Notes on Technic

Generally accepted staining methods using hematoxylin for identification of elastic tissues include Verhoeffs iron-hematoxylin (Verhoeff 1908), Herx-heimer's lithium carbonate-hematoxylin (Herxheimer 1886), Harris' aluminum chloride-hematoxylin (Harris 1902), and Petry's phosphomolybdic acid-hematoxylin (Pearse 1975). Since alum hematoxylin shows little or no affinity for elastic fibers, it is not used for staining elastic fibers.     

Gene affecting longevity of messenger RNA: a mutant of Escherichia coli with altered mRNA stability.

Summary we have screened 897 temperature sensitive growth mutants ofE. coli for mutant strains showing longer mRNA half-life. The fate of pulse-labelled RNA was examined at 42° C after cessation of RNA synthesis and with prior exposure to nonpermissive temperature (42° C). Eight stains showed altered turn-over of RNA (presumably mRNA), and further analysis on mutant strain JE15144 indicated that the stability of pulse-labeled RNA as well as of tryptophan (trp) mRNA increased four to seven fold over its parental strain at 42° C. At 4 min or 10 min after addition of rifampicin, some 70 to 80% of polyribosome in the growing cells could still be conserved in JE15144 cultured at the nonpermissive temperature while little, if any, polyribosomes remained in its parental strain (PA3092) under the same condition. Two generation times were required for complete stoppage of growth of this mutant strain after shifting to 42° C, and protein synthesis continued at a significant, but slightly reduced, rate at 42° C. However, functional decay of mRNA in the mutant strain, with respect to the capacity for producing peptides, appeared to be similar to the parent strain, with half-lives of 3.5 min in PA3092 and 4.7 min in JE15144.     

Activation mechanism of c-Jun amino-terminal kinase in the course of neural differentiation of P19 embryonic carcinoma cells

P19 embryonic carcinoma cells, a model system for studying early development and differentiation, can differentiate into neurons and primitive endoderm-like cells depending on the culture conditions. We have previously reported that the activation of c-Jun amino-terminal kinase (JNK) is required for the retinoic acid-induced neural differentiation of P19 cells. However, the signaling pathway(s) responsible for the activation of JNK has not been known. In this study, we demonstrated that activities of MAPK kinase 4 (MKK4) and TAK1, one of the upstream kinases of MKK4, were enhanced in the neurally differentiating cells. Inhibition of the neural differentiation by an overexpression of protein phosphatase 2Cepsilon, an inactivator of TAK1, suggested a critical role of the TAK1 signaling pathway during the differentiation. Confocal microscopic analysis indicated that TAK1, phospho-MKK4, and phospho-JNK were colocalized with tubulin in the neurites and localized also in the nuclei of the differentiating cells. In contrast, two TAK1-binding proteins, TAB1 and TAB2, which are involved in the activation of TAK1, were localized in the neurites and the nuclei of the differentiating cells, respectively. These results suggest that two distinct TAK1-MKK4-JNK signaling pathways are independently activated at the different intracellular locations and may participate in the regulation of the neural differentiation of P19 cells.     

Simplified sample preparation using frame spotting method for direct counting of total bacteria by fluorescence microscopy

A new preparation method for direct counting of bacteria in liquid samples with fluorescence microscope was developed using a glass slide coated with 3-aminopropyltriethoxy silane and ring-shaped polyester seal as a retainer. The experimental steps of this method were spotting samples onto the coated slides with the seal, drying under vacuum, staining with SYBR Green II, drying and covering with immersion oil and coverslip to allow counting. This simplified method provided consistent results when compared with the conventional filtration method for fluorescence microscopy, and is rapid, inexpensive and reproducible.     

