Effect of opioid peptides on proliferation and differentiation of skeletogenic cells of rabbit bone marrow in vitro

The influence of opioid peptides DSLET and DAGO in doses 10(-5), 10(-7) or 10(-10) mg per 1 ml of the medium on colony formation in the culture of stromal bone marrow fibroblast precursors was investigated 5. 10(-6) bone marrow cells were placed in plastic containers (Costar). 12 day old cell cultures were fixed with ethanol and stained with hematoxyline-eosin. Effectiveness of fibroblast colony formation (EFFC) was detected. Grown fibroblast colonies were stained after Gomory for alkaline phosphatase. Opioid peptides DSLET and DAGO in the used doses exerted no influence on EFFC and percentage phosphatase-positive colonies, which casts doubt on a presumable direct action of opioid peptides on stromal bone marrow cell-precursors. But it does not seem unlikely that opioid peptides may affect stromal bone marrow precursors of fibroblasts through the cell environment, particularly, via macrophages.     

Iron Deposition after Transient Forebrain Ischemia in Rat Brain

In this study, we investigated the iron deposition in the cerebral cortex, hippocampus CA1 area and corpus striatum pars dorsolateralis in a rat model of cerebral ischemia. Forebrain ischemia was induced by four-vessel occlusion for 20 min. Using iron histochemistry, regional changes were examined from 1 to 8 weeks of postischemic recirculation. Neuronal death was demonstrated in pyramidal cells of the hippocampal CA1 area and in the dorsolateral part of the corpus striatum, which are known as areas most vulnerable to ischemia. Iron deposition in hippocampal CA1 area was coupled to delayed pyramidal cell death. Perl's reaction with DAB intensification revealed of the 1 week iron deposits in the CA1 area, which gradually increased and formed clusters by 8 weeks. In the corpus striatum, strong iron staining was observed in injured cellular layer pars dorsolateralis 1 week after recirculation. Granular iron was deposited in the cytoplasm of pyramidal cells in layers III and V of the frontal cortex after 2 weeks of recirculation. In contrast to the hippocampus and striatum, the cerebral cortex did not develop severe neuronal cell death and atrophy immediately after the ischemic insult, which suggest that the neuronal cell death in the cerebral cortex occurs extremely late.     

Stability of bacteriophage T4 short tail fiber

Adsorption of T-even bacteriophages to the E. coli host cell is mediated by long and short tail fibers. Bacteriophage T4 short tail fiber protein p12 was used to investigate the stability against thermal and chemical denaturation. Purified p12 is thermostable with a melting point of 78 degrees C. Guanidinium chloride-induced denaturation displayed strong hysteresis and an intermediate between 2 and 3 M denaturant. The transitions occur at 1.5 and 3.2 M denaturant as revealed by fluorescence spectroscopy and circular dichroism. The data suggest an equilibrium unfolding intermediate with a separate unfolding of the C-terminal knob domain and the shaft region.     

Proteomic characterization of protein phosphatase 1 complexes in ischemia-reperfusion and ischemic tolerance

Serine/threonine protein phosphatase 1 (PP1) regulates multiple cellular processes. Protein phosphorylation-dephosphorylation is largely altered during ischemia and subsequent reperfusion. The brain is particularly vulnerable to stress resulting from ischemia-reperfusion (IR), however, the acquisition of ischemic tolerance (IT) protects against IR stress. We studied PP1 complexes in response to IR stress and IT in brain using proteomic characterization of PP1 complexes in animal models of IR and IT. PP1alpha and PP1gamma were immunoprecipitated and resolved by 2-D. DIGE analysis detected 14 different PP1-interacting proteins that exhibited significant changes in their association with PP1alpha or PP1gamma. These proteins were identified by MALDI-TOF MS. Seven had the PP1-binding RVxF motif. IR altered the interaction of heat shock cognate 71 kDa-protein, creatine kinase B, and dopamine- and cAMP-regulated phosphoprotein 32 kDa (DARPP32) with both PP1alpha and PP1gamma, and the interaction of phosphodiesterase-6B, transitional ER ATPase, lamin-A, glucose-regulated 78 kDa-protein, dihydropyrimidinase-related protein-2, gamma-enolase, neurofilament-L, and ubiquitin ligase SIAH2 with PP1gamma. IT prevented most of the IR-induced effects. This study identifies novel PP1alpha- and PP1gamma-interacting proteins and reveals an in vivo modularity of PP1 holoenzymes in response to physiological ischemic stress. It supports a potential role of PP1 in IR stress and as a target of the endogenous protective mechanisms induced by IT.     

