Identification of the crude drug atractylodes rhizome (Byaku-jutsu) and atractylodes lancea rhizome (So-jutsu) using chloroplast TrnK sequence as a molecular marker.

Novel methods for molecular authentication of Atractylodes-derived crude drugs (Jutsu) were established based on PCR-restriction fragment length polymorphism (RFLP) and direct sequencing of chloroplast trnK. Two regions inside the chloroplast trnK were selected as molecular markers for identification and discrimination of Atractylodes Rhizome (Byaku-jutsu) and Atractylodes Lancea Rhizome (So-jutsu). The Region 1 fragment (260 bp) amplified from So-jutsu and Wa-byaku-jutsu (Atractylodes Rhizome derived from A. japonica) gave 2 bands of 180 bp and 80 bp on agarose gel electrophoresis after digestion with a restriction endonuclease HinfI, whereas the fragment amplified from Kara-byaku-jutsu (Atractylodes Rhizome derived from A. ovata) remained undigested, which allowed unambiguous identification of Kara-byaku-jutsu. By direct sequencing of Region 2 (436 bp) and comparison of the nucleotide sequence data sets we could not only discriminate Byaku-jutsu and So-jutsu but also identify the original plant species of each crude drug specimen. A simple and reliable protocol for rapid preparation of DNA suitable for PCR from as little as 1 mg of Atractylodes-derived crude drugs was also described.

Isolation from beer and structural determination of a potent stimulant of gastrin release.

Beer was subjected to five successive chromatographic procedures to isolate the gastrin release-inducing activity, guided by bioassay of the fractions in anaesthetized Donryu rats. The procedures were: (1) hydrophobic interaction chromatography (aqueous effluent with an HP20 column); (2) weak cation-exchange chromatography (1 M acetic acid eluate with a CM Sephadex C-25 column); (3) gel filtration (methanol eluate with a Sephadex LH-20 column); (4) same as (2); (5) high-performance liquid chromatography (YMC-Pack ODS-AM with 7% acetonitrile-0.01 M HCl). The active component finally isolated had a specific activity approximately 10000 times higher than that of beer. It was identified by means of mass, 1H- and 13C-nuclear magnetic resonance spectral analyses as N-methyltyramine (NMT). The dose of NMT giving maximal gastrin-releasing activity was 25 microg/kg, and the 50% effective dose was approximately 10 microg/kg on oral administration to rats. NMT was isolated and identified as a gastrin release inducer in beer. Its concentration in beer is sufficient to account for most of the activity of beer.

Necrotizing fasciitis caused by Vibrio vulnificus differs from that caused by streptococcal infection.

We reviewed the clinical record of all patients admitted to Saga Medical School Hospital during the most recent 10 years and found that 17 (0.03%) were diagnosed as having necrotizing fasciitis. Bacteriological examination demonstrated that Vibrio vulnificus was the pathogen responsible in five patients (29%). The disease caused by V. vulnificus occurred in the warmer half of the year. All of the patients had underlying chronic liver dysfunction, and three of them had previously consumed raw seafood. In these patients, the predominant skin lesions were oedema and subcutaneous bleeding, such as ecchymosis and purpura, while superficial necrosis was not recognized. Three patients died of systemic complications. By contrast, all of the five patients with necrotizing fasciitis caused by Streptococcus pyogenes had the disorder in winter, and only one of them had chronic liver dysfunction. In skin lesions, subcutaneous bleeding was rare but necrosis was seen often. Despite the high incidence of systemic complications, no patients with streptococcal necrotizing fasciitis died. These findings suggest that the clinical features of necrotizing fasciitis caused by V. vulnificus are different from those of necrotizing fasciitis caused by classical pathogens, and that the two should be differentiated as early as possible to improve the prognosis.

Proliferation and differentiation of organoid hair follicle cells co-cultured with fat cells in collagen gel matrix culture.

