The ganglioside GD1α, IV3Neu5Ac,III6Neu5Ac-GgOse4Cer,is a major disialoganglioside in the highly metastatic murine lymphoreticular tumour cell line MDAY-D2

The aim of the present study was to investigate the ganglioside expression of the highly metastatic murine lymphoreticular tumour cell line MDAY-D2. Cells were propagated under controlled pH conditions and oxygen supply in bioreactors of 1 and 7.5l volumes by repeated batch fermentation. Gangliosides were isolated from 2.7×10¹¹ cells, purified by silica gel chromatography and separated into mono- and disialoganglioside fractions by preparative DEAE anion exchange high performance liquid chromatography. Individual gangliosides were obtained by preparative thin layer chromatography. Their structural features were established by immunostaining, fast atom bombardment and gas chromatography mass spectrometry. In addition to gangliosides of the G_M1a-pathway (G_M2, G_M1a and G_D1a) and G_M1b (IV³Neu5Ac-GgOse₄Cer) and GalNAc-G_M1b of the G_M1b-pathway, the dis8aloganglioside G_D1 (IV³Neu5Ac, III⁶Neu5Ac-GgOse₄Cer) was found in equal amounts compared to G_D1a (IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer). All gangliosides were substituted with C_24:0,24:1 and C_16:0 fatty acids, sphingosine andN-acetylneuraminic acid as the sole sialic acid. Abbreviations: FAB-MS, fast atom bombardment-mass spectrometry; GC-MS, gas chromatography-mass spectrometry; GSL(s), glycosphingolipid(s); HPLC, high performance liquid chromatography; HPTLC, high performance thin layer chromatography; Neu5Ac,N-acetylneuraminic acid; Neu5Gc,N-glycoloylneuraminic acid [57]. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations [58] and the nomenclature of Svennerholm [59]. Gangliotriaosylceramide or GgOse₃Cer, GalNAc1-4Gal1-4Glc1-1Cer; gangliotetraosylceramide or GgOse₄Cer, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer gangliopentaosylceramide or GgOse₅Cer, GalNAc1-4Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; G_M2, II³Neu5Ac-GgOse₃Cer; G_M1a, II³Neu5Ac-GgOse₄Cer; G_M1b, IV³Neu5Ac-GgOse₄Cer; GalNAc-G_M1b, IV³Neu5Ac-GgOse₅Cer; G_D1a, IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer; G_D1b, II³(Neu5Ac)₂-GgOse₄Cer; G_D1 or G_D1e, IV³Neu5Ac, III⁶Neu5AcGgOse₄Cer; G_D1e, IV³(Neu5Ac)₂-GgOse₄Cer; G_T1b, IV³Neu5Ac, II³(Neu5Ac)₂-GgOse₄Cer.     

CTCF-Induced Circular DNA Complexes Observed by Atomic Force Microscopy

The CTCF protein has emerged as a key architectural protein involved in genome organization. Although hypothesized to initiate DNA looping, direct evidence of CTCF-induced DNA loop formation is still missing. Several studies have shown that the 11 zinc finger (11 ZF) domain of CTCF is actively involved in DNA binding. We here use atomic force microscopy to examine the effect of the 11 ZF domain comprising residues 266–579 (11 ZF CTCF) and the 3 ZF domain comprising residues 402–494 (6–8 ZF CTCF) of human CTCF on the DNA morphology. Our results show that both domains alter the DNA architecture from the relaxed morphology observed in control DNA samples to compact circular complexes, meshes, and networks, offering important insights into the multivalent character of the 11 ZF CTCF domain. Atomic force microscopy images reveal quasi-circular DNA/CTCF complexes, which are destabilized upon replacing the 11 ZF CTCF by the 6–8 ZF CTCF domain, highlighting the role of the 11 ZF motif in loop formation. Intriguingly, the formation of circular DNA/CTCF complexes is dominated by non-specific binding, whereby contour length and height profiles suggest a single DNA molecule twice wrapped around the protein.     

nov: a new genetic locus that affects the response of Escherichia coli K-12 to novobiocin

We have identified a new gene locus (nov) affecting the resistance of Escherichia coli K-12 to novobiocin. The gene also affects, although to a lesser extent, tolerance to another gyrase inhibitor coumermycin. Transductional and complementation analysis show that nov is located between att phi 80 and the osmZ (hns) genes at minute 27 of the E. coli K-12 genetic map. In standard laboratory strains of E. coli K-12 nov exists at least in two allelic forms.     