Adaptive differentiations of the skin of the head in a subterranean rodent,Spalax ehrenbergi

The skin of macroscopically distinct regions (hairy skin, vibrissal fields, buccal ridge, and rhinarium) of the head of the blind mole-rat, Spalax ehrenbergi, was studied by routine histological methods. Few guard and several soft vellus hairs are organized into tufts that grow from a group of hair follicles localized in an invaginated compound cavity. We suggest that this hair arrangement may be a burrowing adaptation to match frictional resistance. The follicles and the compound cavity possess either well developed complex striated musculature or errector pili muscles. There are no structural specializations (sweat glands, glomus bodies) to enhance thermo-regulatory (heat dissipative) capacities in the hairy skin of the head. Vibrissae penetrate the epidermal surface as single hairs. They are microscopically normally developed are arranged in vibrissal fields according to a basal mammalian pattern. Most of them are, however, relatively short and inconspicuous. The mystical vibrissal field is horizontally divided by a prominent buccal ridge which is probably involved in bulldozing. The hairs in the ridge leave the compound cavity singularly. The follicles of guard hairs and bristles are equipped with well developed pilo-Ruffini complexes indicating that the buccal ridge may serve also as a tactile organ. The glabrous skin of the rhinarium has a highly interdigitated dermal-epidermal interface. The dermal papillae possess simple lamellated and/or simple Meissner's corpuscles and few Merkel cell-axon-complexes indicating that the skin of the rhinarium may be particularly sensitive to perception of vibrations. J Morphol 233:53–66, 1997. © 1997 Wiley-Liss, Inc.     

The interactions of square platinum(II) complexes with guanine and adenine: a quantum-chemical ab initio study of metalated tautomeric forms

The influence of binding of square planar platinum complexes on tautomeric equilibria of the DNA bases guanine and adenine was investigated using the density functional B3LYP method. Neutral trans-dichloro(amine)-, +1 charged chloro(diamine)-, and +2 charged triamine-platinum(II) species were chosen for coordination to bases. Only the N₇ interaction site of the bases was considered. The calculations demonstrate that the neutral platinum adduct does not change the tautomeric equilibria of the bases. Furthermore, N₇ binding of the neutral Pt adduct moderately reduces the probability of protonation of the N₁ position of adenine. Larger effects can be observed for +1 and mainly +2 adducts, but these can be rationalized by electrostatic effects. Since the electrostatic effects are expected to be efficiently compensated for by a charged backbone of DNA and counterions in a polar solvent, no dramatic increase in mispair formation is predicted for Pt(II) adducts, which is in agreement with experiment. The interaction energies between Pt adducts and the nucleobases were also evaluated. These interaction energies range from ca. 210 kJ/mol for neutral adducts, interacting with both bases and their tautomers, up to 500 kJ/mol for the +2 charged adducts, interacting with the keto-guanine tautomer and the anti-imino-adenine tautomer. The surprisingly large interaction energy for the latter structure is due to the strong H-bond between the NH₃ ligand group of the metal adduct and the N₆ nitrogen atom of the base. Received: 6 July 1999 / Accepted: 7 December 1999     

Transduction with recombinant adeno-associated virus for gene therapy is limited by leading-strand synthesis.

Adeno-associated virus is an integrating DNA parvovirus with the potential to be an important vehicle for somatic gene therapy. A potential barrier, however, is the low transduction efficiencies of recombinant adeno-associated virus (rAAV) vectors. We show in this report that adenovirus dramatically enhances rAAV transduction in vitro in a way that is dependent on expression of early region 1 and 4 (E1 and E4, respectively) genes and directly proportional to the appearance of double-stranded replicative forms of the rAAV genome. Expression of the open reading frame 6 protein from E4 in the absence of E1 accomplished a similar but attenuated effect. The helper activity of adenovirus E1 and E4 for rAAV gene transfer was similarly demonstrated in vivo by using murine models of liver- and lung-directed gene therapy. Our data indicate that conversion of a single-stranded rAAV genome to a duplex intermediate limits transduction and usefulness for gene therapy.     

Does the morphology of the ear of the Chinese bamboo rat (Rhizomys sinensis) show “Subterranean” characteristics?

