Opa-typing: a high-resolution tool for studying the epidemiology of gonorrhoea

A single gonococcus possesses a family of 11 distinct and highly variable opa genes. The extensive variation and rapid evolution of the opa gene repertoire has been exploited to provide a high-resolution typing method for studies of the short-term transmission of gonorrhoea. The 11 opa genes are amplified with a single pair of primers by the polymerase chain reaction, digested with frequently-cutting restriction enzymes, and the fragments are fractionated on polyacrylamide to provide an opa-type. The method appeared to be highly discriminatory as the opa-types of gonococci, isolated world-wide over the last 30 years, were all different. Opa-typing discriminated between isolates of the same auxotype/serovar class. Similarly, there were 41 opa-types among 43 consecutive isolates from a sexually transmitted disease (STD) clinic. The two pairs of isolates from this clinic that gave the same opa-types were identical by other criteria and may have been from unsuspected sexual contacts. With one minor exception, identical opa-types were obtained from gonococci recovered from known sexual contacts. These results suggest that variation in the family of 11 opa genes evolves so rapidly that the opa-types of gonococci are distinguishable, unless the isolates are from sexual contacts or a short chain of disease transmission. The identification of gonococci with identical opa-types is therefore believed to be a good indicator that the individuals from which they were recovered were sexual partners, or part of a short chain of disease transmission.     

Depression of hepatic cytochrome P-450-dependent monooxygenase systems with administered interferon inducing agents.

Cytochrome P-450-dependent monooxygenase activities and cytochrome P-450 levels were depressed in hepatic microsomes from rats treated with 12 interferon inducing agents of various types: small molecules (e.g. tilorone), an RNA virus (Mengo), a fungal mycophage (statolon), liver RNA, a synthetic double-stranded polynucleotide (poly rI · poly rC), a bacterial lipopolysaccharide ($\text{E.}\text{coli}$ endotoxin) and an attenuated bacteria ($\text{B.}\text{pertussis}$ vaccine). The results suggest that the depression of hepatic cytochrome P-450-dependent monooxygenase systems may be a general property of interferon inducing agents.     

Cytoplasmic filaments and cellular wound healing in amoeba proteus

The flexibility and self-healing properties of animal cell surface membranes are well known. These properties have been best exploited in various micrurgical studies on living cells (2, 3), especially in amoebae (7, 20). During nuclear transplantation in amoebae, the hole in the membrane through which a nucleus passes can have a diameter of 20-30 μm, and yet such holes are quickly sealed, although some cytoplasm usually escapes during the transfer. While enucleating amoebae in previous studies, we found that if a very small portion of a nucleus was pushed through the membrane and exposed to the external medium, the amoeba expelled such a nucleus on its own accord. When this happened, a new membrane appeared to form around the embedded portion of the nucleus and no visible loss of cytoplasm occurred during nuclear extrusion. In the present study, we examined amoebae that were at different stages of expelling partially exposed nuclei, to follow the sequence of events during the apparent new membrane formation. Unexpectedly, we found that a new membrane is not formed around the nucleus from inside but a hole is sealed primarily by a constriction of the existing membrane, and that cytoplasmic filaments are responsible for the prevention of the loss of cytoplasm.     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

Composition and ecological drivers of the kwongan scrub and woodlands in the northern Swan Coastal Plain,Western Australia

The nature of community patterns and environmental drivers in kwongan mediterranean‐type shrubland on nutrient‐poor soils occurring in Western Australia remain poorly examined. We aimed to determine whether (i) classification of the kwongan vegetation of the northern Swan Coastal Plain would be ecologically informative and (ii) which environmental drivers underpin the plant community patterns. The study area was positioned on the northern Swan Coastal Plain, locality of Cooljarloo (30°39′ S, 115°22′ E), situated 170 km north of Perth, Western Australia. Compositional (518 species × 337 relevés) and environmental data set (29 variables × 87 relevés) describing time since last fire, soil chemical and physical properties, and terrain characteristics were analysed using classification and ordination techniques. OptimClass assisted in the selection of a robust data transformation, resemblance function and clustering algorithm to identify the vegetation patterns. Major ecological drivers of the vegetation patterns were detected using distance‐based redundancy analysis (db‐RDA). Classification revealed major groupings of Wet Heath and Banksia Woodland distinguishable by the high prevalence of myrtyoid and proteoid taxa, respectively. On floristic‐sociological grounds, we recognised four Wet Heath and two Banksia Woodland communities. The Wet Heath was constrained to areas of higher litter depth (db‐RDA axis 1: 9%). Soil chemical and physical properties explained the highest proportion (17%) of the compositional variance, while the terrain‐ and fire‐related variables explained 2% and <0.001%, respectively. While fire explained little compositional variance overall, a separate db‐RDA analysis found that it may play an important pattern‐structuring role within Banksia Woodlands. Fine‐scale compositional patterns correspond only to a small extent to environmental data; the substantial unexplained variance may be due to slow‐acting neutral and stochastic processes.     

1,5-Disubstituted indole derivatives as selective human neuronal nitric oxide synthase inhibitors

A series of 1,5-disubstituted indole derivatives was designed, synthesized and evaluated as inhibitors of human nitric oxide synthase. A variety of flexible and restricted basic amine side chain substitutions was explored at the 1-position of the indole ring, while keeping the amidine group fixed at the 5-position. Compounds having N-(1-(2-(1-methylpyrrolidin-2-yl)ethyl)- (12, (R)-12, (S)-12 and 13) and N-(1-(1-methylazepan-4-yl)- side chains (14, 15, (-)-15 and (+)-15) showed increased inhibitory activity for the human nNOS isoform and selectivity over eNOS and iNOS isoforms. The most potent compound of the series for human nNOS (IC(50)=0.02 μM) (S)-12 showed very good selectivity over the eNOS (eNOS/nNOS=96-fold) and iNOS (iNOS/nNOS=850-fold) isoforms.     

