Tracking of human cells in mice

Tracking and tracing of transplanted cells in mice is required in many fields of research. Examples are transplantation of stem cells into organs of mice to study their differentiation capacity and injection of tumor cells to examine metastatic behavior. In the present study we tested the lipid dye CM-DiI and red fluorescent nanoparticles Qdot655 for their applicability in tagging and tracing of human cells in mice. Labeling of different cell types, including MCF-7 human breast cancer cells, human cord blood derived cells, human NeoHep cells and human hepatopancreatic precursor cells, is technically easy and did not compromise further cell culture. After transplantation of CM-DiI or Qdot655 marked cells, red fluorescent structures could be detected already in unprocessed paraffin slices of the studied organs, namely liver, lung, pancreas, kidney, spleen and bone marrow. Next, we examined whether the red fluorescent structures represent the transplanted human cells. For this purpose, we established an in situ hybridization (ISH) technique that allows clear-cut differentiation between human and murine nuclei, based on simultaneous hybridization with human alu and mouse major satellite (mms) probes. We observed a high degree of coincidence between CM-DiI-marked cells and alu positive nuclei. However, also some mms positive cells contained CM-DiI, suggesting phagocytosis of the transplanted CM-DiI-marked cells. The degree of such CM-DiI-positive mouse cells depended on the cell type and route of administration. From a technical point of view it was important that CM-DiI-positive structures in paraffin slices remained fluorescent also after ISH. In contrast, Qdot655 positive structures faded during further staining procedures. In conclusion, marking of cells with CM-DiI or Qdot655 prior to transplantation facilitates recovery of human cells, since a high fraction of positive structures in the host's tissue originate from the transplanted cells. However, CM-DiI or Qdot655 positive staining of individual cells in transplanted tissues is not sufficient to prove their human origin. Additional procedures, such as ISH with alu-probes, are essential, when characterizing individual cells.     

Differences in hippocampal protein expression at 3 days, 3 weeks, and 3 months following induction of perinatal asphyxia in the rat

Perinatal asphyxia (PA) was induced in Sprague-Dawley rats; pups were sacrificed 3 days, 3 weeks, and 3 months following the asphyctic insult, and hippocampal protein levels were determined by a gel-based proteomic method. Levels of antioxidant, metabolic, cytoskeleton, signaling, channel, proteasomal, chaperone, splicing, and synaptosomal proteins were dysregulated depending on the age following induction of PA. These proteins are proposed to be potential markers or pharmacological targets for PA.     

Targeting of mitochondria by 10-N-alkyl acridine orange analogues: role of alkyl chain length in determining cellular uptake and localization

10-N-Nonyl acridine orange (NAO) is used as a mitochondrial probe because of its high affinity for cardiolipin (CL). Targeting of NAO may also depend on mitochondrial membrane potential. As the nonyl group has been considered essential for targeting, a systematic study of alkyl chain length was undertaken; three analogues (10-methyl-, 10-hexyl-, and 10-hexadecyl-acridine orange) were synthesized and their properties studied in phospholipid monolayers and breast cancer cells. The shortest and longest alkyl chains reduced targeting, whereas the hexyl group was superior to the nonyl group, allowing very clear and specific targeting to mitochondria at concentrations of 20-100 nM, where no evidence of toxicity was apparent. Additional studies in wild-type and cardiolipin-deficient yeast cells suggested that cellular binding was not absolutely dependent upon cardiolipin.     

Persistence of the Attractiveness of Two Sex-specific Scents in Meadow Voles,Microtus pennsylvanicus

The function of an odour may be reflected in its fade-out time in the environment. In this study, we investigated fade-out times of two specific odours, the anogenital area scent and that of the posterolateral region. These two odours support opposite-sex preferences in male and female meadow voles, Microtus pennsylvanicus, but convey nonidentical information to conspecifics during the breeding season. The first experiment tested whether meadow voles respond preferentially to scents that were aged for 15 min (fresh) to 30 d. Males preferred female anogenital area scent to male anogenital area scent if both scents were ≤ 10 d old. By comparison, females preferred male anogenital area scent to female anogenital area scent if the scents were ≤ 25 d old. However, male and female voles preferred the posterolateral scent of males to that of females if the scents were ≤ 1 d old. Thus, fade-out times for these two scents differ for males and females, suggesting different functions. In the second experiment, male and female voles preferred fresh anogenital area scent and fresh posterolateral region scent compared with those same scents that were older. This result suggests that older scents may have lost information over time about the sex of the donor. Overall, data from both experiments indicate that voles may use specific scents for communication in different social contexts.     

GPI anchor biosynthesis in yeast: phosphoethanolamine is attached to the alpha1,4-linked mannose of the complete precursor glycophospholipid

Cells synthesize the GPI anchor carbohydrate core by successively adding N-acetylglucosamine, three mannoses, and phosphoethanolamine (EtN-P) onto phosphatidylinositol, thus forming the complete GPI precursor lipid which is then added to proteins. Previously, we isolated a GPI deficient yeast mutant accumulating a GPI intermediate containing only two mannoses, suggesting that it has difficulty in adding the third, alpha1,2-linked Man of GPI anchors. The mutant thus displays a similar phenotype as the mammalian mutant cell line S1A-b having a mutation in the PIG-B gene. The yeast mutant, herein named gpi10-1 , contains a mutation in YGL142C, a yeast homolog of the human PIG-B. YGL142C predicts a highly hydrophobic integral membrane protein which by sequence is related to ALG9, a yeast gene required for adding Man in alpha1,2 linkage to N-glycans. Whereas gpi10-1 cells grow at a normal rate and make normal amounts of GPI proteins, the microsomes of gpi10-1 are completely unable to add the third Man in an in vitro assay. Further analysis of the GPI intermediate accumulating in gpi10 shows it to have the structure Manalpha1-6(EtN-P-)Manalpha1-4GlcNalpha1- 6(acyl) Inositol-P-lipid. The presence of EtN-P on the alpha1,4-linked Man of GPI anchors is typical of mammalian and a few other organisms but had not been observed in yeast GPI proteins. This additional EtN-P is not only found in the abnormal GPI intermediate of gpi10-1 but is equally present on the complete GPI precursor lipid of wild type cells. Thus, GPI biosynthesis in yeast and mammals proceeds similarly and differs from the pathway described for Trypanosoma brucei in several aspects.      

