Vaccination against shigellosis: is it the path that is difficult or is it the difficult that is the path?

Following several decades of research, there is not yet a convincing vaccine against shigellosis. It is still difficult, in spite of the breadth of strategies (i.e. live attenuated oral, killed oral, subunit parenteral) to select an optimal option. Two approaches are clearly emerging: (i) live attenuated deletion mutants based on rational selection of genes that are key in the pathogenic process, and (ii) conjugated detoxified polysaccharide parenteral vaccines, or more recently conjugated synthetic carbohydrates. Some of these approaches have already undergone phase I and II clinical trials with promising results, but important issues have also emerged, particularly the discrepancy between colonization and immunogenic potential of live attenuated vaccine candidates depending upon the population concerned (i.e. non endemic vs. endemic areas). Efforts are needed to definitely establish the proof of concept of these approaches, and thus the need for clinical trials which should also soon explore the possibility to associate different serotypes, in response to serotype specific protection against shigellosis. More basic research is also required to improve what we can still consider as first-generation vaccines, and to explore possible new paradigms including the search for cross-protective antigens.     

Does It Matter that Madagascar is an Island?

Being an island has been important for Madagascar's archaeology in two ways. First, its insularity was probably responsible for the lateness and some features of its first human colonizers, as well as for its highly endemic flora and fauna. The earliest coastal communities continued to interact with the Indian Ocean trade network, but the latterly settled interior eventually saw the greatest population increases, eclipsing the coastal communities in power. Second, it may be that as an island, archaeologists have unconsciously been predisposed to interpret Malagasy prehistory in terms of a tree-like model of evolution from a single ancestral culture. Yet if, as seems probable, Malagasy culture has seen contributions from many others, such a single proto-culture may never have existed.     

Insect haemocytes: what type of cell is that?

Classification of insect larvae circulating haemocytes is the subject of controversy, and the terminology used to designate each cellular type is often different from one species to another. However, a survey of the literature on insect haemocytes suggests that there are resemblances for most of the cell types and functions, in different insect species. In this review paper, we compare the structure and functions of circulating haemocytes in those insect species that are, by far, the most often used species for insect physiology studies, i.e. lepidopteran species and Drosophila. We show that there is high degree of homology of haemocyte types and suggest possible synonymies in terminology among species from these taxa.     

Evidence that human α-thrombin is a monovalent cation-activated enzyme

The activity of human α-thrombin (EC 3.4.21.5) on small peptide substrates was enhanced by NaCl or KCl while tetramethylammonium chloride ((CH₃)₄NCl) or choline chloride (HO(CH₂)₂N(CH₃)₃Cl) which were used as ionic strength controls were without effect. The steady-state kinetic parameters of thrombin amidolysis of several peptidyl p-nitroanilide substrates were measured. Na⁺ enhanced thrombin activity by decreasing the K_m,app (0.2 to 0.7-fold) of all substrates, as well as increasing thombin turnover (3.4 to 4.5-fold) of some substrates. The average K_A for Na⁺for the four substrates examined was 3.5 × 10^?2m. A comparison of the effects of Na⁺ vs K⁺ on thrombin hydrolysis of a single substrate indicated that both cations similarly decreased the K_m,app (0.2 to 04.-fold) and increased thek_cat,app (3.1 to 3.4-fold) except that higher K⁺ concentrations (K_A = 2.8 × 10^?1M) were required. The rate of inactivation of thrombin by the active site-directed inhibitor N-p-tosyl-lysine chloromethyl ketone under pseudo-first-order conditions was enhanced 3-fold by saturating NaCl. Also, the fibrinogen clotting activity of thrombin was enhanced by NaCl compared to the choline chloride control. Spectral studies demonstrated that thrombin titration by Na⁺ caused a positive ultraviolet difference spectrum with maxima at 281.5 and 288.5 nm (Δ?_288.5 = +1067). The K_m for Na⁺ was 2.3 × 10^?2m which agrees with the kinetically determined K_A for Na⁺. The results are consistent with Na⁺ binding to thrombin causing a conformational change in the active site. It is concluded that human α-thrombin is a monovalent cation-activated enzyme.     