Role of NMDA Receptors in Pentobarbital Tolerance/Dependence

Effects of continuous pentobarbital administration on binding characteristics of [³H]MK-801 in the rat brain were examined by autoradiography. Animals were rendered tolerant to pentobarbital using i.c.v. infusion of pentobarbital (300g/10l/hr for 7 days) by osmotic minipumps and dependent by abrupt withdrawal from pentobarbital. The levels of [³H]MK-801 binding were elevated in rats 24-hr after withdrawal from pentobarbital while there were no changes except in septum and anterior ventral nuclei in tolerant rats. For assessing the role of NMDA receptor in barbiturate action, an NMDA receptor antagonist (MK-801, 2.7 femto g/10l/hr) was co-infused with pentobarbital. The pentobarbital-infused group had a shorter duration of pentobarbital-induced loss of righting reflex (sleeping time) than that of the control group, and MK-801 alone did not affect the righting reflex. However, co-infusion of MK-801 blocked hyperthermia, and prolonged the onset of convulsions induced by t-butylbicyclophosphorothionate (TBPS) in pentobarbital withdrawal rats. In addition, elevated [³⁵S]TBPS binding was significantly attenuated by co-infusion with MK-801. These results suggest the involvement of NMDA receptor up-regulation in pentobarbital withdrawal and that the development of dependence can be attenuated by the treatment of subtoxic dose of MK-801.     

The plastid aldolase gene fromChlamydomonas reinhardtii: Intron/exon organization,evolution, and promoter structure

Genomic clones encoding the plastidic fructose- 1,6-bisphosphate aldolase ofChlamydomonas reinhardtii were isolated and sequenced. The gene contains three introns which are located within the coding sequence for the mature protein. No introns are located within or near the sequence encoding the transit-peptide, in contrast to the genes for plastidic aldolases of higher plants. Neither the number nor the positions of the three introns of theC. reinhardtii aldolase gene are conserved in the plastidic or cytosolic aldolase genes of higher plants and animals. The 5′ border sequences of introns in the aldolase gene ofC. reinhardtii exhibit the conserved plant consensus sequence. The 3′ acceptor splice sites for introns 1 and 3 show much less similarity to the eukaryotic consensus sequences than do those of intron 2. The plastidic aldolase gene has two tandemly repeated CAAT box motifs in the promoter region. Genomic Southern blots indicate that the gene is encoded by a single locus in theC. reinhardtii genome.     

Haplotype analysis of familial amyloidotic polyneuropathy

Summary Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant genetic disease characterized by systemic accumulation of amyloid fibrils. A major component of FAP anyloid has been identified as variant transthyretin (TTR, also called prealbumin). In particular, a variant with the substitution ³⁰ValMet has been commonly found in FAP of various ethnic groups. To understand the origin and spread of the ValMet mutation, we analyzed DNA polymorphisms associated with the TTR gene in six Japanese FAP families and several Portuguese FAP patients. Three distinct haplotypes associated with the ValMet mutation were identified in Japanese FAP families, one of which was also found in Portuguese patients. On the other hand, it was found that the ValMet mutation can be explained by a C-T transition at the C_pG dinucleotide sequence of a mutation hot spot. Thus, our findings indicate that the ValMet mutation has probably recurred in the human population, to generate FAP families of independent origin.     

Intra-articular hyaluronic acid injection versus oral non-steroidal anti-inflammatory drug for the treatment of knee osteoarthritis: a multi-center,randomized, open-label,non-inferiority trial

Introduction

While many of the commonly used conservative treatments for knee osteoarthritis (OA) have been recognized to be effective, there is still insufficient evidence available. Among the pharmacological treatments for knee OA, oral non-steroidal anti-inflammatory drugs (NSAIDs) act rapidly and are recommended for the management of OA. However, frequent and serious adverse effects of NSAIDs have been recognized. Intra-articular injections of hyaluronic acid (IA-HA) for the treatment of knee OA have been shown to reduce pain and improve joint function. However, there has been no qualified direct comparison study of the efficacy and safety between IA-HA and NSAIDs for patients with knee OA. The aim of this study was to clarify the efficacy and safety of early-phase IA-HA in comparison to those of NSAIDs for patients with knee OA.

Methods

This multicenter, randomized, open-label, parallel-group, non-inferiority comparison study with an oral NSAID involved a total of 200 patients with knee OA. An independent, computer-generated randomization sequence was used to randomly assign patients in a 1:1 ratio to NSAIDs three times per day for five weeks (n = 100) or IA-HA once a week for five weeks (n = 100). The primary endpoint was the percentage change in the patient-oriented outcome measure for knee OA, the Japanese Knee Osteoarthritis Measure (JKOM) score. All patients were questioned regarding any adverse events during treatment. The full analysis set (FAS) was used for analysis. The margin of non-inferiority was 10%.

