Inhibition of Farnesoic Acid Methyl transferase by Sinefungin

Sinefungin inhibited the S-adenosylmethionine-dependent farnesoic acid methyltransferase in a cell-free system containing a homogenate of corpora allata from female locusts, Locusta migratoria. The enzyme catalyzed the penultimate step of juvenile hormone biosynthesis in the insects. Culturing corpora allata in the presence of sinefungin greatly suppressed juvenile hormone production. The following in vivo effects were visible after injection of the inhibitor: increase in mortality and reduction of total haemolymph protein titer and ovary fresh weight, as well as length of terminal oocytes. Attempts to reverse these effects by topical application of the juvenile hormone analog ZR-515 (methoprene) were only partly successful. Therefore, the in vivo effects may be due to a general inhibition of methyltransferase enzymes in the insect. Sinefungin appeared to be of potential interest as the first representative of a new class of insect growth regulators.     

Observations below multiple lower limits of quantification: How to estimate the mean and variance

Multiple lower limits of quantification (MLOQs) result if various laboratories are involved in the analysis of concentration data and some observations are too low to be quantified. For normally distributed data under MLOQs there exists only the multiple regression method of Helsel to estimate the mean and variance. We propose a simple imputation method and two new maximum likelihood estimation methods: the multiple truncated sample method and the multiple censored sample method. A simulation study is conducted to compare the performances of the newly introduced methods to Helsel's via the criteria root mean squared error (RMSE) and bias of the parameter estimates. Two and four lower limits of quantification (LLOQs), various amounts of unquantifiable observations and two sample sizes are studied. Furthermore, the robustness is investigated under model misspecification. The methods perform with decreasing accuracy for increasing rates of unquantified observations. Increasing sample sizes lead to smaller bias. There is almost no change in the performance between two and four LLOQs. The magnitude of the variance impairs the performance of all methods. For a smaller variance, the multiple censored sample method leads to superior estimates regarding the RMSE and bias, whereas Helsel's method is superior regarding the bias for a larger variance. Under model misspecification, Helsel's method was inferior to the other methods. Estimating the mean, the multiple censored sample method performed better, whereas the multiple truncated sample method performs best in estimating the variance. Summarizing, for a large sample size and normally distributed data we recommend to use Helsel's method. Otherwise, the multiple censored sample method should be used to obtain estimates of the mean and variance of data including MLOQs.     

Der Einfluß der Umweltfaktoren auf das Wachstum und den Häutungsrhythmus der StrandkrabbeCarcinides maenas

Zusammenfassung 1. Junge Strandkrabben von 4–16 mm Carapaxbreite wurden bis zur Geschlechtsreife unter konstanten Umweltbedingungen aufgezogen.2. Die Dauer ihrer Häutungsintervalle nimmt bei konstanter Temperatur mit der Körpergröße stetig zu.3. Die Dauer der Häutungsintervalle hängt von der Temperatur und der Ernährung ab. Von der Tageslänge scheint sie weitgehend unabhängig zu sein.4. Der relative Grösßenzuwachs bei jeder Häutung ist im gesamten untersuchten Größenbereich und bei den verschiedenen Temperaturen bei allen Häutungen gleich: Bei den Häutungen verdoppelt sich jeweils das Körpervolumen.5. Augenstielamputationen und Verlust von Extremitäten wirken auf den Häutungsrhythmus in gleicher Weise: Die Schwankungsbreite in der Dauer der Häutungsintervalle ist vermindert. Die Häutungsintervalle sind in 20° C deutlich, in 10° C nur geringfügig verkürzt.6. Durch die Anwesenheit größerer Artgenossen werden die Häutungen verzögert. Die optische Wahrnehmung spielt dabei keine Rolle.7. Aus diesen Ergebnissen wird folgendes geschlossen: Der ausschlaggebende Faktor für die Auslösung von Häutungen ist ein bestimmter Größenzuwachs. Temperatur und Ernährung beeinflussen den Häutungsrhythmus dadurch, daß sie das Tempo des Wachstums bestimmen. Die winterliche Häutungsruhe in Freilandpopulationen wird nicht durch den Kurztag bedingt, sondern durch die Kälte. Diese hemmt lediglich das Wachstum, sie verhindert nicht die Häutungen über das häutungshemmende Hormon. Dieses vermindert vielmehr die Temperaturabhängigkeit des Häutungsrhythmus, indem es die Häutungen im Warmen stärker verzögert als im Kalten. Es gestattet die Anpassung des Häutungstermins an die individuelle Lage der Tiere. Es hemmt in Anwesenheit größerer Artgenossen die Häutung. Beim Verlust mehrerer Gliedmaßen wird seine Sekretion eingestellt, so daß die nächste Häutung vorzeitig erfolgt. Das häutungshemmende Hormon bedingt dementsprechend die große individuelle Variation in der Dauer der Häutungsintervalle.

