A critical evaluation of the use of filipin-permeabilized rat hepatocytes to study functions of the endoplasmic reticulum in situ

We have used transmission (TEM) and scanning electron microscopy (SEM) and leakage of lactate dehydrogenase (LDH; EC 1.1.1.27) to evaluate two published procedures which use filipin to render isolated rat hepatocytes permeable to ionic substrates. Cells treated by the procedure of Jorgenson and Nordlie retained less than 10 per cent of their LDH. TEM revealed severe damage to the internal structure of these cells, which included swelling, disintegration and extensive vesicularization of the endoplasmic reticulum (ER). Hepatocytes treated with filipin by the procedure of Gankema et al. retained 65-75 per cent of their LDH and displayed incomplete but highly variable permeability to Trypan blue. SEM revealed the loss of microvilli, other signs of swelling, and the presence of large lesions in the plasma membrane. TEM revealed signs of cell swelling, but the nuclei and the mitochondria were only moderately altered. The rough ER was not swollen, but significant fragmentation was evident and characteristic stacks of lamellar ER were never seen. We conclude that useful information about the functions of the ER in situ cannot be obtained from studies of filipin-treated cells. Our results indicate that retention of LDH is not a sufficient criterion of preservation of cell morphology and that staining with Trypan blue may significantly underestimate the permeability of cells to small ionic metabolites.     

Clinical features and predictors for disease natural progression in adults with Pompe disease: a nationwide prospective observational study

Background

Due partly to physicians’ unawareness, many adults with Pompe disease are diagnosed with great delay. Besides, it is not well known which factors influence the rate of disease progression, and thus disease outcome. We delineated the specific clinical features of Pompe disease in adults, and mapped out the distribution and severity of muscle weakness, and the sequence of involvement of the individual muscle groups. Furthermore, we defined the natural disease course and identified prognostic factors for disease progression.

Methods

We conducted a single-center, prospective, observational study. Muscle strength (manual muscle testing, and hand-held dynamometry), muscle function (quick motor function test), and pulmonary function (forced vital capacity in sitting and supine positions) were assessed every 3–6 months and analyzed using repeated-measures ANOVA.

Results

Between October 2004 and August 2009, 94 patients aged between 25 and 75 years were included in the study. Although skeletal muscle weakness was typically distributed in a limb-girdle pattern, many patients had unfamiliar features such as ptosis (23%), bulbar weakness (28%), and scapular winging (33%). During follow-up (average 1.6 years, range 0.5-4.2 years), skeletal muscle strength deteriorated significantly (mean declines of ?1.3% point/year for manual muscle testing and of ?2.6% points/year for hand-held dynamometry; both p<0.001). Longer disease duration (>15 years) and pulmonary involvement (forced vital capacity in sitting position <80%) at study entry predicted faster decline. On average, forced vital capacity in supine position deteriorated by 1.3% points per year (p=0.02). Decline in pulmonary function was consistent across subgroups. Ten percent of patients declined unexpectedly fast.

Conclusions

Recognizing patterns of common and less familiar characteristics in adults with Pompe disease facilitates timely diagnosis. Longer disease duration and reduced pulmonary function stand out as predictors of rapid disease progression, and aid in deciding whether to initiate enzyme replacement therapy, or when.

     

Sodium channel beta1 subunit-mediated modulation of Nav1.2 currents and cell surface density is dependent on interactions with contactin and ankyrin

Voltage-gated sodium channels are composed of a pore-forming alpha subunit and at least one auxiliary beta subunit. Both beta1 and beta2 are cell adhesion molecules that interact homophilically, resulting in ankyrin recruitment. In contrast, beta1, but not beta2, interacts heterophilically with contactin, resulting in increased levels of cell surface sodium channels. We took advantage of these results to investigate the molecular basis of beta1-mediated enhancement of sodium channel cell surface density, including elucidating structure-function relationships for beta1 association with contactin, ankyrin, and Nav1.2. beta1/beta2 subunit chimeras were used to assign putative sites of contactin interaction to two regions of the beta1 Ig loop. Recent studies have shown that glutathione S-transferase fusion proteins containing portions of Nav1.2 intracellular domains interact directly with ankyrinG. We show that native Nav1.2 associates with ankyrinG in cells in the absence of beta subunits and that this interaction is enhanced in the presence of beta1 but not beta1Y181E, a mutant that does not interact with ankyrinG. beta1Y181E does not modulate Nav1.2 channel function despite efficient association with Nav1.2 and contactin. beta1Y181E increases Nav1.2 cell surface expression, but not as efficiently as wild type beta1. beta1/beta2 chimeras exchanging various regions of the beta1 Ig loop were all ineffective in increasing Nav1.2 cell surface density. Our results demonstrate that full-length beta1 is required for channel modulation and enhancement of sodium channel cell surface expression.     

