeptide adipokinetic hormone, a decapeptide blocked at the N-terminus by a pGlu residue. Analysis of digests by RP-HPLC and LC-MS revealed that an initial endoproteolytic cleavage step produced a heptapeptide with an unblocked N-terminus that can serve as a substrate for aminopeptidases. The aminopeptidase activity is depressed in the presence of the inhibitor amastatin; the initial product of the endoproteolytic step accumulates during incubation, and expected aminopeptidase product peptides are reduced in amount, as assessed by chromatographic peak size. The absence of some expected peptide fragments in the reaction mixtures suggests that multiple proteases contribute to short peptide half-lives. Comparison of the adipokinetic hormone digestion inC. elegansto that reported previously for insects reveals the same general pattern of peptide fragment production.
Molecular cloning and expression of the full-length tropomyosin gene fromTrichinella spiralis
Authors: Nakada T. , Nagano I. , Wu Z. , Takahashi Y.
Published: April 2003

A clone, designated as TsTM, was selected from the cDNA library of newborn larvae (NBL) ofTrichinella spiralisthrough immunoscreening against infected sera. The clone contained a cDNA transcript of 855 bp in length with a single open reading frame, which encoded 285-amino acids (33 kDa in the estimated molecular weight). A sequence analysis revealed that the clone TsTM encoded the full-length of tropomyosin gene. The phylogenetic analysis of the tropomyosin gene was in good agreement with the classical taxonomical position ofT. spiralis. The fusion proteins encoded by the clone TsTM were produced in anEscherichia coliexpression system and affinity purified, and the antibody was raised against the protein for the following studies. The antibody against the fusion protein positively bound to the hypodermal muscle layer in immunolocalization analysis, and the 35 kDa band in crude extracts of muscle larvae but not in excretory and secretory (ES) products on Western blots. The antigenicity of the clone TsTM was recognized by host mice but exhibited little species specificity.
Pseudobacciger harengulae from the Atlantic herring Clupea harengus: a new host and locality record
Authors: Rahimian H. , Thulin J.
Published: April 2003

During the summer and autumn of 1994, 1995 and 1996, 406 juvenile herring caught off the Swedish west coast were examined for parasites. Amongst those found was the digenean Pseudobacciger harengulae, which represents new host and locality records for this parasite. Pseudobacciger harengulae has been reported from several species of clupeiformes, mostly from tropical and temperate regions of the Atlantic, Pacific and Indian Oceans. The morphology of P. harengulae is described and compared with earlier descriptions of P. harengulae and P. manteri. The possible relationships between P. harengulae and P. manteri are discussed and the validity of the P. manteri is questioned. Most of the specimens (75%) of P. harengulae were found in the pyloric caeca and the remainder (25%) in the intestine.
Larval stages of Brachylaima fuscatum in the terrestrial snail Limicolaria aurora from southern Nigeria
Authors: Awharitoma A.O. , Okaka C.E. , Obaze S.E.
Published: April 2003

Of 150 specimens of the gastropod snail Limicolaria aurora examined from the Edo and Delta states of Nigeria, 63.4% were infected with larval digeneans comprising mother sporocysts (12.1%) daughter sporocysts (20.4%) cercariae (43.1%) and metacercariae (24.5%). Attempts to experimentally infect three 14-day-old chicks (Gallus domesticus) and two laboratory-bred 4-month-old mice (Mus musculus) by oral feeding and peritoneal injection with cercariae were negative, although experimental infections of chicks via a cloacal drop yielded 62 immature and 37 mature worms from the intestinal caeca and ileum. The worms were identified as Brachylaima fuscatum (Trematoda: Brachylaimidae). The study also revealed that L. aurora acts as an intermediate host for B. fuscatum, in addition to Eulota sp., Helix sp., Helicella sp., Oxychilus sp. and Agrolimax sp.
Penetration of human skin by the cercariae of Schistosoma mansoni: an investigation of the effect of multiple cercarial applications
Authors: Ingram R.J. , Bartlett A. , Brown M.B. , Marriott C. , Whitfield P.J.
Published: April 2003

