Frequency of anticardiolipin, antinuclear and anti-beta2 glycoprotein I antibodies in children with epilepsy

A high prevalence of epilepsies in specific immunological diseases suggests that the immune system may play a role in the pathogenesis of epilepsy or might be associated with it. In this study the frequency of anticardiolipin antibodies (aCL), antinuclear antibodies (ANA) and anti-beta2-glycoprotein I antibodies (anti-beta2-GPI) in 40 children with epilepsy and in 38 healthy subjects was determined. Positive aCL was found in 3 patients, and anti-beta2-GPI in 1 patient. In control group they were negative. ANA antibodies were negative in both groups. Duration of epilepsy < 1 year was observed in all three patients with positive aCL. No statistically significant difference was found concerning the presence of these antibodies between patients and controls. There was no statistically significant correlation of age, sex, age at the onset of epilepsy, duration of epilepsy, type of epilepsy, seizure frequency or specific antiepileptic medications with the presence of any measured antibodies.     

A mineralogical characterization of biogenic calcium carbonates precipitated by heterotrophic bacteria isolated from cryophilic polar regions

Precipitation of calcium carbonate (CaCO_3(s)) can be driven by microbial activity. Here, a systematic approach is used to identify the morphological and mineralogical characteristics of CaCO_3(s) precipitated during the heterotrophic growth of micro‐organisms isolated from polar environments. Focus was placed on establishing mineralogical features that are common in bioliths formed during heterotrophic activity, while in parallel identifying features that are specific to bioliths precipitated by certain microbial phylotypes. Twenty microbial isolates that precipitated macroscopic CaCO_3(s) when grown on B4 media supplemented with calcium acetate or calcium citrate were identified. A multimethod approach, including scanning electron microscopy, high‐resolution transmission electron microscopy, and micro‐X‐ray diffraction (μ‐XRD), was used to characterize CaCO_3(s) precipitates. Scanning and transmission electron microscopy showed that complete CaCO_3(s) crystal encrustation of Arthrobacter sp. cells was common, while encrustation of Rhodococcus sp. cells did not occur. Several euhedral and anhedral mineral formations including disphenoid‐like epitaxial plates, rhomboid‐like aggregates with epitaxial rhombs, and spherulite aggregates were observed. While phylotype could not be linked to specific mineral formations, isolates tended to precipitate either euhedral or anhedral minerals, but not both. Three anhydrous CaCO_3(s) polymorphs (calcite, aragonite, and vaterite) were identified by μ‐XRD, and calcite and aragonite were also identified based on TEM lattice‐fringe d value measurements. The presence of certain polymorphs was not indicative of biogenic origin, although several mineralogical features such as crystal‐encrusted bacterial cells, or casts of bacterial cells embedded in mesocrystals are an indication of biogenic origin. In addition, some features such as the formation of vaterite and bacterial entombment appear to be linked to certain phylotypes. Identifying phylotypes consistent with certain mineralogical features is the first step toward discovering a link between these crystal features and the precise underlying molecular biology of the organism precipitating them.     

The isolation, purification and biological activity of a novel antibacterial compound produced by Pseudomonas stutzeri

A novel compound designated zafrin [4beta-methyl-5, 6, 7, 8 tetrahydro-1 (4beta-H)-phenanthrenone] was isolated from a crude extract of a marine bacterium identified as Pseudomonas stutzeri. Zafrin showed strong antibacterial activity against both Gram-positive and Gram-negative bacteria. The compound was purified and its structure was elucidated by spectroscopic methods including 1H-nuclear magnetic resonance (NMR), 13C-NMR, 1D-NMR and 2D-NMR spectroscopy. It could be demonstrated that a purified solution of zafrin was active against several human pathogens, including Staphylococcus aureus, and Salmonella typhi. By contrast, zafrin did not inhibit the growth of eukaryotic organisms Candida albicans and Schizosaccharomyces pombe. The minimal inhibitory concentration for Gram-positive bacteria ranged from 50 to 75 microg mL(-1) and varied between 75 and 125 microg mL(-1) for Gram-negative bacteria. Zafrin lysed Bacillus subtilis cells grown in an osmotically protected medium, suggesting that it does not act upon the cell wall. Further investigation using B. subtilis indicated that the compound is bactericidal and is likely to target the cell membrane.     

