Konstantin Beier

Coevolution of male and female reproductive traits in a simultaneously hermaphroditic land snail

Inter‐ and intraspecific studies in gonochoristic animals reveal a covariation between sperm characteristics and the size of the female reproductive tract, indicating a rapid evolutionary divergence, which is consistent with the theory of post‐copulatory sexual selection. Simultaneous hermaphrodites differ from species with separate sexes (gonochorists) in that they possess both functional male and female reproductive organs at the same time. We investigated whether in hermaphroditic animals intraspecific variation in reproductive traits results from divergent coevolution, by quantifying the variation in male and female traits among six natural populations of the snail Arianta arbustorum and examining the covariation in interacting traits. There was a significant among‐population variation in spermatophore volume, number of sperm transferred and sperm length, as well as in volume of the sperm storage organ (spermatheca) and number of tubules, but not in spermatheca length. We found a positive association between sperm number transferred and spermatheca volume. This result suggests that the same post‐copulatory mechanisms as in gonochorists drive the correlated evolution of reproductive characters in hermaphrodites.

Toxicity of Valproic Acid in Mice with Decreased Plasma and Tissue Carnitine Stores

The aim of this study was to investigate whether a decrease in carnitine body stores is a risk factor for valproic acid (VPA)-associated hepatotoxicity and to explore the effects of VPA on carnitine homeostasis in mice with decreased carnitine body stores. Therefore, heterozygous juvenile visceral steatosis (jvs)+/– mice, an animal model with decreased carnitine stores caused by impaired renal reabsorption of carnitine, and the corresponding wild-type mice were treated with subtoxic oral doses of VPA (0.1 g/g b.wt./day) for 2 weeks. In jvs+/– mice, but not in wild-type mice, treatment with VPA was associated with the increased plasma activity of aspartate aminotransferase and alkaline phosphatase. Furthermore, jvs+/– mice revealed reduced palmitate metabolism assessed in vivo and microvesicular steatosis of the liver. The creatine kinase activity was not affected by treatment with VPA. In liver mitochondria isolated from mice that were treated with VPA, oxidative metabolism of l-glutamate, succinate, and palmitate, as well as β-oxidation of palmitate, were decreased compared to vehicle-treated wild-type mice or jvs+/– mice. In comparison to vehicle-treated wild-type mice, vehicle-treated jvs+/– mice had decreased carnitine plasma and tissue levels. Treatment with VPA was associated with an additional decrease in carnitine plasma (wild-type mice and jvs+/– mice) and tissue levels (jvs+/– mice) and a shift of the carnitine pools toward short-chain acylcarnitines. We conclude that jvs+/– mice reveal a more accentuated hepatic toxicity by VPA than the corresponding wild-type mice. Therefore, decreased carnitine body stores can be regarded as a risk factor for hepatotoxicity associated with VPA.

Postnatal Schwann cell proliferation but not myelination is strictly and uniquely dependent on cyclin-dependent kinase 4 (cdk4)

Peripheral myelin formation depends on axonal signals that tightly control proliferation and differentiation of the associated Schwann cells. Here we demonstrate that the molecular program controlling proliferation of Schwann cells switches at birth. We have analyzed the requirements for three members of the cyclin-dependent kinase (cdk) family in Schwann cells using cdk-deficient mice. Mice lacking cdk4 showed a drastic decrease in the proliferation rate of Schwann cells at postnatal days 2 and 5, but proliferation was unaffected at embryonic day 18. In contrast, ablation of cdk2 and cdk6 had no significant influence on postnatal Schwann cell proliferation. Taken together, these findings indicate that postnatal Schwann cell proliferation is uniquely controlled by cdk4. Despite the lack of the postnatal wave of Schwann cell proliferation, axons were normally myelinated in adult cdk4-deficient sciatic nerves. Following nerve injury, Schwann cells lacking cdk4 were unable to re-enter the cell cycle, while Schwann cells deficient in cdk2 or cdk6 displayed proliferation rates comparable to controls. We did not observe compensatory effects such as elevated cdk4 levels in uninjured or injured nerves of cdk2 or cdk6-deficient mice. Our data demonstrate that prenatal and postnatal Schwann cell proliferation are driven by distinct molecular cues, and that postnatal proliferation is not a prerequisite for the generation of Schwann cell numbers adequate for correct myelination.