Using rat skin, we studied the influence of fat cells on the proliferation and differentiation of organoid hair follicle cells in a three-dimensional collagen gel matrix culture system. We cultured organoid hair follicles embedded in collagen gel under each of the following three conditions: cell-free collagen gel for control experiments (condition 1); co-culture with fat cells in close apposition (condition 2); and co-culture with fat cells in spatial separation (condition 3). Outgrowths of epithelial cells from the organoid hair follicles associated with perifollicular proliferation of fibroblasts were observed under conditions 1 and 3. Under condition 2, proliferation of both organoid hair follicle cells and fibroblasts was inhibited, but differentiation of the hair follicle cells appeared to be accelerated. Fat cells are considered to have an inhibitory effect on the proliferation of perifollicular fibroblasts, which might have resulted in the inhibition of hair follicle cell proliferation and also in the better maintenance of normal follicular structure and integrity, allowing for hair-type differentiation to proceed. A direct accelerating effect of fat cells on hair follicle differentiation may also have been responsible. In a physiological state (co-culture with keratinocytes on the collagen gel), similar results were observed under conditions 1 and 2. The different findings under conditions 2 and 3 may be due to either of two possibilities: either the concentration gradient of the soluble factors released from fat cells, acting on either the hair follicle cells or the perifollicular fibroblasts as an inhibitor of proliferation, caused the difference in the results, or direct contact between the organoid hair follicle cells and fat cells may have influenced the accelerating effect of fat cells on the differentiation of hair follicle cells.

Neurotropic melanoma invading the median nerve.

We report a case of acral lentiginous melanoma of the right thumbnail bed which demonstrated characteristic intraneural invasion and extension along the median nerve. Four years after amputation of the involved thumb, a melanotic tumor recurred on the right thenar. Radiation therapy was given. The tumor invaded the median nerve, however, causing progressive pain and paralysis of the right hand. Despite right arm amputation, the tumor extensively metastasized, and the patient died three years later. Histopathologically, the tumors were characterized by extensive proliferation of spindle-shaped cells forming neuroid fascicles especially prominent in the metastatic region. Tumor cells were positive immunohistochemically with S-100 protein antisera.

Phylogenetic analysis of Atractylodes plants based on chloroplast trnK sequence.

The phylogenetic relationship of Atractylodes lancea, A. chinensis, A. koreana, A. ovata and A. japonica were analyzed by comparing the 2.6 kb sequence in a chloroplast gene trnK encoding tRNALys (UUU). The dried rhizomes of the former three species have been used as the crude drug "So-jutsu" and those of the latter two as "Byaku-jutsu" in Chinese and Japanese traditional medicine ("Kampo-medicine"). The trnK phylogenetic tree revealed that A. ovata is an outgroup of the five Atractylodes species examined and that A. japonica and A. lancea are most closely related. PCR amplification of trnK with HinfI digestion provided us with a simple method to distinguish A. ovata from other Atractylodes species at the molecular level.

Random amplified polymorphic DNA analysis and variation of essential oil components of Atractylodes plants.

Total DNAs were prepared from the leaves of Atractylodes lancea DE CANDOLLE, A. chinensis KOIDZUMI, A. lancea var. simplicifolia KITAMURA, A. japonica KOIDZUMI ex KITAMURA and A. ovata DE CANDOLLE. The DNAs were subjected to random amplified polymorphic DNA (RAPD) analysis. Some primers showed the definitive polymorphic DNA patterns in A. lancea, A. japonica and A. ovata. The RAPD of A. lancea var. simplicifolia and one of A. chinensis gave similar patterns to those of A. lancea, but one of the other A. chinensis gave a similar pattern to A. japonica. Furthermore, quantitative analysis of atractylon, hinesol, beta-eudesmol and atractylodin in the rhizomes was done using gas chromatography. Though atractylon was detected not only in A. japonica and A. ovata but also in some of A. lancea, their RAPD profiles revealed the presence of intraspecific variation with A. lancea.

Three-dimensional demonstration of melanocyte distribution of human hair follicles: special reference to the bulge area.

Melanocytes of human hair follicles were histochemically and immunohistochemically examined in two and three dimensions. EDTA-treated extracted anagen vellus and intermediate hair follicles showed that monoclonal murine antibody (MoAb) NKI/ beteb-reactive melanocytes were distributed from the infundibulum to the bulb. Melanocytes of the infundibulum and bulb were larger and more strongly stained with MoAb NKI/beteb than those of the middle portion below the sebaceous gland. Latter melanocytes showed less dendricity. Dendritic melanocytes were exclusively observed in the bulb as well as the bulge area of vellus and intermediate hair follicles, where they were variably melanized. In longitudinal and transverse sections of adult human scalp some keratinocytes of the outer root sheath of the bulge area were melanized compared to other parts. This phenomenon was independent of the hair cycle. These findings may be characteristic of bulge melanocytes and/or keratinocytes.

Apoptotic pocket-like structures of the bulge of the terminal hair follicles of the human scalp.