HIV-specific T-cells accumulate in the liver in HCV/HIV co-infection

Background and Aims

Hepatitis C Virus (HCV)-related liver disease progresses more rapidly in individuals co-infected with Human Immunodeficiency Virus-1 (HIV), although the underlying immunologic mechanisms are unknown. We examined whether HIV-specific T-cells are identified in the liver of HCV/HIV co-infected individuals and promote liver inflammation through bystander immune responses.

Methods

Ex-vivo intra-hepatic lymphocytes from HCV mono-infected and HCV/HIV co-infected individuals were assessed for immune responses to HIV and HCV antigens by polychromatic flow cytometry.

Results

HCV/HIV liver biopsies had similar frequencies of lymphocytes but lower percentages of CD4⁺ T-cells compared to HCV biopsies. In co-infection, intra-hepatic HIV-specific CD8⁺ and CD4⁺ T-cells producing IFN-γ and TNF-α were detected and were comparable in frequency to those that were HCV-specific. In co-infected individuals, viral-specific CD8⁺ T-cells produced more of the fibrogenic cytokine, TNF-α. In both mono- and co-infected individuals, intra-hepatic HCV-specific T-cells were poorly functional compared to HIV-specific T-cells. In co-infection, HAART was not associated with a reconstitution of intra-hepatic CD4⁺ T-cells and was associated with reduction in both HIV and HCV-specific intra-hepatic cytokine responses.

Conclusion

The accumulation of functional HIV-specific T-cells in the liver during HCV/HIV co-infection may represent a bystander role for HIV in inducing faster progression of liver disease.     

Glutathione and gamma-glutamyl transpeptidase in the adult female rat brain after intraventricular injection of LHRH and somatostatin

Glutathione content and glutamyl transpeptidase activity in different regions of adult female rat brain were determined at 10 and 30 min following intraventricular injection of LHRH and somatostatin. Hypothalamic glutathione levels were significantly elevated at 10 and 30 min after a single injection of a 0.1 micrograms dose of LHRH. On the contrary, glutathione levels significantly decreased in the hypothalamus, cerebral cortex and cerebellum at 10 and 30 min after 0.5 or 1 microgram dose. However, significant decrease in brain stem glutathione was evident at 30 min after 0.5 microgram and 10 min after the 1 microgram dose. Somatostatin at doses of 0.5 microgram and 1 microgram significantly decreased glutathione levels in all four brain regions both at 10 and 30 min following injection into the 3rd ventricle. Gamma-glutamyl transpeptidase activity in the hypothalamus and cerebral cortex was significantly elevated after intraventricular injection of LHRH. However, a significant increase in gamma-glutamyl transpeptidase activity in cerebellum and brain stem was seen only with 0.5 and 1 micrograms doses of LHRH. Somatostatin also significantly increased gamma-glutamyl transpeptidase activity in hypothalamus, cerebral cortex, brain stem and cerebellum. The decrease in glutathione levels with corresponding increase in gamma-glutamyl transpeptidase activity after intraventricular administration of LHRH and somatostatin suggests a possible interaction between glutathione and hypothalamic peptides.     

Studium der schwachen B-Variante in einer Familie

Zusammenfassung Es wird eine Familie beschrieben, in der bei 17 von 31 untersuchten Mitgliedern eine schwache B-Variante vorkommt (B-Grouppe bei 13, AB-Gruppe bei 4 Personen). Die serologischen Eigenschaften des schwachen B-Antigens, ebenso der Prozentsatz der mit Anti-B nichtagglutinierten Erythrocyten, unterschieden sich von Fall zu Fall, so daß die Klassifizierung der B-Variante bei einzelnen Personen unterschiedlich war. Alle Träger des schwachen B-Antigens sind Nichtausscheider der B-Substanzen und Ausscheider von H(A). Bei 2 Familienmitgliedern mit der A- und AB-Blutgruppe wurde eine Abnormalität der phänotypischen Ausprägung des A-Antigens festgestellt; die A_HP-Antigene zeigen eine normale Expressivität. Diese Ergebnisse sprechen für die Unabhängigkeit der A- und A_HP-Gene. Die Rolle der modifizierenden Gene yy für das Auftreten von A- und B-Antigenen an Erythrocyten und im Speichel wird diskutiert.

