Phellilane L, Sesquiterpene Metabolite of Phellinus linteus: Isolation, Structure Elucidation, and Asymmetric Total Synthesis.

A new cyclopropane-containing sesquiterpenoid, phellilane L (1), was isolated from the medicinal mushroom Phellinus linteus ("Meshimakobu" in Japanese), a member of the Hymenochaetaceae family and a well-known fungus that is widely used in East Asia. The planar structure of 1 was determined on the basis of spectroscopic analysis. The authors achieved the first total synthesis of 1. Our protecting group-free synthesis features a highly stereoselective one-pot synthesis involving an intermolecular alkylation/cyclization/lactonization strategy for construction of the key cyclopropane-γ-lactone intermediate. Additionally, our synthesis determined the absolute configuration of phellilane L (1).

Corrigendum: γ-Ionylidene-type sesquiterpenoids possessing antimicrobial activity against Porphyromonas gingivalis from Phellinus linteus and their absolute structure determination.

This corrects the article DOI: 10.1038/ja.2017.35.

Isolation, structure determination, and anti-HIV evaluation of tigliane-type diterpenes and biflavonoid from Stellera chamaejasme.

Five novel tigliane-type diterpenes, stelleracins A-E (3-7), a novel flavanone dimer, chamaeflavone A (8), and six known compounds were isolated from the roots of Stellera chamaejasme. Their structures were elucidated by extensive spectroscopic analyses. The isolated compounds were evaluated for anti-HIV activity in MT4 cells. New compounds 3-5 showed potent anti-HIV activity (EC90 0.00056-0.0068 µM) and relatively low or no cytotoxicity (IC50 4.4-17.2 µM). These new compounds represent promising new leads for development into anti-AIDS clinical trial candidates.

Labdane-type diterpenoids from hairy root cultures of Coleus forskohlii, possible intermediates in the biosynthesis of forskolin.

Significant attention has been devoted to studying hairy root cultures as a promising strategy for production of various valuable secondary metabolites. These offer many advantages, such as high growth rate, genetic stability and being hormone-free. In this study, a detailed phytochemical investigation of the secondary metabolites of Coleus forskohlii hairy root cultures was undertaken and which resulted in the isolation of 22 compounds, including four forskolin derivatives and a monoterpene. Their structures were elucidated by extensive spectroscopic analyses. These compounds could be classified into four groups viz.: labdane-type diterpenes, monoterpenes, triterpenes and phenylpropanoid dimers. Apart from one compound, all labdane type diterpenes are oxygenated at C-11 as in forskolin and a scheme showing their biosynthetic relationships is proposed.

Stelleralides A-C, novel potent anti-HIV daphnane-type diterpenoids from Stellera chamaejasme L.

Three novel 1-alkyldaphnane-type diterpenes, stelleralides A-C (4-6), and five known compounds were isolated from the roots of Stellera chamaejasme L. The structures of 4-6 were elucidated by extensive spectroscopic analyses. Several isolated compounds showed potent anti-HIV activity. Compound 4 showed extremely potent anti-HIV activity (EC(90) 0.40 nM) with the lowest cytotoxicity (IC(50) 4.3 µM) and appears to be a promising compound for development into anti-AIDS clinical trial candidates.

Disc regeneration therapy using marrow mesenchymal cell transplantation: a report of two case studies.

STUDY DESIGN: Marrow mesenchymal cells (MSCs) contain stem cells and possess the ability to regenerate bone, cartilage, and fibrous tissues. Here, we applied this regenerative ability to intervertebral disc regeneration therapy in an attempt to develop a new spinal surgery technique. OBJECTIVE: We analyzed the regenerative restoration ability of autologous MSCs in the markedly degenerated intervertebral discs. SUMMARY OF BACKGROUND DATA: Fusion for lumbar intervertebral disc instability improves lumbago. However, fused intervertebral discs lack the natural and physiologic functions of intervertebral discs. If intervertebral discs can be regenerated and repaired, then damage to adjacent intervertebral discs can be avoided. We verified the regenerative ability of MSCs by animal studies, and for the first time, performed therapeutic intervertebral disc regeneration therapy in patients and obtained favorable findings. METHODS: Subjects were 2 women aged 70 and 67 years; both patients had lumbago, leg pain, and numbness. Myelography and magnetic resonance imaging showed lumbar spinal canal stenosis, and radiograph confirmed the vacuum phenomenon with instability. From the ilium of each patient, marrow fluid was collected, and MSCs were cultured using the medium containing autogenous serum. In surgery, fenestration was performed on the stenosed spinal canal and then pieces of collagen sponge containing autologous MSCs were grafted percutaneously to degenerated intervertebral discs. RESULTS: At 2 years after surgery, radiograph and computed tomography showed improvements in the vacuum phenomenon in both patients. On T2-weighted magnetic resonance imaging, signal intensity of intervertebral discs with cell grafts was high, thus indicating high moisture contents. Roentgenkymography showed that lumbar disc instability improved. Symptom was alleviated in both patients. CONCLUSION: The intervertebral disc regeneration therapy using MSC brought about favorable results in these 2 cases. It seems to be a promising minimally invasive treatment.

