Development of novel elongated fiber-structure in protoplast cultures of Betula platyphylia and Larix leptolepis

Summary Novel elongated fiber-structures were repeatedly found both in leaf protoplast culture of two clones of Betula platyphylla and in protoplast culture of embryogenic cells of Larix leptolepis. Suboptimum culture conditions for cell division appeared to lead to fiber formation when using multi-well plate culture with varying medium compositions The suboptimum conditions for cell divisions were brought about by (1) plant growth regulators: auxins and cytokinins; (2) pH: 3.5, 4.5, 5.8; (3) divalent cations: CaCl₂ and MgCl₂; and (4) sugars: sucrose and mannitol. Divalent cations had the most profound effect on fiber formation. Calcium ions were preferred by Betula and magnesium ions were preferred by Larix. Single fiberpurification and micro-staining methods using a micromanipulator were developed. The fibers fluoresced when stained with Calcofluor White and Aniline Blue, which suggested that they were composed of cell wall component(s), including callose (β-1,3-glucan). Electron microscopy showed that fiber bundles of Larix fibers had helical substructures.     

Influence of lipophilic groups in cationic alpha-helical peptides on their abilities to bind with DNA and deliver genes into cells.

For the purpose of achieving gene transfer into cells mediated by peptides with a short chain length, we employed two kinds of amphiphilic alpha-helix peptides, mastoparan (INLK-ALAA-LAKK-IL-NH2) obtained from wasp venom and an alpha-helix model peptide (LARL-LARL-LARL-NH2). Furthermore, to strengthen the hydrophobicity of the peptide required for the formation of the aggregates with the DNA, we modified these peptides using several lipophilic groups, i.e. acyl groups with a single chain, a dialkylcarbamoyl group and a cholesteryloxycarbonyl group. We examined the ability of the peptides and their derivatives to bind and aggregate with plasmid DNA, the structural change in the peptides caused by binding with the DNA and the in vitro gene transfer abilities into COS-7 cells. As a result, mastoparan was found to acquire the DNA binding ability by introduction of the lipophilic group. The conformational change in the peptides depended on the hydrophobicity of the introduced acyl group. The DNA complex of most lipophilic mastoparan derivatives could be incorporated into the cells via the endocytosis pathway. In the case of the helix model peptide, the acyl group with a moderate chain length was required for the formation of the aggregate which is competent for incorporation into the cells. In this study, we succeeded in giving such short peptides sufficient gene transfer ability by modifying them with some lipophilic groups. However, the influence of the modification by the lipophilic groups on the formation of aggregates with DNA and the gene transfer ability depended on the structure of the peptide portion. These results indicate that consideration of total hydrophobicity balance is needed for the design of an efficient gene carrier peptide.     

The kinesin KIF4 mediates HBV/HDV entry through the regulation of surface NTCP localization and can be targeted by RXR agonists in vitro

Intracellular transport via microtubule-based dynein and kinesin family motors plays a key role in viral reproduction and transmission. We show here that Kinesin Family Member 4 (KIF4) plays an important role in HBV/HDV infection. We intended to explore host factors impacting the HBV life cycle that can be therapeutically addressed using siRNA library transfection and HBV/NLuc (HBV/NL) reporter virus infection in HepG2-hNTCP cells. KIF4 silencing resulted in a 3-fold reduction in luciferase activity following HBV/NL infection. KIF4 knockdown suppressed both HBV and HDV infection. Transient KIF4 depletion reduced surface and raised intracellular NTCP (HBV/HDV entry receptor) levels, according to both cellular fractionation and immunofluorescence analysis (IF). Overexpression of wild-type KIF4 but not ATPase-null KIF4 mutant regained the surface localization of NTCP and significantly restored HBV permissiveness in these cells. IF revealed KIF4 and NTCP colocalization across microtubule filaments, and a co-immunoprecipitation study revealed that KIF4 interacts with NTCP. KIF4 expression is regulated by FOXM1. Interestingly, we discovered that RXR agonists (Bexarotene, and Alitretinoin) down-regulated KIF4 expression via FOXM1-mediated suppression, resulting in a substantial decrease in HBV-Pre-S1 protein attachment to HepG2-hNTCP cell surface and subsequent HBV infection in both HepG2-hNTCP and primary human hepatocyte (PXB) (Bexarotene, IC₅₀ 1.89 ± 0.98 μM) cultures. Overall, our findings show that human KIF4 is a critical regulator of NTCP surface transport and localization, which is required for NTCP to function as a receptor for HBV/HDV entry. Furthermore, small molecules that suppress or alleviate KIF4 expression would be potential antiviral candidates targeting HBV and HDV entry.     