Non-invasive femoropopliteal assessment: is that angiogram really necessary?

A method of non-invasive preoperative assessment of chronically ischaemic legs was developed that used clinical data and data derived from Doppler ultrasonography to produce a numerical score that could be compared with an angiographic score for stenosis of the popliteal artery trifurcation. The two scoring systems were applied retrospectively to 144 legs after femorodistal bypass. A close correlation was observed (r = 0.89, p less than 0.001), and both systems tended to predict the level of grafting undertaken. A prospective comparison was then made in 81 ischaemic legs that were examined by arteriography; the correlation between the two scoring systems remained close (r = 0.89, p less than 0.001), and the level of bypass was correctly predicted by the non-invasive assessment in 44 of 50 legs that were operated on. Use of the non-invasive assessment subsequently greatly reduced the indications for preoperative arteriography in patients requiring femorodistal vascular reconstruction.     

TonB or not TonB: is that the question?

Bacteria are able to survive in low-iron environments by sequestering this metal ion from iron-containing proteins and other biomolecules such as transferrin, lactoferrin, heme, hemoglobin, or other heme-containing proteins. In addition, many bacteria secrete specific low molecular weight iron chelators termed siderophores. These iron sources are transported into the Gram-negative bacterial cell through an outer membrane receptor, a periplasmic binding protein (PBP), and an inner membrane ATP-binding cassette (ABC) transporter. In different strains the outer membrane receptors can bind and transport ferric siderophores, heme, or Fe3+ as well as vitamin B12, nickel complexes, and carbohydrates. The energy that is required for the active transport of these substrates through the outer membrane receptor is provided by the TonB/ExbB/ExbD complex, which is located in the cytoplasmic membrane. In this minireview, we will briefly examine the three-dimensional structure of TonB and the current models for the mechanism of TonB-dependent energy transduction. Additionally, the role of TonB in colicin transport will be discussed.     

Opinion: peroxisomal-protein import: is it really that complex?

Peroxisomal enzymes are synthesized in the cytoplasm and imported post-translationally across the peroxisome membrane. Unlike other organelles with a sealed membrane, peroxisomes can import folded enzymes, and they seem to lack intraperoxisomal chaperones. Here, we propose a mechanistic model for the early steps in peroxisomal-matrix-enzyme import, which might help to explain the unusual features of this process.     

How do we decide that a species is sex-role reversed?

Defining sex roles has been driven by differences in mating systems at the extreme: polygyny and polyandry. Roles may reverse depending on which sex limits the reproductive rate of the other, and it is generally the female that limits the male. Males therefore compete for female mates. But in species in which the male limits the reproductive rate of the female, the female competes for male mates and assumes the masculine role. Complications arise, however, in species with typical roles when males are temporarily limiting, and females then briefly compete for and display to males. Problems also occur among tightly monogamous species with biparental care, where the mates have equal reproductive rates; both males and females compete intrasexually for mates. Despite this, monogamous species have masculine and feminine roles, typically manifested as the male dominating the female. Some monogamous species are nevertheless sex-role reversed. The pervasive behavioral mechanism characterizing the masculine role is dominance through aggression, size, or both. Attending more to behavioral mechanisms will enrich our understanding of sex-role reversal.     

Is a healthy ecosystem one that is rich in parasites?

Historically, the role of parasites in ecosystem functioning has been considered trivial because a cursory examination reveals that their relative biomass is low compared with that of other trophic groups. However there is increasing evidence that parasite-mediated effects could be significant: they shape host population dynamics, alter interspecific competition, influence energy flow and appear to be important drivers of biodiversity. Indeed they influence a range of ecosystem functions and have a major effect on the structure of some food webs. Here, we consider the bottom-up and top-down processes of how parasitism influences ecosystem functioning and show that there is evidence that parasites are important for biodiversity and production; thus, we consider a healthy system to be one that is rich in parasite species.     

