bluuebunny/crossref_metadata_2025 huggingface dataset:
278 MEIOTIC RESUMPTION IN VITRO OF CANINE OOCYTES: COMPARATIVE METHODS
Authors: Bogliolo L. , Ariu F. , Zedda M.T. , Pau S. , Ledda S.
Published: December 2004

In the bitch, mechanisms responsible for the meiotic resumption and progression of the oocytes are not known. In order to better understand cellular signals involved in canine oocyte meiosis, the present study was performed to investigate the ability of bitch oocytes to resume meiosis in vitro (1) after culture with okadaic acid (OA), and (2) after cell fusion with bovine MII oocytes. For this purpose oocytes were collected from ovaries of bitches undergoing ovariectomy. Ovaries were sliced repeatedly to release oocytes; only cumulus-oocytes complexes with two or more dense layer of cumulus cells, darkly granulated cytoplasm, and >110 μm in diameter were selected for experiments. In the first experiment, oocytes were pre-incubated for different times (1, 3, 20 h) in TCM 199 + 20% FCS + 0.5 μM or 2 μM OA and thereafter cultured for 48 h in the same medium without OA. A group of oocytes was matured in TCM 199 + 20% FCS for 72 h as control. At the end of culture, oocytes were stained with glycerol-Hoechst 33342 to evaluate meiotic stage. Results indicated that incubation with 2 μM OA for 1, 3, and 20 h determined a significantly higher (P < 0.001) meiotic resumption (GVBD) of canine oocytes compared to that in the control group, but the percentage of oocytes reaching MI and MII did not increase. Similar results were obtained after culture with 0.5 μM OA for 3 and 20 h. However, meiotic progression to MI and MII was significantly improved (P < 0.05) after incubation with 0.5 μM OA for 1 h. In the second experiment canine oocytes at GV stage were fused with MII bovine oocytes matured in vitro. This experiment was designed to test whether the high activity of MPF of MII bovine oocytes was able to determine modification of GV of canine oocytes. The zonae pellucidae from both GV and MII oocytes were removed using 0.1% pronase. Pairs of oocytes (n = 37) were agglutinated in medium containing 250 mg/mL phytohemoagglutinin placed between two electrodes in 0.5% glucose fusion medium, fused with a single pulse of direct current (1.25 KV/cm for 80 ms) and cultured for 2–3 h in TCM 199 + 10% FCS. After culture fused partners (n = 36) were stained with glycerol-Hoechst 33342 and evaluated. Results indicated that the fusion of MII bovine oocytes to GV reporter canine oocytes failed to induce nuclear membrane disassembly, chromatin condensation, or modification of canine nuclei in all of the fused combinations. These data suggest that OA induces meiotic resumption of canine oocytes. However, the cell fusion results seem to indicate that increased levels of MPF are not able to determine cell cycle progression. Furthermore, incubation times and concentration of okadaic acid could be refined to optimize a system for meiotic maturation of bitch oocytes.

Table 1.
Meiotic progression of bitch oocytes after incubation with okadaic acid

This work was supported by MIUR (ex 40%).
328 SOMATIC CELL ISOLATION FROM SEMEN BY PERCOLL GRADIENTS
Authors: Nel-Themaat L. , Gomez M.C. , Wirtu G. , Cole A. , Bondioli K.R. , Dresser B.L. , Godke R.A. , Pope C.E.
Published: December 2004

We previously isolated epithelial-like cells (ELC) from sheep (Ovis aries) and eland (Taurotragus oryx) ejaculates (Nel-Themaat et al. 2004 Reprod. Fertil. Dev. 16, 152). Success rates were low, and the presence of live sperm during initial culture may have altered medium properties, physically prevented contact between cells and the substrate, and/or damaged somatic cell membranes. Using the complete semen sample requires the sacrifice of valuable live spermatozoa without the certainty of obtaining a somatic cell population. Therefore, the purpose of the present study was to develop a method for isolating somatic cells from semen before culture. Separation by discontinuous Percoll gradient centrifugation was performed. Ejaculates obtained by electro-ejaculation from two Louisiana Native rams and rectal massage of one eland bull were washed, layered on columns of 2.5 mL each of 20% (P-20), 50% (P-50), and 90% (P-90) Percoll in 15-mL conical tubes, and centrifuged for 20 min at 400g. Bands were obtained at the three interfaces, as well as a pellet in the P-90 fraction. Each Percoll layer plus the interface band immediately below it was collected by aspiration. Cells from each fraction were re-suspended in serum-supplemented Minimum Essential Medium Alpha containing 500 IU/mL of penicillin, 0.5 mg/mL of streptomycin and 250 g/mL of gentamycin, and were plated on collagen-1 coated dishes. Trypan blue staining was used to determine cell viability in each fraction. After 24 h, dishes were washed and the culture continued until cells attached and proliferated. For both species, the surface fraction yielded primarily dead ELC that were morphologically flat and angular in shape. In the P-20 band, more viable cells were isolated compared with other fractions, although dead cells were also present. In the P-50 and P-90 fractions, few ELC were observed compared with the surface and P-20 fractions. A negligible number of sperm was observed in the surface and P-20 fractions. In contrast, mostly dead sperm were found at the P-50 and P-90 interface band, whereas live spermatozoa were detected in the P-90 pellet. Epithelial-like cells isolated from semen of both species attached (A, initial 24 h of culture), divided (D, within 3 days after plating), and proliferated (P, after 1 week of culture) (see Table). Ram ELC derived from the surface and from the P-20 and P-50 layers attached, but proliferation was detected only with cells collected in the P-20 fraction. In contrast, eland cells collected from the surface and P-20 layers readily attached and proliferated. In summary, a technique has been developed for the isolation of somatic cells from semen using a three-layer Percoll gradient. This method also allows the isolation of motile spermatozoa, hich are available for further use.
43 DNA SYNTHESIS, PREIMPLANTATION DEVELOPMENT AND Oct-4 EXPRESSION OF BOVINE CLONES RECONSTRUCTED WITH OOCYTES PREACTIVATED OR ENUCLEATED AFTER SPINDLE DISASSEMBLY
Authors: Kurosaka S. , McLaughlin K.J.
Published: December 2004

