Overexpression of a homeodomain protein confers axis-forming activity to uncommitted Xenopus embryonic cells.

The anteroposterior character of mesoderm induced by a peptide growth factor (XTC-MIF) was tested by transplantation into host Xenopus gastrulae. Both retinoic acid and a homeodomain protein were able to override the anteriorizing effect of the growth factor. Microinjection of a posteriorly expressed homeobox mRNA can respecify anteroposterior identity, transforming head mesoderm into tail-inducing mesoderm. Unexpectedly, overexpression of XIHbox 6 protein in the transplanted cells, without addition of growth factors, caused the formation of tail-like structures. The cells overexpressing XIHbox 6 were able to recruit cells from the host into the secondary axis. The results suggest that vertebrate homeodomain proteins are part of the biochemical pathway leading to the generation of the body axis.     

Cell separation from high cell density broths of Alcaligenes eutrophus by using a coagulant

Summary Alcaligenes eutrophus was successfully recovered from high cell density broths by pre-treatment with polyaluminium hydroxide chloride silicate as a coagulant at 36–90 mg Al/l. The optimum pH range for cell coagulation was 10–12. Subsequent centrifugation (45×g) and filtration (pore size 0.5 mm) gave a cell recovery of higher than 90%. The energy demand for cell recovery with the coagulant was only 3–11% of that without it.     

Promoter analysis of the Drosophila melanogaster gene encoding the pterin 4alpha-carbinolamine dehydratase.

Pterin-4alpha-carbinolamine dehydratase (PCD) is a key enzyme in the regeneration pathway of tetrahydrobiopterin. Previously, we isolated and reported the Drosophila melanogaster gene encoding PCD. In the present study, we isolated and characterized the Drosophila virilis gene encoding PCD. The Drosophila virilis PCD gene has two introns and an open reading frame to encode a protein of 101 amino acids. The amino acid sequence of Drosophila virilis PCD shows a 83% homology to that of the Drosophila melanogaster PCD protein. From the alignment of the nucleotide sequence in the 5'-flanking region of the Drosophila melanogaster and Drosophila virilis PCD genes, we found four conserved sequences. Using a transient transfection assay, we showed that one of the conserved sequences (-127 to approximately -115) is critical for expression, also the minimal promoter region between -127 and +51 is necessary for the efficient expression of Drosophila melanogaster PCD.     

Structure-function analysis of the DNA binding domain of Saccharomyces cerevisiae ABF1.

To localize the DNA binding domain of the Saccharomyces cerevisiae Ars binding factor 1 (ABF1), a multifunctional DNA binding protein, plasmid constructs carrying point mutations and internal deletions in the ABF1 gene were generated and expressed in Escherichia coli. Normal and mutant ABF1 proteins were purified by affinity chromatography and their DNA binding activities were analyzed. The substitution of His61, Cys66 and His67 respectively, located in the zinc finger motif in the N-terminal region (amino acids 40-91), eliminated the DNA binding activity of ABF1 protein. Point mutations in the middle region of ABF1, specifically at Leu353, Leu399, Tyr403, Gly404, Phe410 and Lys434, also eliminated or reduced DNA binding activity. However, the DNA binding activity of point mutants of Ser307, Ser496 and Glu649 was the same as that of wild-type ABF1 protein and deletion mutants of amino acids 200-265, between the zinc finger region and the middle region (residues 323-496) retained DNA binding activity. As a result, we confirmed that the DNA binding domain of ABF1 appears to be bipartite and another DNA binding motif, other than the zinc finger motif, is situated between amino acid residues 323 and 496.     

pH-dependent binding of the Epsin ENTH domain and the AP180 ANTH domain to PI(4,5)P2-containing bilayers

Epsin and AP180 are essential components of the endocytotic machinery, which controls internalization of protein receptors and other macromolecules at the cell surface. Epsin and AP180 are recruited to the plasma membrane by their structurally and functionally related N-terminal ENTH and ANTH domains that specifically recognize PtdIns(4,5)P₂. Here, we show that membrane anchoring of the ENTH and ANTH domains is regulated by the acidic environment. Lowering the pH enhances PtdIns(4,5)P₂ affinity of the ENTH and ANTH domains reinforcing their association with lipid vesicles and monolayers. The pH dependency is due to the conserved histidine residues of the ENTH and ANTH domains, protonation of which is necessary for the strong PtdIns(4,5)P₂ recognition, as revealed by liposome binding, surface plasmon resonance, NMR, monolayer surface tension and mutagenesis experiments. The pH sensitivity of the ENTH and ANTH domains is reminiscent to the pH dependency of the FYVE domain suggesting a common regulatory mechanism of membrane anchoring by a subset of the PI-binding domains.     

Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis

The transient gene expression system using Arabidopsis mesophyll protoplasts has proven an important and versatile tool for conducting cell-based experiments using molecular, cellular, biochemical, genetic, genomic and proteomic approaches to analyze the functions of diverse signaling pathways and cellular machineries. A well-established protocol that has been extensively tested and applied in numerous experiments is presented here. The method includes protoplast isolation, PEG-calcium transfection of plasmid DNA and protoplast culture. Physiological responses and high-throughput capability enable facile and cost-effective explorations as well as hypothesis-driven tests. The protoplast isolation and DNA transfection procedures take 6-8 h, and the results can be obtained in 2-24 h. The cell system offers reliable guidelines for further comprehensive analysis of complex regulatory mechanisms in whole-plant physiology, immunity, growth and development.     