Pokeweed antiviral protein region Gly209-Lys225 is critical for RNA N-glycosidase activity of the prokaryotic ribosome.

Pokeweed antiviral protein (PAP) isolated from Phytolacca americana is a ribosome-inactivating protein (RIP) that has RNA N-glycosidase (RNG) activity towards both eukaryotic and prokaryotic ribosomes. In contrast, karasurin-A (KRN), a RIP from Trichosanthes kirilowii var. japonica, is active only on eukaryotic ribosomes. Stepwise selection of chimera proteins between PAP and KRN indicated that the C-terminal region of PAP (residues 209-225) was critical for RNG activity toward prokaryotic ribosomes. When the region of PAP (residues 209-225) was replaced with the corresponding region of KRN the PAP chimera protein, like KRN, was active only on eukaryotic ribosomes. Furthermore, insertion of the region of PAP (residues 209-225) into the KRN chimera protein resulted not only in the detectable RNG activity toward prokaryotic ribosome, but also activity toward the eukaryotic ribosomes as well that was seven-fold higher than for the original KRN. In this study, the possibility of genetic manipulation of the activity and substrate specificity of RIPs is demonstrated.

Wound therapy by marrow mesenchymal cell transplantation.

BACKGROUND: Marrow mesenchymal cells are useful in regenerative medicine because they contain stem cells, but there have been few reports of clinical applications. The authors developed a new wound treatment technique by improving marrow mesenchymal cell culture methods and placing cultured cells in an artificial skin material. This new treatment was useful for tissue regeneration in 20 patients with skin wounds. METHODS: Marrow mesenchymal cells from a 46-year-old man were cultured and placed in artificial dermis made of collagen sponge. This composite graft was implanted subcutaneously into the back of a nude mouse and removed 10 days later; immunohistological analysis confirmed regeneration of subcutaneous tissue using human marrow mesenchymal cells. Next, in 20 patients (nine men and 11 women; average age, 64.8 years; range, 22 to 91 years) with intractable dermatopathies, 10 to 20 ml of bone marrow fluid was aspirated from the ilium and cultured in medium containing either fetal calf or autologous serum. The resulting cultured cells were placed in artificial dermis made of collagen sponge, and this composite graft was used to treat skin wounds. RESULTS: The wound mostly healed in 18 of the 20 patients; the remaining two patients died of causes unrelated to transplantation. In all patients, autologous marrow mesenchymal cell transplantation was shown to be therapeutically effective. CONCLUSIONS: In skin regeneration therapy using a marrow mesenchymal cell/artificial dermis composite graft, skin regeneration is possible with bone marrow aspiration, a minimally invasive procedure. Compared with existing skin grafting techniques, the present technique is practical and much less invasive.

Cloning and functional characterization of Arabidopsis thaliana D-amino acid aminotransferase--D-aspartate behavior during germination.

The understanding of D-amino acid metabolism in higher plants lags far behind that in mammals, for which the biological functions of these unique amino acids have already been elucidated. In this article, we report on the biochemical behavior of D-amino acids (particularly D-Asp) and relevant metabolic enzymes in Arabidopsis thaliana. During germination and growth of the plant, a transient increase in D-Asp levels was observed, suggesting that D-Asp is synthesized in the plant. Administration of D-Asp suppressed growth, although the inhibitory mechanism responsible for this remains to be clarified. Exogenous D-Asp was efficiently incorporated and metabolized, and was converted to other D-amino acids (D-Glu and D-Ala). We then studied the related metabolic enzymes, and consequently cloned and characterized A. thaliana D-amino acid aminotransferase, which is presumably involved in the metabolism of D-Asp in the plant by catalyzing transamination between D-amino acids. This is the first report of cDNA cloning and functional characterization of a D-amino acid aminotransferase in eukaryotes. The results presented here provide important information for understanding the significance of D-amino acids in the metabolism of higher plants.

