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Recent studies identified three begomoviruses infecting soybean and bean crops in northwestern (NW) Argentina, bean golden mosaic virus (BGMV), a virus with high capsid protein identity with Sida mottle virus (SiMoV) and a possible new viral species (isolate A). Analysis of complete DNA-A sequences confirmed that isolate A represents a new viral species for which the authors propose the name soybean blistering mosaic virus (SbBMV), whereas the SiMoV-like virus is actually an isolate of tomato yellow spot virus (ToYSV). Molecular hybridisation-based detection of the three begomoviruses was accomplished using a general probe obtained by mixing full-length DNA-A clones of the three begomoviruses and specific probes comprising part of the common region of each viral genome. These probes were used to test samples collected in NW Argentina from 2004 through 2007. Fifty-three percent of the bean samples were infected with BGMV, 11.5% with ToYSV and 9% with SbBMV. For soybean samples, 33% were infected with SbBMV and 18% with ToYSV. BGMV was not detected in soybean. ToYSV was also detected in the wild species Abutilon theophrasti.

A mild isolate (designated as 10-O) of Sweet potato feathery mottle virus (SPFMV) was obtained from Ipomoea batatas. This isolate rarely caused skin discoloration and did not cause russet crack disease on the storage roots of I. batatas that are typical of infection with the severe strain of SPFMV (SPFMV-S). The yield of the 10-O–infected I. batatas ranged from 92 to 105% of the yield of healthy I. batatas. The coat protein gene of 10-O encodes 315 amino acids and has sequence identities of 91.1% at the nucleotide level and 95.6% at the amino acid level with the corresponding region of SPFMV-S. When I. batatas cuttings were inoculated with 10-O and later challenged with SPFMV-S, russet crack symptoms were much reduced and SPFMV-S was not detected in the challenged plants. These results indicate that 10-O can be an effective biological control agent against russet crack disease.

サツマイモ斑紋モザイクウイルス（SPFMV）は，食用や焼酎用原料として利用されるサツマイモの塊根に帯状粗皮病を発病させ，減収をもたらす．大分県のサツマイモから分離したサツマイモ斑紋モザイクウイルス弱毒株10-Oは，サツマイモにあらかじめ接種することで，帯状粗皮病に対する干渉効果を示し，その感染による減収もない．本研究により，この10-Oの感染による塊根の内容成分と醸造適性に及ぼす影響はなく，焼酎用原料として利用可能であることが明らかにされた．さらに，10-Oが感染すると塊根の形状は細長くなり，熱伝導性や加工作業性を高める可能性が示された．以上から，サツマイモにおける弱毒株10-Oの利用は，帯状粗皮病の防除のみならず，醸造原料としての加工適性の向上にも有用と考えられる．

Nucleotide (nt) sequencing has contributed to the identification of virus species and has also proved diagnostically useful in the control of tomato-infecting begomoviruses disease. We determined the complete nt sequences of the DNA-A genome and its cognate DNAbeta satellite molecules in isolates of Tobacco leaf curl Japan virus, Honeysuckle yellow vein mosaic virus, Eupatorium yellow vein virus in Japan. Pairwise comparison analyses based on the nt sequences of DNA-A from the genetic group of these viruses tentatively named as TbLCJV, HYVMV and EpYVV (TbJV/HYV/EpV) revealed that this group had a significance threshold of 84 % identity. Phylogenetic relationship analyses of the nt sequences of DNA-A and DNAbeta revealed that their isolates were separated into a discrete Far East Asian clade, distinct from all other begomoviruses. This clade was divided into two distinct clusters comprising the subgroups TbJV/HYV and EpV. Furthermore, recombination analysis revealed that members of the TbJV/HYV/EpV group had the genetic variation indicative of many recombination events. Our study demonstrates that this group forms a unique species complex, but that members have discrete lineages depending on their natural perennial host plants.

To study the photoexcitation and subsequent reactions of large molecular ions, anions of 5,10,15,20-tetraphenyl-21H,23H-porphine, which is a typical metal-free porphyrin, were produced and stored in an electrostatic ion storage ring at the Tokyo Metropolitan University. The circulating ions were merged using a pulsed laser, resulting in the production of super-excited anions, which eventually yielded neutral species. The decay profile deviated considerably from t-1 behavior, depending on the laser fluence and excitation wavelength.