MRI Features in a Canine Model of Ischemic Stroke: Correlation between Lesion Volume and Neurobehavioral Status during the Subacute Stage

The purpose of this study was to evaluate the diagnostic value of magnetic resonance imaging (MRI) and assess the correlation between the volume of the ischemic lesion and neurobehavioral status during the subacute stage of ischemic stroke. Ischemic stroke was induced in 6 healthy laboratory beagles through permanent occlusion of the middle cerebral artery (MCAO). T2-weighted and fluid-attenuated inversion recovery (FLAIR) imaging, diffusion-weighted imaging (DWI), measurement of the apparent diffusion coefficient (ADC) ratio, and neurobehavioral evaluation were performed 3 times serially by using a 1.5-T MR system: before and 3 and 10 d after MCAO. Ischemic lesions demonstrated T2 hyperintensity, FLAIR hyperintensity, and DWI hyperintensity. The ADC ratio was decreased initially but then was increased at 10 d after MCAO. Ischemic lesion volumes on T2-weighted and FLAIR imaging were not significantly different from those on DWI. The lesion volume and neurobehavioral score showed strong correlation. Our results suggest that conventional MRI may be a reliable diagnostic tool during the subacute stage of canine ischemic stroke.Abbreviations: ADC, apparent diffusion coefficient; DWI, diffusion-weighted imaging; FLAIR, fluid-attenuated inversion recovery; MCAO, middle cerebral artery occlusion; MRI, magnetic resonance imaging; PWI, perfusion-weighted imagingIn human medicine, stroke is a leading cause of adult mortality and neurologic disability worldwide.¹ Strokes previously were thought to be uncommon in small animals, but the true prevalence is unknown.⁴ These events are now recognized more frequently in dogs because of increased use of magnetic resonance imaging (MRI).^5,14,17Because the infusion of thrombolytic agents, such as urokinase or tissue plasminogen activator, within 3 to 6 h of the onset symptoms is effective in restoring blood flow and improving stroke outcome in humans,¹⁹ the detection of early ischemic changes is now thought to be necessary to improve patient outcome. Computed tomography and conventional MRI are not sufficiently sensitive to predict the presence and extent of ischemic damage during the acute stage after a stroke.^12,20 Therefore several MRI sequences, such as fluid-attenuated inversion recovery (FLAIR), diffusion-weighted imaging (DWI), perfusion-weighted imaging (PWI), and MR angiography, have been developed for early diagnosis and subsequent follow-up of ischemic stroke.³ High-field magnetic strengths (at least 1.5 T) are necessary to perform these sequences.In contrast to the situation in humans, ischemic stroke in many dogs is diagnosed during the subacute stage—24 h to 6 wk after the vascular insult—due to the time lag between the onset of clinical signs to referral and to the lack of standard diagnostic protocols for ischemic stroke in dogs. In most reports of strokes in dogs, the median interval between the onset of neurologic dysfunction and performance of an MRI was more than 2 d.^5,14,17 Whereas DWI has marked sensitivity to very early ischemic changes in the brain, T2-weighted and FLAIR images gradually become more hyperintense later (that is, during the first 24 h after the insult).³ Therefore, hyperintensity on T2-weighted and FLAIR images is believed to be representative of mature lesions.¹⁵ In light of these findings, we hypothesized that conventional MR sequences, such as T2-weighted and FLAIR imaging as well as DWI would be used for the diagnosis of the subacute stage of ischemic stroke in dogs.The purpose of this study was to evaluate the diagnostic value of MRI and assess the correlation between the volume of ischemic lesions and neurobehavioral status during the subacute stage of ischemic stroke in dogs. We therefore investigated the lesion volume of T2-weighted and FLAIR images compared with that on DWI images. Furthermore, we assessed the relationship between the apparent diffusion coefficient (ADC) of the ischemic lesions and the neurobehavioral status of the dogs.     

Genotype of Toxoplasma gondii from Blood of Stray Cats in Gyeonggi-do, Korea

Genotyping of Toxoplasma gondii has been performed in 23 PCR positive blood samples from stray cats in Korea. We used 2 separate PCR-restriction fragment length polymorphism (RFLP) patterns of SAG2 gene, amplifying the 5''and 3''ends of the locus. The results revealed that all samples belonged to the type I clonal lineage. Although T. gondii organisms were not isolated from the samples, the results of the present study represent that stray cats with T. gondii infection should be seriously concerned in our environment. Adequate and continuous control programs of stray cats are needed to reduce the risk of transmission of T. gondii as a zoonotic infection threatening the public health.     

Overexpression of the downward leaf curling (DLC) gene from melon changes leaf morphology by controlling cell size and shape in Arabidopsis leaves

A plant-specific gene was cloned from melon fruit. This gene was named downward leaf curling (CmDLC) based on the phenotype of transgenic Arabidopsis plants overexpressing the gene. This expression level of this gene was especially upregulated during melon fruit enlargement. Overexpression of CmDLC in Arabidopsis resulted in dwarfism and narrow, epinastically curled leaves. These phenotypes were found to be caused by a reduction in cell number and cell size on the adaxial and abaxial sides of the epidermis, with a greater reduction on the abaxial side of the leaves. These phenotypic characteristics, combined with the more wavy morphology of epidermal cells in overexpression lines, indicate that CmDLC overexpression affects cell elongation and cell morphology. To investigate intracellular protein localization, a CmDLC-GFP fusion protein was made and expressed in onion epidermal cells. This protein was observed to be preferentially localized close to the cell membrane. Thus, we report here a new plant-specific gene that is localized to the cell membrane and that controls leaf cell number, size and morphology.