Occurrence of an Environmental Acinetobacter baumannii Strain Similar to a Clinical Isolate in Paleosol from Croatia

Over the past decade, bacteria of the genus Acinetobacter have emerged as a leading cause of hospital-acquired infections. Outbreaks of Acinetobacter infections are considered to be caused exclusively by contamination and transmission in hospital environments. The natural habitats of clinically important multiresistant Acinetobacter spp. remain to be defined. In this paper, we report an incidental finding of a viable multidrug-resistant strain of Acinetobacter baumannii, related to clinical isolates, in acid paleosol from Croatia. The environmental isolate of A. baumannii showed 87% similarity to a clinical isolate originating from a hospital in this geographic area and was resistant to gentamicin, trimethoprim-sulfamethoxazole, ciprofloxacin, and levofloxacin. In paleosol, the isolate was able to survive a low pH (3.37), desiccation, and a high temperature (50°C). The probable source of A. baumannii in paleosol is illegally disposed waste of external origin situated in the abandoned quarry near the sampling site. The bacteria could have been leached from waste by storm water and thus infiltrated the paleosol.     

Identification of the gate regions in the primary structure of the secretin pIV

Secretins are a family of large bacterial outer membrane channels that serve as exit ports for folded proteins, filamentous phage and surface structures. Despite the large size of their substrates, secretins do not compromise the barrier function of the outer membrane, implying a gating mechanism. The region in the primary structure that forms the putative gate has not previously been determined for any secretin. To identify residues involved in gating the pIV secretin of filamentous bacteriophage f1, we used random mutagenesis of the gene followed by positive selection for mutants with compromised barrier function (‘leaky’ mutants). We identified mutations in 34 residues, 30 of which were clustered into two regions located in the centre of the conserved C‐terminal secretin family domain: GATE1 (that spanned 39 residues) and GATE2 (that spanned 14 residues). An internal deletion constructed in the GATE2 region resulted in a severely leaky phenotype. Three of the four remaining mutations are located in the region that encodes the N‐terminal, periplasmic portion of pIV and could be involved in triggering gate opening. Two missense mutations in the 24‐residue region that separates GATE1 and GATE2 were also constructed. These mutant proteins were unstable, defective in multimerization and non‐functional.     

Control of pathogens and microbiota by innate lymphoid cells

Innate lymphoid cells (ILCs) are the innate counterpart of T cells. Upon infection or injury, ILCs react promptly to direct the developing immune response to the one most adapted to the threat facing the organism. Therefore, ILCs play an important role early in resistance to infection, but also to maintain homeostasis with the symbiotic microbiota following perturbations induced by diet and pathogens. Such roles of ILCs have been best characterized in the intestine and lung, mucosal sites that are exposed to the environment and are therefore colonized with diverse but specific types of microbes. Understanding the dialogue between pathogens, microbiota and ILCs may lead to new strategies to re-inforce immunity for prevention, vaccination and therapy.     

Glutathione cycle in stable chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation and oxidant/antioxidant imbalance. Glutathione is the most abundant cellular low‐molecular weight thiol and the glutathione redox cycle is the fundamental component of the cellular antioxidant defence system. Concentration of total glutathione and catalytic activities of glutathione peroxidase and glutathione reductase were determined in peripheral blood of patients (n = 109) and healthy subjects (n = 51). Concentration of total glutathione in patients was not changed in comparison to healthy controls. However, we found statistically significant difference between patients with moderate and severe disease stages. Glutathione reductase activity was increased, while glutathione proxidase activity was decreased in the patients with COPD, when compared to healthy controls. We found no significant difference in glutathione peroxidase and glutathione reductase activities between stages. Patients who smoked had lower concentration of total glutathione compared with former smokers and never‐smoking patients. Lung function parameters were inversely associated with glutathione level. Evidence is presented for differential modulation of glutathione peroxidase and glutathione reductase activities in peripheral blood of patients with stable COPD. We suppose that in addition to glutathione biosynthesis, glutathione reductase‐dependent regulation of the glutathione redox state is vital for protection against oxidative stress. Copyright © 2010 John Wiley & Sons, Ltd.     