Konjak mosaic virus (KoMV) belongs to the genus Potyvirus, family Potyviridae. The complete nucleotide sequence of KoMV F isolate (KoMV F) was determined. The genome is 9,544 nucleotides long excluding the 3' terminal poly A tail and encodes a typical potyviral 350-kDa polyprotein of 3,087 amino acids. Phylogenetic analysis using known potyvirus polyproteins shows that KoMV constitutes a branch with yam mosaic virus, close to another branch including Japanese yam mosaic virus, turnip mosaic virus, scallion mosaic virus and lettuce mosaic virus. The 3' terminal 1,842 nucleotides of a different isolate of KoMV, K-2, was also determined, covering the C-terminal 292 amino acids of the nuclear inclusion protein b (NIb), coat protein (CP), and the 3' untranslated region. The amino acid sequences of the KoMV F CP and the nucleotide sequences of the KoMV F 3' untranslated region showed 92.5 and 90.5% identity to the corresponding genes of K-2, 88.7-96.8 and 92.7-94.4% to those of Zantedeschia mosaic virus (ZaMV) isolates, 87.5-89.7% and 85.5-90.3% to those of Japanese hornwort mosaic virus (JHMV) isolates. These results showed that KoMV is a distinct potyvirus and that KoMV, ZaMV, and JHMV are members of the same potyvirus species. Considering that KoMV was the first of these to be described, ZaMV and JHMV may be considered isolates of KoMV.

The nucleotide sequence of melon yellow spot virus (MYSV) M RNA segment was determined. The M RNA segment contains one open reading frame (ORF) encoding 308 amino acids (aa) in the sense orientation and another ORF encoding 1,127 aa in the complementary orientation, which were homologous to the NSm protein and G1/G2 glycoprotein precursor (Gp) protein, respectively. Amino acid sequences identities with the other tospovirus suggested that MYSV is closely related to groundnut bud necrosis virus and watermelon silver mottle virus. To analyze subgenomic RNA of the M RNA segment, RNA transcripts corresponding to the NSm and Gp genes were specifically amplified, and the nucleotide sequence of the 5' terminal region was determined. Sequence analysis of the NSm and Gp transcripts showed that they had a non-viral sequence 12-18 and 10-18 nucleotides long, respectively. Although these sequences varied considerably, in more than half of the cases, a cytosine residue was observed at the 3' end of the non-viral leader sequence, which suggests that the viral transcriptase prefers certain cap-donor sequences harboring a 3'CA dinucleotide.

Alstroemeria plants (Alstroemeria sp. and A. ligtu) with necrotic spots and streaks on leaves were found in Chiba prefecture, Japan. Tospovirus-like enveloped, spherical particles 80-100nm in diameter and filamentous particles 750-850nm in length were observed together in the infected leaves. The enveloped particles were identified as Iris yellow spot virus (IYSV) by RT-PCR. Deduced amino acid sequences of the nucleocapsid gene of the two IYSV isolates (designated CbAlsD1 and Als1) were compared with those of IYSV isolates reported previously; the isolates were genetically similar to an isolate from Dutch iris (Iris hollandica) in the Netherlands. A local lesion isolate of CbAlsD1 reproduced the original symptoms on alstroemeria seedlings without the presence of filamentous virions. These results showed that IYSV was the causal virus of necrotic spots and streaks. Naturally infected A. ligtu plants produced new shoots that appeared healthy when grown under thrips-free conditions. Although IYSV could not be detected in these symptomless shoots by RT-PCR alone, the virus was detected by Southern blotting of the RT-PCR products. Therefore, a low level of IYSV virions exist in the symptomless shoots that developed from infected alstroemeria plants.

Iris yellow spot virus (IYSV) was isolated from onion with streaks on leaves in Saga and Chiba prefectures, Japan. Phylogenetic analysis based on the amino acid sequence identities of the nucleocapsid genes showed the presence of two strains, as represented by an isolate from onion in Brazil (IYSVBR) and that from Dutch iris (Iris hollandica) in the Netherlands (IYSVNL), on onion in Japan. Pathogenicity was tested on 11 cultivars of onion and five of leaf onion by mechanical inoculation with either of the two IYSV isolates, which were genetically similar with IYSVBR and IYSVNL, respectively. Both isolates caused the same type of streaks only on inoculated leaves of all onion cultivars tested, and the virus was not detected in upper leaves. None of the leaf onions developed symptoms on either inoculated or upper leaves, even though IYSV was detected in the inoculated leaves. Onion thrips (Thrips tabaci) transmission to onion, leaf onion, wakegi (Allium wakegi) and shallot (Allium grayi) was examined, and IYSV was detected from onion leaves where thrips fed.