---

Study of the weak B variant in a family

Summary A family is described, in which a weak B variant occurred in 17 from 31 examined members (B group in 13 cases, AB group in 4 persons). Both the serological properties of the weak B antigen and the percent of the red cells non-agglutinated with anti-B differed as the case may be; the classification of the weak B antigen varied therefore in single persons. All the carriers of the weak B were non-secretors of B and secretors of H(A). By 2 members of the family of the A and AB group an abnormality concerning the phenotype expression of the A antigen was found, whereas the A_HP receptors had a normal antigenicity. This result proves an independence of the A and A_HP genes. The role of the suppression genes yy for the phenotypic expresion of the A and B antigens in the red cells and in the saliva is discussed.

     

Temporal shedding patterns and virulence factors of Escherichia coli serogroups O26, O103, O111, O145, and O157 in a cohort of beef calves and their dams

This study investigated the shedding of Escherichia coli O26, O103, O111, O145, and O157 in a cohort of beef calves from birth over a 5-month period and assessed the relationship between shedding in calves and shedding in their dams, the relationship between shedding and scouring in calves, and the effect of housing on shedding in calves. Fecal samples were tested by immunomagnetic separation and by PCR and DNA hybridization assays. E. coli O26 was shed by 94% of calves. Over 90% of E. coli O26 isolates carried the vtx(1), eae, and ehl genes, 6.5% carried vtx(1) and vtx(2), and one isolate carried vtx(2) only. Serogroup O26 isolates comprised seven pulsed-field gel electrophoresis (PFGE) patterns but were dominated by one pattern which represented 85.7% of isolates. E. coli O103 was shed by 51% of calves. Forty-eight percent of E. coli O103 isolates carried eae and ehl, 2% carried vtx(2), and none carried vtx(1). Serogroup O103 isolates comprised 10 PFGE patterns and were dominated by two patterns representing 62.5% of isolates. Shedding of E. coli O145 and O157 was rare. All serogroup O145 isolates carried eae, but none carried vtx(1) or vtx(2). All but one serogroup O157 isolate carried vtx(2), eae, and ehl. E. coli O111 was not detected. In most calves, the temporal pattern of E. coli O26 and O103 shedding was random. E. coli O26 was detected in three times as many samples as E. coli O103, and the rate at which calves began shedding E. coli O26 for the first time was five times greater than that for E. coli O103. For E. coli O26, O103, and O157, there was no association between shedding by calves and shedding by dams within 1 week of birth. For E. coli O26 and O103, there was no association between shedding and scouring, and there was no significant change in shedding following housing.     

Modulation of Calcium Oxalate Dihydrate Growth by Selective Crystal-face Binding of Phosphorylated Osteopontin and Polyaspartate Peptide Showing Occlusion by Sectoral (Compositional) Zoning