It has previously been postulated that L-arginine emitted by penetrating Schistosoma mansoni cercariae serves as an intraspecific signal guiding other cercariae to the penetration site. It was suggested that penetrating in groups offers a selective advantage. If this hypothesis is correct and group penetration at one site on the host offers an advantage, it would follow that at such a site, successive groups of cercariae would be able to penetrate skin in either greater numbers or at a faster rate. This prediction was tested by the use of an in vitro model of cercarial penetration based on the Franz cell and using human skin. It was demonstrated that there was no increase in the percentage of cercariae able to penetrate the skin with subsequent exposures. Consequently, it seems unlikely that the release of L-arginine by cercariae during penetration could have evolved as a specific orientation system based on a selective advantage offered by group penetration.
Increased susceptibility of Atlantic salmon Salmo salar to infections with Gyrodactylus derjavini induced by dexamethasone bath treatment
Authors: Nielsen C.V. , Buchmann K.
Published: April 2003

Dexamethasone, a known immunosuppressant, was administered by bath or injection to Atlantic salmon Salmo salar (Conon stock) to study if this treatment could affect the susceptibility of fish to infections with a Danish strain of Gyrodactylus derjavini (Monogenea). Three groups of S. salar (Conon stock) were immersion treated either with 10, 60 or 240 μg dexamethasone l-1 water, respectively. In addition, one group (positive control) was treated intraperitoneally with 200 μg dexamethasone per fish and one negative control group was kept untreated. A single G. derjavini parasite was placed on the anal fin of each fish and the infection was subsequently monitored weekly for 6 weeks. An increase in parasite populations on the salmon was positively correlated with the amount of immunosuppressant used. Infection levels in the group immersion treated with dexamethasone (240 μg l-1 water) and in the i.p. treated positive control group were significantly higher compared to the untreated control group.
Setaria cervi: in vitro released collagenases and their inhibition by Wuchereria bancrofti infected sera
Authors: Singh R.N. , Rathaur S.
Published: April 2003

In vitro released products of adult Setaria cervi females, microfilariae and extracts showed considerable amounts of collagenase activity. On the basis of per mg protein released in vitro, the products of both microfilariae and adult females exhibited comparable activity but this was much higher than that of extract of microfilariae and adult females. Two collagenase enzymes with molecular masses of 50 kDa and 70 kDa were separated using DEAE-sepharose CL6B and Sephadex G-100 column chromatography. The 50 kDa and 70 kDa collagenase exhibited pH optima of 5.2 and 7.0, respectivly. Considering specific activity, the 50 kDa enzyme was found to contribute about ten times more collagenase activity as compared to the 70 kDa enzyme. An inhibition study revealed obvious differences between them. Thiol group inhibitors such as N-ethylmaleimide and leupeptin inhibited the 50 kDa enzyme but this was strongly activated by dithiothreitol, a thiol group stabilizer. Alternatively, the 70 kDa enzyme showed a sensitivity to a metal chelator and a serine group inhibitor indicating its metalloserine protease nature. The antifilarial drug diethylcarbamazine did not demonstrate any inhibition under in vitro conditions. Both enzymes were significantly inhibited by antibody IgG separated from Wuchereria bancrofti infected human sera, showing a possible immunoprotective role.
Distributions of corank 1 and their characteristic vector fields
Authors: Jakubczyk B. , Zhitomirskii M.
Published: April 2003

We prove that any 1-parameter family of corank 1 distributions (or Pfaff equations) on a compact manifold

M

n

M^{n}

is trivializable, i.e., transformable to a constant family by a family of diffeomorphisms, if all distributions of the family have the same characteristic line field. The characteristic line field is a field of tangent lines which is invariantly assigned to a corank one distribution. It is defined on

M

n

M^{n}

, if

n
=
2
k

n=2k

, or on a subset of

M

n

M^{n}

called the Martinet hypersurface, if

n
=
2
k
+
1

n=2k+1

. Our second main result states that if two corank one distributions have the same characteristic line field and are close to each other, then they are equivalent via a diffeomorphism. This holds under a weak assumption on the singularities of the distributions. The second result implies that the abnormal curves of a distribution determine the equivalence class of the distribution, among distributions close to a given one.
A classification and examples of rank one chain domains
Authors: Brungs H. , Dubrovin N.
Published: April 2003

A chain order of a skew field

D
D

is a subring

R
R

of

D
D

so that

d
∈
D
∖
R

d\in D\backslash R

implies

d

−
1

∈
R
.

d^{-1}\in R.