Beyond patient reported pain: perfusion magnetic resonance imaging demonstrates reproducible cerebral representation of ongoing post-surgical pain

Development of treatments for acute and chronic pain conditions remains a challenge, with an unmet need for improved sensitivity and reproducibility in measuring pain in patients. Here we used pulsed-continuous arterial spin-labelling [pCASL], a relatively novel perfusion magnetic-resonance imaging technique, in conjunction with a commonly-used post-surgical model, to measure changes in regional cerebral blood flow [rCBF] associated with the experience of being in ongoing pain. We demonstrate repeatable, reproducible assessment of ongoing pain that is independent of patient self-report. In a cross-over trial design, 16 participants requiring bilateral removal of lower-jaw third molars underwent pain-free pre-surgical pCASL scans. Following extraction of either left or right tooth, repeat scans were acquired during post-operative ongoing pain. When pain-free following surgical recovery, the pre/post-surgical scanning procedure was repeated for the remaining tooth. Voxelwise statistical comparison of pre and post-surgical scans was performed to reveal rCBF changes representing ongoing pain. In addition, rCBF values in predefined pain and control brain regions were obtained. rCBF increases (5-10%) representing post-surgical ongoing pain were identified bilaterally in a network including primary and secondary somatosensory, insula and cingulate cortices, thalamus, amygdala, hippocampus, midbrain and brainstem (including trigeminal ganglion and principal-sensory nucleus), but not in a control region in visual cortex. rCBF changes were reproducible, with no rCBF differences identified across scans within-session or between post-surgical pain sessions. This is the first report of the cerebral representation of ongoing post-surgical pain without the need for exogenous tracers. Regions of rCBF increases are plausibly associated with pain and the technique is reproducible, providing an attractive proposition for testing interventions for on-going pain that do not rely solely on patient self-report. Our findings have the potential to improve our understanding of the cerebral representation of persistent painful conditions, leading to improved identification of specific patient sub-types and implementation of mechanism-based treatments.     

Rapid Acyl-Homoserine Lactone Quorum Signal Biodegradation in Diverse Soils

Signal degradation impacts all communications. Although acyl-homoserine lactone (acyl-HSL) quorum-sensing signals are known to be degraded by defined laboratory cultures, little is known about their stability in nature. Here, we show that acyl-HSLs are biodegraded in soils sampled from diverse U.S. sites and by termite hindgut contents. When amended to samples at physiologically relevant concentrations, ¹⁴C-labeled acyl-HSLs were mineralized to ¹⁴CO₂ rapidly and, at most sites examined, without lag. A lag-free turf soil activity was characterized in further detail. Heating or irradiation of the soil prior to the addition of radiolabel abolished mineralization, whereas protein synthesis inhibitors did not. Mineralization exhibited an apparent K_m of 1.5 μM acyl-HSL, ca. 1,000-fold lower than that reported for a purified acyl-HSL lactonase. Under optimal conditions, acyl-HSL degradation proceeded at a rate of 13.4 nmol·h⁻¹·g of fresh weight soil⁻¹. Bioassays established that the final extent of signal inactivation was greater than for its full conversion to CO₂ but that the two processes were well coupled kinetically. A most probable number of 4.6 × 10⁵ cells·g of turf soil⁻¹ degraded physiologically relevant amounts of hexanoyl-[1-¹⁴C]HSL to ¹⁴CO₂. It would take chemical lactonolysis months to match the level of signal decay achieved in days by the observed biological activity. Rapid decay might serve either to quiet signal cross talk that might otherwise occur between spatially separated microbial aggregates or as a full system reset. Depending on the context, biological signal decay might either promote or complicate cellular communications and the accuracy of population density-based controls on gene expression in species-rich ecosystems.     

Consequences of Fragmentation of Tropical Moist Forest for Birds and Their Role in Predation of Herbivorous Insects

The consequences of habitat alteration on the role of understory insectivorous birds as predators of herbivorous insects in tropical forests are poorly understood. To examine whether fragmentation may affect the top–down controls of herbivory, we compared the number of species, individuals, and the community structure of insectivorous birds between fragments and continuous tropical moist forest in Mexico. We also registered insect herbivore abundances and conducted a larvae predation experiment to evaluate the potential role of insectivorous birds as predators of herbivorous insects. We recorded 63 bird species from 22 families, 43 percent of which were insectivorous birds. Species richness, abundance, and diversity of the avian community were higher in continuous forest compared with forest fragments. For insectivorous birds in particular, there was low similarity in avian insectivore communities between forest types, and forest fragments had more heavily dominated communities of avian insectivores. During the dry season, forest fragments presented significantly higher predation rates on artificial caterpillars, and lower abundance of herbivorous Lepidoptera larvae, compared with continuous forest. Furthermore, there was a significant negative correlation between artificial caterpillar predation rate and larval Lepidoptera abundance, with higher rates of predation in sample sites of low Lepidoptera abundance. Hence, the potentially greater light in the dry season combined with a more dominated avian insectivore community in forest fragments may facilitate increased predation by avian insectivores, resulting in a decline in abundance of larval Lepidoptera, with implications for the process of insect‐driven herbivory in forest fragments.