Phylogeny and adaptive radiation within the Anomopoda: a preliminary exploration

The distinctness of the Anomopoda and the polyphyletic nature of the so-called Cladocera are emphasized.An attempt is made to reconstruct the ancestral anomopod, which probably lived in Palaeozoic times. This task is facilitated by the availability of detailed information on extant forms, which includes functional as well as purely morphological considerations and enables us to understand the means whereby complex mechanisms were transformed during evolution. Comparative studies on the ecology and habits of extant forms also throw light on the probable way of life of the ancestral anomopod.Adaptive radiation within the Anomopoda is briefly surveyed and an outline of the suggested phylogeny of the order is indicated.Institute of Environmental and Biological Sciences, University of Lancaster     

Catalytic components of proteasomes and the regulation of proteinase activity

The proteasome (multicatalytic proteinase complex) is a large multimeric complex which is found in the nucleus and cytoplasm of eukaryotic cells. It plays a major role in both ubiquitin-dependent and ubiquitin-independent nonlysosomal pathways of protein degradation. Proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the and subunits of the simpler proteasome isolated fromThermoplasma acidophilum. Proteasomes have a cylindrical structure composed of four rings of seven subunits. The 26S form of the proteasome, which is responsible for ubiquitin-dependent proteolysis, contains additional regulatory complexes. Eukaryotic proteasomes have multiple catalytic activities which are catalysed at distinct sites. Since proteasomes are unrelated to other known proteases, there are no clues as to which are the catalytic components from sequence alignments. It has been assumed from studies with yeast mutants that -type subunits play a catalytic role. Using a radiolabelled peptidyl chloromethane inhibitor of rat liver proteasomes we have directly identified RC7 as a catalytic component. Interestingly, mutants in Prel, the yeast homologue of RC7, have already been reported to have defective chymotrypsin-like activity. These results taken together confirm a direct catalytic role for these -type subunits. Proteasome activities are sensitive to conformational changes and there are several ways in which proteasome function may be modulatedin vivo. Our recent studies have shown that in animal cells at least two proteasome subunits can undergo phosphorylation, the level of which is likely to be important for determining proteasome localization, activity or ability to form larger complexes. In addition, we have isolated two isoforms of the 26S proteinase.     

Effect of inhA and katG on isoniazid resistance and virulence of Mycobacterium bovis

Isoniazid (INH) resistance of the Mycobacterium tuberculosis Complex (MtbC) is associated with both loss of catalase activity and mutation of the inhA gene. However, the relative contributions of these changes to resistance and to the loss of virulence for guinea-pigs is unknown. In this study, a virulent strain of Mycobacterium bovis, a member of the MtbC., was exposed to increasing concentrations of INH. Two INH-resistant strains were produced which had lost catalase activity. Strain WAg405, which had a higher resistance to INH, also had a mutation in the inhA gene. This demonstrated that loss of catalase activity and mutation of inhA had a cumulative effect on INH resistance. When a functional katG gene was integrated into the genome of WAg405 the INH resistance was greatly reduced. This indicated that most of the resistance had been caused by loss of catalase activity. While the parent INH-sensitive strain was virulent for guinea-pigs, the INH-resistant strains were significantly less virulent. Integration of a functional katG gene into the most resistant strain restored full virulence. This clearly established that katG is a virulence factor for M. bovis and that mutation of the inhA gene has no effect on virulence.     

