Autologous fat injection therapy including a high concentration of adipose-derived regenerative cells in a vocal fold paralysis model: animal pilot study.

OBJECTIVES: To verify the effectiveness and safety of the addition of adipose-derived regenerative cells to autologous fat injection therapy. METHODS: Unilateral vocal fold paralysis models were made by cutting the right recurrent laryngeal nerve in two pigs. At day 30, 0.5 ml adipose-derived regenerative cells mixed with 1 ml autologous fat was injected into the right vocal fold of one pig, with the other receiving 0.5 ml Ringer's solution mixed with 1 ml autologous fat. At day 120, fibrescopy, laser Doppler flowmeter, computed tomography, vocal function evaluation and histological assessment were conducted. RESULTS: Although histological assessment revealed atrophy of the thyroarytenoid muscle fibre in both pigs, there was remarkable hypertrophy of the thyroarytenoid muscle fibre in the area surrounding the adipose-derived regenerative cells injection site. CONCLUSION: The addition of a high concentration of adipose-derived regenerative cells to autologous fat injection therapy has the potential to improve the treatment outcome for unilateral vocal fold paralysis.

Occult hepatitis B infections among blood donors in Lao PDR.

BACKGROUND AND OBJECTIVES: In Lao People's Democratic Republic, hepatitis B virus is highly endemic. However, blood donations are only screened for HBsAg, leaving a risk of transmission by HBsAg-negative occult infected donors. Here, we characterized first-time blood donors to assess prevalence of hepatitis B virus infections and occult infected donors. MATERIALS AND METHODS: Sera were screened for HBsAg, HBeAg and anti-HBs, anti-HBc and anti-HBe antibodies. Occult HBV infections (OBIs) were assessed in HBsAg-negative sera by PCR, and sera of HBsAg positive and occult infected donors were phylogenetically characterized. RESULTS: 9·6% of the donors were HBsAg positive, and 45.5% were positive for at least one of the hepatitis B virus serum markers. More than 40% HBsAg carriers were HBeAg positive, with HBeAg seroconversion occurring around 30 years of age. Furthermore, 10·9% of HBsAg-negative, anti-HBc and/or anti-HBs-positive donors were occult infected with hepatitis B virus. Thus, at least 3·9% of blood donations would potentially be unsafe, but hepatitis B virus DNA copy numbers greatly varied between donors. CONCLUSION: In Lao People's Democratic Republic, a sizable proportion of HBsAg-negative and anti-HBc antibody-positive blood donations are potentially DNA positive and infective for hepatitis B.

High genetic diversity including potential new subtypes of hepatitis C virus genotype 6 in Lao People's Democratic Republic.

Sera from 105 anti-HCV-positive first-time blood donors collected in 2004, 2005 and 2008 in different provinces in Laos were investigated by PCR. Forty-five samples were positive for HCV (42.86%); two belonged to subtype 1b (2/45, 4.4%) and all others to genotype 6 (43/45, 95.6%), including subtypes 6b, 6h, 6k, 6l, 6n and 6q. Three groups of sequences were not clearly attributable to any genotype 6 subtype, two of which may be regarded as candidates for new subtypes of genotype 6. Two samples were mixed infected with different subtypes or clusters of genotype 6 viruses.

Detection of Human papillomavirus and cellular regulators p16INK4a, p53, and NF-kappaB in penile cancer cases in Kenya.

Human papillomavirus (HPV) E6 and E7 gene products play a central role in the induction of benign proliferation and malignant transformation by interacting with several cellular regulatory proteins such as p53, p16(INK4a), and nuclear factor kappaB (NF-kappaB). In this study, HPV DNA was detected by in situ hybridization (ISH) and p53, p16(INK4a), and NF-kappaB by immunochemistry in 22 penile cancer cases in Kenya. HPV DNA was found in 68.2% of the cases. There was no difference in the p53- and p16(INK4a)-positivities in HPV DNA-positive and HPV DNA-negative cases. In HPV DNA-positive cases, the NF-kappaB positivity in the nucleus, cytoplasm, and nucleus and/or cytoplasm amounted to 73.3%, 93.3%, and 100%, respectively, while in HPV DNA-negative cases, a 28.7% NF-kappaB positivity of in the nucleus and/or cytoplasm was observed. It is concluded that NF-kappaB in penile cancer is expressed more frequently in the presence of HPV infection than in its absence.