Enucleation procedures applied in mammalian cloning remove not only the oocyte's chromosomes but presumably also the spindle-associated factors. If these factors are beneficial for reprogramming, alternative protocols that limit enucleation of factors in addition to the chromosomes may improve cloning efficiency. In this study, we evaluated the enucleation in combination with various activation protocols on clone development and gene expression. Clones produced by nuclear transfer into pre-activated bovine oocytes rather than non-activated oocytes can develop in vitro (Kurosaka et al. 2002 Biol. Reprod. 67, 643–647; Tani et al. 2003 Biol. Reprod. 69, 1890–1894). We produced bovine clones using four different nuclear transfer protocols and, in clones of all groups, examined timing of DNA synthesis in the first cell cycle, pre-implantation development, and gene expression at the blastocyst stage. Protocols applied were: (A) donor cells were fused with non-activated oocytes; (B) donor cells were fused with oocytes at 2 hours after activation with ethanol (7%, 7 min); (C) oocytes were enucleated after spindle disassembly with nocodazole treatment (0.3 μg/mL, 30 min) and donor cells were fused with non-activated oocytes; and (D) oocytes were enucleated after spindle disassembly and donor cells were fused with oocytes at 2 h after activation. Fused couplets in all treatment groups were treated with 10 μg/mL cycloheximide for 6 h, and cultured in vitro in SOF supplemented with fetal bovine serum at 39°C in an atmosphere of 5% CO2, 5% O2, and 90% N2. The onset of DNA synthesis was determined by an immunofluorescence assay of 5-bromo-deoxyuridine incorporation at 6 and 9 h post-fusion (hpf). Oct-4 mRNA distribution in clone blastocysts was examined by whole mount in situ hybridization using a bovine Oct-4-specific antisense riboprobe. Data were statistically analyzed with Student's t-test. In the majority of clones DNA synthesis had not commenced 6 hpf but had initiated 9 hpf. Although the cell cycle of activated oocytes (protocols B and D) was 2 hours advanced compared to non-activated oocytes (protocols A and C), clones produced by all protocols had a similar onset of DNA synthesis at 6 to 9 h post-fusion. Developmental rates to the blastocyst stage of clones were not significantly different between the four protocols (48.5%–57.7%, P < 0.05). Oct-4 distribution in clones produced by all four protocols was not different from that of IVF embryos used as a control in that Oct-4 mRNA signal was typically restricted to the ICM (87.0%–100.0%, P < 0.05). We conclude that in bovine clones produced in this study, nocodazole-treated enucleation and activation status of recipient oocyte did not influence the pre-implantation development and spatial pattern of Oct-4 expression.
This work was supported by the Lalor Foundation.
91 EMBRYO DEVELOPMENT AFTER ICSI OF EQUINE OOCYTES VITRIFIED BEFORE AND AFTER IVM
Authors: Merlo B. , Iacono E. , Colleoni S. , Dell'Aquila E. , Galli C. , Mari G.
Published: December 2004

Vitrification has proven to be the method of choice for cryopreservation of mammalian oocytes. In this study, we evaluated in vitro embryonic developmental competence of equine oocytes, vitrified before and after IVM, and fertilized by ICSI. The benefits of the interaction between Naloxone (Nx) and endogenous opioids peptide receptors in different conditions of cellular stress have already been demonstrated (Sheu et al. 1997 Biochem. Biophys. Res. Comm. 231, 12–16). In this study we determined whether addition of Nx to the vitrification solutions can limit the oocyte's damages. COCs collected April to June from abattoir ovaries were: (1) vitrified immediately after recovery (PREM) or (2) matured for 24 h in TCM 199 (Galli et al. 2002 Theriogenology 58, 705–708) before vitrification (POSTM). Half of the oocytes of the two groups were vitrified using solutions supplemented with 10−8 M Nx. Cryoprotectants were loaded in three steps as reported by Maclellan et al. (2002 Theriogenology 58, 911–919). Oocytes were placed on a nylon cryoloop (Hampton Research, Laguna Niguel, CA, USA) and immediately plunged into liquid nitrogen. Oocytes were thawed by immersing the loop sequentially in 0.25 M, 0.188 M, and 0.125 M sucrose in HEPES synthetic oviductal fluid (HSOF) for 30 s per step. PREM oocytes were subjected to 24 h IVM, POSTM were cultured 2–3 h after thawing. Matured oocytes, as assessed by the presence of the first polar body, underwent ICSI. Frozen semen was separated over a discontinuous Percoll gradient and denuded oocytes were injected with a single spermatozoon. Non-vitrified oocytes matured under the same conditions were used as a control. Injected oocytes were cultured in SOFaa until Day 9 (Day 0 day of ICSI). Vitrification was done in five replicates and all oocytes were injected on the same day. Chi-square test was used for statistical analysis (Statistica for Windows; Stat Soft, Inc., Tulsa, OK, USA); significance was assessed at P < 0.05. Results are reported in Table 1. The number of degenerated oocytes and the cleavage rates were not significantly different among treatments (P > 0.05). Within vitrified COCs, only those with Nx in the vitrification solutions reached the blastocyst stage at Day 9; because of the low number of oocytes used in this work, blastocyst rate was not different among treatments. Further studies are needed to evaluate the benefits of adding Nx to oocyte vitrification solutions.

