Selecting representative model micro-organisms

Background  

Micro-biological research relies on the use of model organisms that act as representatives of their species or subspecies, these are frequently well-characterized laboratory strains. However, it has often become apparent that the model strain initially chosen does not represent important features of the species. For micro-organisms, the diversity of their genomes is such that even the best possible choice of initial strain for sequencing may not assure that the genome obtained adequately represents the species. To acquire information about a species' genome as efficiently as possible, we require a method to choose strains for analysis on the basis of how well they represent the species.     

High-affinity [3H]inositol uptake by dissociated brain cells and cultured fibroblasts from fetal mice

The accumulation of [³H]inositol by mechanically dissociated brain cells and cultured skin fibroblasts from fetal mice was examined. Uptake by both tissues was strongly dependent on temperature and the presence of sodium ions. Brain and fibroblast uptake also responded similarly to inhibition by inositol isomers and phloridzin. At lower concentrations of inositol, both tissues exhibited high-affinity uptake kinetics with apparent K_m values near 30 M, similar to values observed previously in human fibroblasts and other cultured cells. The activity of brain high-affinity uptake was nearly an order of magnitude lower than that of fibroblasts, however, and was in part confounded by the presence of a low-affinity or simple diffusion system operating at inositol concentrations above 100M. Brain preparation from adult mice also showed evidence of high-affinity, Na⁺ dependent uptake, but its activity was significantly diminished relative to that of fetal brain preparations. Our results demonstrate that a high-affinity inositol transport system closely resembling that found in cultured cells is expressed in the developing mouse brain.     

Modelling expected trout ranges under current and future water temperature regimes in the Eastern Cape,South Africa

Different values have resulted in conflicts between anglers and conservation lobbies in the management of trout in South Africa. Key to the conflict is the demarcation of boundaries to areas in which brown trout Salmo trutta and rainbow trout Oncorhynchus mykiss currently occur, or are likely to establish following stocking for angling. To provide a longer-term perspective on these areas, we developed models to link salmonid biological thermal thresholds to elevation. These, when applied spatially using a digital elevation model with a probability of occurrence model, provided the basis for estimating potentially available thermal habitat for these two cold water species. Here, we acknowledge that other variables (stocking history; river connectivity) also play a role in understanding trout distributions. Using a simple scenario of an increase in mean daily water temperatures of 2 °C, we demonstrated that both brown and rainbow trout are likely to exhibit considerable range reductions in the future. Because it is possible that these range restrictions will result in an increasing desire to introduce trout into areas above their current distribution limits for the maintenance of angling opportunities, conservation managers should prioritise these areas, with management interventions seeking to understand what will help to limit introductions.     

Predicted warming and browning affect timing and magnitude of plankton phenological events in lakes: a mesocosm study

1. Aquatic ecosystems in Northern Europe are expected to face increases in temperature and water colour (TB) in future. While effects of these factors have been studied separately, it is unknown whether and how a combination of them might affect phenological events and trophic interactions. 2. In a mesocosm study, we combined both factors to create conditions expected to arise during the coming century. We focused on quantifying effects on timing and magnitude of plankton spring phenological events and identifying possible mismatches between resources (phytoplankton) and consumers (zooplankton). 3. We found that the increases in TB had important effects on timing and abundance of different plankton groups. While increased temperature led to an earlier peak in phytoplankton and zooplankton and a change in the relative timing of different zooplankton groups, increased water colour reduced chlorophyll‐a concentrations. 4. Increased TB together benefitted cladocerans and calanoid copepods and led to stronger top‐down control of algae by zooplankton. There was no sign of a mismatch between primary producers and grazers as reported from other studies. 5. Our results point towards an earlier onset of plankton spring growth in shallow lakes in future with a stronger top‐down control of phytoplankton by zooplankton grazers.     