Identification Of A Synthetic Peptide Inducing Cross-reactive Antibodies Binding To Rhipicephalus (boophilus) Decoloratus, Rhipicephalus (boophilus) Microplus, Hyalomma Anatolicum Anatolicum And Rhipicephalus Appendiculatus Bm86 Homologues

The BM86 antigen, originally identified in Rhipicephalus (Boophilus) microplus, is the basis of the only commercialized anti-tick vaccine. The long-term goal of our study is to improve BM86 based vaccines by induction of high levels of tick gut binding antibodies that are also cross-reactive with a range of BM86 homologues expressed in other important tick species. Here we have used a BD86 derived synthetic peptide, BD86-3, to raise a series of mouse monoclonal antibodies. One of these mAbs, named 12.1, recognized BM86 homologues in immuno-histochemical analyses in four out of five tick species including R. (B.) microplus, Rhipicephalus (Boophilus) decoloratus, Hyalomma anatolicum anatolicum and Rhipicephalus appendiculatus. Our results indicate that broadly cross-reactive tick gut binding antibodies can be induced after immunization with a synthetic peptide derived from the protein BD86.

Effect of carnitine deprivation on carnitine homeostasis and energy metabolism in mice with systemic carnitine deficiency

Background/Aims: Juvenile visceral steatosis (jvs–/–) mice lack the activity of the carnitine transporter OCTN2 and are dependent on carnitine substitution. The effects of carnitine deprivation on carnitine homeostasis and energy metabolism are not known in jvs–/– mice. Methods: jvs–/– mice were studied 3, 6 and 10 days after carnitine deprivation, and compared to jvs–/– mice substituted with carnitine, wild-type (jvs+/+) and jvs+/– mice. Carnitine concentrations were assessed radioenzymatically. Results: Compared to wild-type mice, carnitine-treated jvs–/– mice had decreased plasma β-hydroxybutyrate levels and showed hepatic fat accumulation. The carnitine levels in plasma, liver and skeletal muscle were decreased by 58, 16 and 17%, respectively. After ten days of carnitine deprivation, the plasma carnitine concentration had fallen by 87% (to 2.3 µmol/l) and the tissue carnitine levels by ≈50% compared to carnitine-treated jvs–/– mice. Carnitine deprivation was associated with a further drop in plasma β-hydroxybutyrate and increased hepatic fat. Skeletal muscle glycogen stores decreased and lactate levels increased with carnitine deprivation, whereas tissue ATP levels were maintained. Conclusions: In jvs–/– mice, tissue carnitine stores are more resistant than carnitine plasma concentrations to carnitine deprivation. Metabolic changes (liver steatosis and loss of muscle glycogen stores) appear also early after carnitine deprivation.

Losartan but not enalaprilat acutely reduces reperfusion ventricular tachyarrhythmias in hypertrophied rat hearts after low‐flow ischaemia

Based on clinical and experimental studies, angiotensin II receptor blockers and angiotensin converting enzyme inhibitors have been proposed to exert acute anti‐arrhythmic effects in heart failure patients. Therefore, the goal of this study was to assess acute anti‐arrhythmic effects of losartan and enalaprilat in hypertrophied rat hearts during low‐flow ischaemia and reperfusion. In dose‐finding experiments in non‐hypertrophied isolated perfused hearts, we performed dose‐response curves of losartan and enalaprilat studying monophasic action potential duration at 90% repolarisation (MAPD90%) and ventricular fibrillation (VF) threshold. Subsequently, we determined the effects of losartan and enalaprilat (in therapeutically relevant concentrations) on ventricular tachyarrhythmias induced by low‐flow ischaemia/reperfusion in hearts demonstrating left ventricular (LV) hypertrophy 70 days after aortic banding. We found that neither drug significantly affected MAPD90% (1 nm‐1 mm) or VF threshold (1 μm losartan and 10 μm enalaprilat) in non‐hypertrophied hearts. Similarly in hypertrophied hearts, neither drug significantly affected the incidence or the duration of ventricular tachyarrhythmias (ventricular tachycardia and VF) during low‐flow ischaemia. However, 1 μm losartan significantly reduced the duration of ventricular tachyarrhythmias during reperfusion. In conclusion, neither losartan nor enalaprilat is acutely anti‐arrhythmic in hypertrophied rat hearts during low‐flow ischaemia. During reperfusion, however, losartan but not enalaprilat exerts acute anti‐arrhythmic effects.

