Virulence studies on chromosomal α-toxin and Θ-toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of α-toxin in Clostridium perfringens-mediated gas gangrene

The pathogenesis of clostridial myonecrosis, or gas gangrene, involves the growth of the anaerobic bacterium Clostridium perfringens in the infected tissues and the elaboration of numerous extracellular toxins and enzymes. The precise role of each of these toxins in tissue invasion and necrosis has not been determined. To enable genetic approaches to be used to study C. perfringens pathogenesis we developed an allelic exchange method which involved the transformation of C. perfringens cells with a suicide plasmid carrying a gene insertionally inactivated with an erythromycin-resistance determinant. The frequency with which double reciprocal crossover events were observed was increased to a workable level by increasing the amount of homologous DNA located on either side of the inactivated gene. Allelic exchange was used to isolate mutations in the‘chromosomal pfoA gene, which encodes an oxygen-labile haemolysin known as Θ-toxin or perfringolysin O. and in the chromosomal pic gene, which encodes the α-toxin or phospholipase C. The resultant mutants failed to produce detectable Θ-toxin or α-toxin activity, respectively, and could be complemented by recombinant plasmids that carried the respective wild-type genes. The resultant strains were virulence tested in a mouse myonecrosis model. The results showed that the pic mutants had demonstrably reduced virulence and therefore provided definitive genetic evidence for the essential role of α-toxin in gas gangrene or clostridial myonecrosis.     

Haematology of the Australian eastern quoll, Dasyurus viverrinus--II. Red cell enzymes and metabolic intermediates

1. The activity of 21 red cell enzymes and three red cell metabolic intermediates were measured in adult Dasyurus viverrinus and compared with published data on other marsupials. 2. Phosphofructokinase (PFK), glyceraldehyde dehydrogenase (GAPD) and phosphoglycerate kinase (PGK) were elevated in comparison to other marsupials. 3. Enolase (ENO) and 2,3-diphosphoglycerate (2,3 DPG) were lower than in other marsupials.     

Regulation of endomembrane biogenesis in arabidopsis by phospatidic acid hydrolase

Coordination of membrane lipid biosynthesis is important for cell function during plant growth and development. Here we summarize our recent work on PHOSPHATIDIC ACID PHOSPHOHYDROLASE (PAH) which suggests that this enzyme is a key regulator of phosphaticylcholine (PC) biosynthesis in Arabidopsis thaliana. Disruption of PAH activity elevates phosphatidic acid (PA) levels and stimulates PC biosynthesis and biogenesis of the endoplasmic reticulum (ER). Furthermore, the activity of PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE (CCT), which is the key enzyme controlling the rate of PC biosynthesis, is directly stimulated by PA and expression of a constitutively active version of CCT replicates the effects of PAH disruption. Hence PAH activity can control the abundance of PA, which in turn can modulate CCT activity to govern the rate of PC biosynthesis. Crucially it is not yet clear how PAH activity is regulated in Arabidopsis but there is evidence that PAH1 and PAH2 are both phosphorylated and further work will be required to investigate whether this is functionally significant.     

Carrion flies of forensic interest: a study of seasonal community composition and succession in Lisbon,Portugal

Information on Diptera community, seasonality and successional patterns in every geographical region is fundamental for the use of flies as forensic indicators of time of death. In order to obtain these data from the Lisbon area (Portugal), experiments were conducted during the four seasons of the year, using piglet carcasses as animal models. Five stages were recognized during the decomposition process. The stages, besides visually defined, could be separated taking into account the occurrence and abundance of the specific groups of Diptera collected. In general, the bloated stage recorded higher abundance and species‐richness values. Seventy‐one species were identified, belonging to 39 families, in a total of 20 144 adult Diptera collected. Autumn yielded the highest values of species richness, whereas winter had the lowest. In all seasons of the year, Calliphoridae was the dominant family; Muscidae and Fanniidae were very abundant as well. Calliphora vicina Robineau‐Desvoidy, Calliphora vomitoria (Linnaeus), Chrysomya albiceps (Wiedemann), Lucilia ampullacea Villeneuve, Lucilia caesar (Linnaeus), Lucilia sericata (Meigen) (Calliphoridae), Hydrotaea ignava (Harris), Muscina prolapsa (Harris), Synthesiomyia nudiseta (van der Wulp) (Muscidae), Piophila megastigmata McAlpine, Stearibia nigriceps (Meigen) (Piophilidae) and Nemopoda nitidula (Fallén) (Sepsidae) were revealed to be very important members of the Diptera community collected. The necrophagous behaviour, demonstrated by their immatures, using carrion as a food source makes them useful forensic indicator species. Also of relevance is the presence of Chrysomya megacephala (Fabricius), S. nudiseta and P. megastigmata, foreign species established in the local carrion communities. This study also marks the first record of S. nudiseta in Portugal.     

