Comparative atomic force and scanning electron microscopy: An investigation of structural differentiation of hepatic stellate cells

The molecular mechanism leading to the transdifferentiation of hepatic stellate cells (HSC) into myofibroblast-like cells following liver injury is not well understood. The state of cultured rat HSCs was determined using primarily fluorescence microscopy (UV), immunofluorescence (IF) (Glial fibrillary acidic protein (GFAP), Desmin, alpha-smooth muscle actin (alpha-SMA), F-actin) and immunocytochemistry (ICC) (GFAP, Desmin, alpha-SMA, Fibulin-2). Additionally, tapping-mode atomic force microscopy (TM-AFM) and field-emission scanning electron microscopy (FE-SEM) with low-resistivity indium-tin-oxide (ITO) thin-film were performed to observe the micro-morphological character of cells during HSC differentiation. Quiescent HSCs changed to the activated state were identified via UV, IF, and ICC observations. Normal rat HSCs (NHSCs) and thioacetamide-induced rat HSCs (THSCs) were demonstrated to be UV⁻, GFAP⁺, Desmin⁺, alpha-SMA⁺ and Fibulin-2⁻. After F-actin staining, lamellipodia and filopodia were found in both NHSCs and THSCs, but membrane ruffles were only seen in THSCs. The micro-structures of lamellipodia and filopodia in both NHSCs and THSCs were confirmed using FE-SEM and TM-AFM with ITO; in contrast, the micro-projection was not found. Moreover, “aerial root” structures were observed for the first time in the filopodia of THSCs using TM-AFM. These results reveal that HSC transdifferentiation to a myofibroblastic-like cell (activated HSC) from thioacetamide-induced rat HSC induces extensive changes in the cytoskeleton.     

Identification and biochemical characterization of a unique Mn2+-dependent UMP kinase from Helicobacter pylori

Uridine monophosphate (UMP) kinase converts UMP to the corresponding UDP in the presence of metal ions and ATP and is allosterically regulated by nucleotides such as UTP and GTP. Although the UMP kinase reported to date is Mg²⁺-dependent, we found in this study that the UMP kinase of Helicobacter pylori had a preference for Mn²⁺ over Mg²⁺, which may be related to a conformational difference between the Mn²⁺-bound and Mg²⁺-bound UMP kinase. Similar to previous findings, the UMP kinase activity of H. pylori UMP kinase was inhibited by UTP and activated by GTP. However, a relatively low GTP concentration (0.125 mM) was required to activate H. pylori UMP kinase to a level similar to other bacterial UMP kinases using a higher GTP concentration (0.5 mM). In addition, depending on the presence of either Mg²⁺ or Mn²⁺, a significant difference in the level of GTP activation was observed. It is therefore hypothesized that the Mg²⁺-bound and Mn²⁺-bound H. pylori UMP kinase may be activated by GTP through different mechanisms.     

BRIZ1 and BRIZ2 Proteins Form a Heteromeric E3 Ligase Complex Required for Seed Germination and Post-germination Growth in Arabidopsis thaliana

Ubiquitin pathway E3 ligases are an important component conferring specificity and regulation in ubiquitin attachment to substrate proteins. The Arabidopsis thaliana RING (Really Interesting New Gene) domain-containing proteins BRIZ1 and BRIZ2 are essential for normal seed germination and post-germination growth. Loss of either BRIZ1 (At2g42160) or BRIZ2 (At2g26000) results in a severe phenotype. Heterozygous parents produce progeny that segregate 3:1 for wild-type:growth-arrested seedlings. Homozygous T-DNA insertion lines are recovered for BRIZ1 and BRIZ2 after introduction of a transgene containing the respective coding sequence, demonstrating that disruption of BRIZ1 or BRIZ2 in the T-DNA insertion lines is responsible for the observed phenotype. Both proteins have multiple predicted domains in addition to the RING domain as follows: a BRAP2 (BRCA1-Associated Protein 2), a ZnF UBP (Zinc Finger Ubiquitin Binding protein), and a coiled-coil domain. In vitro, both BRIZ1 and BRIZ2 are active as E3 ligases but only BRIZ2 binds ubiquitin. In vitro synthesized and purified recombinant BRIZ1 and BRIZ2 preferentially form hetero-oligomers rather than homo-oligomers, and the coiled-coil domain is necessary and sufficient for this interaction. BRIZ1 and BRIZ2 co-purify after expression in tobacco leaves, which also requires the coiled-coil domain. BRIZ1 and BRIZ2 coding regions with substitutions in the RING domain are inactive in vitro and, after introduction, fail to complement their respective mutant lines. In our current model, BRIZ1 and BRIZ2 together are required for formation of a functional ubiquitin E3 ligase in vivo, and this complex is required for germination and early seedling growth.     