Such a ring

R
R

has rank one if

J
(
R
)

J(R)

, the Jacobson radical of

R
,

R,

is its only nonzero completely prime ideal. We show that a rank one chain order of

D
D

is either invariant, in which case

R
R

corresponds to a real-valued valuation of

D
,

D,

or

R
R

is nearly simple, in which case

R
,

R,

J
(
R
)

J(R)

and

(
0
)

(0)

are the only ideals of

R
,

R,

or

R
R

is exceptional in which case

R
R

contains a prime ideal

Q
Q

that is not completely prime. We use the group

M

(
R
)

\mathcal {M}(R)

of divisorial

R
R

-ideals of

D
D

with the subgroup

H

(
R
)

\mathcal {H}(R)

of principal

R
R

-ideals to characterize these cases. The exceptional case subdivides further into infinitely many cases depending on the index

k
k

of

H

(
R
)

\mathcal {H}(R)

in

M

(
R
)
.

\mathcal {M}(R).

Using the covering group

G

\mathbb {G}

of

SL
⁡
(
2
,

R

)

\operatorname {SL}(2,\mathbb {R})

and the result that the group ring

T

G

T\mathbb {G}

is embeddable into a skew field for

T
T

a skew field, examples of rank one chain orders are constructed for each possible exceptional case.
When are the tangent sphere bundles of a Riemannian manifold reducible?
Authors: Boeckx E.
Published: April 2003

We determine all Riemannian manifolds for which the tangent sphere bundles, equipped with the Sasaki metric, are local or global Riemannian product manifolds.
Extracellular ATP Causes ROCK I-dependent Bleb Formation in P2X7-transfected HEK293 Cells
Authors: Morelli Anna , Chiozzi Paola , Chiesa Anna , Ferrari Davide , Sanz Juana M. , Falzoni Simonetta , Pinton Paolo , Rizzuto Rosario , Olson Michael F. , Di Virgilio Francesco
Published: April 2003

The P2X7ATP receptor mediates the cytotoxic effect of extracellular ATP. P2X7-dependent cell death is heralded by dramatic plasma membrane bleb formation. Membrane blebbing is a complex phenomenon involving as yet poorly characterized intracellular pathways. We have investigated the effect of extracellular ATP on HEK293 cells transfected with the cytotoxic/pore-forming P2X7receptor. Addition of ATP to P2X7-transfected, but not to wt P2X7-less, HEK293 cells caused massive membrane blebbing within 1–2 min. UTP, a nucleotide incapable of activating P2X7, had no early effects on cell shape and bleb formation. Bleb formation triggered by ATP was reversible and required extracellular Ca2+and an intact cytoskeleton. Furthermore, it was completely prevented by preincubation with the P2X blocker oxidized ATP. It was recently observed that the ROCK protein is a key determinant of bleb formation. Preincubation of HEK293-P2X7cells with the ROCK blocker Y-27632 completely prevented P2X7-dependent blebbing. Although ATP triggered cleavage of the ROCK I isoform in P2X7-transfected HEK293 cells, the wide range caspase inhibitor z-VAD-fluoromethylketone had no effect. These observations suggest that P2X7-dependent plasma membrane blebbing depends on the activation of the serine/threonine kinase ROCK I.
Human MPS1 Kinase Is Required for Mitotic Arrest Induced by the Loss of CENP-E from Kinetochores
Authors: Liu Song-Tao , Chan Gordon K.T. , Hittle James C. , Fujii Gregory , Lees Emma , Yen Tim J.
Published: April 2003

We have determined that the previously identified dual-specificity protein kinase TTK is the human orthologue of the yeast MPS1 kinase. Yeast MPS1 (monopolar spindle) is required for spindle pole duplication and the spindle checkpoint. Consistent with the recently identified vertebrate MPS1 homologues, we found that hMPS1 is localized to centrosomes and kinetochores. In addition, hMPS1 is part of a growing list of kinetochore proteins that are localized to nuclear pores. hMPS1 is required by cells to arrest in mitosis in response to spindle defects and kinetochore defects resulting from the loss of the kinesin-like protein, CENP-E. The pattern of kinetochore localization of hMPS1 in CENP-E defective cells suggests that their interaction with the kinetochore is sensitive to microtubule occupancy rather than kinetochore tension. hMPS1 is required for MAD1, MAD2 but not hBUB1, hBUBR1 and hROD to bind to kinetochores. We localized the kinetochore targeting domain in hMPS1 and found that it can abrogate the mitotic checkpoint in a dominant negative manner. Last, hMPS1 was found to associate with the anaphase promoting complex, thus raising the possibility that its checkpoint functions extend beyond the kinetochore.
Morphology and Dynamics of Clathrin/GGA1-coated Carriers Budding from theTrans-Golgi Network
Authors: Puertollano Rosa , van der Wel Nicole N. , Greene Lois E. , Eisenberg Evan , Peters Peter J. , Bonifacino Juan S.
Published: April 2003