Mechanisms of P(i) regulation of the skeletal muscle SR Ca(2+) release channel

Inorganic phosphate(P_i) accumulates in the fibers of actively working musclewhere it acts at various sites to modulate contraction. To characterizethe role of P_i as a regulator of the sarcoplasmic reticulum(SR) calcium (Ca²⁺) release channel, we examined the actionof P_i on purified SR Ca²⁺ release channels,isolated SR vesicles, and skinned skeletal muscle fibers. In singlechannel studies, addition of P_i to the cis chamberincreased single channel open probability (P_o;0.079 ± 0.020 in 0 P_i, 0.157 ± 0.034 in 20 mMP_i) by decreasing mean channel closed time; mean channelopen times were unaffected. In contrast, the ATP analog,,-methyleneadenosine 5'-triphosphate (AMP-PCP), enhancedP_o by increasing single channel open time anddecreasing channel closed time. P_i stimulation of[³H]ryanodine binding by SR vesicles wassimilar at all concentrations of AMP-PCP, suggesting P_i andadenine nucleotides act via independent sites. In skinned musclefibers, 40 mM P_i enhanced Ca²⁺-inducedCa²⁺ release, suggesting an in situ stimulation ofthe release channel by high concentrations of P_i. Ourresults support the hypothesis that P_i may be an importantendogenous modulator of the skeletal muscle SR Ca²⁺ releasechannel under fatiguing conditions in vivo, acting via a mechanismdistinct from adenine nucleotides.

     

Reduced threshold for luminal Ca2+ activation of RyR1 underlies a causal mechanism of porcine malignant hyperthermia

Naturally occurring mutations in the skeletal muscle Ca(2+) release channel/ryanodine receptor RyR1 are linked to malignant hyperthermia (MH), a life-threatening complication of general anesthesia. Although it has long been recognized that MH results from uncontrolled or spontaneous Ca(2+) release from the sarcoplasmic reticulum, how MH RyR1 mutations render the sarcoplasmic reticulum susceptible to volatile anesthetic-induced spontaneous Ca(2+) release is unclear. Here we investigated the impact of the porcine MH mutation, R615C, the human equivalent of which also causes MH, on the intrinsic properties of the RyR1 channel and the propensity for spontaneous Ca(2+) release during store Ca(2+) overload, a process we refer to as store overload-induced Ca(2+) release (SOICR). Single channel analyses revealed that the R615C mutation markedly enhanced the luminal Ca(2+) activation of RyR1. Moreover, HEK293 cells expressing the R615C mutant displayed a reduced threshold for SOICR compared with cells expressing wild type RyR1. Furthermore, the MH-triggering agent, halothane, potentiated the response of RyR1 to luminal Ca(2+) and SOICR. Conversely, dantrolene, an effective treatment for MH, suppressed SOICR in HEK293 cells expressing the R615C mutant, but not in cells expressing an RyR2 mutant. These data suggest that the R615C mutation confers MH susceptibility by reducing the threshold for luminal Ca(2+) activation and SOICR, whereas volatile anesthetics trigger MH by further reducing the threshold, and dantrolene suppresses MH by increasing the SOICR threshold. Together, our data support a view in which altered luminal Ca(2+) regulation of RyR1 represents a primary causal mechanism of MH.     

Expression of polypeptide GalNAc-transferases in stratified epithelia and squamous cell carcinomas: immunohistological evaluation using monoclonal antibodies to three members of the GalNAc-transferase family

Mucin-type O-glycosylation is initiated by a large family of UDP- GalNAc: polypeptide N -acetyl-galactosaminyltransferases (GalNAc- transferases). Individual GalNAc-transferases appear to have different functions and Northern analysis indicates that they are differently expressed in different organs. This suggests that O-glycosylation may vary with the repertoire of GalNAc-transferases expressed in a given cell. In order to study the repertoire of GalNAc-transferases in situ in tissues and changes in tumors, we have generated a panel of monoclonal antibodies (MAbs) with well defined specificity for human GalNAc-T1, -T2, and -T3. Application of this panel of novel antibodies revealed that GalNAc- transferases are differentially expressed in different cell lines, in spermatozoa, and in oral mucosa and carcinomas. For example, GalNAc-T1 and -T2 but not -T3 were highly expressed in WI38 cells, and GalNAc-T3 but not GalNAc-T1 or -T2 was expressed in spermatozoa. The expression patterns in normal oral mucosa were found to vary with cell differentiation, and for GalNAc-T2 and -T3 this was reflected in oral squamous cell carcinomas. The expression pattern of GalNAc-T1 was on the other hand changed in tumors to either total loss or expression in cytological poorly differentiated tumor cells, where the normal undifferentiated cells lacked expression. These results demonstrate that the repertoire of GalNAc-transferases is different in different cell types and vary with cellular differentiation, and malignant transformation. The implication of this is not yet fully understood, but it suggests that specific changes in sites of O-glycosylation of proteins may occur as a result of changes in the repertoire of GalNAc-transferases.      

