UPTAKE OF GLUCOSE ANALOGUES BY RAT BRAIN CORTEX SLICES: Na+-INDEPENDENT MEMBRANE TRANSPORT

Abstract— The glucose analogues 3-O-methyl-D-glucose and α-methyl-D-glucoside were not metabolized in brain tissue.
The uptake of these two sugars into the intracellular compartment of brain cortex slices was investigated using media with normal and low Na⁺ concentration (replacement of all NaCl with choline Cl). The cellular transport was not Na⁺-dependent. The transport mechanism clearly distinguished between the two sugars in both normal and low Na⁺ media.     

Effect of postnatal development on calcium currents and slow charge movement in mammalian skeletal muscle

Single- (whole-cell patch) and two-electrode voltage-clamp techniques were used to measure transient (Ifast) and sustained (Islow) calcium currents, linear capacitance, and slow, voltage-dependent charge movements in freshly dissociated fibers of the flexor digitorum brevis (FDB) muscle of rats of various postnatal ages. Peak Ifast was largest in FDB fibers of neonatal (1-5 d) rats, having a magnitude in 10 mM external Ca of 1.4 +/- 0.9 pA/pF (mean +/- SD; current normalized by linear fiber capacitance). Peak Ifast was smaller in FDB fibers of older animals, and by approximately 3 wk postnatal, it was so small as to be unmeasurable. By contrast, the magnitudes of Islow and charge movement increased substantially during postnatal development. Peak Islow was 3.6 +/- 2.5 pA/pF in FDB fibers of 1-5-d rats and increased to 16.4 +/- 6.5 pA/pF in 45-50-d-old rats; for these same two age groups, Qmax, the total mobile charge measurable as charge movement, was 6.0 +/- 1.7 and 23.8 +/- 4.0 nC/microF, respectively. As both Islow and charge movement are thought to arise in the transverse-tubular system, linear capacitance normalized by the area of fiber surface was determined as an indirect measure of the membrane area of the t-system relative to that of the fiber surface. This parameter increased from 1.5 +/- 0.2 microF/cm2 in 2-d fibers to 2.9 +/- 0.4 microF/cm2 in 44-d fibers. The increases in peak Islow, Qmax, and normalized linear capacitance all had similar time courses. Although the function of Islow is unknown, the substantial postnatal increase in its magnitude suggests that it plays an important role in the physiology of skeletal muscle.     

Genetic variations at the human growth hormone receptor (GHR) gene locus are associated with idiopathic short stature

GH plays an essential role in the growing child by binding to the growth hormone receptor (GHR) on target cells and regulating multiple growth promoting and metabolic effects. Mutations in the GHR gene coding regions result in GH insensitivity (dwarfism) due to a dysfunctional receptor protein. However, children with idiopathic short stature (ISS) show growth impairment without GH or GHR defects. We hypothesized that decreased expression of the GHR gene may be involved. To test this, we investigated whether common genetic variants (microsatellites, SNPs) in regulatory regions of the GHR gene region were associated with the ISS phenotype. Genotyping of a GT‐repeat microsatellite in the GHR 5′UTR in a Montreal ISS cohort (n = 37 ISS, n = 105 controls) revealed that the incidence of the long/short (L/S) genotype was 3.3× higher in ISS children than controls (P = 0.04, OR = 3.85). In an Italian replication cohort (n = 143 ISS, n = 282 controls), the medium/short (M/S) genotype was 1.9× more frequent in the male ISS than controls (P = 0.017, OR = 2.26). In both ISS cohorts, logistic regression analysis of 27 SNPs showed an association of ISS with rs4292454, while haplotype analysis revealed specific risk haplotypes in the 3′ haploblocks. In contrast, there were no differences in GT genotype frequencies in a cohort of short stature (SS) adults versus controls (CARTaGENE: n = 168 SS, n = 207 controls) and the risk haplotype in the SS cohort was located in the most 5′ haploblock. These data suggest that the variants identified are potentially genetic markers specifically associated with the ISS phenotype.     

Binding of transmissible gastroenteritis coronavirus to cell surface sialoglycoproteins

The surface glycoprotein S of transmissible gastroenteritis virus (TGEV) has two binding activities. (i) Binding to porcine aminopeptidase N (pAPN) is essential for the initiation of infection. (ii) Binding to sialic acid residues on glycoproteins is dispensable for the infection of cultured cells but is required for enteropathogenicity. By comparing parental TGEV with mutant viruses deficient in the sialic acid binding activity, we determined the contributions of both binding activities to the attachment of TGEV to cultured cells. In the presence of a functional sialic acid binding activity, the amount of virus bound to two different porcine cell lines was increased sixfold compared to the binding of the mutant viruses. The attachment of parental virus was reduced to levels observed with the mutants when sialic acid containing inhibitors was present or when the cells were pretreated with neuraminidase. In virus overlay binding assays with immobilized cell surface proteins, the mutant virus only recognized pAPN. In addition, the parental virus bound to a high-molecular-mass sialoglycoprotein. The recognition of pAPN was sensitive to reducing conditions and was not dependent on sialic acid residues. On the other hand, binding to the sialic acid residues of the high-molecular-mass glycoprotein was observed regardless of whether the cellular proteins had been separated under reducing or nonreducing conditions. We propose that binding to a surface sialoglycoprotein is required for TGEV as a primary attachment site to initiate infection of intestinal cells. This concept is discussed in the context of other viruses that use two different receptors to infect cells.     