Transgenic mouse models of amyotrophic lateral sclerosis

The discovery of missense mutations in the gene coding for the Cu/Zn superoxide dismutase 1 (SOD1) in subsets of familial cases was rapidly followed by the generation of transgenic mice expressing various forms of SOD1 mutants. The mice overexpressing high levels of mutant SOD1 mRNAs do develop motor neuron disease but unraveling the mechanisms of pathogenesis has been very challenging. Studies with mouse lines suggest that the toxicity of mutant SOD1 is unrelated to copper-mediated catalysis but rather to propensity of a subfraction of mutant SOD1 proteins to form misfolded protein species and aggregates. However, the mechanism of toxicity of SOD1 mutants remains to be elucidated. Involvement of cytoskeletal components in ALS pathogenesis is supported by several mouse models of motor neuron disease with neurofilament abnormalities and with genetic defects in microtubule-based transport. Here, we describe how transgenic mouse models have been used for understanding pathogenic pathways of motor neuron disease and for pre-clinical drug testing.     

Contact allergy and sociodemographic characteristics

The aim of the study was to determine the frequency of positive patch test reaction to different contact allergens according to patients age, sex, occupation and clinical features. Between 1999 and 2003, patch testing was performed in 3,293 patients with respective clinical diagnoses. Patch testing was done by the standard technique proposed by the International Contact Dermatitis Research Group (ICDRG). Study results showed statistically significant differences in patch test response according to sex and age for three allergens (cobalt chloride, nickel sulphate and thiomersal); according to occupation for nine allergens (cobalt chloride, nickel sulphate, balsam of Peru, fragrance mix, thiuram mix, wood tars, neomycin sulphate, thiomersal and detergents), and clinical diagnosis for two allergens (nickel sulphate, and wood tars). The most common and relevant allergens were: nickel sulphate, cobalt chloride and carba mix. They were found in all examinees regardless of age, sex, occupation and diagnoses. The increased awareness of allergens and their potential sources may help to limit the usage of these chemicals in manufacture of consumer products.     

Body mass index and nutritional status of the Bayash Roma from eastern Croatia

This study examines anthropometrically assessed nutritional status of the Bayash, the Roma population from the eastern Croatian region of Baranya, and compares it to the non-Roma general population of eastern Croatia. The analysis of nutritional status and diets is a segment of multidisciplinary anthropological and epidemiological survey of the Roma minority population in Croatia began in 2005. The Bayash are an ethnic group that arrived to Croatia from Romania most likely in the 19th century and speaks a distinct archaic dialect of the Romanian language. The Roma population of Baranya approximates 1,000 according to the 2001 census. The Bayash sample comprised 227 adults aged 18-65yrs. The women fall below the Croatian 10th percentile for stature and men track about the 10th percentile. Both sexes approximate the 25th percentile for body weight. Despite their diminutive size, the Bayash appear to have adequate nutritional status until the age of 35yrs after which their average BMI exceeds the value of 25 kg/m(2) and falls in the overweight category. However, 8% of Bayash are underweight (BMI<18.5) in contrast to 1% of the majority population in the region. Underweight rates are especially high in women (11%) compared to men (4%). The prevalence of overweight (BMI 25.0 to 29.9) of 30% is considerably lower than in the majority population (42%) while the prevalence of obesity (BMI>or=30.0) of 23% is approximately equal. Overall unsatisfactory nutritional status of the Bayash merits attention. It appears to be the product of unhealthy dietary habits and their socio-economic deprivation that resulted from their poor education and extremely high unemployment.