Calcium oxalate dihydrate (COD) mineral and the urinary protein osteopontin/uropontin (OPN) are commonly found in kidney stones. To investigate the effects of OPN on COD growth, COD crystals were grown with phosphorylated OPN or a polyaspartic acid-rich peptide of OPN (DDLDDDDD, poly-Asp^86–93). Crystals grown with OPN showed increased dimensions of the {110} prismatic faces attributable to selective inhibition at this crystallographic face. At high concentrations of OPN, elongated crystals with dominant {110} faces were produced, often with intergrown, interpenetrating twin crystals. Poly-Asp^86–93 dose-dependently elongated crystal morphology along the {110} faces in a manner similar to OPN. In crystal growth studies using fluorescently tagged poly-Asp^86–93 followed by imaging of crystal interiors using confocal microscopy, sectoral (compositional) zoning in COD was observed resulting from selective binding and incorporation (occlusion) of peptide exclusively into {110} crystal sectors. Computational modeling of poly-Asp^86–93 adsorption to COD {110} and {101} surfaces also suggests increased stabilization of the COD {110} surface and negligible change to the natively stable {101} surface. Ultrastructural, colloidal-gold immunolocalization of OPN by transmission electron microscopy in human stones confirmed an intracrystalline distribution of OPN. In summary, OPN and its poly-Asp^86–93 sequence similarly affect COD mineral growth; the {110} crystallographic faces become enhanced and dominant attributable to {110} face inhibition by the protein/peptide, and peptides can incorporate into the mineral phase. We, thus, conclude that the poly-Asp^86–93 domain is central to the OPN ability to interact with the {110} faces of COD, where it binds to inhibit crystal growth with subsequent intracrystalline incorporation (occlusion).Calcium oxalate is the major mineral phase of human renal calculi, constituting roughly 70% by weight of the stones (1). Two polymorphs of calcium oxalate, calcium oxalate monohydrate (COM)³ and calcium oxalate dihydrate (COD), are the most abundant mineral types, but others may exist in smaller amounts, including calcium phosphate minerals. It has been reported that the occurrence of COM, the more thermodynamically stable polymorph of calcium oxalate, is often at the core of most kidney stones and is approximately twice as frequent as COD (2), although both crystal types typically exist to some degree in most stones (3, 4). COM is commonly found in the urine of “stone formers,” but seldom is seen in healthy urine; on the other hand, COD crystals are typically found in the urine of both healthy people and stone formers and are routinely excreted during urination (5–7). Importantly, in patients with severe uremia and hypercalciuria, elongated, large rod-shaped COD crystals are not only often observed but are the sole mineral phase present in the kidneys in these pathologies (8, 9).In comparing physiologic differences between calcium oxalate polymorphs, one study has shown that for a given amount of added crystals, ∼50% more COM than COD crystals bound to inner medullary collecting duct cells in vitro (10). Other studies have reported that COD crystals are less prone than COM crystals to adhere to cell surfaces, suggesting that COD might, thus, contribute to a lesser degree than COM to the retention of mineral in the renal collecting ducts leading to kidney stone formation (5, 11). This is supported by the fact that COM crystals are large cationic particulates, presenting more calcium ions than COD crystals at their surface that would have a stronger affinity for anionic molecules on renal epithelial cell membranes (10, 12). Further to this, COM and COD crystals bind to cultured renal cells with different face-selective affinities (13–16), and COM crystals are known to be more injurious to cell membranes than COD crystals (17). In this regard, Wesson et al. (1) proposed that the preferential formation of COD crystals in vivo protects against urolithiasis because they are less likely to adhere to renal tubular cells and are, thus, more readily excreted. This notion is supported by direct experimental measurements of the macromolecular adhesion force on specific crystal faces of COM or COD at the near-molecular level (13, 14). Given this, conversely, inhibition of the formation of COD crystals could lead to preferential COM deposition and the formation of kidney stones.Although calcium and oxalate ionic concentrations are frequently supersaturated with respect to both COM and COD mineral polymorphs, normal human urine likely contains factors that can modulate calcium oxalate crystallization into COD (10). In this context the presence of urinary macromolecular inhibitors of crystal growth can cause preferential crystallization of COD, rather than COM, from a supersaturated solution of calcium chloride and sodium oxalate (10). Substantial elegant work has been performed on COM growth and the effect of citrate and peptides/proteins as crystal growth modifiers. Some macromolecules, including urinary osteopontin (OPN), contain polyanionic regions and net negative charges that have been shown to inhibit calcium oxalate crystallization (14, 18–21) and influence calcium oxalate growth in favor of COD (3, 10, 22, 23). Although there appears to be preferential inhibition of COM, several studies present evidence for higher affinity of OPN binding to COD, and here we investigate this further to show the effects of OPN (and a peptide of OPN) on COD crystal growth and provide information on peptide/protein occlusion that might facilitate crystal dissolution as originally proposed by Ryall et al. (see below) (23, 24).OPN is a highly acidic, glycosylated phosphoprotein produced by many types of epithelial cells and can be found in normal plasma and in various body fluids such as bile, urine, and milk (25, 26). In normal kidneys, OPN is secreted by the thin and thick ascending limbs of the loop of Henle and distal nephrons (27–32). OPN contains a 15–20% aspartic acid residue content, and the mineral binding and inhibitory properties of OPN have often been partly attributed to an aspartic acid-rich sequence within this protein (10, 26, 33, 34). Likewise, post-translational phosphorylation of OPN has been shown to markedly enhance the mineral binding and inhibitory ability of this protein (34, 35). Furthermore, OPN also contains sialic acid (26, 27), which may play an indirect role in crystal binding by forming a bridge between transiently expressed crystal binding molecules and the cell surface (12). Several studies have shown that OPN has a higher affinity for COD than COM in normal urinal precipitates, with some evidence given for the incorporation of protein into calcium oxalate crystals (1, 23, 36).OPN consistently localizes to kidney stones (37) and at physiologically relevant concentrations applied in vitro, acts as a potent inhibitor of the nucleation, growth, and aggregation of calcium oxalate crystals (27, 30, 34). In a rat model of urolithiasis, although increased OPN mRNA expression was associated with increased renal calculi formation, the urinary excretion level of OPN was less than in controls, discussed as being attributable to incorporation of OPN into stones (37, 38). In general, the inclusion of OPN plus other urinary macromolecules into renal calculi has been suggested to be part of a cellular defense mechanism designed to inhibit crystal growth and limit the growth of kidney stones, to interrupt inorganic crystal structure of the calcium oxalate minerals, and to provide an organic volume whose degradation by permeating proteases creates channels facilitating dissolution of the mineral phase (23, 24). Given these possibilities, our aim was to determine whether full-length phosphorylated OPN and a poly-Asp peptide of OPN affect COD crystal growth and morphology, and if so, we further aimed to identify the contribution of face-specific binding and intracrystalline incorporation (occlusion) of OPN peptide into COD. Combining experimental and computational approaches, we have identified preferential binding and unique occlusion of a peptide of OPN at a specific crystallographic face of COD. Furthermore, we present a possible adsorption mechanism in a model where multiple peptide carboxylate groups bind calcium atoms at the COD {110} surface. Taken together, our findings provide new information on the pathogenesis of renal calculi by describing specific actions of phosphorylated full-length OPN and a short peptide sequence of OPN (not having post-translational modifications) on specific COD crystal faces that modulate calcium oxalate growth and crystal morphologies.     

