Synthesis of phosphorylated 3,4-branched trisaccharides corresponding to LPS inner core structures of Neisseria meningitidis and Haemophilus influenzae

2-(N-Benzyloxycarbonyl)aminoethyl 7-O-acetyl-6-O-allyl-2-O-benzoyl-4-O-benzyl-3-O-chloroacetyl-l-glycero-α-d-manno-heptopyranosyl-(1→3)-[2,3,4,6-tetra-O-benzoyl-β-d-glucopyranosyl-(1→4)]-6,7-di-O-acetyl-2-O-benzyl-l-glycero-α-d-manno-heptopyranoside, a spacer-equipped protected derivative of the common 3,4-branched diheptoside trisaccharide structure of the lipopolysaccharide core of Neisseria meningitidis and Haemophilus influenzae has been synthesized. The protecting group pattern installed allows regioselective introduction of phosphoethanolamine residues in the 3- and 6-position of the second heptose unit in accordance with native structures. From this intermediate the 3-and 6-monophosphoethanolamine as well as the non-phosphorylated deprotected trisaccharides have been synthesized to be used in evaluation of antibody binding specificity and in investigation of the substrate specificity of glycosyl transferases involved in the biosynthesis of LPS core structures.     

A Toxoplasma type 2C serine-threonine phosphatase is involved in parasite growth in the mammalian host cell

Toxoplasma gondii is a human protozoan parasite that belongs to the phylum of Apicomplexa and causes toxoplasmosis. As the other members of this phylum, T. gondii obligatory multiplies within a host cell by a peculiar type of mitosis that leads to daughter cell assembly within a mother cell. Although parasite growth and virulence have been linked for years, few molecules controlling mitosis have been yet identified and they include a couple of kinases but not the counteracting phosphatases. Here, we report that in contrast to other animal cells, type 2C is by far the major type of serine threonine phosphatase activity both in extracellular and in intracellular dividing parasites. Using wild type and transgenic parasites, we characterized the 37 kDa TgPP2C molecule as an abundant cytoplasmic and nuclear enzyme with activity being under tight regulation. In addition, we showed that the increase in TgPP2C activity significantly affected parasite growth by impairing cytokinesis while nuclear division still occurred. This study supports for the first time that type 2C protein phosphatase is an important regulator of cell growth in T. gondii.     

The binding of pentaammineruthenium (III) to RNase B and RNase A+d(pA)4 in the crystalline state

The binding of pentaammineruthenium (III) to ribonuclease A and B both free and complexed with d(pA)₄ has been examined in the crystalline state through the application of X-ray diffraction and difference Fourier techniques. In crystals of native RNase B, the reagent was observed to have many binding sites, some entirely electrostatic in nature and others consistent with coordination to histidine residues. The primary histidine in the latter case was 105 with 119 also partially substituted. In crystals of RNase A+d(pA)₄ complex only a single, extremely strong site of substitution was observed, and this was 2.4 Å from the native position of the imidazole ring of histidine 105. Thus, the results of these X-ray diffraction studies appear to be quite consistent with the findings of earlier NMR studies and with the results obtained in crystals of the gene 5 DNA binding protein.     

Accuracies in Po-210 determination for lead-210 dating

Various laboratory techniques have been utilized worldwide for measuring lead-210 in sub-recent deposits through its grand-daughter product polonium-210. Isotope dilution alpha spectrometry proved a suitable tool for absolute determination of lead-210 for the dating of aquatic deposits. Moreover, isotope dilution alpha spectrometry along with speciation experiments can be used to resolve depositional anomalies arising from supported lead-210/Ra-226 disequilibrium levels and unsupported lead-210 mobile fractions. Isotope dilution alpha spectrometry of sub-recent sediment and peat deposits has been critically evaluated for more than ten years. Our results show that type, size and composition of deposits analyzed as well as radiochemical procedures used, together with alpha counting techniques, are important factors influencing lead-210 determinations and tailing corrections using its granddaughter product polonium-210. Optimization of these parameters is of prime importance to achieve economic and accurate analyses, especially at low lead-210 concentrations and small sample sizes.     