Table 1.
Embryo development after ICSI of vitrified equine oocytes

This research was funded by MIUR Cofin PRIN 2003.
Populations of photoinactivated photosystem II reaction centers characterized by chlorophyllafluorescence lifetimein vivo
Authors: Matsubara Shizue , Chow Wah Soon
Published: December 2004

Photosystem (PS) II centers, which split water into oxygen, protons, and electrons during photosynthesis, require light but are paradoxically inactivated by it. Prolonged light exposure concomitantly decreased both the functional fraction of PSII reaction centers and the integral PSII chlorophyll (Chl)afluorescence lifetime in leaf segments ofCapsicum annuumL. Acceleration of photoinactivation of PSII by a pretreatment with the inhibitors/uncoupler lincomycin, DTT, or nigericin further reduced PSII Chlafluorescence lifetimes. A global analysis of fluorescence lifetime distributions revealed the presence of at least two distinct populations of photoinactivated PSII centers, one at 1.25 ns, and the other at 0.58 ns. Light treatment first increased the 1.25-ns component, a weak quencher, at the expense of a component at 2.22 ns corresponding to functional PSII centers. The 0.58-ns component, a strong quencher, emerged later than the 1.25-ns component. The strongly quenching PSII reaction centers could serve to avoid further damage to themselves and protect their functional neighbors by acting as strong energy sinks.
The long-range organization of a native photosynthetic membrane
Authors: Frese Raoul N. , Siebert C. Alistair , Niederman Robert A. , Hunter C. Neil , Otto Cees , van Grondelle Rienk
Published: December 2004

Photosynthesis relies on the delicate interplay between a specific set of membrane-bound pigment–protein complexes that harvest and transport solar energy, execute charge separation, and conserve the energy. We have investigated the organization of the light-harvesting (LH) and reaction-center (RC) complexes in native bacterial photosynthetic membranes of the purple bacterium
Rhodobacter sphaeroides
by using polarized light spectroscopy, linear dichroism (LD) on oriented membranes. These LD measurements show that in native membranes, which contain LH2 as the major energy absorber, the RC–LH1–PufX complexes are highly organized in a way similar to that which we found previously for a mutant lacking LH2. The relative contribution of LH1 and LH2 light-harvesting complexes to the LD spectrum shows that LH2 preferentially resides in highly curved parts of the membrane. Combining the spectroscopic data with our recent atomic force microscopy (AFM) results, we propose an organization for this photosynthetic membrane that features domains containing linear arrays of RC–LH1–PufX complexes interspersed with LH2 complexes and some LH2 found in separate domains. The study described here allows the simultaneous assessment of both global and local structural information on the organization of intact, untreated membranes.
Lack of delta waves and sleep disturbances during non-rapid eye movement sleep in mice lacking α1
G
-subunit of T-type calcium channels
Authors: Lee Jungryun , Kim Daesoo , Shin Hee-Sup
Published: December 2004

T-type calcium channels have been implicated as a pacemaker for brain rhythms during sleep but their contribution to behavioral states of sleep has been relatively uncertain. Here, we found that mice lacking α1
G
T-type Ca
2+
channels showed a loss of the thalamic delta (1–4 Hz) waves and a reduction of sleep spindles (7–14 Hz), whereas slow (<1 Hz) rhythms were relatively intact, when compared with the wild-type during urethane anesthesia and non-rapid eye movement (NREM) sleep. Analysis of sleep disturbances, as defined by the occurrence of brief awakening (BA) episodes during NREM sleep, revealed that mutant mice exhibited a higher incidence of BAs of >16 sec compared with the wild-type, whereas no difference was seen in BAs of <16 sec between the two genotypes. These results are consistent with the previous idea of the distinct nature of delta oscillations and sleep spindles from cortically generated slow waves. These results also suggest that the α1
G
-subunit of T-type calcium channels plays a critical role in the genesis of thalamocortical oscillations and contributes to the modulation of sleep states and the transition between NREM sleep and wake states.
Estrogen mediates Aurora-A overexpression, centrosome amplification, chromosomal instability, and breast cancer in female ACI rats
Authors: Li Jonathan J. , Weroha S. John , Lingle Wilma L. , Papa Dan , Salisbury Jeffrey L. , Li Sara Antonia
Published: December 2004