Molecular evolution of plant β-glucan endohydrolases

The evolutionary relationships of two classes of plant β-glucan endohydrolases have been examined by comparison of their substrate specificities, their three-dimensional conformations and the structural features of their corresponding genes. These comparative studies provide compelling evidence that the (1→3)-β-glucanases and (1→3,1→4)-β-glucanases from higher plants share a common ancestry and, in all likelihood, that the (1→3,1→4)-β-glucanases diverged from the (1→3)-β-glucanases during the appearance of the graminaceous monocotyledons. The evolution of (1→3,1→4)-β-glucanases from (1→3)-β-glucanases does not appear to have invoked ‘modular’ mechanisms of change, such as those caused by exon shuffling or recombination. Instead, the shift in specificity has been acquired through a limited number of point mutations that have resulted in amino acid substitutions along the substrate-binding cleft. This is consistent with current theories that the evolution of new enzymic activity is often achieved through duplication of the gene encoding an existing enzyme which is capable of performing the required chemistry, in this case the hydrolysis of a glycosidic linkage, followed by the mutational alteration and fine-tuning of substrate specificity. The evolution of a new specificity has enabled a dramatic shift in the functional capabilities of the enzymes. (1→3)-β-Glucanases that play a major role, inter alia, in the protection of the plant against pathogenic microorganisms through their ability to hydrolyse the (1→3)-β-glucans of fungal cell walls, appear to have been recruited to generate (1→3,1→4)-β-glucanases, which quite specifically hydrolyse plant cell wall (1→3,1→4)-β-glucans in the graminaecous monocotyledons during normal wall metabolism. Thus, one class of β-glucan endohydrolase can degrade β-glucans in fungal walls, while the other hydrolyses structurally distinct β-glucans of plant cell walls. Detailed information on the three-dimensional structures of the enzymes and the identification of catalytic amino acids now present opportunities to explore the precise molecular and atomic details of substrate-binding, catalytic mechanisms and the sequence of molecular events that resulted in the evolution of the substrate specificities of the two classes of enzyme.     

Characterisation of ionisations that influence the redox potential of mitochondrial cytochrome c and photosynthetic bacterial cytochromes c2

Several cytochromes c₂ from the Rhodospirillaceae show a pH dependence of redox potential in the physiological pH range which can be described by equations involving an ionisation in the oxidised form (pK_o) and one in the reduced form (pK_r). These cytochromes fall into one of two groups according to the degree of separation of pK_o and pK_r. In group A, represented here by the Rhodomicrobium vannielii cytochrome c₂, the separation is approx. one pH unit and the ionisation is that of a haem propionic acid. Members of this group are unique among both cytochromes c₂ and mitochondrial cytochromes c in lacking the conserved residue Arg-38. We propose that the role of Arg-38 is to lower the pK of the nearby propionic acid, so that it lies out of the physiological pH range. Substitution of this residue by an uncharged amino acid leads to a raised pK for the propionic acid. In group B, represented here by Rhodopseudomonas viridis cytochrome c₂, the separation between pK_o and pK_r is approx. 0.4 pH unit and the ionisable group is a histidine at position 39. This was established by NMR spectroscopy and confirmed by chemical modification. Only a few other members of the cytochrome c₂/mitochondrial cytochrome c family have a histidine at this position and of these, both Crithidia cytochrome c-557 and yeast cytochrome c were found to have a pH-dependent redox potential similar to that of Rps. viridis cytochrome c₂. Using Coulomb's law, it was found that the energy required to separate pK_o and pK_r could be accounted for by simple electrostatic interactions between the haem iron and the ionisable group.     

Studies in cryoprotection II: Propylene glycol and glycerol

Propylene glycol (PG), glycerol (G), or a combination of both were introduced into and eluted from 116 rabbit kidneys. The function of the kidneys was then studied on an ex vivo shunt by determining bloodflow and creatinine clearance. The cryoprotective agents (CPAs) were introduced and eluted at the rate of about 30 mM/min in an albumin-Ringer's lactate perfusate. In this system, 1.75 to 2.25 M cryoprotectant could be introduced and removed with no demonstrable loss of function. Significant injury was incurred at and above 2.5 M concentrations. When a combination of PG and G (equal proportions) was utilized, both 2.5 and 2.75 M total concentrations were tolerated by the kidney, with no loss of function. These studies demonstrate that in this very rigorous test system, cryoprotectant-induced injury can be significantly diminished by combining two agents of low toxicity. This suggests that osmotic injury may be enhanced by specific toxicities of cryoprotective agents, which can be minimized by using combinations, such as was done in these studies. Also, it has been shown that PG is at least as low in toxicity in the rabbit kidney as is G.     

Positive cooperativity in the adherence between elementary bodies of Chlamydia trachomatis strain UW-31 and HeLa cells

Abstract The adherence of purified elementary bodies of Chlamydia trachomatis strain UW-31 to monolayer cultures of HeLa 229 cells exhibited kinetic evidence of positive cooperativity. An abrupt increase in the rate of adherence occurred as chlamydial dose was increased. Only freshly isolated chlamydiae showed this behavior. In the presence of the lectin wheat germ agglutinin, the stimulated adherence showed typical Michaelis-Menten kinetics. The results suggest that chlamydiae may promote their own binding to the host-cell surface, and the lectin, when cell-bound, may provide additional chlamydiae-binding sites.