Suppressive effect of alpha2 Heremans-Schmid glycoprotein on in vitro calcification of osteogenesis.

BACKGROUND: alpha(2) Heremans-Schmid glycoprotein (alpha(2)HS glycoprotein) is predominantly found in bone. To date, we have investigated plasma alpha(2)HS levels in immature babies and neonates as well as the histological distribution in various neonatal tissues in order to clarify its physiological significance. In an effort to understand the physiological function of alpha(2)HS glycoprotein in bones, we studied the effects of alpha(2)HS glycoprotein in cultured osteogenesis model using rat marrow cells. METHODS: We added different concentrations of alpha(2)HS glycoprotein to cultured marrow cells, including osteoblasts in the presence of dexamethasone, in an attempt to elucidate the effects of alpha(2)HS glycoprotein on osteoblast growth and bone calcification in vitro. RESULTS: The results showed that total DNA content was significantly increased with 0.2-20 nM (f.c.) alpha(2)HS glycoprotein, but was neither suppressed nor increased with 200 nM (f.c.) alpha(2)HS glycoprotein. Although ALP activity increased with 0.2 or 2 nM (f.c.) alpha(2)HS glycoprotein, it decreased with 20 or 200 nM (f.c.) alpha(2)HS glycoprotein. While 0.2 nM (f.c.) alpha(2)HS glycoprotein had no effect on calcium or osteocalcin content, 2 nM (f.c.) alpha(2)HS glycoprotein decreased both calcium content and osteocalcin content by about half, and no calcium or osteocalcin was observed with 20 or 200 nM (f.c.). Calcium staining of cultured marrow cells revealed that the number of stained cell tubercles decreased in a concentration-dependent manner. CONCLUSION: These findings suggest that alpha(2)HS glycoprotein regulates the growth of osteoblasts and acts as an inhibitory factor in the regulation of bone calcification.

Bone formation-promoting effect of genistein on marrow mesenchymal cell culture.

The effect of genistein, a soybean isoflavone, on new bone formation by bone marrow cells from mature rats and humans was examined. Bone marrow cells were collected from the femoral diaphysis of 7-week-old Fisher rats, cultured in MEM containing fetal calf serum and then cultured with or without the addition of dexamethasone to the bone-forming medium. Genistein was added at concentrations of 10(-5),10(-6),10(-7) or 10(-8) M. Bone formation was examined 2 weeks after culture. After informed consent was obtained from a 55-year-old woman with lumbar spondylosis deformans, bone marrow cells were collected from her ilium for culture by the same process, and bone formation investigated. In both rats and humans, when dexamethasone was added to the bone-forming medium, genistein (10(-7) M and 10(-8) M) caused a significant increase in the levels of calcium, alkaline phosphatase, and DNA compared with cells not cultured in genistein. In conclusion, genistein was found to promote bone formation at lower concentrations across species, and thus may be useful as a bone formation-promoting factor.

Clinical and pathological features of non-alcoholic steatohepatitis.

Clinical and pathological features were reviewed in 76 Japanese patients with non-alcoholic steatohepatitis (NASH). Forty-one were male and 35 were female with the mean age of 49.7 years old (range 15-75 years old, males; 46.3, females; 53.7 years old). Fifty-four percent of patients were preobese with a body mass index (BMI) between 25 and 30, while 16% of the patients were non-obese, and only 30% of the cases were morbidly obese, indicating that Japanese have a greater tendency to develop insulin-resistance and fatty liver disease than Western people. Hyperlipidemia was found in 51%, diabetes mellitus in 38%, and hypertension in 33% of the patients. Abnormally elevated liver function tests were found in one-third to two-thirds of the patients and were characteristically mild with 2- to 3-fold elevation from the normal range in the majority of the cases. Histological features of the liver were similar or identical to those reported in English literature and were characterized by fatty change, perivenular and pericellular fibrosis in zone 3, hepatocyte ballooning and necrosis with occasional Mallory's body formation and polymorphonuclear leukocyte infiltration. Mallory's bodies were found in 39% of patients and were characteristically small and poorly formed compared with those in alcoholic hepatitis. Eosinophilic granular or dirty foggy aggregated, not sufficient to be identified as Mallory's bodies, were a rather characteristic cytoplasmic expression in NASH patients. Portal inflammation and fibrosis were not found in the early stage of NASH, but were found as the disease progresses with formation of C-C and/or P-C bridging fibrosis, and eventually resulting in liver cirrhosis.