Sorting of transmembrane proteins and their ligands at various compartments of the endocytic and secretory pathways is mediated by selective incorporation into clathrin-coated intermediates. Previous morphological and biochemical studies have shown that these clathrin-coated intermediates consist of spherical vesicles with a diameter of 60–100 nm. Herein, we report the use of fluorescent imaging of live cells to demonstrate the existence of a different type of transport intermediate containing associated clathrin coats. Clathrin and the adaptors GGA1 and adaptor protein-1, labeled with different spectral variants of the green fluorescent protein, are shown to colocalize to the trans-Golgi network and to a population of vesicles and tubules budding from it. These intermediates are highly pleiomorphic and move toward the peripheral cytoplasm for distances of up to 10 μm with average speeds of ∼1 μm/s. The labeled clathrin and GGA1 cycle on and off membranes with half-times of 10–20 s, independently of vesicle budding. Our observations indicate the existence of a novel type oftrans-Golgi network-derived carriers containing associated clathrin, GGA1 and adaptor protein-1 that are larger than conventional clathrin-coated vesicles, and that undergo long-range translocation in the cytoplasm before losing their coats.
The Microtubule Plus-End Proteins EB1 and Dynactin Have Differential Effects on Microtubule Polymerization
Authors: Ligon Lee A. , Shelly Spencer S. , Tokito Mariko , Holzbaur Erika L.F.
Published: April 2003

Several microtubule-binding proteins including EB1, dynactin, APC, and CLIP-170 localize to the plus-ends of growing microtubules. Although these proteins can bind to microtubules independently, evidence for interactions among them has led to the hypothesis of a plus-end complex. Here we clarify the interaction between EB1 and dynactin and show that EB1 binds directly to the N-terminus of the p150Gluedsubunit. One function of a plus-end complex may be to regulate microtubule dynamics. Overexpression of either EB1 or p150Gluedin cultured cells bundles microtubules, suggesting that each may enhance microtubule stability. The morphology of these bundles, however, differs dramatically, indicating that EB1 and dynactin may act in different ways. Disruption of the dynactin complex augments the bundling effect of EB1, suggesting that dynactin may regulate the effect of EB1 on microtubules. In vitro assays were performed to elucidate the effects of EB1 and p150Gluedon microtubule polymerization, and they show that p150Gluedhas a potent microtubule nucleation effect, whereas EB1 has a potent elongation effect. Overall microtubule dynamics may result from a balance between the individual effects of plus-end proteins. Differences in the expression and regulation of plus-end proteins in different cell types may underlie previously noted differences in microtubule dynamics.
IGF-I–induced Differentiation of L6 Myogenic Cells Requires the Activity of cAMP-Phosphodiesterase
Authors: De Arcangelis Vania , Coletti Dario , Conti Marco , Lagarde Michel , Molinaro Mario , Adamo Sergio , Nemoz Georges , Naro Fabio
Published: April 2003

Inhibition of type 4 cAMP-specific phosphodiesterase (PDE4) activity in L6-C5 and L6-E9 abolished myogenic differentiation induced by low-serum medium and IGF-I. L6-C5 cells cultured in low-serum medium displayed a PDE4 activity higher than cells cultured in serum-free medium, a condition not sufficient to induce differentiation. In the presence of serum, PDE4D3, the major isoform natively expressed in L6-C5 cells, translocated to a Triton-insoluble fraction, which increased the PDE specific activity of the fraction, and exhibited a Mr shift typical of phosphorylation of this isoform. Furthermore, serum promoted the localization of PDE4D3 to a vesicular subcellular compartment. In L6-C5 cells, IGF-I is a stronger inducer of myogenic differentiation in the presence than in absence of serum. Its ability to trigger differentiation in the absence of serum was restored by overexpressing wild-type PDE4D3, but not a phosphorylation-insensitive mutant. This finding was confirmed in single cells overexpressing a GFP-PDE4D3 fusion protein by assessing nuclear accumulation of myogenin in both L6-C5 and L6-E9. Overexpression of other PDE isoforms was less efficient, confirming that PDE4D3 is the physiologically relevant phosphodiesterase isoform in the control of myogenesis. These results show that downregulation of cAMP signaling through cAMP-phosphodiesterase stimulation is a prerequisite for induction of myogenesis.
Extracellular Signal-regulated Kinase Mediates Phosphorylation of Tropomyosin-1 to Promote Cytoskeleton Remodeling in Response to Oxidative Stress: Impact on Membrane Blebbing
Authors: Houle François , Rousseau Simon , Morrice Nick , Luc Mario , Mongrain Sébastien , Turner Christopher E. , Tanaka Sakae , Moreau Pierre , Huot Jacques
Published: April 2003