Estrogens play a crucial role in the causation and development of sporadic human breast cancer (BC). Chromosomal instability (CIN) is a defining trait of early human ductal carcinoma
in situ
(DCIS) and is believed to precipitate breast oncogenesis. We reported earlier that 100% of female ACI (August/Copenhagen/Irish) rats treated with essentially physiological serum levels of 17β-estradiol lead to mammary gland tumors with histopathologic, cellular, molecular, and ploidy changes remarkably similar to those seen in human DCIS and invasive sporadic ductal BC. Aurora-A (Aur-A), a centrosome kinase, and centrosome amplification have been implicated in the origin of aneuploidy via CIN. After 4 mo of estradiol treatment, levels of Aur-A and centrosomal proteins, γ-tubulin and centrin, rose significantly in female ACI rat mammary glands and remained elevated in mammary tumors at 5–6 mo of estrogen treatment. Centrosome amplification was initially detected at 3 mo of treatment in focal dysplasias, before DCIS. At 5–6 mo, 90% of the mammary tumor centrosomes were amplified. Comparative genomic hybridization revealed nonrandom amplified chromosome regions in seven chromosomes with a frequency of 55–82% in 11 primary tumors each from individual rats. Thus, we report that estrogen is causally linked via estrogen receptor α to Aur-A overexpression, centrosome amplification, CIN, and aneuploidy leading to BC in susceptible mammary gland cells.
Activation of Soluble Guanylyl Cyclase at the Leading Edge duringDictyosteliumChemotaxis
Authors: Veltman Douwe M. , Roelofs Jeroen , Engel Ruchira , Visser Antonie J.W.G. , Van Haastert Peter J.M.
Published: December 2004

Dictyostelium contains two guanylyl cyclases, GCA, a 12-transmembrane enzyme, and sGC, a homologue of mammalian soluble adenylyl cyclase. sGC provides nearly all chemoattractant-stimulated cGMP formation and is essential for efficient chemotaxis toward cAMP. We show that in resting cells the major fraction of the sGC-GFP fusion protein localizes to the cytosol, and a small fraction is associated to the cell cortex. With the artificial substrate Mn2+/GTP, sGC activity and protein exhibit a similar distribution between soluble and particulate fraction of cell lysates. However, with the physiological substrate Mg2+/GTP, sGC in the cytosol is nearly inactive, whereas the particulate enzyme shows high enzyme activity. Reconstitution experiments reveal that inactive cytosolic sGC acquires catalytic activity with Mg2+/GTP upon association to the membrane. Stimulation of cells with cAMP results in a twofold increase of membrane-localized sGC-GFP, which is accompanied by an increase of the membrane-associated guanylyl cyclase activity. In a cAMP gradient, sGC-GFP localizes to the anterior cell cortex, suggesting that in chemotacting cells, sGC is activated at the leading edge of the cell.
Lipopolysaccharide Is a Cofactor for Malondialdehyde‐Acetaldehyde Adduct–Mediated Cytokine/Chemokine Release by Rat Sinusoidal Liver Endothelial and Kupffer Cells
Authors: Duryee Michael J. , Klassen Lynell W. , Freeman Thomas L. , Willis Monte S. , Tuma Dean J. , Thiele Geoffrey M.
Published: December 2004

Background: The nonparenchymal cells of the liver have been suggested to play a significant role in the inflammatory processes observed in the development and/or progression of alcoholic liver disease. Our laboratories have shown that malondialdehyde‐acetaldehyde (MAA)‐modified proteins can induce immune responses, cytokine/chemokine secretion, and antigen processing and presentation by liver sinusoidal endothelial cells (SECs). Another molecule that has been shown to induce similar types of responses in Kupffer cells (KCs) is lipopolysaccharide (LPS). Because these materials induce similar responses, it was the purpose of this study to investigate the relationship between LPS and MAA‐modified proteins in the development of proinflammatory responses by SECs and KCs.

Methods: For these studies, SECs and KCs were isolated from chow‐fed, pair‐fed, and ethanol‐fed rats. Cells were stimulated with media alone, bovine serum albumin (Alb), or MAA‐modified Alb (MAA‐Alb) in the presence or absence of LPS 1 ng/ml, and the supernatants were assayed by enzyme‐linked immunosorbent assay for tumor necrosis factor α, macrophage chemotactic protein 1, and macrophage inhibitory protein.

Results: All three cytokines/chemokines were 3 to 5 times higher when SECs or KCs were stimulated by MAA‐Alb in the presence of LPS, in contrast to cells stimulated with Alb or media in the presence of LPS. Chronic ethanol consumption (6 weeks) had variable effects on the secretion of these cytokines/chemokines but in general did not alter the increased secretion in response to MAA‐Alb in the presence of LPS.

Conclusions: These studies strongly suggest that the sensitization of SECs and KCs by LPS plays a significant role in the development and/or progression of alcoholic liver disease, and the subsequent activation by MAA‐modified proteins may be a mechanism by which proinflammatory processes are initiated.
Comorbidity of Select Anxiety and Affective Disorders With Alcohol Dependence in Southwest California Indians
Authors: Gilder David A. , Wall Tamara L. , Ehlers Cindy L.
Published: December 2004

Background: Native Americans, overall, have the highest prevalence of alcohol dependence of any US ethnic group. In several large national surveys, alcohol dependence has been significantly associated with higher rates of anxiety and affective disorders (comorbidity). However, the frequencies of these disorders and their comorbidity with alcohol dependence in Native American populations are relatively unknown.

Methods: Demographic information and DSM‐III‐R diagnoses were obtained by using the Semi‐Structured Assessment for the Genetics of Alcoholism developed for the Collaborative Study on the Genetics of Alcoholism from 483 Southwest California Indian adults residing on contiguous reservations. The Semi‐Structured Assessment for the Genetics of Alcoholism allowed differentiation of each anxiety and affective disorder into one of three types: independent of substance use, concurrent with alcohol use, and concurrent with drug use.