Cervical laminoplasty combined with muscle release in patients with athetoid cerebral palsy.

STUDY DESIGN: A retrospective study comparing cervical laminoplasty with or without muscle release for the treatment of cervical myelopathy resulting from athetoid cerebral palsy. OBJECTIVE: To assess the effectiveness of muscle release in the treatment of athetoid cerebral palsy. SUMMARY OF BACKGROUND DATA: While anterior and/or posterior spinal fusion has been generally accepted as necessary in surgical treatment for cervical myelopathy due to athetoid cerebral palsy, several studies have shown relatively favorable results following laminoplasty. Better results can be obtained by combining muscle release. METHODS.: Study participants were 10 patients who underwent cervical laminoplasty combined with muscle release (mean age, 44.6 years) and 15 patients who underwent cervical laminoplasty alone (mean age, 48.2 years). Therapeutic outcomes 1 year after surgery, as assessed by Kurokawa's methods and JOA scores, were compared between groups. RESULTS: Recovery rate 1 year after surgery was significantly higher for the muscle release group than for the control group. In both groups, recovery rates were significantly better for patients who could walk before surgery. CONCLUSIONS: Cervical laminoplasty combined with muscle release for the treatment of cervical myelopathy due to athetoid cerebral palsy is effective in simplifying postoperative therapy and improving JOA scores.

Stimulatory effects of statins on bone marrow-derived mesenchymal stem cells. Study of a new therapeutic agent for fracture.

Recent studies have reported that statins, inhibitors of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, increase bone formation in osteoblasts in vitro, suggesting that statins may have a new therapeutic application in the treatment of osteoporosis. During the reparative phase of healing of bone fractures, bone marrow-derived mesenchymal stem cells differentiate into osteoblasts or chondrocytes to form callus. If statins also stimulate bone formation in bone marrow-derived mesenchymal stem cells they may have beneficial effects in the treatment of bone fractures. In this study, we assessed the effect of statins on bone formation in rat bone marrow-derived mesenchymal stem cells in vitro. The statins fluvastatin, simvastatin and pravastatin did not significantly enhance mineralization, alkaline phosphatase (ALP) activity and bone gra protein (BGP, osteocalcin). These findings suggest that statins do not increase bone formation in bone marrow-derived mesenchymal stem cells.

In vitro bone formation induced by immunosuppressive agent tacrolimus hydrate (FK506).

When rat bone marrow cells were cultured with an immunosuppressive agent, tacrolimus hydrate (FK506), as well as with beta-glycerophosphate and vitamin C, numerous cell clusters became positive for alkaline phosphatase activity. Scanning electron microscopy revealed mineralized bone matrix in the cell clusters, which was identical to that of living bone. High levels of alkaline phosphatase (ALP), indicating osteoblastic activity, and high levels of osteocalcin (Oc) and calcium were found in the mature bone matrix of the cultures. There was significantly increased expression of mRNAs for ALP and Oc. These results indicate that the cultures contained both bone matrix and high osteoblastic activity, suggesting that FK506 induces ossification.

Bio-artificial periosteum for severe open fracture--an experimental study of osteogenic cell/collagen sponge composite as a bio-artificial periosteum.

In an attempt to reduce complications in cases of severe open fracture, we developed a bio-artificial periosteum composed of osteogenic cells and collagen sponge. In the present study, we evaluated the osteogenic potential of the bio-artificial periosteum in vivo and in vitro. After 4-week incubation in vitro, the bio-artificial periosteum had high alkaline phosphatase activity and osteocalcin content. Moreover, energy dispersive X-ray analysis revealed numerous crystal structures consisting of P and Ca on the surface of the bio-artificial periosteum. Using a rat model for severe bone injury, we examined the bone formation process in defect sites covered with the bio-artificial periosteum. New bone formation occurred in the central part of the bone defect as well as at the bone edge. We conclude that by using the bio-artificial periosteum, the fracture site benefited from an improved osteogenic environment. These results indicate that a clinical trial to further evaluate this technique should be conducted.