Terminal hair follicles of the human scalp of all ages showed apoptotic pocket-like structures in the outer root sheath of the bulge area at anagen, but not telogen phase. The occurrence of these hole structures was roughly estimated in 15% of anagen terminal hair follicles of the human scalp. The size of these apoptotic pockets was variable, ranging from pin hole-like spaces to larger structures filled with homogeneous black materials. These unusual variations were often co-localized with apoptotic degenerations and exclusively present in the presumptive bulge of anagen terminal hair follicles where arrector pili muscles were seen in the vicinity. In fact, these vacuolated structures tended to be present on the side where the major part of the arrector pili muscles anchored.

Micropropagation ofPanax notoginseng by somatic embryogenesis and RAPD analysis of regenerated plantlets.

Somatic embryogenesis was induced in callus tissues derived from young flower buds ofPanax notoginseng via callus within 18 weeks of culture. The mature somatic embryos were germinated on half-strength Murashige and Skoog's (MS) medium supplemented with gibberellic acid A3(GA) and 6-benzyladenine (BA). The most suitable medium for optimal root formation proved to be MS medium supplemented with 1-naphthaleneacetic acid (NAA). Total DNA was extracted from the leaves of the regenerated plantlets ofP. notoginseng. Analysis of random-amplified polymorphic DNA (RAPD) using 21 arbitrary oligonucleotide 10-mers, showed the genetic homogeneity ofP. notoginseng. The amplification products were monomorphic for all of the plantlets ofP. notoginseng regenerated by embryogenesis, suggesting that somatic embryogenesis can be used for clonal micropropagation of this plant.

Immune response to a differentiation antigen induced by altered antigen: a study of tumor rejection and autoimmunity.

Recognition of self is emerging as a theme for the immune recognition of human cancer. One question is whether the immune system can actively respond to normal tissue autoantigens expressed by cancer cells. A second but related question is whether immune recognition of tissue autoantigens can actually induce tumor rejection. To address these issues, a mouse model was developed to investigate immune responses to a melanocyte differentiation antigen, tyrosinase-related protein 1 (or gp75), which is the product of the brown locus. In mice, immunization with purified syngeneic gp75 or syngeneic cells expressing gp75 failed to elicit antibody or cytotoxic T-cell responses to gp75, even when different immune adjuvants and cytokines were included. However, immunization with altered sources of gp75 antigen, in the form of either syngeneic gp75 expressed in insect cells or human gp75, elicited autoantibodies to gp75. Immunized mice rejected metastatic melanomas and developed patchy depigmentation in their coats. These studies support a model of tolerance maintained to a melanocyte differentiation antigen where tolerance can be broken by presenting sources of altered antigen (e.g., homologous xenogeneic protein or protein expressed in insect cells). Immune responses induced with these sources of altered antigen reacted with various processed forms of native, syngeneic protein and could induce both tumor rejection and autoimmunity.

Necrotizing fasciitis due to group A streptococci: a clinicopathological study of six patients.

The recent worldwide appearance of invasive group A streptococcal infections has again called attention to streptococcal necrotizing fasciitis. However, in contrast to polymicrobial necrotizing fasciitis, the streptococcal form has not been thoroughly studied clinically. The objective of the study was to elucidate the characteristic features of recent cases of necrotizing fasciitis due exclusively to pure group A streptococci. We encountered six patients with these criteria at a single hospital in Japan during the last 12 years. A clinicopathological analysis was performed in these six patients. In three patients, the clinical signs and the laboratory findings were characteristic of systemic toxicity. In this group, the clinical presentation was a pale or blue-gray lesion associated with severe intravascular coagulation histologically involving the vessels in the lesion. In the three patients without signs of systemic toxicity, a swollen, erythematous skin lesion persisted for as long as one week; histologically, the intravascular coagulation within these lesions was mild. In clinicopathological terms, the entity in these six patients could be clearly classified as either fulminant or subacute. In the fulminant type, immediate surgical debridement of necrotic fascia is required; in the subacute type, incision and drainage alone are sufficient.

Familial atypical mole syndrome manifesting as polypoid melanoma: a case report and review of the Japanese literature.