Kinetics of violaxanthin de-epoxidation by violaxanthin de-epoxidase, a xanthophyll cycle enzyme, is regulated by membrane fluidity in model lipid bilayers.

This paper describes violaxanthin de-epoxidation in model lipid bilayers. Unilamellar egg yolk phosphatidylcholine (PtdCho) vesicles supplemented with monogalactosyldiacylglycerol were found to be a suitable system for studying this reaction. Such a system resembles more the native thylakoid membrane and offers better possibilities for studying kinetics and factors controlling de-epoxidation of violaxanthin than a system composed only ofmonogalactosyldiacylglycerol and is commonly used in xanthophyll cycle studies. The activity of violaxanthin de-epoxidase (VDE) strongly depended on the ratio of monogalactosyldiacylglycerol to PtdCho in liposomes. The mathematical model of violaxanthin de-epoxidation was applied to calculate the probability of violaxanthin to zeaxanthin conversion at different phases of de-epoxidation reactions. Measurements of deepoxidation rate and EPR-spin label study at different temperatures revealed that dynamic properties of the membrane are important factors that might control conversion of violaxanthin to antheraxanthin. A model of the molecular mechanism of violaxanthin de-epoxidation where the reversed hexagonal structures (mainly created by monogalactosyldiacylglycerol) are assumed to be required for violaxanthin conversion to zeaxanthin is proposed. The presence of monogalactosyldiacylglycerol reversed hexagonal phase was detected in the PtdCho/monogalactosyldiacylglycerol liposomes membrane by 31P-NMR studies. The availability of violaxanthin for de-epoxidation is a diffusion-dependent process controlled by membrane fluidity. The significance of the presented results for understanding themechanism of violaxanthin de-epoxidation in native thylakoid membranes is discussed.     

Structure and function of the cochlea in the African mole rat (Cryptomys hottentotus): evidence for a low frequency acoustic fovea

Summary The cochlea of the mole rat Cryptomys hottentotus was investigated with physiological and anatomical methods. In order to reveal the place-frequency map of the cochlea, iontophoretic HRP-applications were made in the cochlear nucleus at physiologically characterized locations. Subsequent HRP-transport in auditory nerve fibres and labeling patterns of spiral ganglion cells within the cochlea were evaluated.A cochlear place-frequency map was constructed from 17 HRP-applications in the cochlear nucleus at positions where neurons had characteristic frequencies between 0.1 and 12.6 kHz. As in other mammals, high frequencies were found to be represented at the cochlear base, low frequencies at the cochlear apex. The placefrequency map had three distinct parts which were characterized by their different slopes. A clear overrepresentation of the frequencies between 0.6 and 1 kHz was revealed, in this frequency range the slope of the place-frequency map amounted to 5.3 mm/octave. As calculated from the regression analysis, below 0.6 kHz the slope of the cochlear place-frequency map amounted to 0.24 mm/octave, above 1 kHz to 0.9 mm/octave.As in other mammals width of the basilar membrane (BM) increased from the cochlear base towards the cochlear apex. Also in concordance with the findings in other mammals, BM-thickness decreased from the cochlear base to the apex. However, it was remarkable to find that there was no or little change in BM-width and thickness between 40 and 85% BM-length. It was also revealed that scala tympani was only 1/10th the size found in the rat or other mammals of similar body size.On the basis of the cochlear place-frequency map and the morphological findings we speculate that in Cryptomys hottentotus an acoustic fovea is present in the frequency range between 0.6 and 1 kHz. In analogy to echolocating bats, about half of the cochlea is devoted to the analysis of a narrow frequency band within the hearing range.Abbreviations BM basilar membrane - CF characteristic frequency - CN cochlear nucleus     

The effect of preconditioning on the iron deposition after transient forebrain ischemia in rat brain

In this study we investigated iron deposition in the hippocampus CA1 area and the corpus striatum pars dorsolateralis in a rat model of cerebral ischemia and ischemic tolerance. Forebrain ischemia was induced by four-vessel occlusion for 5-min as ischemic preconditioning. Two days after the preconditioning or the sham operation, a second ischemia was induced for 20-min. With the use of iron histochemistry, regional changes were examined after 2 to 8 weeks of recirculation following the 20-min ischemia with or without preconditioning. Perl's reaction with DAB intensification demonstrated iron deposits in the CA1 area and in the corpus striatum pars dorsolateralis after 2 weeks of recirculation. These iron deposits gradually increased in density and formed clusters by the 8th week. When the rats were exposed to 5-min ischemia 2 days before lethal 20-min ischemia, the deposition of iron in the CA1 region of the hippocampus and also in the corpus striatum pars dorsolateralis was decreased and produced a minimal number of iron-containing cells between the second and the 8th week of recirculation. Preconditioning with sublethal 5-min ischemia followed by 2 days of reperfusion also prevented the neuronal destruction of the hippocampal CA1 region induced by 20-min ischemia.