Chimeric RNA/DNA oligonucleotide-directed gene targeting in rice.

Site-specific mutagenesis in a rice genome was obtained by introducing chimeric RNA/DNA oligonucleotides (COs) by means of particle bombardment. Three COs were designed to target the independent codons for Pro-171, Trp-548 and Ser-627 of the endogenous rice acetolactate synthase (ALS) gene so it would confer resistance to ALS-inhibiting herbicides. Sequencing of the ALS gene of herbicide-resistant plants demonstrated that the ALS sequence was modified in a site-specific fashion. The efficiency of gene conversion mediated by COs was estimated to be 1x10(-4). These results demonstrate that CO-directed gene targeting is feasible in rice.

Visualization of somatic deletions mediated by R/RS site-specific recombination and induction of germinal deletions caused by callus differentiation and regeneration in rice.

A transgenic rice plant expressing the recombinase of Zygosaccharomyces rouxii under the control of the CaMV 35S promoter was crossed with a transgenic plant carrying a cryptic (beta-glucuronidase) GUS reporter gene, which was activated by recombinase-mediated deletions between two specific recombination sites ( RSs). In F(1) plants, GUS activity was observed as blue spots and stripes in vascular bundles in several parts of the leaves. GUS expression was detected in all of the calli induced from F(1) seeds and throughout the regenerated plants. DNA analysis using the polymerase chain reaction and Southern blotting showed that R/ RS-mediated deletions occurred in all of the cells of the regenerated plants. Stable GUS expression was confirmed in the progeny resulting from self-pollination. Thus, the deletions obtained in the regenerated plants were genetically equivalent to the germinal deletions. These results indicate that the induction of callus differentiation and shoot regeneration is an effective manner to activate the R/ RS system and to produce plants with chromosomal deletions.

RET tyrosine kinase enhances hair growth in association with promotion of melanogenesis.

We first demonstrated that c-Ret protein is transiently expressed mainly in the inner and outer root sheaths of hair follicles soon after birth in the skin of normal C57BL/6 and BALB/c mice. A longer-lasting expression of activated RET protein overlapped the c-Ret expression with some preferential expression in the outer root sheath in close association with increase in the number of S-100 protein-containing cells in the area and excess melanogenesis in and around hair bulbs in the skin of RFP-RET-transgenic mice on a C57BL/6 background (RFP-RET/B6). Hair follicles in the skin of the transgenic mice continuously showed histology of the anagen phase, and the recovery period for the hair of the transgenic mice after shaving was shortened. Such growth promotion was not observed in the case of white hairs of RFP-RET-transgenic mice on a BALB/c background. These results suggest that RET works to extend the anagen phase in association with upregulation of melanin production.

Hypoxic enhancement of type IV collagen secretion accelerates adipose conversion of 3T3-L1 fibroblasts.

Hypoxic modulation of collagen metabolism appears to be related to pathogenesis of many diseases such as fibrosis of connective tissue after injury and scleroderma. Since most of our understanding of how procollagen assembles within the cell has come from studies on cells cultured under normoxia, it may not be helpful for the etiology of the diseases observed in peripheral tissues under hypoxic conditions. As an experimental model for the hypoxic modulation of collagen metabolism, we cultured 3T3-L1 fibroblasts under low partial oxygen pressure and found that hypoxia enhances secretion of type IV collagen 10-fold and accelerates adipose conversion of the cells. The enhanced secretion of type IV collagen was not accompanied by an appreciable increase of alpha1(IV) and alpha2(IV) mRNAs. Prolyl 4-hydroxylase alpha increased only 3-fold under hypoxia. We suggest that hypoxia creates an environment of prolyl 4-hydroxylase alpha(2)beta(2) tetramers favorable for the folding of type IV procollagen which has many interruptions of the Gly-Xaa-Yaa repeat.

Dioctophymatid nematode larva found from human skin with creeping eruption.

A female dioctophymatid nematode larva, presumably belonging to the genus Dioctophyme, was found in a dermal granuloma accompanied by creeping eruption in the left inner thigh of a 26-yr-old Chinese woman who had stayed in Japan for 4 yr. Morphology of the sectioned worm is described in detail. This is the fourth case of dermal infection with dioctophymatid larva in humans.

Frequency and pattern of transposition of the maize transposable element Ds in transgenic rice plants.