Results: Sixty‐six percent of the men and 53% of the women sampled had a lifetime diagnosis of alcohol dependence. Fourteen percent of the sample had a lifetime independent anxiety disorder, and 14% of the sample had a lifetime independent affective disorder. Alcohol‐ and/or drug‐concurrent major depression occurred in 8%, and other alcohol‐ and/or drug‐concurrent anxiety and affective disorders each occurred in less than 1.1% of the sample. No significant comorbidity was found between alcohol dependence and independent agoraphobia, social phobia, or major depressive disorder.

Conclusions: In this Southwest California Indian sample, rates of anxiety and affective disorders were substantially similar to those reported in the National Comorbidity Survey; however, comorbidity of independent disorders with alcohol dependence was not as pervasive as in the National Comorbidity Survey. Rates of concurrent anxiety and affective disorders were low. These data support the hypothesis that despite high rates of alcohol dependence, Southwest California Indians do not have higher rates of anxiety and affective disorders or comorbidity of these disorders with alcohol dependence than those reported in large surveys of non–American Indian populations.
Increased Ethanol Drinking After Repeated Chronic Ethanol Exposure and Withdrawal Experience in C57BL/6 Mice
Authors: Becker Howard C. , Lopez Marcelo F.
Published: December 2004

Background: The development of dependence may have significant motivational consequences regarding continued use and abuse of ethanol. We have developed a mouse model of ethanol dependence and repeated withdrawals that demonstrates sensitization of seizures and other symptoms of withdrawal. It is unclear whether such experience influences ethanol drinking behavior. The present series of experiments were designed to examine whether repeated cycles of chronic ethanol exposure and withdrawal has an impact on subsequent motivation to voluntarily self‐administer ethanol.

Methods: With the use of a modified sucrose‐fading procedure, adult male C57BL/6J mice were trained to drink 15% (v/v) ethanol in a limited access procedure (2 hr/day). The animals were not food or water deprived at any time during the experiments. Once stable baseline intake was established, mice were exposed to four cycles of 16 hr of ethanol vapor (or air) in inhalation chambers separated by 8‐hr periods of withdrawal. At 32 hr after the last cycle of ethanol exposure, all mice were tested for ethanol intake under limited access conditions for 5 consecutive days. The animals then received a second series of chronic ethanol exposure and withdrawal followed by another 5‐day test period for ethanol drinking.

Results: Stable daily baseline intake was established in mice that drank 15% ethanol combined with 5% sucrose (experiment 1), 15% ethanol alone (experiment 2), 5% sucrose alone (experiment 3), or 15% ethanol when presented as a choice with water (experiment 4). After repeated cycles of chronic ethanol exposure and withdrawal experience, consumption of ethanol solutions increased over baseline levels and in comparison with control (air‐exposed) groups. However, sucrose consumption did not change in mice that were trained to drink 5% sucrose. The increase in ethanol consumption after chronic ethanol exposure and withdrawal experience resulted in a significant increase in resultant blood ethanol levels.

Conclusions: Once the positive reinforcing properties of ethanol were established, chronic ethanol exposure and withdrawal experience resulted in a significant increase in voluntary ethanol drinking that yielded a >2‐fold increase in resultant blood ethanol levels. This increase in ethanol intake occurred whether ethanol was presented in combination with sucrose, alone (unadulterated), or as a choice with tap water. Furthermore, this effect seems to be selective for ethanol in that animals that were trained to drink a sucrose solution did not exhibit a change in their intake after similar chronic ethanol exposure. As such, this model may be useful in studying the mechanisms and conditions in which chronic ethanol treatment influences motivation to resume drinking after a period of abstinence (relapse).
Transmissible spongiform encephalopathy strain, PrP genotype and brain region all affect the degree of glycosylation of PrPSc
Authors: Somerville Robert A. , Hamilton Scott , Fernie Karen
Published: December 2004

Transmissible spongiform encephalopathies (TSEs), sometimes known as prion diseases, are caused by an infectious agent whose molecular properties have not been determined. Traditionally, different strains of TSE diseases are characterized by a series of phenotypic properties after passage in experimental animals. More recently it has been recognized that diversity in the degree to which an abnormal form of the host protein PrP, denoted PrPSc, is glycosylated and the migration of aglycosyl forms of PrPSc on immunoblots may have some differential diagnostic potential. It has been recognized that these factors are affected by the strain of TSE agent but also by other factors, e.g. location within the brain. This study shows in some cases, but not others, that host PrP genotype has a major influence on the degree of PrPSc glycosylation and migration on gels and provides further evidence of the effect of brain location. Accordingly both the degree of glycosylation and the apparent molecular mass of PrPSc may be of some value for differential diagnosis between TSE strains, but only when host effects are taken into account. Furthermore, the data inform the debate about how these differences arise, and favour hypotheses proposing that TSE agents affect glycosylation of PrP during its biosynthesis.
Type 1 and type 2 human PrPSc have different aggregation sizes in methionine homozygotes with sporadic, iatrogenic and variant Creutzfeldt–Jakob disease
Authors: Kobayashi Atsushi , Satoh Sakae , Ironside James W. , Mohri Shirou , Kitamoto Tetsuyuki
Published: December 2004