Genome size and ploidy of Paracoccidioides brasiliensis reveals a haploid DNA content: flow cytometry and GP43 sequence analysis

The aim of this study was to evaluate genome size and ploidy of the dimorphic pathogenic fungus Paracoccidioides brasiliensis. The cell cycle analysis of 10 P. brasiliensis isolates by flow cytometry (FCM) revealed a genome size ranging from 26.3+/-0.1Mb (26.9+/-0.1fg) to 35.5+/-0.2Mb (36.3+/-0.2fg) per uninucleated yeast cell. The DNA content of conidia from P. brasiliensis ATCC 60855-30.2+/-0.8Mb (30.9+/-0.8fg) -showed no significant differences with the yeast form, possibly excluding the occurrence of ploidy shift during morphogenesis. The ploidy of several P. brasiliensis isolates was assessed by comparing genome sizing by FCM with the previously described average haploid size obtained from electrophoretic karyotyping. The analysis of intra-individual variability of a highly polymorphic P. brasiliensis gene, GP43, indicated that only one allele seems to be present. Overall, the results showed that all analysed isolates presented a haploid, or at least aneuploid, DNA content and no association was detected between genome size/ploidy and the clinical-epidemiological features of the studied isolates. This work provides new knowledge on P. brasiliensis genetics/genomics, important for future research in basic cellular/molecular mechanisms and for the development/design of molecular techniques in this fungus.     

OESTROGEN EFFECTS ON BRAIN AND PITUITARY ENZYME ACTIVITIES

Abstract— Ovariectomized female rats were treated daily with oestradiol-17β benzoate for intervals up to one week and enzyme activities were measured in the pituitary and various brain regions. Brain regions were selected for study on the basis of their previously demonstrated content of putative oestradiol receptor sites. (1) Pituitary showed oestrogen-dependent increases in glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH) and lactic dehydrogenase (LDH), and no change in NADP⁺-dependent isocitric dehydrogenase (ICDH), NADP⁺-dependent malic dehydrogenase (MDH) or hexokinase (HK). MDH and ICDH were elevated in whole hypothalamus. Enzyme activities did not change significantly in whole amygdala, cerebral cortex, or hippocampus. (2) Sub-regions of the preoptic area, hypothalamus and amygdala were dissected to obtain more highly concentrated populations of cells containing putative oestrogen receptor sites. In the basomedial sub-region of hypothalamus, activities of MDH, ICDH and G6PDH were elevated by oestrogen treatment. In the corticomedial sub-region of amygdala, MDH and ICDH were elevated by oestrogen treatment. No change was observed in any of the six enzymes in medial preoptic area. (3) Increases in enzyme activities were related to the total in vivo dose of oestradiol benzoate given. (4) Hypophysectomy or adrenalectomy did not prevent the enzymatic responses to oestrogen. (S) Oestrogen added directly to the enzyme incubation medium did not change enzyme activities. (6) Weight loss in ovariectomized rats due to reduced food intake did not increase enzyme activities. (7) In the pituitary, good correlation was obtained between the known receptor binding properties of various oestrogenic and non-oestrogenic steroids and the elevation in G6PDH activity. The results indicate that oestradiol acts directly to cause changes in activities of some brain and pituitary enzymes. The possibility is discussed that these changes may result from oestrogen interaction with putative receptor sites found in pituitary and certain brain regions.     

A survey of state insurance commissioners concerning genetic testing and life insurance.

Rapid advances in genetic testing have stimulated growing concern about the potential for misuse of genetic data by insurance companies, employers, and other third parties. Thus far, reports of genetically based discrimination in life insurance have been anecdotal. Reasoning that state insurance commissioners were likely to be aware of (1) the extent of current use of and interest in genetic tests by life insurers and (2) consumer complaints about insurance being denied because of genetic condition or because of genetic test results, we conducted a survey of that group. We received responses from 42 of the 51 jurisdictions. Our results suggest (1) that those who regulate the life insurance industry do not yet perceive genetic testing to pose a significant problem in how insurers rate applicants, (2) that life insurers have much legal latitude to require genetic tests, and (3) that so far few consumers have formally complained to commissioners about the use of genetic data by life insurers.     

Effects of synthetic cohesin-containing scaffold protein architecture on binding dockerin-enzyme fusions on the surface of Lactococcus lactis

Background

In the last years, the biotechnological production of platform chemicals for fuel components has become a major focus of interest. Although ligno-cellulosic material is considered as suitable feedstock, the almost inevitable pretreatment of this recalcitrant material may interfere with the subsequent fermentation steps. In this study, the fungus Ustilago maydis was used to produce itaconic acid as platform chemical for the synthesis of potential biofuels such as 3-methyltetrahydrofuran. No studies, however, have investigated how pretreatment of ligno-cellulosic biomass precisely influences the subsequent fermentation by U. maydis. Thus, this current study aims to first characterize U. maydis in shake flasks and then to evaluate the influence of three exemplary pretreatment methods on the cultivation and itaconic acid production of this fungus. Cellulose enzymatically hydrolysed in seawater and salt-assisted organic-acid catalysed cellulose were investigated as substrates. Lastly, hydrolysed hemicellulose from fractionated beech wood was applied as substrate.