Two kinds of T-DNA constructs, I-RS/dAc-I-RS and Hm(R)Ds, carrying a non-autonomous transposable element of Ac of maize were introduced into rice plants by Agrobacterium-mediated gene transfer. Six transgenic rice plants identified as containing a single copy of the element were crossed with two transgenic rice plants carrying a gene for Ac transposase under the control of the cauliflower mosaic virus 35S promoter. In F2 progenies, excision of the element was detected by PCR analysis and re-integration of the element was investigated by Southern blot analysis. The frequency of the excision of the element was found to vary from 0 to 70% depending on the crossing combination. The frequency of the number of individual transposition events out of the total number of F2 plants with germinal excision was 44% in one crossing combination and 38% in the other. In the most efficient case, 10 plants with independent transposition were obtained out of the 49 F2 plants tested. Linkage analysis of the empty donor site and the transposed Ds-insertion site in F3 plants demonstrated that one of five Ds-insertion sites was not linked to the empty donor site. The transgenic rice obtained in this study can be used for functional genomics of rice.

Expression of Bra r 1 gene in transgenic tobacco and Bra r 1 promoter activity in pollen of various plant species.

Bra r 1 encodes a Ca2+-binding protein specifically expressed in anthers of Brassica rapa. In this study, we isolated a genomic clone of Bra r 1 and found sequences similar to Pollen Box core motifs and LAT56/59 box, pollen-specific cis-acting element, in the 5' upstream region of Bra r 1. Reporter gene fusion revealed that the Bra r 1 promoter directs male gametophytic expression in Nicotiana tabacum, Arabidopsis thaliana and B. napus, showing strong expression in mature pollen grains similar to that of endogenous Bra r 1. Genomic DNA of Bra r 1 was introduced into tobacco plants and the highest accumulation of Bra r 1 protein was observed in mature pollen in the same manner as reporter gene expression. Using in vitro-germinated pollen tubes of transgenic tobacco, we firstly demonstrated the subcellular localization of Bra r 1 in pollen tubes. Bra r 1 protein was distributed throughout the pollen tube of transgenic tobacco and slightly intense signals of Bra r 1 were observed in the tip region. In long-germinated pollen tubes, Bra r 1 was detected only in the cytoplasmic compartments while no signals were observed in the empty part of the pollen tube, indicating that cytoplasmic movement toward the tube tip is accompanied by Bra r 1. Hence, we suggest that Bra r 1 is involved in pollen germination and pollen tube growth.

A monolayer complex of Cu2(OH)3C12H25SO4 directly precipitated from an aqueous SDS solution.

A layered complex with the chemical composition of Cu2(OH)3C12H25OSO3 was obtained through direct precipitation from a sodium dodecyl sulfate micellar solution of Cu(II) at a nearly neutral condition (pH approximately equals 6.8). From the XRD, 13C CPMAS NMR, and the spin probe ESR observations, a modified botallackite structure with a monolayer alkyl phase was proposed for the complex. Magnetic susceptibility measurements for the complex showed that there exist at least two exchange couplings: one is positive, and its magnitude is larger than that of the other, which is negative (J approximately equals -8 K). Although the cupric ions of the complex are paramagnetic, most of those are ESR silent and only the ESR signals from defects could be observed even at 298 K. A spin clustering model has been employed to explain the magnetic properties of the complex.

Functional transformation of plant peroxisomes.

Peroxisomes in higher plant cells are known to differentiate into at least three different classes, namely, glyoxysomes, leaf peroxisomes, and unspecialized peroxisomes, depending on the cell types. In germinating fatty seedlings, glyoxysomes that first appear in the etiolated cotyledonary cells are functionally transformed into leaf peroxisomes during greening. Subsequently, the organelles are transformed back into glyoxysomes during senescence of the cotyledons. Flexibility of function is a distinct feature of plant peroxisomes. This article briefly describes recent studies of the regulatory mechanisms involved in the changes of the function of plant peroxisomes.

Introduction of SLG (S locus glycoprotein) alters the phenotype of endogenous S haplotype, but confers no new S haplotype specificity in Brassica rapa L.