Beryllium is an inhibitor of cellular GSK-3β that is 1,000-fold more potent than lithium

Glycogen synthase kinase 3β (GSK-3β) is a key regulator in signaling networks that control cell proliferation, metabolism, development, and other processes. Lithium chloride is a GSK-3 family inhibitor that has been a mainstay of in vitro and in vivo studies for many years. Beryllium salt has the potential to act as a lithium-like inhibitor of GSK-3, but it is not known whether this agent is effective under physiologically relevant conditions. Here we show that BeSO₄ inhibits endogenous GSK-3β in cultured human cells. Exposure to 10 µM Be²⁺ produced a decrease in GSK-3β kinase activity that was comparable to that produced by 10 mM Li⁺, indicating that beryllium is about 1,000-fold more potent than the classical inhibitor when treating intact cells. There was a statistically significant dose-dependent reduction in specific activity of GSK-3β immunoprecipitated from cells that had been treated with either agent. Lithium inhibited GSK-3β kinase activity directly, and it also caused GSK-3β in cells to become phosphorylated at serine-9 (Ser-9), a post-translational modification that occurs as part of a well-known positive feedback loop that suppresses the kinase activity. Beryllium also inhibited the kinase directly, but unlike lithium it had little effect on Ser-9 phosphorylation in the cell types tested, suggesting that alternative modes of feedback inhibition may be elicited by this agent. These results indicate that beryllium, like lithium, can induce perturbations in the GSK-3β signaling network of treated cells.     

Ursolic acid is a PPAR-α agonist that regulates hepatic lipid metabolism

In this study, we confirmed that ursolic acid, a plant triterpenoid, activates peroxisome proliferator-activated receptor (PPAR)-α in vitro. Surface plasmon resonance and time-resolved fluorescence resonance energy transfer analyses do not show direct binding of ursolic acid to the ligand-binding domain of PPAR-α; however, ursolic acid enhances the binding of PPAR-α to the peroxisome proliferator response element in PPAR-α-responsive genes, alters the expression of key genes in lipid metabolism, significantly reducing intracellular triglyceride and cholesterol concentrations in hepatocytes. Thus, ursolic acid is a PPAR-α agonist that regulates the expression of lipid metabolism genes, but it is not a direct ligand of PPAR-α.     

How is it that microsatellites and random oligonucleotides uncover DNA fingerprint patterns?

Minisatellites, microsatellites, and short random oligonucleotides all uncover highly polymorphic DNA fingerprint patterns in Southern analysis of genomic DNA that has been digested with a restriction enzyme having a 4-bp specificity. The polymorphic nature of the fragments is attributed to tandem repeat number variation of embedded minisatellite sequences. This explains why DNA fingerprint fragments are uncovered by minisatellite probes, but does not explain how it is that they are also uncovered by microsatellite and random oligonucleotide probes. To clarify this phenomenon, we sequenced a large bovine genomic BamHI restriction fragment hybridizing to the Jeffreys 33.6 minisatellite probe and consisting of small and large Sau3A-resistant subfragments. The large Sau3A subfragment was found to have a complex architecture, consisting of two different minisatellites, flanked and separated by stretches of unique DNA. The three unique sequences were characterized by sequence simplicity, that is, a higher than chance occurrence of tandem or dispersed repetition of simple sequence motifs. This complex repetitive structure explains the absence of Sau3A restriction sites in the large Sau3A subfragment, yet provides this subfragment with the ability to hybridize to a variety of probe sequences. It is proposed that a large class of interspered tracts sharing this complex yet simplified sequence structure is found in the genome. Each such tract would have a broad ability to hybridize to a variety of probes, yet would exhibit a dearth of restriction sites. For each restriction enzyme having 4-bp specificity, a subclass of such tracts, completely lacking the corresponding restriction sites, will be present. On digestion with the given restriction enzyme, each such tract would form a large fragment. The largest fragments would be those that contained one or more long minisatellite tracts. Some of these large fragments would be highly polymorphic by virtue of the included minisatellite sequences; by virtue of their complex structure, all would be capable of hybridizing to a wide variety of probes, uncovering a DNA fingerprint pattern.     