Loci controlling lymphocyte production of interferon γ after alloantigen stimulation in vitro and their co-localization with genes controlling lymphocyte infiltration of tumors and tumor susceptibility

Low infiltration of lymphocytes into cancers is associated with poor prognosis, but the reasons why some patients exhibit a low and others a high infiltration of tumors are unknown. Previously we mapped four loci (Lynf1–Lynf4) controlling lymphocyte infiltration of mouse lung tumors. These loci do not encode any of the molecules that are involved in traffic of lymphocytes. Here we report a genetic relationship between these loci and the control of production of IFNγ in allogeneic mixed lymphocyte cultures (MLC). We found that IFNγ production by lymphocytes of O20/A mice is lower than by lymphocytes of OcB-9/Dem mice (both H2 ^pz ) stimulated in MLC by irradiated splenocytes of C57BL/10SnPh (H2 ^b ) or BALB/cHeA (H2 ^d ) mice, or by ConA. IFNγ production in MLCs of individual (O20 × OcB-9)F₂ mice stimulated by irradiated C57BL/10 splenocytes and genotyped for microsatellite markers revealed four IFNγ-controlling loci (Cypr4-Cypr7), each of which is closely linked with one of the four Lynf loci and with a cluster of susceptibility genes for different tumors. This suggests that inherited differences in certain lymphocyte responses may modify their propensity to infiltrate tumors and their capacity to affect tumor growth.     

Promoting Smoke-Free Homes: A Novel Behavioral Intervention Using Real-Time Audio-Visual Feedback on Airborne Particle Levels

Interventions are needed to protect the health of children who live with smokers. We pilot-tested a real-time intervention for promoting behavior change in homes that reduces second hand tobacco smoke (SHS) levels. The intervention uses a monitor and feedback system to provide immediate auditory and visual signals triggered at defined thresholds of fine particle concentration. Dynamic graphs of real-time particle levels are also shown on a computer screen. We experimentally evaluated the system, field-tested it in homes with smokers, and conducted focus groups to obtain general opinions. Laboratory tests of the monitor demonstrated SHS sensitivity, stability, precision equivalent to at least 1 µg/m³, and low noise. A linear relationship (R² = 0.98) was observed between the monitor and average SHS mass concentrations up to 150 µg/m³. Focus groups and interviews with intervention participants showed in-home use to be acceptable and feasible. The intervention was evaluated in 3 homes with combined baseline and intervention periods lasting 9 to 15 full days. Two families modified their behavior by opening windows or doors, smoking outdoors, or smoking less. We observed evidence of lower SHS levels in these homes. The remaining household voiced reluctance to changing their smoking activity and did not exhibit lower SHS levels in main smoking areas or clear behavior change; however, family members expressed receptivity to smoking outdoors. This study established the feasibility of the real-time intervention, laying the groundwork for controlled trials with larger sample sizes. Visual and auditory cues may prompt family members to take immediate action to reduce SHS levels. Dynamic graphs of SHS levels may help families make decisions about specific mitigation approaches.     

Plasma FA composition in familial LCAT deficiency indicates SOAT2-derived cholesteryl ester formation in humans

Mutations in the LCAT gene cause familial LCAT deficiency (Online Mendelian Inheritance in Man ID: #245900), a very rare metabolic disorder. LCAT is the only enzyme able to esterify cholesterol in plasma, whereas sterol O-acyltransferases 1 and 2 are the enzymes esterifying cellular cholesterol in cells. Despite the complete lack of LCAT activity, patients with familial LCAT deficiency exhibit circulating cholesteryl esters (CEs) in apoB-containing lipoproteins. To analyze the origin of these CEs, we investigated 24 carriers of LCAT deficiency in this observational study. We found that CE plasma levels were significantly reduced and highly variable among carriers of two mutant LCAT alleles (22.5 [4.0–37.8] mg/dl) and slightly reduced in heterozygotes (218 [153–234] mg/dl). FA distribution in CE (CEFA) was evaluated in whole plasma and VLDL in a subgroup of the enrolled subjects. We found enrichment of C16:0, C18:0, and C18:1 species and a depletion in C18:2 and C20:4 species in the plasma of carriers of two mutant LCAT alleles. No changes were observed in heterozygotes. Furthermore, plasma triglyceride-FA distribution was remarkably similar between carriers of LCAT deficiency and controls. CEFA distribution in VLDL essentially recapitulated that of plasma, being mainly enriched in C16:0 and C18:1, while depleted in C18:2 and C20:4. Finally, after fat loading, chylomicrons of carriers of two mutant LCAT alleles showed CEs containing mainly saturated FAs. This study of CEFA composition in a large cohort of carriers of LCAT deficiency shows that in the absence of LCAT-derived CEs, CEs present in apoB-containing lipoproteins are derived from hepatic and intestinal sterol O-acyltransferase 2.