Self-incompatibility (SI) in Brassicaceae is genetically controlled by the S locus complex in which S locus glycoprotein (SLG) and S receptor kinase (SRK) genes have been identified, and these two genes encoding stigma proteins are believed to play important roles in SI recognition reaction. Here we introduced the SLG43 gene of Brassica rapa into a self-incompatible cultivar, Osome, of B. rapa, and examined the effect of this transgene on the SI behavior of the transgenic plants. Preliminary pollination experiments demonstrated that Osome carried S52 and S60, and both were codominant in stigma, but S52 was dominant to S60 in pollen. S43 was found to be recessive to S52 and codominant with S60 in stigma. The nucleotide sequence of SLG43 was more similar to that of SLG52 (87.8% identity) than to that of SLG60 (74.8% identity). Three of the ten primary transformants (designated No. 1 to No. 10) were either completely (No. 9) or partially (No. 6 and No. 7) self-compatible; the SI phenotype of the stigma was changed from S52S60 to S60, but the SI phenotype of the pollen was not altered. In these three plants, the mRNA and protein levels of both SLG43 and SLG52 were reduced, whereas those of SLG60 were not. All the plants in the selfed progeny of No. 9 and No. 6 regained SI and they produced a normal level of SLG52. These results suggest that the alteration of the SI phenotype of the stigma in the transformants Nos. 6, 7, and 9 was the result of specific co-suppression between the SLG43 transgene and the endogenous SLG52 gene. Three of the transformants (Nos. 5, 8 and 10) produced SLG43 protein, but their SI phenotype was not altered. The S60 homozygotes in the selfed progeny of No. 10 which produced the highest level of SLG43 were studied because S43 was codominant with S60 in the stigma. They produced SLG43 at approximately the same level as did S43S60 heterozygotes, but did not show S43 haplotype specificity at the stigma side. We conclude that SLG is necessary for the expression of the S haplotype specificity in the stigma but the introduction of SLG alone is not sufficient for conferring a novel S haplotype specificity to the stigma.

Accumulation of a fusion protein containing 2S albumin induces novel vesicles in vegetative cells of Arabidopsis.

We have previously reported that precursor-accumulating (PAC) vesicles found exclusively in developing seeds are involved in a transport of seed storage proteins, such as 2S albumin, from the endoplasmic reticulum to protein-storage vacuoles. Here, we constructed chimeric genes that encode fusion proteins consisting of both various lengths of polypeptides derived from pumpkin 2S albumin and a selectable marker enzyme, phosphinothricin acetyltransferase. The chimeric genes were expressed in transgenic Arabidopsis in order to investigate the mechanism of the PAC vesicle formation. A fusion protein expressed by one of the chimeric genes is accumulated as a proprotein-precursor form, and localized in novel vesicles of vegetative cells. The vesicles show distinct features that well much to the PAC vesicles. Despite of the accumulation of the fusion protein, the transgenic Arabidopsis is still sensitive to phosphinothricin. Phosphinothricin acetyltransferase contained in the fusion protein is obviously compartmentalized in the PAC-like vesicles that do not permit the detoxification of this herbicide. These results indicate that the PAC-like vesicle can be induced in vegetative cells by the ectopic expression of the protein that is destined to be compartmentalized into the PAC vesicles.

Expression of midkine in normal and burn sites of rat skin.

Expression of midkine (MK), a retinoic acid-inducible heparin binding growth factor, was examined immunohistochemically in normal and burn sites of rat skin. In the normal skin, MK was localized in the epidermis and dermal appendages such as hair follicles and sebaceous glands. Mast cells in the subdermal connective tissue also accumulated MK. After burn injury, MK-positive cells began to infiltrate into subdermal connective tissue, and the number of MK-positive cells in the region increased to a maximum at postburn day two and then decreased gradually. Western blotting analysis of both normal and postburn skin revealed a 30 kDa band reactive with anti-MK antibody; this band was concluded to be a dimer of MK. These findings were discussed from the viewpoint of the possible role of MK in wound healing.

Localization of the Ca(2+)-binding protein, Bra r 1, in anthers and pollen tubes.