A family with atypical mole syndrome which manifested as polypoid melanoma in one member is reported. A 46-year-old man presented with a polypoid tumor on his lower back, which was excised under clinical diagnosis of soft fibroma. The tumor proved to be polypoid melanoma. He was referred to our clinic, and examination revealed that he had the atypical mole syndrome phenotype. The clinicopathological screening of his first-degree relatives confirmed that his 58-year-old sister had the same phenotype. In addition to our discussion of the clinical significance of polypoid melanoma, we also attempted to elucidate the characteristics of the reported Japanese cases of melanomas with atypical moles from a survey of the literature, which revealed the following characteristics: 1) younger mean age than that of total Japanese melanoma cases, 2) sun sensitive skin, 3) unexpectedly and frequently observed familial incidence (Kraemer's D1 and D2), 4) frequent site of the melanoma on the trunk and none on the sole of the foot, and 5) high incidence of superficial spreading melanoma and nodular melanoma, and 6) no cases of acral lentiginous melanoma.

Primary cutaneous T-cell lymphoma with an angiocentric growth pattern: association with Epstein-Barr virus.

We report a case of primary cutaneous T-cell lymphoma with an angiocentric growth pattern. The lesions had been confined for about 2.5 years to the skin, but there had been a gradual progression of the disease both clinically and histologically. We assessed the neoplastic clonality and the presence of Epstein-Barr (EB) virus genome in this case using immunohistochemistry, Southern blot analysis and RNA in situ hybridization. Clonal proliferation of a CD4+ alpha beta T-cell phenotype was demonstrated. In addition, the clonal population harboured the EB virus genome, which suggested that the virus was involved in the pathogenesis of the disease. The patient has remained in remission for 10 months, and has received treatment with cyclophosphamide and prednisone.

A case of Bednar tumor recurring after 23 years.

We reported a patient with a Bednar tumor who developed local recurrence 23 years later. The recurrent Bednar tumor showed the histopathological features dermatofibrosarcoma protuberans (DFSP), and expressed CD34. We favor the term "Bednar tumor" rather than pigmented DFSP and consider that Bednar tumor is an intermediate type between cellular blue nevus and DFSP.

Accordion-like structure of terminal hair follicles of the human scalp.

Terminal hair follicles extracted after ethylenediaminetetraacetic acid treatment preserved the basal cell surface of the outer root sheath well enough to examine the outer surface in detail. Light microscopic observations of whole mount specimens revealed several sawtooth-like structures of follicular epithelium in the lower portion of terminal hair follicle. Vertical sections of the human scalp also showed several invaginations of the outer root sheath of terminal hair follicles in the lower portion, not in association with the arrector pili muscle. Scanning electron microscopy of the same specimens demonstrated accordion-like structures below the sebaceous gland in the lower half of anagen terminal hair follicles and surrounding the entire circumference of the follicles. Sawtooth-like structures were unilaterally observed at the level of the middle or lower portion below the sebaceous gland. With transmission electron microscopy, these structures were seen as undulations of basal cells which preserved hemidesmosomes, although the basal lamina had been peeled off during the extraction procedure. Thus, this accordion-like structure is a genuine structural variation of some human anagen hair follicles and not an artifact.

Clonal propagation of Cnidium officinale by shoot tip culture.

Multiple shoots were induced from the apical domes of shoot tips of Cnidium officinale Makino (Apiaceae) by culturing them on Murashige and Skoog (MS) 1 static media solidified with 0.2% gelrite and supplemented with 6-benzylaminopurine (BAP) 10 (-6)M. An average of 5.3 shoots per segment were obtained within 6 weeks and this ability did not decline even after two years of subculture. Subsequent transfer of these regenerated shoots on MS media supplemented with alpha-naphthaleneacetic acid (NAA) 10 (-7)M and BAP 10 (-7)M resulted in root formation. Rooted plantlets were able to grow in soil after a short period of acclimatization. Cytological observation in root tip cells of cultivated, as well as in vitro propagated plantlets revealed that in both cases the cells had 2n = 22 chromosomes indicating the homogeneity of the clonally propagated plants.

Restriction fragment length polymorphisms of rDNA and variation of essential oil composition in Atractylodes plants.

Total DNA was extracted from the leaves of Atractylodes lancea DE CANDOLLE, A. ovata DE CANDOLLE and A. japonica KOIDZUMI ex KITAMURA of various origins and hybridized with digoxigenin-labeled rice ribosomal DNA after digestion with eight different restriction endonucleases. The resulting restriction fragment length polymorphism (RFLP) profiles allowed us to distinguish the three Atractylodes species when DNA was digested with Sac I. Although atractylon was detected in the rhizomes of some of the cultivated strains of A. lancea, their RFLP profiles clearly indicate that these plants are not hybrids of A. ovata or A. japonica. RFLP analysis also revealed the presence of intraspecific variation in DNA sequence of rRNA locus among A. lancea as well as A. japonica.