Metal reduction and iron biomineralization by a psychrotolerant Fe(III)-reducing bacterium, Shewanella sp. strain PV-4

A marine psychrotolerant, dissimilatory Fe(III)-reducing bacterium, Shewanella sp. strain PV-4, from the microbial mat at a hydrothermal vent of Loihi Seamount in the Pacific Ocean has been further characterized, with emphases on metal reduction and iron biomineralization. The strain is able to reduce metals such as Fe(III), Co(III), Cr(VI), Mn(IV), and U(VI) as electron acceptors while using lactate, formate, pyruvate, or hydrogen as an electron donor. Growth during iron reduction occurred over the pH range of 7.0 to 8.9, a sodium chloride range of 0.05 to 5%, and a temperature range of 0 to 37 degrees C, with an optimum growth temperature of 18 degrees C. Unlike mesophilic dissimilatory Fe(III)-reducing bacteria, which produce mostly superparamagnetic magnetite (<35 nm), this psychrotolerant bacterium produces well-formed single-domain magnetite (>35 nm) at temperatures from 18 to 37 degrees C. The genome size of this strain is about 4.5 Mb. Strain PV-4 is sensitive to a variety of commonly used antibiotics except ampicillin and can acquire exogenous DNA (plasmid pCM157) through conjugation.     

Amplification and demultiplexing in insulin-regulated Akt protein kinase pathway in adipocytes

Akt plays a major role in insulin regulation of metabolism in muscle, fat, and liver. Here, we show that in 3T3-L1 adipocytes, Akt operates optimally over a limited dynamic range. This indicates that Akt is a highly sensitive amplification step in the pathway. With robust insulin stimulation, substantial changes in Akt phosphorylation using either pharmacologic or genetic manipulations had relatively little effect on Akt activity. By integrating these data we observed that half-maximal Akt activity was achieved at a threshold level of Akt phosphorylation corresponding to 5-22% of its full dynamic range. This behavior was also associated with lack of concordance or demultiplexing in the behavior of downstream components. Most notably, FoxO1 phosphorylation was more sensitive to insulin and did not exhibit a change in its rate of phosphorylation between 1 and 100 nm insulin compared with other substrates (AS160, TSC2, GSK3). Similar differences were observed between various insulin-regulated pathways such as GLUT4 translocation and protein synthesis. These data indicate that Akt itself is a major amplification switch in the insulin signaling pathway and that features of the pathway enable the insulin signal to be split or demultiplexed into discrete outputs. This has important implications for the role of this pathway in disease.