In Creutzfeldt–Jakob disease (CJD), the type (type 1 or 2) of abnormal isoform of the prion protein (PrPSc) in the brain and the genotype at codon 129 of the PrP gene are major determinants of clinicopathological phenotype. Little is known about the difference in biochemical properties between the two types of PrPSc, except for the different proteinase K cleavage sites. To investigate the size of aggregates formed by PrPSc types 1 and 2, brain homogenates from various cases of CJD with the same genotype (homozygous for methionine at codon 129) were passed through filters with a mean pore size of 72±4 nm. Type 2 PrPSc was efficiently removed from the filtrates by the filters, in contrast to type 1. Even type 2 PrPSc from a patient without amyloid plaques was removed more efficiently than type 1 from patients with amyloid plaques. These results indicate that type 2 PrPSc has a larger aggregation size than type 1, irrespective of the existence of amyloid plaques.
Genetic variability and population structure of Grapevine leafroll-associated virus 3 isolates
Authors: Turturo Camilla , Saldarelli Pasquale , Yafeng Dong , Digiaro Michele , Minafra Angelantonio , Savino Vito , Martelli G. P.
Published: December 2004

The genetic variability and population structure of a collection of 45 Grapevine leafroll-associated virus 3 (GLRaV-3) isolates were studied by single-stranded conformation polymorphism (SSCP) and sequence analysis of the RNA-dependent RNA polymerase (RdRp), heat-shock protein 70 homologue (HSP-70) and coat protein (CP) genes. The distribution of SSCP profiles was not correlated with the geographical origin of the isolates, indicating that GLRaV-3 is a single, undifferentiated population. The majority of the isolates showed an SSCP profile and a population structure that were composed of a single predominant variant. However, 10 % of the isolates for the RdRp and HSP-70 genes and 15 % for the CP gene were composed of a combination of two or more variants. Estimation of genetic diversity and phylogenetic analysis disclosed the possible existence of vines with mixed infections by diverging sequence variants, showing, in some cases, possible recombination events. Furthermore, differences in the genetic diversity and constraints existing in the three regions analysed indicated a higher variability in the CP gene. The epidemiological and biological implications of this finding are discussed.
Polymorphisms at codons 141 and 154 in the ovine prion protein gene are associated with scrapie Nor98 cases
Authors: Moum Truls , Olsaker Ingrid , Hopp Petter , Moldal Torfinn , Valheim Mette , Moum Torbjørn , Benestad Sylvie L.
Published: December 2004

Until June 2004, thirty-eight scrapie cases with unusual features, designated Nor98, have been diagnosed in Norway. This study investigated the distribution of PrP genotypes among Nor98 cases, their flock-mates and a random sample of Norwegian slaughtered sheep. The PrP genotype distribution of Nor98 cases differed markedly from that of previous cases of classical scrapie. A leucine/phenylalanine polymorphism at codon 141 with hitherto unknown significance to scrapie was strongly associated with Nor98 cases. Twenty of 38 (52·6 %) cases were either homozygous or heterozygous for phenylalanine at codon 141. In contrast, this allele was present in only 10·5 % of the flock-mates and 4·5 % of the random sample of slaughtered sheep. Moreover, the H154 allele was represented in 24 of 38 (63·2 %) of Nor98 cases, as opposed to 27·0 % of Nor98 flock-mates and 17·0 % of the slaughtered sheep.
Role of the protein kinase PKR in the inhibition of varicella-zoster virus replication by beta interferon and gamma interferon
Authors: Desloges Nathalie , Rahaus Markus , Wolff Manfred H.
Published: December 2004

Varicella-zoster virus (VZV) is sensitive to type I and type II interferons (IFNs), which mediate antiviral effects. In this study, it was demonstrated that IFN-β and IFN-γ inhibited the replication of VZV in vitro. Although IFN-β was more effective than IFN-γ, the level of inhibition of VZV replication achieved by the combination of both IFNs was more than additive and it was concluded that these two cytokines acted synergistically. Expression of the IFN-induced, double-stranded RNA-activated protein kinase PKR and its phosphorylation level were not modulated strongly during ongoing replication of VZV. However, in the presence of IFN-β, but not IFN-γ, PKR expression and its phosphorylation were increased, explaining in part the inhibition of virus replication by IFNs. The expression of herpes simplex virus Us11, a viral protein with several functions, including prevention of PKR activation, strongly increased the level of VZV replication.
The arginine-rich carboxy-terminal domain of the hepatitis B virus core protein mediates attachment of nucleocapsids to cell-surface-expressed heparan sulfate
Authors: Vanlandschoot Peter , Van Houtte Freya , Serruys Benedikte , Leroux-Roels Geert
Published: December 2004

Binding of hepatitis B virus nucleocapsids to mouse B cells leads to production of nucleocapsid-specific antibodies, class II presentation of peptides and the generation of T helper-1 immunity. This T-cell-independent activation of B cells is thought to result from cross-linking of cell-surface immunoglobulin molecules, if these contain a specific motif in the framework region 1–complementarity determining region 1 junction. In the present study, it was observed that nucleocapsids bound to different B-cell lines, an interaction that was not dependent on cell-surface-expressed immunoglobulins. Furthermore, binding to several non-B-cell lines was observed. Capsids that lacked the carboxy-terminal protamine-like domains did not bind to cells. Treatment of nucleocapsids with ribonucleases enhanced the attachment of nucleocapsids to cells. Various soluble glycosaminoglycans inhibited attachment of nucleocapsids, while treatment of cells with heparinase I also reduced binding. These observations demonstrated that the arginine-rich protamine-like regions of the core proteins are responsible for the attachment of nucleocapsids to glycosaminoglycans expressed on the plasma membranes of cells.
Substitution of proline 306 in the reverse transcriptase domain of hepatitis B virus regulates replication
Authors: Lin Xu , Ma Zhang-Mei , Yao Xin , He Li-Fang , Yuan Zheng-Hong , Ding Jian-Ping , Wen Yu-Mei
Published: December 2004