Osteogenesis with cryopreserved marrow mesenchymal cells.

Rat marrow cells were collected from the femurs of 7-week-old male rats (Fischer 344), cultured in 75-cm2 flasks for 10 days, released with trypsin, and then frozen and stored at -196 degrees C in liquid nitrogen. Three months later, the cryopreserved marrow cells were rapidly thawed and cultured in porous hydroxyapatite (HA) blocks in osteogenic medium containing 10 mM sodium beta-glycerophosphate, vitamin C phosphate (82 microg/mL), and 10 nM dexamethasone. After 2 weeks of subculture, cultured cells-HA constructs were subcutaneously implanted into syngeneic rats. The constructs were harvested 2 and 4 weeks postimplantation and examined by histological, biochemical, and genetic analyses. Histological examination showed extensive bone formation in the HA pores. High alkaline phosphatase (ALP) activity and high osteocalcin content were detected in the constructs. Expression of ALP and osteocalcin mRNA was observed at both 2 and 4 weeks. These results indicate that artificial bone prepared with cryopreserved cells had a marked osteogenic capacity.

An apoptotic inducer, aralin, is a novel type II ribosome-inactivating protein from Aralia elata.

We recently found that aralin, a novel cytotoxic protein consisting of two subunits, from Aralia elata selectively induces apoptosis in transformed cells as compared to normal cells. Here we report that aralin is a lectin specific for galactose (Gal) and its derivatives, and possesses RNA N-glycosidase activity as a new type II ribosome-inactivating protein (RIP). The RNA N-glycosidase activity of aralin was detected in cell-free and whole cell systems by the generation of an R-fragment from 28S rRNA. Coinciding with appearance of the R-fragment in aralin-treated cells, significant inhibition of protein synthesis was observed prior to the onset of apoptosis. Aralin-evoked cell death was efficiently repressed by the addition of Gal and its derivatives. Interestingly, melibiose preferentially protected normal cells from apoptosis as compared with transformed cells. Using rhodamine-coupled aralin, the aralin receptor could be clearly detected around the cell surface of transformed cells, but to a lesser extent on normal cells. Receptor binding was suppressed by Gal. These results indicate that aralin is incorporated into cells via its Gal-containing cell surface receptor and induces apoptosis through its RIP activity. Moreover, the expression level and/or structural changes of the aralin receptor may affect the sensitivity toward aralin.

Osteogenic potential of cultured bone/ceramic construct: comparison with marrow mesenchymal cell/ceramic composite.

Osteogenesis occurs in porous hydroxyapatite (HA) when porous HA blocks combined with marrow mesenchymal cells are grafted in vivo. In vitro bone formation occurs in HA pores when HA combined with marrow cells is cultured in osteogenic medium containing dexamethasone. This cultured bone/HA construct possesses higher osteogenic ability when it is grafted in vivo. In the present study, we compared the osteogenic potential of a cultured bone/HA construct with that of a marrow mesenchymal cell/HA composite. Marrow cells were obtained from the femoral bone shaft of 7-week-old, male Fischer 344 rats and were cultured in T-75 flasks. Cells were concentrated, then frozen and stored in liquid nitrogen for 6 months. The cryopreserved cells were then thawed and prepared for subculture in porous HA (5 x 5 x 5 mm, Interpore 500) and for implantation with porous HA. After 2 weeks of subculture, three cultured bone/HA constructs were separately implanted in the right side of the back of each syngeneic 7-week-old male Fischer rat, and three thawed cell/HA composites (without subculture) were separately implanted in the left side. These implants were harvested at 2 or 4 weeks postimplantation, and prepared for histological, biochemical, and genetic analysis. Alkaline phosphatase activity and osteocalcin content of cultured bone/HA constructs were much higher than those of the cell/HA composites at 2 and 4 weeks postimplantation. Histological examination and gene expression data agreed with these findings. The culture technique discussed herein should facilitate the development of biosynthetic bone implants with higher osteogenic capacity.