Distinct Roles for Dectin-1 and TLR4 in the Pathogenesis of Aspergillus fumigatus Keratitis

Aspergillus species are a major worldwide cause of corneal ulcers, resulting in visual impairment and blindness in immunocompetent individuals. To enhance our understanding of the pathogenesis of Aspergillus keratitis, we developed a murine model in which red fluorescent protein (RFP)-expressing A. fumigatus (Af293.1RFP) conidia are injected into the corneal stroma, and disease progression and fungal survival are tracked over time. Using Mafia mice in which c-fms expressing macrophages and dendritic cells can be induced to undergo apoptosis, we demonstrated that the presence of resident corneal macrophages is essential for production of IL-1β and CXCL1/KC, and for recruitment of neutrophils and mononuclear cells into the corneal stroma. We found that β-glucan was highly expressed on germinating conidia and hyphae in the cornea stroma, and that both Dectin-1 and phospho-Syk were up-regulated in infected corneas. Additionally, we show that infected Dectin-1^−/− corneas have impaired IL-1β and CXCL1/KC production, resulting in diminished cellular infiltration and fungal clearance compared with control mice, especially during infection with clinical isolates expressing high β-glucan. In contrast to Dectin 1^−/− mice, cellular infiltration into infected TLR2^−/−, TLR4^−/−, and MD-2^−/− mice corneas was unimpaired, indicating no role for these receptors in cell recruitment; however, fungal killing was significantly reduced in TLR4^−/− mice, but not TLR2^−/− or MD-2^−/− mice. We also found that TRIF^−/− and TIRAP^−/− mice exhibited no fungal-killing defects, but that MyD88^−/− and IL-1R1^−/− mice were unable to regulate fungal growth. In conclusion, these data are consistent with a model in which β-glucan on A.fumigatus germinating conidia activates Dectin-1 on corneal macrophages to produce IL-1β, and CXCL1, which together with IL-1R1/MyD88-dependent activation, results in recruitment of neutrophils to the corneal stroma and TLR4-dependent fungal killing.     

In vitro micropropagation and ex vitro acclimation of Bupleurum kaoi-an endangered medicinal plant native to Taiwan

Summary This study reports an improved protocol for in vitro-shoot multiplication and ex vitro acclimation of Bupleurum kaoi, an endangered medicinal herb. Nodal segments were cultured in half-strength Murashige and Skoog (MS) basal medium supplemented with different concentrations of benzyladenine (BA) and kinetin. The presence of 0.25 mg l⁻¹ BA induced the highest number of shoots per explant after 8 wk of culture. Although BA was more effective than kinetin on shool multiplication, it induced hyperhydric shoots at all concentrations tested. The use of dispense paper (DP) instead of aluminum foil (AF) for container closure was found to reduce hyperhydricity and improve ex vitro acclimation. The best survival rate (61%) was obtained when plantlets were grown in MS basal medium containing 0.5 mg l⁻¹ indole-3-butyric acid and 0.1–0.2 mg l⁻¹ α-naphthaleneacetic acid using DP as container closure. Leaves of the plant treated with AF6 (two layers of AF as container closure and 6 wk of incubation) lacked epicuticular wax and possessed larger stomata, higher stomata density, and fewer functional stomata compared to those of plants treated with AF2+DP4 (two layers of AF for 2 wk, then replaced AF by three layers of DP for 4wk) and ex vitro-acclimated plantlets.     

Alveolar diffusion-perfusion interactions during high-altitude residence in guinea pigs.

