In vitro inhibition of human influenza A virus replication by chloroquine

Inhibition of the human cytomegalovirus UL97 kinase by maribavir is thought to be responsible for the antiviral activity of this compound. Some mutations that confer resistance to maribavir map to UL97, however additional mutations that also confer resistance to the drug were mapped to UL27. These open reading frames share a low level of homology, yet the function of pUL27 remains unknown. A recombinant virus with a deletion in the UL27 open reading frame was reported previously to exhibit a slight replication deficit, but a more important function in vivo was hypothesized given its homology to the UL97 kinase. The potential for an important function in vivo was investigated by determining if these knockout viruses could replicate in human tissue implanted in SCID mice. None of the AD169 derived viruses replicated well in the implanted thymus/liver tissue, and is consistent with previous observations, although all of the viruses replicated to some degree in retinal tissue implants. Replication of the parent viruses was observed at 7 days post inoculation, whereas no replication was detected with any of the recombinant viruses with deletions in UL27. By day 14, replication was detected in two of the three knockout viruses and in all of the viruses by day 42. These data are consistent with minimal defects observed in cell culture, but are not consistent with an important role for UL27 in vivo. We conclude that UL27 is not required for viral replication in vivo.     

Biogenesis of mitochondria: a mutation in the 5''-untranslated region of yeast mitochondrial oli1 mRNA leading to impairment in translation of subunit 9 of the mitochondrial ATPase complex.

A temperature-conditional mit- mutant of Saccharomyces cerevisiae has been characterized; the mutant strain h45 cannot grow at 36 degrees C on nonfermentable substrates yet appears to be normal at 28 degrees C. The mutation in strain h45 maps genetically to the oli1 region of the mitochondrial DNA (mtDNA) genome, and prevents the synthesis at 36 degrees C of the oli1 gene product, subunit 9 of the mitochondrial ATPase complex. Since the level of oli1 mRNA in mutant h45 is close to normal at 36 degrees C, it is concluded that there is a specific block in translation of this mRNA at the non-permissive temperature. DNA sequence analysis of mtDNA from strain h45 reveals an additional T residue inserted 88 bp upstream of the oli1 coding region, in the A,T-rich sequence that is transcribed into the 5'-untranslated region of the oli1 mRNA. Sequence data on two revertants show that one returns to wild-type parental (J69-1B) mtDNA sequence, whilst the other contains an inserted A residue adjacent to the T inserted in the original h45 mutant. The results are discussed in terms of the stability of folds in RNA upstream of putative ribosome-binding sites in mitochondrial mRNA, and the potential action of nuclear-coded proteins that might be activators of the translation of specific mitochondrial mRNAs in yeast mitochondria.     

Cytoplasmic expression of the Achromobacter xylosoxidans blue copper nitrite reductase in Escherichia coli and characterisation of the recombinant protein

The gene of the Achromobacter xylosoxidans (DSM 2402) blue copper-containing nitrite reductase was amplified using the polymerase chain reaction. DNA sequence analysis reveals that the amino acid sequence is identical to those of the GIFU1051 and the NCIMB11015 A. xylosoxidans nitrite reductases. The gene encoding the mature coding region for DSM 2402 nitrite reductase was cloned into a pET-vector, overexpressed in the cytoplasm of Escherichia coli BL21(DE3), and the expressed holoprotein was purified to apparent homogeneity by cation-exchange chromatography. The recombinant blue copper-containing nitrite reductase was obtained in high yields of 70mgL(-1) of culture. The specific catalytic activity as well as the electronic absorption and electron paramagnetic resonance spectra agree with corresponding data for the native protein. Mass spectroscopic analysis of the recombinant nitrite reductase gave a molecular weight of 36659.1Da for the apo-protein monomer, in agreement with the expected molecular mass based on the amino acid sequence.     