Veränderungen der Oenozyten und ihre Funktion während der Metamorphose bei Schwärmern und Zahnspinnern

Zusammenfassung VonCerura vinula undSphinx ligustri wurden die Oenozyten im letzten Larvenstadium und in der Puppe untersucht und ihre Struktur beschrieben. Ihre Aktivitätsphasen liegen zur Zeit der beiden Häutungen (Larven- und Puppenhäutung) und im letzten Larveninstar vor der Umfärbung, einem äußerlich sichtbaren Metamorphoseschritt, und vor der Puppenhäutung im Färbungsstadium III. Sie stehen mit den Umwandlungsprozessen, die in den Raupen zu diesem Zeitpunkt stattfinden in deutlichem Zusammenhang. —2–4 Monate nach der Puppenhäutung sind in den diapausierenden Puppen noch aktive larvale Oenozyten vorhanden. — Aktivitätsphasen sind charakterisiert durch viele große und kleine Vakuolen neben kanalartigen Strukturen im Zytoplasma, stark verzweigte Kerne und weitreichende Zellaus- und-einbuchtungen.Im Vorpuppenstadium (Färbungsstadium IV) entstehen die imaginalen Oenozyten aus der Epidermis, sie werden erst kurz vor der Adulthäutung aktiv.Haemozyten, neurosekrethaltig, legen sich dicht an die Oenozyten an und dringen zwischen Zelleinfaltungen ins Innere vor.Lipide, besonders reichlich in aktiven Phasen vorhanden, konnten sowohl im Zytoplasma nachgewiesen werden, als auch ihr Übertritt aus dem Fettgewebe, das den Drüsen eng anliegt.Glykogen tritt ebenfalls in den Oenozyten auf, seine Menge steht aber in keinem merklichen. Zusammenhang mit den Zellrhythmen. Physiologische Versuche beweisen, daß die Oenozyten und auch die Prothorakaldrüsen in aktiven Phasen das Häutungs- und Metamorphosehormon abgeben. Sie lösen beide den Umfärbungsprozeß aus. Gehirne mit neurosekretorischen Zellen in aktiver Phase oder Cholesterin können die Prothorakaldrüsen und z.T. auch die Oenozyten zur Abgabe ihres Hormons anregen.

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Changes of oenocytes and their function during metamorphosis of sphingidae and notodontidae

Summary Oenocytes of the last larval instar and the pupa ofCerura vinula andSpinx ligustri have been examined, and their structure described. The activity phases of the oenocytes at the time of both moultings, as well as during the last larval instar prior to an externally visible color change and prior to the pupal ecdysis i.e. during color change stage III) were clearly related to the process of metamorphosis, which was occurring in the larvae at this time 2–4 months after pupal ecdysis, diapausing pupae still show active larval oenocytes. Activity phases are characterized by many large and small vacuoles in addition to channel-like cytoplasmatic structures, heavily branched nuclei and extensive cell processes and infoldings of the cell membrane. In the pharate pupal stage (colour change stage IV) the imaginal oenocytes originate from the hypodermis, becoming active just prior to the adult ecdysis.Haemocytes containing neurosecretory material attach themselves to the oenocytes and enter through infoldings of the cell membrane. Lipids, which are particularly abundant during active phases, could be demonstrated in the cytoplasm as well as passing from the fatty tissue closely surrounding the glands. Glycogen was also present in the oenocytes. There was, however, no noticeable relation of these materials to the rhythm.Physiological experiments demonstrated that oenocytes as well as prothoracic glands, when active, secrete the moulting and metamorphosis hormone. Both glands initiate the process of colour change. Brain tissue, containing active neurosecretory cells, or cholesterol, may stimulate the prothoracic galnds and the oenocytes to secrete their hormone.