We previously reported in weanling guinea pigs raised at high altitude (HA; 3,800 m) an elevated lung diffusing capacity estimated by morphometry from alveolar-capillary surface area, harmonic mean blood-gas barrier thickness, and pulmonary capillary blood volume (Vc) compared with litter-matched control animals raised at an intermediate altitude (IA; 1,200 m) (Hsia CCW, Polo Carbayo JJ, Yan X, Bellotto DJ. Respir Physiol Neurobiol 147: 105-115, 2005). To determine if HA-induced alveolar ultrastructural changes are associated with improved alveolar function, we measured lung diffusing capacity for carbon monoxide (DLCO), membrane diffusing capacity for carbon monoxide (DMCO), Vc, pulmonary blood flow, and lung volume by a rebreathing technique in litter-matched male weanling Hartley guinea pigs raised at HA or IA for 4 or 12 mo. Separate control animals were also raised and studied at sea level (SL). Resting measurements were obtained in the conscious nonsedated state. In HA animals compared with corresponding IA or SL controls, lung volume and hematocrit were significantly higher while pulmonary blood flow was lower. At a given pulmonary blood flow, DLCO and DMCO were higher in HA-raised animals than in control animals without a significant change in Vc. We conclude that 1) HA residence enhanced physiological diffusing capacity corresponding to that previously estimated on the basis of structural adaptation, 2) adaptation in diffusing capacity and its components should be interpreted with respect to pulmonary blood flow, and 3) this noninvasive rebreathing technique could be used to follow adaptive responses in small animals.     

DNA microarray analysis of region-specific gene expression in the mouse epididymis

Microarray analysis was carried out to identify genes with enriched expression in the initial segment region of the mouse epididymis. A set of approximately 15 000 clones developed at the National Institutes for Aging and consisting of expressed sequence tags (ESTs) derived from pre- and peri-implantation embryos, Embryonic Day 12.5 female gonad/mesonephros, and newborn ovary were hybridized with probes generated against the initial segment (epididymal region 1) and the remainder of the epididymis (epididymal regions 2-5). The median values for the normalized ratios of region 1 to regions 2-5 from three independent experiments were averaged for each gene/EST using Genespring 5.0 software. The majority of clones showed a ratio of 1.0, suggesting they were expressed at similar levels in all epididymal regions. In addition, 123 clones exhibited 2-fold or higher expression in the initial segment, including Cres3, prostein, lipocalin 2, ALEX3, synaptotagmin-like 4, erm, and milk fat globule factor, whereas 216 clones, including elafin-like 1, lactotransferrin, Sin3B, zinc-finger protein 91, and membrane-type frizzled-related protein, showed 2-fold or higher expression in epididymal regions 2-5. Northern blot analyses of 12 clones predicted by microarray analysis to be either enriched in the initial segment (n = 8), enriched in epididymal regions 2-5 (n = 2), or similar in all regions (n = 2) were carried out. All clones exhibited the expected region-specific expression, thus confirming the microarray results. The studies presented here show a global survey of region-specific gene expression in the epididymis, identifying 15287 sequences, the majority of which have not previously been shown to be expressed in this organ.     

Genome sequence of Chlamydophila caviae (Chlamydia psittaci GPIC): examining the role of niche-specific genes in the evolution of the Chlamydiaceae