Evidence that Dopamine is Acting at α-Adrenoceptors of the Rat Vas Deferens

Abstract

In the rat vas deferens, a vast number of experiments have shown that the α-adrenoceptors present are of two types: α₁ and α₂. This series of experiments with the isolated rat vas deferens was designed to probe by pharmacological means, the nature of the responses elicited by neurogenic transmural stimulation and also those responses evoked by exogenous NE and DA. The methodology required the production of chemical denervation, neurotransmitter depletion, and the use of specific adrenoceptor blockers. The results obtained with the blocking agents, yohimbine or prazosin versus NE and DA, were pA2 values that were virtually interchangeable. The effects of chemical alteration with 6-OH-DA or reserpine point to a certain similarity and interdependence of the mechanism of action for the two neurotransmitters. Therefore, it is suggested that these two transmitters act at the same receptor site or share a common receptive microenvironment in the rat vas deferens.     

Evidence that the conformational stability of ‘aged’ organophosphate-inhibited cholinesterase is altered

In order to determine whether a structural modification at the active center of cholinesterase may alter the conformational stability of the enzyme we compared the urea-induced unfolding of the tetrameric form of non-inhibited and irreversibly inhibited human plasma cholinesterase (acylcholine acylhydrolase, EC 3.1.1.8). We studied enzyme inhibited by methanesulfonyl fluoride, diisopropylfluorophosphonate (DFP) and racemic soman. DFP- and soman-inhibited cholinesterases are converted spontaneously into non-reactivable forms called ‘aged’ enzymes through a process involving dealkylation of the bound organophosphate residue. The unfolding was followed by transverse urea-gradient polyacrylamide electrophoresis at various temperatures ranging from 0 to 60°C. Unfolding of cholinesterase appears to be a complex process. The denaturation patterns showed that partially unfolded states are thermodynamically unstable, but that several intermediates are involved; the lifetime of these depends on the temperature at which electrophoreses are carried out. Cholinesterase inhibited by methanesulfonyl fluoride behaved like the non-inhibited enzyme. On the other hand, small but significant differences in stability between non-inhibited and aged enzymes were observed. Whatever the temperature, the urea concentration at the mid-point of transition was always greater for aged enzyme than for the non-inhibited enzyme. In addition, aged enzymes showed more complex denaturation patterns at the lower temperatures (under 20°C). These findings suggest that the overall stability of aged-cholinesterases is slightly increased as compared with the stability of non-inhibited or methanesulfonyl fluoride-inhibited enzymes. The denaturation pattern obtained at 0°C for soman-inhibited cholinesterase under non-aging conditions (inhibition at 0°C, pH 10.7) was similar to that of non-inhibited enzyme at this temperature, although splitting in two of the denaturation curve over the transition zone reflects the heterogeneity of soman-inhibited enzyme. The slight difference in denaturation behavior between these species may be due to stereoisomerism in soman. The differences in electrophoretic behavior and apparent stability observed between non-inhibited and aged enzymes were interpreted as the result of a conformational change induced by the dealkylation reaction of enzyme-inhibitor conjugates.     

Why is the Pathology of Falciparum Worse than that of Vivax Malaria?

Here, Ian Clark and Bill Cowden summarize new evidence suggesting that nitric oxide (NO) generated by inducible NO synthase (iNOS) provides a functional link between the previously competing approaches to malarial disease pathogenesis: ischaemic hypoxia and NO. When combined with the newly recognized roles of iNOS in renal and pulmonary function and glucose metabolism, synergy between inflammatory cytokines and hypoxia in iNOS induction provides a framework to help explain, at a molecular level, the differences in the pathology seen in falciparum and vivax malaria. Thus sequestration, through localized hypoxia, might contribute to pathology by enhancing cytokine-induced iNOS. Generalized hypoxia might have the same effect.