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Mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Alteration of the Thymic T Cell Repertoire by Rotavirus Infection Is Associated with Delayed Type 1 Diabetes Development in Non-Obese Diabetic Mice

Rotaviruses are implicated as a viral trigger for the acceleration of type 1 diabetes in children. Infection of adult non-obese diabetic (NOD) mice with rotavirus strain RRV accelerates diabetes development, whereas RRV infection in infant NOD mice delays diabetes onset. In this study of infant mice, RRV titers and lymphocyte populations in the intestine, mesenteric lymph nodes (MLN) and thymus of NOD mice were compared with those in diabetes-resistant BALB/c and C57BL/6 mice. Enhanced intestinal RRV infection occurred in NOD mice compared with the other mouse strains. This was associated with increases in the frequency of CD8αβ TCRαβ intraepithelial lymphocytes, and their PD-L1 expression. Virus spread to the MLN and T cell numbers there also were greatest in NOD mice. Thymic RRV infection is shown here in all mouse strains, often in combination with alterations in T cell ontogeny. Infection lowered thymocyte numbers in infant NOD and C57BL/6 mice, whereas thymocyte production was unaltered overall in infant BALB/c mice. In the NOD mouse thymus, effector CD4⁺ T cell numbers were reduced by infection, whereas regulatory T cell numbers were maintained. It is proposed that maintenance of thymic regulatory T cell numbers may contribute to the increased suppression of inflammatory T cells in response to a strong stimulus observed in pancreatic lymph nodes of adult mice infected as infants. These findings show that rotavirus replication is enhanced in diabetes-prone mice, and provide evidence that thymic T cell alterations may contribute to the delayed diabetes onset following RRV infection.     

Improved method for transport of living cell cultures

An easy and cost-effective method for transport of living cell cultures which avoids the use of dry ice and prevents bacterial contamination is described. Cells are suspended in buffered culture medium in sealed and insulated 2 ml cryovials and are able to grow and survive in substantial numbers during several days of storage and shipment at ambient temperature. Replating results in an identical repopulation in all cell lines. Not only tumor cells but also fibroblasts seem to tolerate well this improved method for shipment.     

Selecting the quality of mule duck fatty liver based on near-infrared spectroscopy

Background

“Foie gras” is produced predominantly in France and about 90% of the commercialized product is obtained from male mule ducks. The melting rate (percentage of fat released during cooking) is the main criterion used to determine the quality of “foie gras”. However, up to now the melting rate could not be predicted without causing liver damage, which means that selection programs could not use this criterion.

Methods

Fatty liver phenotypes were obtained for a population of over 1400 overfed male mule ducks. The phenotypes were based on two types of near-infrared spectra (on the liver surface and on ground liver) in order to predict the melting rate and liver composition (ash, dry matter, lipid and protein contents). Genetic parameters were computed in multiple traits with a “sire-dam” model and using a Gibbs sampling approach.

Results

The estimates for the genetic parameters show that the measured melting rate and the predicted melting rate obtained with two near-infrared spectrometer devices are genetically the same trait: genetic correlations are very high (ranging from +0.89 to +0.97 depending on the mule duck parental line and the spectrometer) and heritabilities are comparable. The predictions based on the spectra of ground liver samples using a laboratory spectrometer correlate with those based on the surface spectra using a portable spectrometer (from +0.83 to +0.95 for dry matter, lipid and protein content) and are particularly high for the melting rate (higher than +0.95). Although less accurate than the predictions obtained using the spectra of ground liver samples, the phenotypic prediction of the melting rate based on surface spectra is sufficiently accurate to be used by “foie gras” processors.

Conclusions

Near-infrared spectrometry is an efficient tool to select liver quality in breeding programs because animals can be ranked according to their liver melting rate without damaging their livers. Thus, these original results will help breeders to select ducks based on the liver melting rate, a crucial criterion that defines the quality of the liver and for which there was previously no accurate predictor.     

Polar invasion and translocation of Neisseria meningitidis and Streptococcus suis in a novel human model of the blood-cerebrospinal fluid barrier

Acute bacterial meningitis is a life-threatening disease in humans. Discussed as entry sites for pathogens into the brain are the blood-brain and the blood-cerebrospinal fluid barrier (BCSFB). Although human brain microvascular endothelial cells (HBMEC) constitute a well established human in vitro model for the blood-brain barrier, until now no reliable human system presenting the BCSFB has been developed. Here, we describe for the first time a functional human BCSFB model based on human choroid plexus papilloma cells (HIBCPP), which display typical hallmarks of a BCSFB as the expression of junctional proteins and formation of tight junctions, a high electrical resistance and minimal levels of macromolecular flux when grown on transwell filters. Importantly, when challenged with the zoonotic pathogen Streptococcus suis or the human pathogenic bacterium Neisseria meningitidis the HIBCPP show polar bacterial invasion only from the physiologically relevant basolateral side. Meningococcal invasion is attenuated by the presence of a capsule and translocated N. meningitidis form microcolonies on the apical side of HIBCPP opposite of sites of entry. As a functionally relevant human model of the BCSFB the HIBCPP offer a wide range of options for analysis of disease-related mechanisms at the choroid plexus epithelium, especially involving human pathogens.