Improved microarray methods for profiling the Yeast Knockout strain collection

A remarkable feature of the Yeast Knockout strain collection is the presence of two unique 20mer TAG sequences in almost every strain. In principle, the relative abundances of strains in a complex mixture can be profiled swiftly and quantitatively by amplifying these sequences and hybridizing them to microarrays, but TAG microarrays have not been widely used. Here, we introduce a TAG microarray design with sophisticated controls and describe a robust method for hybridizing high concentrations of dye-labeled TAGs in single-stranded form. We also highlight the importance of avoiding PCR contamination and provide procedures for detection and eradication. Validation experiments using these methods yielded false positive (FP) and false negative (FN) rates for individual TAG detection of 3–6% and 15–18%, respectively. Analysis demonstrated that cross-hybridization was the chief source of FPs, while TAG amplification defects were the main cause of FNs. The materials, protocols, data and associated software described here comprise a suite of experimental resources that should facilitate the use of TAG microarrays for a wide variety of genetic screens.     

Identification and characterization of leukotriene D4 receptors and signal transduction processes in rat basophilic leukemia cells

The leukotriene D4 (LTD4) receptor on rat basophilic leukemia (RBL-1) cell membranes was characterized using a radioligand binding assay. [3H]LTD4 binding to RBL-1 membrane receptors was stereoselective, specific, and saturable. The binding affinity and maximum binding density of [3H]LTD4 to RBL-1 membrane receptors were 0.9 +/- 0.2 nM and 800 +/- 125 fmol/mg protein, respectively. Binding of [3H]LTD4 to the receptors was enhanced by divalent cations (Ca2+, Mg2+, and Mn2+) and inhibited by guanine nucleotides and sodium ions, specifically, indicating that a guanine nucleotide-binding protein may regulate the agonist-receptor interaction. LTD4, LTE4 agonist and antagonist analogs competed with the radioligand in binding to the RBL-1 LTD4 receptors. The binding affinities of these analogs correlated with (a) those determined from the guinea pig lung LTD4 receptors and (b) the pharmacological activities in smooth muscle contraction. LTD4 and related agonists also induced time- and concentration-dependent phosphatidylinositol hydrolysis in RBL-1 cells. The LTD4 induction of inositol 1-phosphate was potent, stereoselective, specific, and was blocked by LTD4 receptor antagonists. The rank order potency of agonist-induced inositol 1-phosphate formation in RBL-1 cells was equivalent to the receptor binding affinity determined using either RBL-1 cell or guinea pig lung membranes. These studies have demonstrated the G protein coupled LTD4 receptors on RBL-1 cell membranes. Binding of agonists to the receptor may activate the G protein-regulated phospholipase C to induce hydrolysis of phosphatidylinositol. The hydrolytic products of phosphatidylinositol, possibly inositol trisphosphate and diacylglycerol, may be the intracellular messengers for LTD4 receptors in RBL-1 cells.     

Dengue virus activates polyreactive, natural IgG B cells after primary and secondary infection

Background

Dengue virus is transmitted by mosquitoes and has four serotypes. Cross-protection to other serotypes lasting for a few months is observed following infection with one serotype. There is evidence that low-affinity T and/or B cells from primary infections contribute to the severe syndromes often associated with secondary dengue infections. such pronounced immune-mediated enhancement suggests a dengue-specific pattern of immune cell activation. This study investigates the acute and early convalescent B cell response leading to the generation of cross-reactive and neutralizing antibodies following dengue infection.

Methodology/Principal Findings

We assayed blood samples taken from dengue patients with primary or secondary infection during acute disease and convalescence and compared them to samples from patients presenting with non-dengue related fever. Dengue induced massive early plasmablast formation, which correlated with the appearance of polyclonal, cross-reactive IgG for both primary and secondary infection. Surprisingly, the contribution of IgG to the neutralizing titer 4–7 days after fever onset was more than 50% even after primary infection.

Conclusions/Significance

Poly-reactive and virus serotype cross-reactive IgG are an important component of the innate response in humans during both primary and secondary dengue infection, and “innate specificities” seem to constitute part of the adaptive response in dengue. While of potential importance for protection during secondary infection, cross-reactive B cells will also compete with highly neutralizing B cells and possibly interfere with their development.     