Oxidative stress induces in endothelial cells a quick and transient coactivation of both stress-activated protein kinase-2/p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases. We found that inhibiting the ERK pathway resulted, within 5 min of oxidative stress, in a misassembly of focal adhesions characterized by mislocalization of key proteins such as paxillin. The focal adhesion misassembly that followed ERK inhibition with the mitogen-activated protein kinase kinase (MEK) inhibitor PD098059 (2′-amino-3′-methoxyflavone) or with a kinase negative mutant of ERK in the presence of H2O2resulted in a quick and intense membrane blebbing that was associated with important damage to the endothelium. We isolated by two-dimensional gel electrophoresis a PD098059-sensitive phosphoprotein of 38 kDa that we identified, by mass spectrometry, as tropomyosin-1. In fact, H2O2induced a time-dependent phosphorylation of tropomyosin that was sensitive to inhibition by PD098059 and UO126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butanediane). Tropomyosin phosphorylation was also induced by expression of a constitutively activated form of MEK1 (MEKCA), which confirms that its phosphorylation resulted from the activation of ERK. In unstimulated cells, tropomyosin-1 was found diffuse in the cells, whereas it quickly colocalized with actin and stress fibers upon stimulation of ERK by H2O2or by expression of MEKCA. We propose that phosphorylation of tropomyosin-1 downstream of ERK by contributing to formation of actin filaments increases cellular contractility and promotes the formation of focal adhesions. Incidentally, ML-7 (1-[5iodonaphthalene-1-sulfonyl]homopiperazine, HCl), an inhibitor of cell contractility, inhibited phosphorylation of tropomyosin and blocked the formation of stress fibers and focal adhesions, which also led to membrane blebbing in the presence of oxidative stress. Our finding that tropomyosin-1 is phosphorylated downstream of ERK, an event that modulates its interaction with actin, may lead to further understanding of the role of this protein in regulating cellular functions associated with cytoskeletal remodeling.
etramps, a NewPlasmodium falciparumGene Family Coding for Developmentally Regulated and Highly Charged Membrane Proteins Located at the Parasite–Host Cell Interface
Authors: Spielmann Tobias , Fergusen David J. P. , Beck Hans-Peter
Published: April 2003

After invasion of erythrocytes, the human malaria parasitePlasmodium falciparum resides within a parasitophorous vacuole and develops from morphologically and metabolically distinct ring to trophozoite stages. During these developmental phases, major structural changes occur within the erythrocyte, but neither the molecular events governing this development nor the molecular composition of the parasitophorous vacuole membrane (PVM) is well known. Herein, we describe a new family of highly cationic proteins from P. falciparum termed early transcribed membrane proteins (ETRAMPs). Thirteen members were identified sharing a conserved structure, of which six were found only during ring stages as judged from Northern and Western analysis. Other members showed different stage-specific expression patterns. Furthermore, ETRAMPs were associated with the membrane fractions in Western blots, and colocalization and selective permeabilization studies demonstrated that ETRAMPs were located in the PVM. This was confirmed by immunoelectron microscopy where the PVM and tubovesicular extensions of the PVM were labeled. Early expressed ETRAMPs clearly defined separate PVM domains compared with the negatively charged integral PVM protein EXP-1, suggesting functionally different domains in the PVM with an oppositely charged surface coat. We also show that the dynamic change of ETRAMP composition in the PVM coincides with the morphological changes during development. The P. falciparum PVM is an important structure for parasite survival, and its analysis might provide better understanding of the requirements of intracellular parasites.
Autoinhibition of Jak2 Tyrosine Kinase Is Dependent on Specific Regions in Its Pseudokinase Domain
Authors: Saharinen Pipsa , Vihinen Mauno , Silvennoinen Olli
Published: April 2003