Fucosterol decreases angiotensin converting enzyme levels with reduction of glucocorticoid receptors in endothelial cells

The modulation of angiotensin converting enzyme (ACE) levels was studied using fucosterol, one of phytosterols, in cultured bovine carotid endothelial cells. Addition of fucosterol to the culture medium resulted in the decrease of ACE activity of endothelial cells; however, fucosterol did not directly inhibit ACE activity. Dexamethasone elevated the levels of ACE in normal cells, but this effect was not seen in the fucosterol-treated cells. Receptor assays showed that the amount of glucocorticoid receptors in fucosterol-treated cells decreased to an undetectable level. These results indicate that fucosterol lowers the ACE levels on the endothelial cells by inhibiting the synthesis of glucocorticoid receptors involved in the regulation of ACE levels.     

Purification and characterization of angiotensin-binding protein from porcine liver cytosolic fraction

A protein that binds angiotensins with high affinity was found in porcine liver cytosol, purified to apparent homogeneity and characterized. The protein was named soluble angiotensin-binding protein (sABP) to distinguish it from angiotensin II receptors present on plasma membranes. Purification of the protein was achieved by a combination of ammonium sulfate fractionation, hydrophobic chromatography, ion-exchange chromatography, hydroxylapatite column chromatography and Mono Q ion-exchange chromatography. Specific angiotensin-binding activity, as measured using 125I-angiotensin II, was enriched more than 3400-fold. SDS/polyacrylamide gel electrophoresis of the purified sABP yielded a single 75-kDa protein band, in good agreement with the molecular mass estimated by affinity labeling. sABP was very similar to the angiotensin II receptor in its sensitivity to reducing agents and in its affinities for angiotensin analogues ([Sar1, Ala8]angiotensin II greater than angiotensin III greater than angiotensin II greater than angiotensin I), suggesting a possible similarity between the ligand-binding sites of sABP and the angiotensin II receptor. To obtain a clue to its physiological role(s), we examined the tissue distribution of sABP and found that this protein is widely distributed not only in the peripheral organs but also in the brain.     

Bimodal induction of sister-chromatid exchanges by luminol,an inhibitor of poly(ADP-ribose) synthetase,during the S-phase of the cell cycle

The cell cycle dependence of sister chromatid exchanges (SCEs) induced by luminol, a new potent inhibitor of poly(ADP-ribose) synthetase, was studied in Chinese hamster V79 cells. Continuous treatment with luminol during two whole cell cycles in the presence of 5-bromo-2-deoxyuridine (BrdUrd), or in the first or second cycle induced SCEs very efficiently in a linear dosedependent manner. However, no enhancement of SCE levels was observed after luminol treatment in a cycle preceding BrdUrd treatment, in contrast to results found with other strong SCE inducers such ascis-diammine-dichloroplatinum (II) (CDDP) and mitomycin C (MMC). Luminol was about ten times as effective in inducing SCEs as 3-aminobenzamide (3AB), an inhibitor of the NAD⁺ site of poly(ADP-ribose) synthetase. The induction of SCEs by luminol was restricted to the Sphase of the cell cycle with peaks at an early and a late stage, corresponding to the biphasic replication of DNA. The mechanism of SCE appears to be the same at the early and late stages of S-phase for luminol-induced SCE formation.     

Probable assignment of soluble isocitrate dehydrogenase (IDH1) to 2q33.3

Summary Gene dosage effects for soluble isocitrate dehydrogenase (IDH₁) were investigated in four unrelated cases with abnormalities involving the long arm of chromosome 2. Case 1 was trisomic for 2q33.3qter, Case 2 monosomic for 2q33.3q35, Case 3 trisomic for 2q11.2q24.2, and Case 4 monosomic for 2q23q24.2. These abnormalities were de novo except in Case 1, where trisomy 2q resulted from a maternal translocation. The red cell IDH₁ levels were significantly reduced in Cases 1 (41.4% of normal value) and 2 (51.9%), while they were normal in Cases 3 and 4. The low IDH₁ level also in the father of Case 1 (43.6%), together with the common electrophoretic phenotype of IDH₁ in red cells as well as leukocytes, led us to suppose that Case 1 was really heterozygous for common and probable null alleles, and that the IDH₁ gene locus could be excluded from 2q33.3qter. On the other hand, normal IDH₁ values in the parents of Case 2 were consistent with the hemizygosity for this locus in Case 2. The results suggested that the IDH₁ locus could be assigned to the 2q33.3 band, especially the proximal portion of it.     

