Function of the Herpes Simplex Virus 1 Small Capsid Protein VP26 Is Regulated by Phosphorylation at a Specific Site

Replacement of the herpes simplex virus 1 small capsid protein VP26 phosphorylation site Thr-111 with alanine reduced viral replication and neurovirulence to levels observed with the VP26 null mutation. This mutation reduced VP26 expression and mislocalized VP26 and its binding partner, the major capsid protein VP5, in the nucleus. VP5 mislocalization was also observed with the VP26 null mutation. Thus, we postulate that phosphorylation of VP26 at Thr-111 regulates VP26 function in vitro and in vivo.     

Evaluating experimental cerebral malaria using oxidative stress indicator OKD48 mice

Cerebral malaria is a fatal complication of malaria. Conventional methods for evaluating experimental cerebral malaria have several drawbacks. Therefore, we aimed to develop an easy-to-use method for evaluating experimental cerebral malaria using OKD48 (Keap1-dependent Oxidative stress Detector, No-48-luciferase) mice to evaluate oxidative stress. OKD48 mice infected with Plasmodium berghei ANKA strain (PbA) suffered from experimental cerebral malaria and oxidative stress was successfully detected in the brains of living OKD48 mice developing experimental cerebral malaria. Oxidative stress in the brain was dependent on the development of experimental cerebral malaria, as prevention of experimental cerebral malaria did not elicit oxidative stress. We provide a novel evaluation method for experimental cerebral malaria using oxidative stress indicator OKD48 mice.     

The Murine Stem Cell Virus Promoter Drives Correlated Transgene Expression in the Leukocytes and Cerebellar Purkinje Cells of Transgenic Mice

The murine stem cell virus (MSCV) promoter exhibits activity in mouse hematopoietic cells and embryonic stem cells. We generated transgenic mice that expressed enhanced green fluorescent protein (GFP) under the control of the MSCV promoter. We obtained 12 transgenic founder mice through 2 independent experiments and found that the bodies of 9 of the founder neonates emitted different levels of GFP fluorescence. Flow cytometric analysis of circulating leukocytes revealed that the frequency of GFP-labeled leukocytes among white blood cells ranged from 1.6% to 47.5% across the 12 transgenic mice. The bodies of 9 founder transgenic mice showed various levels of GFP expression. GFP fluorescence was consistently observed in the cerebellum, with faint or almost no fluorescence in other brain regions. In the cerebellum, 10 founders exhibited GFP expression in Purkinje cells at frequencies of 3% to 76%. Of these, 4 mice showed Purkinje cell-specific expression, while 4 and 2 mice expressed GFP in the Bergmann glia and endothelial cells, respectively. The intensity of the GFP fluorescence in the body was relative to the proportion of GFP-positive leukocytes. Moreover, the frequency of the GFP-expressing leukocytes was significantly correlated with the frequency of GFP-expressing Purkinje cells. These results suggest that the MSCV promoter is useful for preferentially expressing a transgene in Purkinje cells. In addition, the proportion of transduced leukocytes in the peripheral circulation reflects the expression level of the transgene in Purkinje cells, which can be used as a way to monitor transgene expression properties in the cerebellum without invasive techniques.     

Relationship between the flexibility and the motility of actin filaments: effects of pH

Both the sliding velocity of fluorescently labeled actin filament and its persistence length as an index of the bending flexibility of the filament were examined in the motility assay as varying the pH values of the solution for preparing actin filaments. When the pH value was varied from 5.0 to 9.0 in the solution in which actin filaments were formed from the constituent monomers, the motile performance of Mg²⁺ bound actin filaments (Mg-F-actin) was apparently suppressed compared to the case of Ca²⁺ bound ones (Ca-F-actin). The persistence length for Ca-F-actin gradually increased with the increase of the pH value while the similar length for Mg-F-actin remained rather independent of the value. The largest sliding velocity of the filament, on the other hand, obtained at the persistence length of roughly 6 μm for both cases of Mg-F-actin and Ca-F-actin.     

Espresso coffee foam delays cooling of the liquid phase

Espresso coffee foam, called crema, is known to be a marker of the quality of espresso coffee extraction. However, the role of foam in coffee temperature has not been quantitatively clarified. In this study, we used an automatic machine for espresso coffee extraction. We evaluated whether the foam prepared using the machine was suitable for foam analysis. After extraction, the percentage and consistency of the foam were measured using various techniques, and changes in the foam volume were tracked over time. Our extraction method, therefore, allowed consistent preparation of high-quality foam. We also quantitatively determined that the foam phase slowed cooling of the liquid phase after extraction. High-quality foam plays an important role in delaying the cooling of espresso coffee.     

Loop-inserted and thermostabilized structure of P1-P1' cleaved ovalbumin mutant R339T.

Ovalbumin is a member of a superfamily of serine proteinase inhibitors, known as the serpins. It is, however, non-inhibitory towards serine proteinases, and lacks the loop insertion mechanism common to the serpins due to unknown structural factors. Mutant ovalbumin, R339T, in which the P14 hinge residue is replaced, was produced and analyzed for its thermostability and three-dimensional structure. Differential scanning calorimetry revealed that the mutant ovalbumin, but not the wild-type protein, undergoes a marked thermostabilization (DeltaT(m)=15.8 degrees C) following the P1-P1' cleavage. Furthermore, the crystal structure, solved at 2.3 A resolution, clearly proved that the P1-P1' cleaved form assumes the fully loop-inserted conformation as seen in serpin that possess inhibitory activity. We therefore conclude that ovalbumin acquires the structural transition mechanism into the loop-inserted, thermostabilized form by the single hinge mutation. The mutant protein does not, however, possess inhibitory activity. The solved structure displays the occurrence of specific interactions that may prevent the smooth motion, relative to sheet A, of helices E and F and of the loop that follows helix F. These observations provide crucial insights into the question why R339T is still non-inhibitory.     

Tri-dimensional morphometric analysis of astrocytic processes with high voltage electron microscopy of thick Golgi preparations

A characteristic feature of the astrocytic processes is to assume the form of shin sheets or lamellate coverings of other brain constituents. We analyzed the extensive and finely divided processes of the protoplasmic astrocyte in the molecular layer of the rat dentate gyrus by means of computer electron tomography and stereo-photogrammetry using tilted high voltage electron microscope images of thick Golgi preparations. The surface area and volume of the astrocytic processes were measured and the surface/volume ratios were estimated. The surface/volume ratios of astrocytic processes in the neuropile ranged from 18.9 to 33.0 per μm, and the mean value was 26.2 ± 5.0 per μm. The values were roughly comparable to those previously reported for the microdomain of Bergmann glia cell terminal processes in the rat cerebellum, which were estimated from reconstructions using thin serial section electron microscope images. The large surface to volume ratio of the astrocytic processes in the neuropile resulted from the lamellar nature of the processes interposed between other cellular elements, and may reflect the functional activities of the astrocyte. The results suggest the usefulness of the electron tomography and stereo-photogrammetry for three-dimensional morphometrical analysis of the astrocytic processes, although both techniques can be expected to be refined further in order to provide more precise measurements of these complicated processes.