The genome of Chlamydophila caviae (formerly Chlamydia psittaci, GPIC isolate) (1 173 390 nt with a plasmid of 7966 nt) was determined, representing the fourth species with a complete genome sequence from the Chlamydiaceae family of obligate intracellular bacterial pathogens. Of 1009 annotated genes, 798 were conserved in all three other completed Chlamydiaceae genomes. The C.caviae genome contains 68 genes that lack orthologs in any other completed chlamydial genomes, including tryptophan and thiamine biosynthesis determinants and a ribose-phosphate pyrophosphokinase, the product of the prsA gene. Notable amongst these was a novel member of the virulence-associated invasin/intimin family (IIF) of Gram-negative bacteria. Intriguingly, two authentic frameshift mutations in the ORF indicate that this gene is not functional. Many of the unique genes are found in the replication termination region (RTR or plasticity zone), an area of frequent symmetrical inversion events around the replication terminus shown to be a hotspot for genome variation in previous genome sequencing studies. In C.caviae, the RTR includes several loci of particular interest including a large toxin gene and evidence of ancestral insertion(s) of a bacteriophage. This toxin gene, not present in Chlamydia pneumoniae, is a member of the YopT effector family of type III-secreted cysteine proteases. One gene cluster (guaBA-add) in the RTR is much more similar to orthologs in Chlamydia muridarum than those in the phylogenetically closest species C.pneumoniae, suggesting the possibility of horizontal transfer of genes between the rodent-associated Chlamydiae. With most genes observed in the other chlamydial genomes represented, C.caviae provides a good model for the Chlamydiaceae and a point of comparison against the human atherosclerosis-associated C.pneumoniae. This crucial addition to the set of completed Chlamydiaceae genome sequences is enabling dissection of the roles played by niche-specific genes in these important bacterial pathogens.     

Applying the Taguchi robust design to the optimization of the asymmetric reduction of ethyl 4-chloro acetoacetate by bakers' yeast

This study examined the characteristics and operational parameters of the asymmetric reduction of ethyl 4-chloro acetoacetate by bakers’ yeast in order to produce S-4-chloro-3-hydroxybutyric acid ethyl ester. Eight operational variables were also optimized using the Taguchi method with consideration of the freshness of yeast cells as a noise factor. An L₁₈ orthogonal array was used to design the experiments. The reaction yield and the product's optical purity were considered as two product quality variables. A desirability function was applied to combine these two qualities as a single objective function. Additionally, the signal-to-noise (SN) ratio was used to estimate the variability in product quality. Optimization was undertaken not only to yield the best performance, but also to minimize the variation in quality. The confirmation experiments indicated that the reaction performance and the robustness of the product quality under the optimized conditions were higher than those obtained in other experiments in this study. Our results further demonstrate that the product's optical purity could be increased to >95% by adjusting the operational level of the main factors.     

Nuclear localization of basic fibroblast growth factor is mediated by heparan sulfate proteoglycans through protein kinase C signaling

Understanding the process of wound healing will provide valuable insight for the development of new strategies to treat diseases associated with improper regeneration, such as blindness induced by corneal scarring. Heparan sulfate proteoglycans (HSPG) are not normally expressed in the corneal stroma, but their presence at sites of injury suggests their involvement in the wound healing response. Primary cultured corneal stromal fibroblasts constitutively express HSPG and represent an injured phenotype. Recently, nuclear localization of HSPG was shown to increase in corneal stromal fibroblasts plated on fibronectin (FN), an extracellular matrix protein whose appearance in the corneal stroma correlates with injury. One possible role for the nuclear localization of HSPG is to function as a shuttle for the nuclear transport of heparin-binding growth factors, such as basic fibroblast growth factor (FGF-2). Once in the nucleus, these growth factors might directly modulate cellular activities. To investigate this hypothesis, cells were treated with (125)I-labelled FGF-2 under various conditions and fractionated. Our results show that nuclear localization of FGF-2 was increased in cells plated on FN compared to those on collagen type I (CO). Interestingly, FGF-2-stimulated proliferation was increased in cells plated on FN compared to CO and this effect was absent in the presence of heparinase III. Furthermore, pre-treatment with heparinase III decreased nuclear FGF-2, and CHO cells defective in the ability to properly synthesize heparan sulfate chains showed reduced nuclear FGF-2 indicating that the heparan sulfate chains of HSPG are critical for this process. HSPG signaling, particularly through the cytoplasmic tails of syndecans, was investigated as a potential mechanism for the nuclear localization of FGF-2. Treatment with phorbol 12-myristate-13-acetate (PMA), under conditions that caused downregulation of protein kinase Calpha (PKCalpha), decreased nuclear FGF-2. Using pharmacological inhibitors of specific PKC isozymes, we elucidated a potential mode of regulation whereby PKCalpha mediates the nuclear localization of FGF-2 and PKCdelta inhibits it. Our studies suggest a novel mechanism in which FGF-2 translocates to the nucleus in response to injury.