The proline residue at position 306 in hepatitis B virus (HBV) reverse transcriptase (rtP306) has been suggested to constrain the conformation of the α-helices in the thumb subdomain that interacts with the viral DNA template-primer. To study the impact of residue rt306 in HBV replication further, 11 site-directed mutants were constructed that substituted rtP306 with different amino acids. The replicative competencies of these mutants were assayed by HepG2 cell transfection and real-time PCR. When rtP306 was substituted with glycine or threonine, the replication competency of these mutants was drastically reduced to 1·96 and 4·51 % of the wild-type HBV level, respectively. When rtP306 was substituted with glutamic acid, the replicative competency of the mutant increased up to 9·4-fold compared with wild-type virus. The results also showed that changes in the replicative competency of these constructed mutants were not associated with functional changes of HBV enhancer I. These results indicate the importance of amino acid(s) at the interface between the thumb and palm subdomains in modulating the replicative competency of HBV isolates. This regulatory residue(s) could serve as a new target for the development of anti-HBV drugs.
Sleep apnea and hormone replacement therapy: a pilot study and a literature review
Authors: Wesström Jan , Ulfberg Jan , Nilsson Staffan
Published: December 2004

Background.  Sleep apnea syndrome (SAS) is a common condition and a risk factor of cardiovascular and cerebrovascular diseases. The purpose of this pilot study was to investigate the effect of a gestagen (trimegeston) and estradiol hormone combination on perimenopausal and postmenopausal women with SAS.

Methods.  Four postmenopausal and one perimenopausal women were studied by polysomnography before and after treatment with hormone replacement therapy (HRT).

Results.  The subjects had a mean reduction of the severity of their sleep apnea by 75% measured by apnea/hypopnea index.

Conclusions.  HRT might be an alternative in the treatment of SAS.
Threshold values of maternal blood glucose in early diabetic pregnancy – prediction of fetal malformations
Authors: Wender‐Ożegowska E. , Wróblewska K. , Zawiejska A. , Pietryga M. , Szczapa J. , Biczysko R.
Published: December 2004

Background.  The prevention of congenital malformations in the newborns of diabetic mothers still constitutes one of the main problems in this group of patients.

Aim.  The aim of this study was to analyze the prevalence of fetal malformations in diabetic pregnancies, as well as detection of the cut‐off points for the first‐trimester glycemia levels, relating to diabetes‐induced fetal malformations.

Methods.  The data for analysis were collected retrospectively from the case histories of diabetic pregnant women and their newborns, treated in our departments. For the evaluation of maternal diabetes control, the whole‐day glycemia profiles as well as glycated hemoglobin (HbA1C) levels were registered. To establish the glucose cut‐off values for malformations, we have used receiver operating characteristic (ROC) curves for fasting, 1‐hr, and 2‐hr postprandial glucose levels. To determine how metabolic control influences the risk of giving birth to a malformed infant, we followed 198 newborns of diabetic mothers and 4700 infants born of healthy mothers (control group).

Results.  We detected malformations in the infants of 8.6% (n = 17) of diabetic mothers and 3.8% of the control (odds ratio: 2.35, 95% CI = 1.40–3.96). We compared this group of diabetic patients to another diabetic pregnancy group, analyzed over a period of 1988–93 (n = 209), in which 13 newborns (6.2%) manifested congenital malformations (odds ratio: 1.41, 95% CI = 0.67–2.99) (the difference was statistically insignificant). HbA1C level during organogenesis was not significantly higher in women whose infants were malformed. We proved, however, that the risk of malformations was higher, when HbA1C value exceeded 9.3%. The malformation rate in diabetes classes D–H (according to White) was higher than in classes B and C, but the difference was not significant. A wide spectrum of anomalies has been observed in the newborns of diabetic mothers.

Conclusions.  Our results confirm the view that diabetic pregnancy, despite the improved metabolic control, is still a strong risk factor for alterations in fetal development, particularly in patients with a tendency to brittle glycemia during first trimester of pregnancy. It seems that keeping fasting glucose levels in first trimester below 5.8 mmol/l and postprandial glucose levels below 9.1 mmol/l can contribute to decreasing number of fetal malformations in pregestational diabetes mellitus (PGDM) pregnancy. The ROC curves appear to be useful and adequate tool for the analysis of factors influencing fetal development in diabetic pregnancy.
Can cesarean section be performed without the woman's consent?
Authors: Lyng Kristin , Syse Aslak , E. Børdahl Per
Published: December 2004

Occasionally, an obstetrician can experience conflicts between the mother and her fetus. In a situation where the life of the fetus is in danger, religious, cultural, or other convictions may cause the mother to refuse the physician's recommendations for delivery. When there is a medical indication to perform cesarean section, but the mother refuses, has the obstetrician then a right to perform a cesarean section to save the fetus from possible death or serious injury? In 1987, The Norwegian Directorate of Health made a statement on cesarean section without consent, but later, new legislation on patient rights has been introduced. In Norway, no scientific medical articles have been published on this topic, and no cases have yet been brought to court. Cases of court‐ordered cesarean section have been reported from the UK and the USA. In this report, we discuss some of the ethical, moral, and legal aspects of forced cesarean sections.
Maternity wards or emergency obstetric rooms? Incidence of near‐miss events in African hospitals
Authors: Filippi Veronique , Ronsmans Carine , Gohou Valerie , Goufodji Sourou , Lardi Mohamed , Sahel Amina , Saizonou Jacques , De Brouwere Vincent
Published: December 2004

Background.  This study examines near‐miss obstetric events in African hospitals as to the frequency, nature, and ratio of near miss to death and considers whether these could become useful indicators for monitoring the performance of obstetric services in Africa.