In spite of the growing interest in rodents with subterranean activity in general and the spalacids (Spalacidae) in particular, little is known about the biology of most members of this clade, such as the Chinese bamboo rat (Rhizomys sinensis). Here, we analyzed the ear morphology of R. sinensis with respect to hearing specialization for subterranean or aboveground modes of communication. It is well‐known that ecology and style of life of a particular species can be reflected in morphology of its ear, its hearing and vocalization, so we expect that such information could provide us insight into its style of life and its sensory environment. The ratio between the eardrum and stapedial footplate areas, which influences the efficiency of middle ear sound transmission, suggests low hearing sensitivity, as is typical for subterranean species. The cochlea had 3.25 coils and resembled species with good low frequency hearing typical for subterranean mammals. The length of the basilar membrane was 18.9 ± 0.8 mm and its width slowly increased towards the cochlear apex from 60 to 85 μm. The mean density of outer hair cells was 344 ± 22 and of inner hair cells 114 ± 7.3 per 1 mm length of the organ of Corti, and increased apically. These values (except for relatively low hair cell density) usually characterize ears specialized for low frequency hearing. There was no evidence for an acoustic fovea. Apart of low hair cell density which is common in aboveground animals, this species has also relatively large auricles, suggesting the importance of sound localization during surface activity. The ear of the Chinese bamboo rat thus contains features typical for both aboveground and subterranean mammals and suggests that this spalacid has fossorial habits combined with regular aboveground activity. J. Morphol. 277:575–584, 2016. © 2016 Wiley Periodicals, Inc.     

Plasmodium berghei MAPK1 Displays Differential and Dynamic Subcellular Localizations during Liver Stage Development

Mitogen-activated protein kinases (MAPKs) regulate key signaling events in eukaryotic cells. In the genomes of protozoan Plasmodium parasites, the causative agents of malaria, two genes encoding kinases with significant homology to other eukaryotic MAPKs have been identified (mapk1, mapk2). In this work, we show that both genes are transcribed during Plasmodium berghei liver stage development, and analyze expression and subcellular localization of the PbMAPK1 protein in liver stage parasites. Live cell imaging of transgenic parasites expressing GFP-tagged PbMAPK1 revealed a nuclear localization of PbMAPK1 in the early schizont stage mediated by nuclear localization signals in the C-terminal domain. In contrast, a distinct localization of PbMAPK1 in comma/ring-shaped structures in proximity to the parasite’s nuclei and the invaginating parasite membrane was observed during the cytomere stage of parasite development as well as in immature blood stage schizonts. The PbMAPK1 localization was found to be independent of integrity of a motif putatively involved in ATP binding, integrity of the putative activation motif and the presence of a predicted coiled-coil domain in the C-terminal domain. Although PbMAPK1 knock out parasites showed normal liver stage development, the kinase may still fulfill a dual function in both schizogony and merogony of liver stage parasites regulated by its dynamic and stage-dependent subcellular localization.     

Transient Forebrain Ischemia Impact on Lymphocyte DNA Damage, Glutamic Acid Level, and SOD Activity in Blood

Aims Brain ischemia–reperfusion injury remains incompletely understood but appears to involve a complex series of interrelated biochemical pathways caused mainly by a burst of reactive oxygen species (ROS). In the present work we studied the impact of postischemic condition in the early phase of reperfusion on plasma and blood cells. Methods Transient forebrain ischemia was induced in Wistar rats by four-vessel occlusion model. Blood samples collected during postischemic reperfusion 20, 40, 60, 90, and 120 min after ischemia were used for assessing breaks of lymphocyte DNA, fluorimetric measurement of whole blood glutamate concentration, and spectrophotometrical determination of SOD activity in plasma and blood cells. Results Our results showed the most interesting changes of all observed parameters mainly at 40 and 120 min of reperfusion, when we observed peak DNA damage of lymphocytes and highest glutamate level and total and Cu/Zn SOD activity. At those time points, Mn SOD activity was low in plasma, as well as in blood cells. On the contrary, at 60 and 90 min, all studied parameters were approximately at the level of control. Conclusion Ischemia/reperfusion injury has influence on blood cells and has at least two waves of impact on DNA damage of peripheral lymphocytes, affects activity of major antioxidant enzymes SODs, as well as blood glutamic acid level. Elevation of Mn SOD activity probably plays an important role in the processes of elimination of postischemic damage in blood cells.