Urbanization in Southeast Asia: Assessing Policy Process and Progress toward Sustainability

In investigating the relationship between urbanization and sustainability in cities of developing countries, many researchers have looked to rising incomes as a driver for environmental improvement. This article challenges the transition and evolutionary models of urban environmental development that suggest that as cities grow in per capita income, their local environmental problems will diminish. The transition model is outcomes-based, and a competing model based on greater attention to participation in setting sustainability goals and assessing the progress toward those goals is presented.
Consensus on appropriate sustainability indicators is a key element, albeit a challenging one, in the task of pursuing urban sustainability. This is due to the contested nature of the concept of sustainability and also the fact that much of the work done on sustainability has been conducted among countries or on a national scale. A brief review of health, urban transport, air quality, and sewerage indicators suggests that cities do not necessarily see more progress as the per capita income increases. Low-income cities in Southeast Asia that are performing well are likely to see a deterioration in standards with rapid economic and population growth.     

Methamphetamine enhances paced mating behaviors and neuroplasticity in the medial amygdala of female rats

Methamphetamine (METH) is a psychomotor stimulant strongly associated with increases in sexual drive and behavior in women and men. Even though men and women are equally as likely to be addicted to or use METH, studies of sexual behavior often focus on male users. The paucity in studies examining the effect of METH in women is of great concern, when one considers the high correlation with sexually transmitted diseases such as HIV/AIDS and unplanned pregnancies. In fact, why METH so profoundly increases sexual drive is unknown. We have demonstrated that repeated exposure to METH enhances both receptivity and proceptivity in hormonally primed female rats. The current study examined whether a repeated exposure to METH enhanced female-initiated sexual behaviors in hormonally primed rats. In a paced mating paradigm, METH treatment significantly decreased the female's return latency following a mount (57%) and an ejaculation (44%), and the likelihood to leave the male following an intromission (37%) compared to controls. The METH-induced changes in paced mating behavior were accompanied by a 60% increase in spinophilin levels in the medial amygdala following hormonal priming and METH treatment. Taken together, these findings suggest that METH increases female sexual motivation and behavior in the rat potentially via changes in the neural substrate that require repeated exposure to the drug.     

Exercise prior to fat ingestion lowers fasting and postprandial VLDL and decreases adipose tissue IL-6 and GIP receptor mRNA in hypertriacylglycerolemic men

Fasting and postprandial triacylglycerol (TAG) concentrations are risk factors for cardiovascular disease. This study evaluated whether interleukin-6 (IL-6) and incretin hormones [gastric inhibitory peptide (GIP) and glucagon-like peptide-1 (GLP-1) (active)] were associated with fasting and postprandial TAG in response to an oral lipid load, including very-low-density lipoprotein (VLDL) and chylomicron (CM) TAG, following one bout of exercise in nine men (age, 59±2 years; body mass index, 34±2 kg/m²; waist circumference, 113±3 cm) with high fasting TAG (2.9±0.2 mmol/L). Subjects completed two oral fat tolerance tests (OFTTs), randomized 1 week apart, that consisted of 1g fat/kg body weight emulsified lipids in the absence of carbohydrate and protein. Approximately 16 h prior to one OFTT, subjects completed 60 min of treadmill walking (estimated 55% VO₂ peak; heart rate, 122±4 beats/min). No exercise was performed on the day before the other OFTT. Fasted (0 h) and postprandial (1, 2, 3, 4, 5 and 6 h) blood samples were taken for analysis of TAG, IL-6 and incretins. Subcutaneous adipose tissue biopsies were taken at 0 and 6 h after OFTT ingestion for IL-6 and GIP receptor (GIPr) mRNA quantification. Exercise lowered fasting and postprandial TAG (P<.05) and VLDL TAG (P<.05), while postprandial CM TAG were similar in both OFTT trials (P>.05). Fasting and postprandial plasma IL-6, GIP and GLP-1 did not differ between rest and exercise OFTT trials (P>.05). Exercise reduced IL-6 and GIPr mRNA (P<.05) in adipose tissue. Our results suggest that the reduction in VLDL TAG following an acute bout of exercise is not associated with circulating IL-6 or incretin concentrations, despite reductions in the adipose tissue expression of IL-6 and GIPr.