Calcium plays an essential role during pollen development and pollen tube growth, and several Ca(2+)-binding proteins are expressed in anthers. We have previously reported that Brassica pollen allergens encoded by Bra r 1 and Bra r 2 show sequence similarities to Ca(2+)-binding proteins [Toriyama et al. (1995) Plant Mol. Biol. 29: 1157]. Herein, we report that both genes are expressed in the diploid tapetum and haploid microspores, as detected by in situ RNA hybridization. Immunoblot analysis revealed that Bra r 1 and Bra r 2 were accumulated in anthers during pollen development. When pollen grains were suspended in an aqueous solution, both proteins were mainly detected in the pollen extracellular fraction, indicating that Bra r 1 and Bra r 2 are released from the pollen upon hydration. Localization of Bra r 1 was further investigated in sections of anthers and pollen tubes. Bra r 1 was detected in the tapetum, microspores and pollen grains. In longitudinal sections of cross-pollinated pistils. Bra r 1 was detected throughout pollen tubes elongating in transmitting-tissue. These findings suggest that Bra r 1 may be involved in pollen-pistil interaction and pollen tube growth.

Reconstituted basement membrane potentiates in vivo adipogenesis of 3T3-F442A cells.

Adipocytes forming fat pad in vivo are surrounded by well developed basement membranes. Synthesis of basement membrane is enhanced during in vitro differentiation of preadipocyte line. In order to know the role of basement membrane in adipogenesis in vivo, we injected 3T3-F442A preadipocytes subcutaneously into nude mice together with or without the reconstituted basement membrane, Matrigel. Histological sections of the fat pads newly formed by injecting the cell alone showed dense population of immature adipocytes and microvessels within 2 weeks and they matured rapidly. In contrast, injection of the cells together with Matrigel showed sparse adipocytes after 2 weeks and they matured slowly over the period of 6 weeks. Quantification of the process by measuring the weight, DNA content, triglyceride content and glycerophosphate dehydrogenase (GPDH) activity of the fat pads showed that injection of the cell alone resulted in early maturation of adipose tissue with fewer adipocytes while the presence of Matrigel decelerated but potentiated the maturation of adipose tissue with 2 fold contents of DNA, triglyceride and GPDH activity. We thus showed that reconstituted basement membrane (Matrigel) supported the survival and maturation of adipocytes.

A comparison of epidemiologic, histologic, and virologic studies on Hodgkin's disease in western Kenya and Nagasaki, Japan.

Rapid progress in molecular technologies has enabled the detection of several oncogenic viruses in various types of tumors. The pathogenesis of Hodgkin's disease is suggested to have a strong association with Epstein-Barr virus (EBV). However, Hodgkin's disease related to EBV shows a wide geographic variation in epidemiology. These variations among different populations suggest an interaction of environmental factors and a direct role of EBV infection. Therefore, we performed a comparative study on epidemiologic, histologic, and virologic features of Hodgkin's disease among those in the western part of Kenya and in Nagasaki, Japan. The age distribution of Hodgkin's disease showed a distinct peak in the 0-9-year-old age group in Kenya, and a higher and lower peak in the 60-69- and 30-39-year-old age groups, respectively, in Japan. The most common subtype of Hodgkin's disease in both countries was mixed cellularity, followed by nodular sclerosis, lymphocyte depletion, and lymphocyte predominance. Mixed cellularity showed a significantly high prevalence among Kenyan children nine years of age or younger. Using the in situ hybridization method, EBV-encoded RNA (EBER-1) was detected in 79% of the Kenyan cases and 59% of the Japanese cases, with the mixed cellularity subtype showing a strong correlation with EBER-1. There was 100% positivity in both countries in those less than nine years old. These results suggest that EBV plays a more direct role in the pathogenesis of Hodgkin's diseases in Kenya, especially in cases among young children and also in Japanese children. Environmental and/or genetic factors may have a role, in addition to EBV, in the pathogenesis of Hodgkin's disease, especially in Nagasaki, Japan.

[An autopsy case of rheumatoid arthritis accompanied with acute exacerbation of interstitial pneumonia].

An autopsy case of rheumatoid arthritis (RA) with acute exacerbation of interstitial pneumonia is reported. A 57-year-old woman with longstanding RA was admitted to our hospital because of progressive dyspnea. On chest roentogenogram, diffuse interstitial shadow was confirmed in both lungs. Chest computed tomography (CT) showed diffuse lesion of elevated density of CT level in both lung. She was diagnosed as an acute exacerbation of interstitial pneumonia, and treated by methylpredonisolone pulse therapy (1,000 mg/day). Although cyclosporin A (2 mg/kg/day) was combined to steroid therapy, she was died of progressive respiratory failure. The histological findings of the lung showed extensive fibrosis with alveolar damage associated with hyaline membranes, edema and hemorrhage in alveolar space.