Effects of gasoline and ethanol-gasoline exhaust exposure on human bronchial epithelial and natural killer cells in vitro

Air pollution exposure, including passenger car emissions, may cause substantial respiratory health effects and cancer death. In western countries, the majority of passenger cars are driven by gasoline fuel. Recently, new motor technologies and ethanol fuels have been introduced to the market, but potential health effects have not been thoroughly investigated. We developed and verified a coculture model composed of bronchial epithelial cells (ECs) and natural killer cells (NKs) mimicking the human airways to compare toxic effects between pure gasoline (E0) and ethanol-gasoline-blend (E85, 85% ethanol, 15% gasoline) exhaust emitted from a flexfuel gasoline car. We drove a steady state cycle, exposed ECs for 6h and added NKs. We assessed exhaust effects in ECs alone and in cocultures by RT-PCR, flow cytometry, and oxidative stress assay. We found no toxic effects after exposure to E0 or E85 compared to air controls. Comparison between E0 and E85 exposure showed a weak association for less oxidative DNA damage after E85 exposure compared to E0. Our results indicate that short-term exposure to gasoline exhaust may have no major toxic effects in ECs and NKs and that ethanol as part of fuel for gasoline cars may be favorable.

An immunolabelling technique to track sperm from different mates in the female reproductive organs of terrestrial gastropods

ABSTRACT The mechanisms of sperm transfer, storage, utilization and digestion are crucial for understanding processes of postcopulatory sexual selection. Previous studies analysing postcopulatory processes have generally focused only on the ultimate outcome of the interactions between male and female sexual selection (paternity patterns). For a mechanistic understanding of the fate of received sperm and the involved patterns of postcopulatory sexual selection new techniques are required. Here we present an improved immunolabelling technique to track the fate of 5-bromo-2′-deoxyuridine (BrdU)-labelled sperm in the female reproductive organs of gastropods. The technique was tested in individuals of the simultaneously hermaphroditic land snail Arianta arbustorum (Linnaeus, 1758). We determined the percentage of labelled sperm in spermato-phores delivered and assessed the reliability of detecting labelled sperm in the spermatheca (sperm storage organ) and bursa copulatrix (sperm digestion organ) of the recipients. In our tests, the proportion of sperm labelled among the sperm produced by an individual averaged 99.3%. Furthermore, labelled sperm could be consistently visualized in both the sperm storage and sperm digestion organ of all recipients examined. Combined with a traditional sperm staining technique, we showed that the BrdU-labelling technique allows usto distinguish between sperm from two males in the female reproductive tract of double-mated snails. In a controlled growth experiment, we found that repeated BrdU-application slightly retarded shell growth of juvenile snails, but did not influence their adult size, survival, number of sperm delivered and mating frequency. The technique presented could be adjusted to other gastropod species providing insight into mechanisms of sperm competition and cryptic female choice.

Mechanism of liver steatosis in rats with systemic carnitine deficiency due to treatment with trimethylhydraziniumpropionate

Rats with systemic carnitine deficiency induced by treatment with trimethylhydraziniumpropionate (THP) develop liver steatosis. This study aims to investigate the mechanisms leading to steatosis in THP-induced carnitine deficiency. Rats were treated with THP (20 mg/100 g) for 3 or 6 weeks and were studied after starvation for 24 h. Rats treated with THP had reduced in vivo palmitate metabolism and developed mixed liver steatosis at both time points. The hepatic carnitine pool was reduced in THP-treated rats by 65% to 75% at both time points. Liver mitochondria from THP-treated rats had increased oxidative metabolism of various substrates and of beta-oxidation at 3 weeks, but reduced activities at 6 weeks of THP treatment. Ketogenesis was not affected. The hepatic content of CoA was increased by 23% at 3 weeks and by 40% at 6 weeks in THP treated rats. The cytosolic content of long-chain acyl-CoAs was increased and the mitochondrial content decreased in hepatocytes of THP treated rats, compatible with decreased activity of carnitine palmitoyltransferase I in vivo. THP-treated rats showed hepatic peroxisomal proliferation and increased plasma VLDL triglyceride and phospholipid concentrations at both time points. A reduction in the hepatic carnitine pool is the principle mechanism leading to impaired hepatic fatty acid metabolism and liver steatosis in THP-treated rats. Cytosolic accumulation of long-chain acyl-CoAs is associated with increased plasma VLDL triglyceride, phospholipid concentrations, and peroxisomal proliferation.