Functional MRI detection of activation in the primary gustatory cortices in humans

Magnetoencephalography (MEG) has recently revealed that the transitions between the parietal operculum (Pop) and the insula (area G) and the ventral end of the central sulcus (cs) were activated with the shortest latency by instrumental gustatory stimulation, which suggests that the location of the primary gustatory area is in these two regions. However, studies using other noninvasive brain-imaging methods such as positron-emission tomography or functional magnetic resonance imaging (fMRI) with manual application of tastants into the mouth have been unable to confirm this. The present study examined cortical activation by repetitive stimulation of the tongue tip with 1 M NaCl with a computer-controlled stimulator and used fMRI to detect it. In individual brains, activations were detected with multiple comparisons (false discovery rate) across the whole brain corrected (threshold at P < 0.05) at both area G and frontal operculum (Fop) in 8 of 11 subjects and at the rolandic operculum (Rop) in 7 subjects. Activations were also found at the ventral end of the cs (n = 3). Group analysis with random-effect models (multiple comparison using familywise error in regions of interest, P < 0.02) revealed activation at area G in both hemispheres and in the Fop, Rop, and ventral end of the cs on the left side. The present study revealed no activation on the gyrus of the external cerebral surface except for the Rop. Taking MEG findings into consideration, the present findings strongly indicate that the primary gustatory area is present at both the transition between the Pop and insula and the Rop including the gray matter within a ventral part of the cs.     

Immunolocalization of proteoglycans and bone-related noncollagenous glycoproteins in developing acellular cementum of rat molars

To elucidate the roles of proteoglycans (PGs), bone sialoprotein (BSP), and osteopontin (OPN) in cementogenesis, their distribution was investigated in developing and established acellular cementum of rat molars by an immunoperoxidase method. To characterize PGs, antibodies against five species of glycosaminoglycans (GAGs), chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S), unsulfated chondroitin (C0S), dermatan sulfate (DS), and keratan sulfate (KS) were used. Routine histological staining was also applied. With onset of dentin mineralization, the initial cementum appeared on the dentin surface as a hematoxylin-stained fibril-poor layer. Subsequently, primitive principal fibers attached to the initial cementum. As the acellular cementum containing extrinsic fibers covered the initial cementum, the initial cementum formed the cemento-dentinal junction. Following immunohistochemistry at the earliest time of cementogenesis, the initial cementum was intensely immunoreactive for C4S, C6S, C0S, BSP, and OPN. After the initial cementum was embedded, neither the cemento-dentinal junction nor the cementum was immunoreactive for any GAG species. However, the cementum and cemento-dentinal junction were consistently immunoreactive for BSP. Although the cemento-dentinal junction was consistently immunoreactive for OPN, the remaining cementum showed no significant immunoreactivity. Thus, initial acellular cementogenesis requires a dense accumulation of PGs, BSP, and OPN, which may be associated with the mineralization process independently of collagen fibrils and initial principal fiber attachment.     

Effects of cholesterol in chylomicron remnant models of lipid emulsions on apoE-mediated uptake and cytotoxicity of macrophages

Chylomicron remnants have been suggested to be involved in the development of atherosclerosis. To investigate the mechanisms of chylomicron remnant-induced atherosclerosis, we prepared cholesterol (Chol)-containing emulsion particles as models for chylomicron remnants. Chol markedly increased the apolipoprotein E (apoE) binding maximum of emulsions without changing the binding affinity and thereby promoted emulsion uptake by J774 macrophages. Fluorescence measurements showed that Chol increased acyl chain order and head group hydration of the surface phospholipid (PL) layer of emulsions. The binding maximum of apoE was closely correlated with the hydration and the increase in the PL head group separation at the emulsion surface. From experiments using inhibitors for lipoprotein receptors, heparan sulfate proteoglycans and low density lipoprotein receptor-related protein were found to be the major contributors to the uptake of Chol-containing emulsions. Trypan blue dye exclusion revealed that the uptake of Chol-containing emulsions induced cytotoxicity to J774 macrophages. This study proposes a mechanism of atherosclerosis induced by chylomicron remnants.