Jak tyrosine kinases have a unique domain structure containing a kinase domain (JH1) adjacent to a catalytically inactive pseudokinase domain (JH2). JH2 is crucial for inhibition of basal Jak activity, but the mechanism of this regulation has remained elusive. We show that JH2 negatively regulated Jak2 in bacterial cells, indicating that regulation is an intrinsic property of Jak2. JH2 suppressed basal Jak2 activity by lowering the Vmaxof Jak2, whereas JH2 did not affect the Kmof Jak2 for a peptide substrate. Three inhibitory regions (IR1–3) within JH2 were identified. IR3 (residues 758–807), at the C terminus of JH2, directly inhibited JH1, suggesting an inhibitory interaction between IR3 and JH1. Molecular modeling of JH2 showed that IR3 could form a stable α-helical fold, supporting that IR3 could independently inhibit JH1. IR2 (725–757) in the C-terminal lobe of JH2, and IR1 (619–670), extending from the N-terminal to the C-terminal lobe, enhanced IR3-mediated inhibition of JH1. Disruption of IR3 either by mutations or a small deletion increased basal Jak2 activity, but abolished interferon-γ–inducible signaling. Together, the results provide evidence for autoinhibition of a Jak family kinase and identify JH2 regions important for autoregulation of Jak2.
Synthetic Lethal Analysis Implicates Ste20p, a p21-activated Protein Kinase, in Polarisome Activation
Authors: Goehring April S. , Mitchell David A. , Tong Amy Hin Yan , Keniry Megan E. , Boone Charles , Sprague George F.
Published: April 2003

The p21-activated kinases Ste20p and Cla4p carry out undefined functions that are essential for viability during budding inSaccharomyces cerevisiae. To gain insight into the roles of Ste20p, we have used a synthetic lethal mutant screen to identify additional genes that are required in the absence of Cla4p. Altogether, we identified 65 genes, including genes with roles in cell polarity, mitosis, and cell wall maintenance. Herein, we focus on a set that defines a function carried out by Bni1p and several of its interacting proteins. We found that Bni1p and a group of proteins that complex with Bni1p (Bud6p, Spa2p, and Pea2p) are essential in acla4Δ mutant background. Bni1p, Bud6p, Spa2, and Pea2p are members of a group of polarity determining proteins referred to as the polarisome. Loss of polarisome proteins from acla4Δ strain causes cells to form elongated buds that have mislocalized septin rings. In contrast, other proteins that interact with or functionally associate with Bni1p and have roles in nuclear migration and cytokinesis, including Num1p and Hof1p, are not essential in the absence of Cla4p. Finally, we have found that Bni1p is phosphorylated in vivo, and a substantial portion of this phosphorylation is dependent on STE20. Together, these results suggest that one function of Ste20p may be to activate the polarisome complex by phosphorylation of Bni1p.
Membrane Targeting of Grb2-associated Binder-1 (Gab1) Scaffolding Protein through Src Myristoylation Sequence Substitutes for Gab1 Pleckstrin Homology Domain and Switches an Epidermal Growth Factor Response to an Invasive Morphogenic Program
Authors: Maroun Christiane R. , Naujokas Monica A. , Park Morag
Published: April 2003

The hepatocyte growth factor receptor tyrosine kinase Met promotes cell dissociation and the inherent morphogenic program of epithelial cells. In a search for substrates downstream from Met, we have previously identified the Grb2-associated binder-1 (Gab1) as critical for the morphogenic program. Gab1 is a scaffold protein that acts to diversify the signal downstream from the Met receptor through its ability to couple with multiple signal transduction pathways. Gab1 contains a pleckstrin homology (PH) domain with specificity for phosphatidylinositol 3,4,5-trisphosphate. The phospholipid binding capacity of the Gab1 PH domain is required for the localization of Gab1 at sites of cell-cell contact in colonies of epithelial cells and for epithelial morphogenesis, suggesting that PH domain-dependent subcellular localization of Gab1 is a prerequisite for function. We have investigated the requirement for membrane localization of Gab1 for biological activity. We show that substitution of the Gab1 PH domain with the myristoylation signal from the c-Src protein is sufficient to replace the Gab1 PH domain for epithelial morphogenesis. The membrane targeting of Gab1 enhances Rac activity in the absence of stimulation and switches a nonmorphogenic noninvasive response to epidermal growth factor to a morphogenic invasive program. These results suggest that the subcellular localization of Gab1 is a critical determinant for epithelial morphogenesis and invasiveness.
Differential Induction of Nuclear Factor-κB and Activator Protein-1 Activity after CD40 Ligation Is Associated with Primary Human Hepatocyte Apoptosis or Intrahepatic Endothelial Cell Proliferation
Authors: Choudhury Jalal Ahmed , Russell Clare L. , Randhawa Satinder , Young Lawrence S. , Adams David H. , Afford Simon C.
Published: April 2003