Results

U. maydis was characterized on shake flask level regarding its itaconic acid production on glucose. Nitrogen limitation was shown to be a crucial condition for the production of itaconic acid. For itaconic acid concentrations above 25 g/L, a significant product inhibition was observed. Performing experiments that simulated influences of possible pretreatment methods, U. maydis was only slightly affected by high osmolarities up to 3.5 osmol/L as well as of 0.1 M oxalic acid. The production of itaconic acid was achieved on pretreated cellulose in seawater and on the hydrolysed hemicellulosic fraction of pretreated beech wood.

Conclusion

The fungus U. maydis is a promising producer of itaconic acid, since it grows as single cells (yeast-like) in submerged cultivations and it is extremely robust in high osmotic media and real seawater. Moreover, U. maydis can grow on the hemicellulosic fraction of pretreated beech wood. Thereby, this fungus combines important advantages of yeasts and filamentous fungi. Nevertheless, the biomass pretreatment does indeed affect the subsequent itaconic acid production. Although U. maydis is insusceptible to most possible impurities from pretreatment, high amounts of salts or residues of organic acids can slow microbial growth and decrease the production. Consequently, the pretreatment step needs to fit the prerequisites defined by the actual microorganisms applied for fermentation.     

House-level risk factors associated with the colonization of broiler flocks with Campylobacter spp. in Iceland, 2001 – 2004

Background

The most predominant beta2-integrin lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18, alphaLbeta2), expressed on all leukocytes, is essential for many adhesive functions of the immune system. Interestingly, RTX toxin-producing bacteria specifically target this leukocyte beta2-integrin which exacerbates lesions and disease development.

Results

This study reports the sequencing of the wild boar beta2-integrin CD11a and CD18 cDNAs. Predicted CD11a and CD18 subunits share all the main structural characteristics of their mammalian homologues, with a larger interspecies conservation for the CD18 than the CD11a. Besides these strong overall similarities, wild boar and domestic pig LFA-1 differ by 2 (CD18) and 1 or 3 (CD11a) substitutions, of which one is located in the crucial I-domain (CD11a, E168D).

Conclusion

As most wild boars are seropositive to the RTX toxin-producing bacterium Actinobacillus pleuropneumoniae and because they have sustained continuous natural selection, future studies addressing the functional impact of these polymorphisms could bring interesting new information on the physiopathology of Actinobacillus pleuropneumoniae-associated pneumonia in domestic pigs.     

Randomised trial of personalised computer based information for cancer patients

ObjectiveTo compare the use and effect of a computer based information system for cancer patients that is personalised using each patient''s medical record with a system providing only general information and with information provided in booklets.DesignRandomised trial with three groups. Data collected at start of radiotherapy, one week later (when information provided), three weeks later, and three months later.Participants525 patients started radical radiotherapy; 438 completed follow up.InterventionsTwo groups were offered information via computer (personalised or general information, or both) with open access to computer thereafter; the third group was offered a selection of information booklets.OutcomesPatients'' views and preferences, use of computer and information, and psychological status; doctors'' perceptions; cost of interventions.ResultsMore patients offered the personalised information said that they had learnt something new, thought the information was relevant, used the computer again, and showed their computer printouts to others. There were no major differences in doctors'' perceptions of patients. More of the general computer group were anxious at three months. With an electronic patient record system, in the long run the personalised information system would cost no more than the general system. Full access to booklets cost twice as much as the general system.ConclusionsPatients preferred computer systems that provided information from their medical records to systems that just provided general information. This has implications for the design and implementation of electronic patient record systems and reliance on general sources of patient information.     

Age-specific threats induce CRF expression in the paraventricular nucleus of the hypothalamus and hippocampus of young rats

Young animals respond to threatening stimuli in an age-specific way. Their endocrine and behavioral responses reflect the potential threat of the situation at a given age. The aim of the present study was to determine whether corticotropin-releasing factor (CRF) is involved in the endocrine and behavioral responses to threat and their developmental changes in young rats. Preweaning 14-day-old and postweaning 26-day-old rats were exposed to two age-specific threats, cat odor and an adult male rat. The acute behavioral response was determined during exposure. After exposure, the time courses of the corticosterone response and of CRF expression in the paraventricular nucleus of the hypothalamus (PVN) and in extrahypothalamic areas were assessed. Preweaning rats became immobile when exposed to cat odor or the male rat, whereas postweaning rats became immobile to cat odor only. Male exposure increased serum corticosterone levels in 14-day-old rats, but cat odor failed to increase levels at either age. Exposure induced elevation of CRF mRNA levels in the PVN that paralleled changes in corticosterone levels. CRF may thus play a role in endocrine regulation and its developmental changes during early life. Neither cat odor nor the adult male altered CRF mRNA levels in the bed nucleus of the stria terminalis (BNST) or the amygdala, but both stimuli increased levels in the hippocampus. Hippocampal CRF mRNA expression levels did not parallel cat odor or male-induced immobility, indicating that CRF is not involved in this response in young rats but may be involved in aspects of learning and memory.