---

The effect of environmental factors on growth and moulting rhythm in the shore crab,Carcinides maenas

Young crabs (carapace width 4 to 16 mm) were raised under controlled conditions in the laboratory. The time intervals between subsequent moults increase at all test temperatures with increasing body size. The length of intermoult periods varies with temperature and feeding. It is not affected by day length. Moulting takes place as soon as a certain increase in size is attained. In comparable size groups, the amount of this increase is identical in all test temperatures. Moreover, the relation of increase to initial size is constant over the whole size range investigated. The body volume doubles at each moult. Eyestalk amputations and loss of extremities have similar effects: They shorten the intermoult periods at 20° C considerably, but at 10° C they do so only slightly; furthermore, the amplitude of fluctuations is narrowed. The presence of large specimens tends to retard moulting in smaller ones; this response is independent of visual stimuli. The following assumptions are made: Low temperatures retard the moulting rhythm directly by slowing down growth. They are not acting via the moult inhibiting hormone. Loss of several extremities causes a stop of hormone delivery resulting in shortened intermoult periods. Recognition by touch of a larger specimen causes increased hormone delivery and thus retardation of the subsequent moulting process.

     

The concerted conformational changes during human rhinovirus 2 uncoating

Delivery of the rhinovirus genome into the cytoplasm involves a cooperative structural modification of the viral capsid. We have studied this phenomenon for human rhinovirus serotype 2 (HRV2). The structure of the empty capsid has been determined to a resolution of better than 15 A by cryo-electron microscopy, and the atomic structure of native HRV2 was used to examine conformational changes of the capsid. The two proteins around the 5-fold axes make an iris type of movement to open a 10 A diameter channel which allows the RNA genome to exit, and the N terminus of VP1 exits the capsid at the pseudo 3-fold axis. A remarkable modification occurs at the 2-fold axes where the N-terminal loop of VP2 bends inward, probably to detach the RNA.     

Use of trypsin for rapid and efficient purification of murine sarcoma and leukemia virus

A method is described for purification of MSV-MuLV from culture supernatant of chronically infected 78A₁ rat embryo cell line. This method involves direct polyethylene glycol-NaCl precipitation of the low speed supernatant of culture fluid followed by digestion of the pellet with trypsin. This procedure efficiently disrupts large aggregates which normally entrap most of the virus. Highly purified virus can be obtained in very good yield by a combination of sedimentation velocity and isopycnic centrifugation : yields up to 100 A₂₈₀ units (17 mg of protein) of purified virus per liter of culture fluid can be observed. This procedure appears well suited for large scale isolation of virion associated enzymatic activities.     

Mnl2, a novel component of the ER associated protein degradation pathway

In eukaryotes, membrane and soluble proteins of the secretory pathway enter the endoplasmic reticulum (ER) after synthesis in an unfolded state. Directly after entry, most proteins are modified with glycans at suitable glycosylation sites and start to fold. A protein that cannot fold properly will be degraded in a process called ER associated degradation (ERAD). Failures in ERAD, either by loss of function or by premature degradation of proteins, are a cause of severe diseases. Therefore, the search for novel ERAD components to gain better insight in this process is of high importance. Carbohydrate trimming is a relevant process in ER quality control. In this work a novel putative yeast mannosidase encoded by the open reading frame YLR057W was identified and named Mnl2. Deletion of MNL2 diminished the degradation efficiency of misfolded CPY^* in the absence of the cognate mannosidase Mnl1, indicating a specific role in ERAD.     