CD40, a tumor necrosis factor receptor superfamily member, is up-regulated on intraheptatic endothelial cells (IHEC) and epithelial cells during inflammatory liver disease, and there is evidence that the functional outcome of CD40 ligation differs between cell types. Ligation of CD40 on cholangiocytes or hepatocytes results in induction of Fas-mediated apoptosis, whereas ligation of IHEC CD40 leads to enhanced chemokine secretion and adhesion molecule expression. We now report that differential activation of two transcription factors, nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), in primary human hepatocytes or IHEC, is associated with and may explain, in part, the different responses of these cell types to CD40 ligation. CD40 ligation induced a rise in NF-κB activity in hepatocytes ,which peaked at 2 h and returned to baseline by 24 h; however, IHEC CD40 ligation resulted in a sustained up-regulation of NF-κB (>24 h). In hepatocytes, CD40 ligation led to sustained up-regulation of AP-1 activity >24 h associated with increased protein levels of RelA (p65), c-Jun, and c-Fos, whereas no induction of AP-1 activity was observed in IHECs. Analysis of mitogen-activated protein kinase phosphorylation (phospho-extracellular signal-regulated kinase 1/2 and phospho-c-Jun NH2-terminal kinase 1/2) and expression of inhibitor κBα were entirely consistent, and thus confirmed the profiles of NF-κB and AP-1 signaling and the effects of the selective inhibitors assessed using electrophoretic mobility shift assay or Western immunoblotting. CD40 ligation resulted in induction of apoptosis in hepatocytes after 24 h, but on IHECs, CD40 ligation resulted in proliferation. Inhibition of (CD40-mediated) NF-κB activation prevented IHEC proliferation and led to induction of apoptosis. Selective extracellular signal-regulated kinase and c-Jun NH2-terminal kinase inhibitors reduced levels of apoptosis in (CD40-stimulated) hepatocytes by ∼50%. We conclude that differential activation of these two transcription factors in response to CD40 ligation is associated with differences in cell fate. Transient activation of NF-κB and sustained AP-1 activation is associated with apoptosis in hepatocytes, whereas prolonged NF-κB activation and a lack of AP-1 activation in IHECs result in proliferation.
ELL and EAF1 are Cajal Body Components That Are Disrupted in MLL-ELL Leukemia
Authors: Polak Paul E. , Simone Federico , Kaberlein Joseph J. , Luo Roger T. , Thirman Michael J.
Published: April 2003

The (11;19)(q23;p13.1) translocation in acute leukemia results in the formation of a chimeric MLL-ELL fusion protein. ELL is an RNA Polymerase II (Pol II) transcriptional elongation factor that interacts with the recently identified EAF1 protein. Here, we show that ELL and EAF1 are components of Cajal bodies (CBs). Although ELL and EAF1 colocalize with p80 coilin, the signature protein of CBs, ELL and EAF1 do not exhibit a direct physical interaction with p80 coilin. Treatment of cells with actinomycin D, DRB, or α-amanitin, specific inhibitors of Pol II, disperses ELL and EAF1 from CBs, indicating that localization of ELL and EAF1 in CBs is dependent on active transcription by Pol II. The concentration of ELL and EAF1 in CBs links the transcriptional elongation activity of ELL to the RNA processing functions previously identified in CBs. Strikingly, CBs are disrupted in MLL-ELL leukemia. EAF1 and p80 coilin are delocalized from CBs in murine MLL-ELL leukemia cells and in HeLa cells transiently transfected with MLL-ELL. Nuclear and cytoplasmic fractionation revealed diminished expression of p80 coilin and EAF1 in the nuclei of MLL-ELL leukemia cells. These studies are the first demonstration of a direct role of CB components in leukemogenesis.