Results

The analyses of primary endpoint included 98 patients in the IA-HA group and 86 patients in the NSAID group. The difference in the percentage changes of the JKOM score between the two intervention arms (IA-HA; -34.7% (P<0.001), NSAID; -32.2% (P<0.001)) was -2.5% (95% confidence interval (CI): -14.0 to 9.1), indicating IA-HA was not inferior to NSAID. The frequency of both withdrawal and adverse events in the IA-HA group were significantly lower than those in the NSAID group (P = 0.026 and 0.004, respectively).

Conclusions

The early efficacy of IA-HA is suggested to be not inferior to that of NSAIDs, and that the safety of the early phase of IA-HA is superior to that of NSAIDs for patients with knee OA.

Trial registration

UMIN Clinical Trials Registry (UMIN-CTR), UMIN000001026.     

Physiological changes in gentian axillary buds during two-step preculturing with sucrose that conferred high levels of tolerance to desiccation and cryopreservation

BACKGROUND AND AIMS: Induction of dehydration tolerance is a key to achieving high survival rates in cryopreservation of plant specimens. It has been reported previously that two-step preculturing with sucrose effectively increased desiccation tolerance in axillary buds of gentian (Gentiana scabra), which allow the buds to survive cryopreservation. This study is aimed at characterizing each step of this preculturing and to elucidate physiological changes induced during this preculturing. METHODS: In standard two-step preculture, excised gentian axillary buds were incubated for 11 d on MS medium with 0.1 m sucrose at 25 degrees C (first step: mild osmotic stress was given) and the subsequent incubation on MS medium with 0.4 m and 0.7 m sucrose for 1 d each (second step). The levels of abscisic acid (ABA), proline and soluble sugars in gentian buds during the preculture were determined. Effects of various combinations of two-step preculturing and of exogenous ABA and proline were studied. KEY RESULTS: During the first preculture step, there was a transient increase in ABA content peaking on day 4, which declined to a background level at the end of the first and second step preculturing. Proline level increased steadily during the first preculture step and increased further in the second preculture step. Incubating buds with medium containing proline, instead of the two-step preculturing, did not allow them to survive desiccation. Incubating buds with ABA instead of 0.1 m sucrose-preculturing effectively increased desiccation tolerance only when it was followed by the second preculture step. Fluridone, an ABA synthesis inhibitor included in the two-step preculture medium, reduced desiccation tolerance of the buds. The normal first-step preculture increased the levels of soluble sugars 2.4-fold, especially sucrose and raffinose. Buds treated with the second preculture step had greatly increased sucrose levels. CONCLUSIONS: These observations lead to the hypothesis that the first preculture step involves ABA-mediated cellular changes and the second step induces loading of sucrose in the gentian buds.     

Chondroitin Sulfate Synthase-2/Chondroitin Polymerizing Factor Has Two Variants with Distinct Function

Chondroitin sulfate (CS) is a polysaccharide consisting of repeating disaccharide units of N-acetyl-d-galactosamine and d-glucuronic acid residues, modified with sulfated residues at various positions. To date six glycosyltransferases for chondroitin synthesis have been identified, and the complex of chondroitin sulfate synthase-1 (CSS1)/chondroitin synthase-1 (ChSy-1) and chondroitin sulfate synthase-2 (CSS2)/chondroitin polymerizing factor is assumed to play a major role in CS biosynthesis. We found an alternative splice variant of mouse CSS2 in a data base that lacks the N-terminal transmembrane domain, contrasting to the original CSS2. Here, we investigated the roles of CSS2 variants. Both the original enzyme and the splice variant, designated CSS2A and CSS2B, respectively, were expressed at different levels and ratios in tissues. Western blot analysis of cultured mouse embryonic fibroblasts confirmed that both enzymes were actually synthesized as proteins and were localized in both the endoplasmic reticulum and the Golgi apparatus. Pulldown assays revealed that either of CSS2A, CSS2B, and CSS1/ChSy-1 heterogeneously and homogeneously interacts with each other, suggesting that they form a complex of multimers. In vitro glycosyltransferase assays demonstrated a reduced glucuronyltransferase activity in CSS2B and no polymerizing activity in CSS2B co-expressed with CSS1, in contrast to CSS2A co-expressed with CSS1. Radiolabeling analysis of cultured COS-7 cells overexpressing each variant revealed that, whereas CSS2A facilitated CS biosynthesis, CSS2B inhibited it. Molecular modeling of CSS2A and CSS2B provided support for their properties. These findings, implicating regulation of CS chain polymerization by CSS2 variants, provide insight in elucidating the mechanisms of CS biosynthesis.