Markers of enhanced cholesterol absorption are a strong predictor for cardiovascular diseases in patients without diabetes mellitus

Objective

Hypercholesterolemia is a major risk factor for cardiovascular disease (CVD), and diabetes mellitus and statin treatment affect cholesterol metabolism. The aim of the present study was to evaluate markers of cholesterol metabolism and determine their relationship with CVD in patients without diabetes mellitus who were not receiving statin treatment.

Methods

In addition to conventional CVD risk factors, plasma levels of campesterol and sitosterol (indicators of cholesterol absorption) and lathosterol (an indicator of cholesterol synthesis) were determined in 835 consecutive patients referred for coronary angiography. Coronary artery disease was evaluated by coronary angiograms, carotid atherosclerosis and peripheral vascular disease were assessed by Doppler ultrasound, and cerebrovascular accidents and transient ischemic attacks were identified by medical history.

Results

After excluding patients with known diabetes mellitus and those receiving statin treatment, 177 patients were included in the analysis. Compared to patients without CVDs (n = 111), patients with concomitant CVDs (n = 66) had a reduced lathosterol-to-cholesterol ratio (1.25 ± 0.61 vs. 1.38 ± 0.63, P < 0.05) and an increased campesterol-to-cholesterol ratio (1.81 ± 1.04 vs. 1.50 ± 0.69, P < 0.05), indicating that enhanced absorption and reduced synthesis of cholesterol is associated with CVD development. Logistic regression analysis including all established cardiovascular risk factors (age, sex, total cholesterol, arterial hypertension, body mass index and smoking) revealed that campesterol and the campesterol-to-cholesterol ratio were significant predictors of concomitant CVD in this patient population.

Conclusion

In patients without diabetes mellitus, markers of enhanced cholesterol absorption were a strong predictor for concomitant CVD.     

Sexual signalling in female crested macaques and the evolution of primate fertility signals

Background

Host-parasite coevolution can lead to local adaptation of either parasite or host if there is specificity (GxG interactions) and asymmetric evolutionary potential between host and parasite. This has been demonstrated both experimentally and in field studies, but a substantial proportion of studies fail to detect such clear-cut patterns. One explanation for this is that adaptation can be masked by counter-adaptation by the antagonist. Additionally, genetic architecture underlying the interaction is often highly complex thus preventing specific adaptive responses. Here, we have employed a reciprocal cross-infection experiment to unravel the adaptive responses of two components of fitness affecting both parties with different complexities of the underlying genetic architecture (i.e. mortality and spore load). Furthermore, our experimental coevolution of hosts (Tribolium castaneum) and parasites (Nosema whitei) included paired replicates of naive hosts from identical genetic backgrounds to allow separation between host- and parasite-specific responses.

Results

In hosts, coevolution led to higher resistance and altered resistance profiles compared to paired control lines. Host genotype × parasite genotype interactions (G_H × G_P) were observed for spore load (the trait of lower genetic complexity), but not for mortality. Overall parasite performance correlated with resistance of its matching host coevolution background reflecting a directional and unspecific response to strength of selection during coevolution. Despite high selective pressures exerted by the obligatory killing parasite, and host- and parasite-specific mortality profiles, no general pattern of local adaptation was observed, but one case of parasite maladaptation was consistently observed on both coevolved and control host populations. In addition, the use of replicate control host populations in the assay revealed one case of host maladaptation and one case of parasite adaptation that was masked by host counter-adaptation, suggesting the presence of complex and probably dynamically changing fitness landscapes.

Conclusions

Our results demonstrate that the use of replicate naive populations can be a useful tool to differentiate between host and parasite adaptation in complex and dynamic fitness landscapes. The absence of clear local adaptation patterns during coevolution with a sexual host showing a complex genetic architecture for resistance suggests that directional selection for generality may be more important attributes of host-parasite coevolution than commonly assumed.