Methods.  Prospective or retrospective reviews of medical records were conducted in nine referral hospitals in three countries (Benin, Côte d'Ivoire, and Morocco). We calculated the incidence of near‐miss obstetric events, near‐miss cases, and maternal deaths related to hemorrhage, hypertensive diseases of pregnancy, dystocia, infections, and anemia and analyzed these according to hospital and timing relative to admission.

Results.  The incidence of near‐miss cases was varied, and in some hospitals extremely large: from 1% to almost a quarter of all deliveries were near misses. Near‐miss cases were 15 times more common than deaths (ranging from a ratio of 9 : 1–108 : 1). Most of the women with near‐miss events (NMEs) (83%) were already in a critical condition on arrival at the hospital (range 54–90%), and two in three were referred from another facility. The most frequent types of NMEs were hemorrhage and hypertensive diseases of pregnancy, but anemia was the leading cause in three first referral level hospitals in Benin and Côte d'Ivoire. Near‐miss events due to infections were rare.

Conclusions.  Near‐miss events are extremely common in some African hospitals, with a high proportion arriving in critical conditions. Near‐miss events must be estimated separately for those already in a critical condition on arrival and those developing after admission; the first as a good indicator of the effectiveness of emergency referrals and the second as a potential tool for monitoring the performance of obstetric services.
Growth rate and nutritional status of wild and released reared juvenile turbot in southern Kattegat, Denmark
Authors: Paulsen H. , Støttrup J. G.
Published: December 2004

More than 800 000 reared juvenile turbot Psetta maxima were released in Danish waters during the period 1990 to 2002, as part of a stock enhancement programme. Performance of the released fish has been investigated using a combination of surveys and experimental studies. A total of 1278 wild fish and 439 alizarine marked released fish were caught in 16 surveys from 1993 to 1997. Recaptures were made over a period of c. 2 years after release. Results of growth analysis and measurements of nutritional status (condition factor, liver index, liver dry matter and otolith residual mass) showed only minor differences between wild and released fish. Growth rates of wild and of released fish were comparable to fish kept in the laboratory at the same temperature (specific length growth rate c. 0·5% day−1 at 75–185 mm total length and 12·5° C). The results indicated sufficient food in the release area, but significant differences in growth and in nutritional status, between  years, were also observed. Nutritional status of both wild and released fish was comparable to fed fish in the experiments and significantly higher than in fish starved for  > 1 week. The effects of the transport and release procedures used were examined by simulated releases in laboratory tanks. The fish were observed to be feeding and growing normally during the first week after simulated release, suggesting this species to be relatively robust and unaffected by the handling and release procedures.
Predator‐induced hyperventilation in wild and hatchery Atlantic salmon fry
Authors: Hawkins L. A. , Armstrong J. D. , Magurran A. E.
Published: December 2004

Following exposure to a predator stimulus (a brown trout Salmo trutta), the opercular rate of Atlantic salmon Salmo salar fry increased by 35·3 ± 11·0%(mean ± 95% CI). The time taken for opercular rate to decline to baseline levels depended upon the occurrence of any associated locomotory activity. Opercular rates of fish that dashed when exposed remained elevated for 38·2 ± 20·6 min, whereas those of individuals that did not move (‘freezers’) recovered within 7·2 ± 2·9 min. The duration that opercular rate remained elevated was positively correlated with the magnitude of the elevation, which was higher in ‘dashers’ than freezers. The maximum opercular rate in ‘freezers’ was similar between wild fry and hatchery‐reared fry (from wild parents). There was a significant delay, however, in hatchery compared with wild fry in the time until peak ventilatory response and onset in the decline phase. This difference in opercular response suggests that hatchery fish were slower to realize fully the potential danger from the predator. Any delay in response could be directly attributed to the effect of hatchery‐rearing environment, rather than domestication or hatchery selection effects.
Differences in the pattern of antipredator behaviour between hatchery‐reared and wild European sea bass juveniles
Authors: Malavasi S. , Georgalas V. , Lugli M. , Torricelli P. , Mainardi D.
Published: December 2004

Shoals of hatchery‐reared and wild sea bass juvenilesDicentrarchus labraxwere tested for differences in their antipredator responses towards a potential live predator, the eelAnguilla anguilla. Eight experimental shoals (i.e. replicates), each composed of 15 individuals from the same stock of juveniles (i.e. wild or hatchery), were video recorded for 5 min before and after predator exposure. A set of behavioural variables were measured during the pre‐stimulus and stimulus phases of each test and compared between the two groups of replicates. Results showed that in both hatchery‐reared and wild juveniles predator exposure elicited a significant increase in the mean level of shoal cohesiveness and mean shoal distance from the predator, and a significant decrease in the mean shoal distance from the bottom. Shoals of wild juveniles, however, aggregated more quickly and reached higher shoal cohesiveness within the first 20 s of the stimulus period than shoals of hatchery‐reared fish. During this period, the wild fish also reached the highest peak in shoal cohesiveness, which then decreased gradually towards the levels observed before predator exposure. Another component of the antipredator response, the predator inspection behaviour, was fully developed in both wild and hatchery fish. Wild fish, however, tended to inspect the predator at a closer distance than hatchery fish.