Fate of Surviving Low-birth-weight Infants with Coagulation Deficiencies on the First Day of Life

Fifty-two surviving low-birth-weight infants who had low Thrombotest (Owren) results on the first day, together with the same number of matched controls with higher Thrombotest results, were examined for the integrity of their central nervous system. Gross abnormalities were found in 13·5% of the low Thrombotest group compared with 1·9% in the higher group. Minor brain damage syndromes were more common in the low Thrombotest group. The combined brain damage syndromes were 23·1% in the low Thrombotest group compared with 3·8% in the higher group.It is suggested that the causes of the brain damage in the low Thrombotest group are either non-fatal cerebral haemorrhage or intravascular fibrin deposition associated with disseminated intravascular coagulation.     

Hydration effects on dynamics of polyglycine and sodium poly(L-glutamate).

Solid state nmr methods were applied to the study of the motions and structural heterogeneity in polyglycine, sodium poly(L-glutamate), and poly(L-alanine). The response of both the main-chain and side-chain resonances to the addition of water was studied using static and magic-angle sample-spinning line shapes as well as the carbon spin-lattice relaxation times, the proton spin-lattice relaxation time in the rotating frame, and the proton-carbon cross-polarization time. The polyglycine motions are not drastically affected by the addition of water when the polymer is in the 3(1)-helix or the beta-sheet structure. The sodium poly(L-glutamate), however, responds to increased hydration with little motion in the main-chain carbon atoms, but considerable flexibility of the side-chain atoms. The greatest motions are reported for the C5 carbon with rotational amplitudes about the C4-C5 bond of about of 50 degrees. In addition, motions somewhat less than half this size are required closer to the main chain.     

An Experimental study of reduced parental effort and future reproductive success in the puffin, Fratercula arctica

1. The puffin, a long-lived seabird, was studied on the Isle of May, East Scotland between 1990 and 1992. During two of these years, parental effort was experimentally decreased by supplementary feeding of young. This aimed to identify inter-year reproductive costs, and show whether they took the form of reduced adult survival, reduced fledging success and/or a reduction in the 'quality' of offspring in the following year.
2. The feeding treatment significantly reduced the daily number of feeds delivered by experimental parents by 67% in 1990 and 87% in 1991.
3. The proportions of experimental and control parents returning to the colony in the year following manipulation did not differ significantly, although in 1991, 2·5 times as many controls (young unfed) as experimental birds (young fed) failed to return.
4. The fledging success of experimental pairs in the year following manipulation (68%) was significantly higher than that of controls (24%).
5. Experimental pairs raised young with significantly higher body condition (Residual Peak Mass) than that of controls in the year following manipulation (1992).
6. Experimental parents did not differ from controls in their body condition (Lipid Reserve Mass) or rate of reserve depletion, either in the year of manipulation or in the following breeding season; hence there was no evidence for a role of the measured component of body condition in the cost mechanism.
7. The study demonstrated inter-year reproductive costs for puffins and supported the hypothesis that long-lived species reduce the 'quality' of their offspring or abandon a breeding attempt rather than compromise their survival and future opportunities to reproduce.     

Deletion of the terminal sequences from cytochromes c

A 12-residue helical segment was deleted from the amino terminus of human heart cytochrome c by cleavage with cyanogen bromide. The resultant hemepeptide (13-104)H is rather insoluble in aqueous solution at neutral pH. Analysis of the crystallographic model with the terminal segment removed as well as qualitative spectral analysis of a transiently soluble sample of (13-104)H suggests that the hemepeptide retains little of the conformation of the native protein. By contrast, hemepeptide (1-80)H obtained by deletion of the carboxyl terminus is quite soluble at neutral pH. Spectral and hydrodynamic measurements suggest that the heme is encapsulated in an apolar cluster stabilized in part by axial ligation of the heme iron.     

Observations and comments on the reliability of muscle reconstruction in fossil vertebrates

In Canis and Ursus the largest proportion of attachments of muscles of the shoulder and brachium on the scapula and humerus is direct; fewer attachments are aponeurotic or tendinous. In both genera most attachments can be associated with superficial osteological features (scars or delimitable surfaces); attachments that lack such features are direct. Most aponeurotic attachments are associated with rugose scarring whereas tendinous attachments are often associated with smooth surfaces. Although most attachments can be associated with osteological features the areal extent of attachment is often not inferrable from the bone. The inference of muscle size or functional significance from osteological features is problematic. The amount of myological information that can be deciphered from the osteology in Canis and Ursus is greater than that reported for particular members of other vertebrate groups which suggests that there may be differences in the degree to which muscles can be reconstructed from superficial osteology alone. Nonetheless, even in mammals such as the Carnivora, detailed muscular reconstructions in extinct taxa cannot be achieved without reference to the musculature of extant relatives. Such reconstructions rely on assumptions, that often have not been adequately tested, regarding the similarity of musculature in closely related taxa. This testing and well corroborated hypotheses of phylogenetic relationship are essential for the evaluation of the accuracy of reconstructions of the musculature in fossil vertebrates.