Calmodulin oxidation and methionine to glutamine substitutions reveal methionine residues critical for functional interaction with ryanodine receptor-1

Calmodulin (CaM) binds to the skeletal muscle ryanodine receptor Ca(2+) release channel (RyR1) with high affinity, and it may act as a Ca(2+)-sensing subunit of the channel. Apo-CaM increases RyR1 channel activity, but Ca(2+)-CaM is inhibitory. Here we examine the functional effects of CaM oxidation on RyR1 regulation by both apo-CaM and Ca(2+)-CaM, as assessed via determinations of [(3)H]ryanodine and [(35)S]CaM binding to skeletal muscle sarcoplasmic reticulum vesicles. Oxidation of all nine CaM Met residues abolished functional interactions of CaM with RyR1. Incomplete CaM oxidation, affecting 5-8 Met residues, increased the CaM concentration required to modulate RyR1, having a greater effect on the apo-CaM species. Mutating individual CaM Met residues to Gln demonstrated that Met-109 was required for apo-CaM activation of RyR1 but not for Ca(2+)-CaM inhibition of the channel. Furthermore, substitution of Gln for Met-124 increased the apo- and Ca(2+)-CaM concentrations required to regulate RyR1. These results thus identify Met residues critical for the productive association of CaM with RyR1 channels and suggest that oxidation of CaM may contribute to altered regulation of sarcoplasmic reticulum Ca(2+) release during oxidative stress.     

Effects of brown and turbid water on piscivore–prey fish interactions along a visibility gradient

1. Environmental changes such as eutrophication and increasing inputs of humic matter (brownification) may have strong effects on predator–prey interactions in lakes through a reduction in the visual conditions affecting foraging behaviour of visually oriented predators. 2. In this experiment, we studied the effects of visual range (25–200 cm) in combination with optically deteriorating treatments (algae, clay or brown humic water) on predator–prey interactions between pike (Esox lucius) and roach (Rutilus rutilus). We measured effects on reaction distance and strike distance for pike and escape distance for roach, when pike individuals were exposed to free‐swimming roach as well as to roach held in a glass cylinder. 3. We found that reaction distance decreased with decreasing visual range caused by increasing levels of algae, clay or humic matter. The effect of reaction distance was stronger in turbid water (clay, algae) than in the brown water treatment. 4. Strike distance was neither affected by visual range nor by optical treatment, but we found shorter strike distances when pike attacked roach using visual cues only (roach held in a cylinder) compared to when pike could use multiple senses (free‐swimming roach). Escape distance for roach was longer in turbid than in brown water treatments. 5. Changes in environmental drivers, such as eutrophication and brownification, affecting the optical climate should thus have consequences for the strength of predator–prey interactions through changes in piscivore foraging efficiency and prey escape behaviour. This in turn may affect lake ecosystems through higher‐order interactions.     

Regulation of the RYR1 and RYR2 Ca2+ release channel isoforms by Ca2+-insensitive mutants of calmodulin

Calmodulin (CaM) may function as a regulatory subunit of ryanodine receptor (RYR) channels, modulating both channel activation and inhibition by Ca2+; however, mechanisms underlying differences in CaM regulation of the RYR isoforms expressed in skeletal muscle (RYR1) and cardiac muscle (RYR2) are poorly understood. Here we use a series of CaM mutants deficient in Ca2+ binding to compare determinants of CaM regulation of the RYR1 and RYR2 isoforms. In submicromolar Ca2+, activation of the RYR1 isoform by each of the single-point CaM mutants was similar to that by wild-type apoCaM, whereas in micromolar Ca2+, RYR1 inhibition by Ca2+CaM was abolished by mutations targeting CaM's C-terminal Ca2+ sites. In contrast to the RYR1, no activation of the cardiac RYR2 isoform by wild-type CaM was observed, but rather CaM inhibited the RYR2 at all Ca2+ concentrations (100 nM to 1 mM). Consequently, whereas the apparent Ca2+ sensitivity of the RYR1 isoform was enhanced in the presence of CaM, the RYR2 displayed the opposite response (RYR2 Ca2+ EC50 increased 7-10-fold in the presence of 5 microM wild-type CaM). CaM inhibition of the RYR2 was nonetheless abolished by each of four mutations targeting individual CaM Ca2+ sites. Furthermore, a mutant CaM deficient in Ca2+ binding at all four Ca2+ sites significantly activated the RYR2 and acted as a competitive inhibitor of RYR2 regulation by wild-type Ca2+CaM. We conclude that Ca2+ binding to CaM determines the effect of CaM on both RYR1 and RYR2 channels and that isoform differences in CaM regulation reflect the differential tuning of Ca2+ binding sites on CaM when bound to the different RYRs. These results thus suggest a novel mechanism by which CaM may contribute to functional diversity among the RYR isoforms.