1998年1月及び1999年6月に静岡県で,また2001年には佐賀県で施設栽培のトルコギキョウに,葉にえそやえそ輪紋等の症状が発生した.これら発病株からウイルスを分離し,T2, T3, SGA及びSG1分離株を得た.4分離株の汁液接種によりトルコギキョウで原病徴が再現された.T2及びT3分離株の宿主範囲は狭く,13科25種の植物のうちで,3科4種のみに全身感染した.T2及びSG1分離株はミカンキイロアザミウマでは媒介されず,ネギアザミウマのみにより媒介された.発病株を電子顕微鏡で観察した結果,平均粒子径85nmの球状粒子が認められた.DAS-ELISA法でT2及びT3分離株ともにIYSV抗体と強く反応したが,TSWV及びINSV抗体には反応しなかった.4分離株のヌクレオキャプシドタンパク質のアミノ酸配列を解析した結果,T2及びSG1分離株はオランダで発生したIYSVと相同性がそれぞれ97.8%, 97.1%であり,T3及びSGA分離株はブラジルで発生したIYSVとのそれが97.4%, 97.8%であった.以上から,4分離株をIYSVと同定した.本病害をえそ輪紋病と命名する.IYSVのトルコギキョウへの病原性は系統により多少異なることと,IYSVに対するトルコギキョウの感受性には品種間差があることが明らかとなった.

Melon yellow spot virus (MYSV) and Watermelon silver mottle virus (WSMoV) are distinct virus species belonging to the genus Tospovirus in the Family Bunyaviridae. They have quasi-spherical virions with lipid envelopes where three single-stranded RNA segments are contained, and are persistently transmitted by Thrips palmi. WSMoV was first identified in 1982, and it has been causing problems on watermelon, wax gourd, melon, cucumber and bitter gourd productions in Japan and Taiwan. MYSV was first identified in 1992, and it has been causing severe damages on melon and cucumber productions in Japan. Nucleotide sequences of the smallest RNA segments of WSMoV and MYSV were analyzed to examine genetic relationships. Identities of the nucleocapsid protein genes of WSMoV or MYSV with other tospovirus species suggested that MYSV and Physalis severe mottle virus (PYSV) were considerably similar. Identities of the nucleocapsid protein genes and the non-structural genes of two isolates of MYSV from cucumber plants with those of reported MYSV with those with PSMV were more than 96%. Therefore, PSMV and MYSV should be classified as the same species. Comparison of Japanese isolates and Taiwanese isolates of WSMoV suggested that they are genetically related each other, except for a 20 nucleotide insertion sequence was found in Japanese isolates. RT-PCR assay with specific primers of WSMoV or MYSV were examined. RT-PCR with WSMoV specific primers WN5 (5' -GCACACACATAGGTATAACT-3') and WN3 (5' -TCCTTTGTAAACACCATG-3') detected approximately 1.1 kb DNA fragment from WSMoV-infected samples. RT-PCR with MYSV specific primers MN5 (5' TATTTCATTCAACTAGTTAA3') and MN3 (5' ATTCAACATCAGCAAGTCAA3') detected approximately 1.0 kb DNA fragment only from MYSV-infected samples. No significant amplification was detected from other tospovirus -infected samples or healthy samples.

Viral diseases of sweet potato are very prevalent and often seriously damaging to the plants. In particular, the severe strain of the sweet potato feathery mottle virus (SPFMV-S) causes obizyo-sohi' disease in Japan. In order to confer viral resistance against SPFMV using current biotechnology, a transgenic sweet potato has been produced, introducing hygromycin-resistant (hpt) and SPFMV-S coat protein (CP) genes, which have shown a significant resistance to SPFMV-S. In the breeding programme, it is important to confirm that the viral resistance conferred in T 0 plants can be inherited by their progeny. In the present study, progeny were obtained from crosses between the transgenic T 0 and a non-transgenic variety of sweet potato. The results showed that the CP gene was inherited by the next generation and that the stability of viral resistance was also confirmed. Thus, this production system for the virus-resistant transgenic sweet potato is useful in practical breeding.

One of the most-serious diseases of sweet potato [Ipomoea batatas (L.) Lam] is russet crack disease caused by sweet potato feathery mottle virus (SPFMV). We constructed an expression vector carrying the coat protein (CP) and hygromycin phosphotransferase (hpt) genes driven by cauliflower mosaic virus 35 S promoters. Accordingly, we introduced the expression vector into sweet potato variety Chikei 682-11 by the electroporation method. Among 449 calli obtained after antibiotic selection, 19 plants from seven independent calli grew to form adventitious shoots. Three transgenic lines were obtained from independent calli, based on analysis of the CP and hpt genes. The transcription and translation of the CP gene were shown in these transgenic lines by Northern- and Western-blot analyses. To assay the virus resistance of the transgenic lines, each line was vegetatively propagated and then grafted with morning glory (Ipomoea nil) that had been infected with SPFMV-S. A PAS-ELISA assay with polyclonal antiserum of the CP demonstrated that virus accumulation 3 months after grafting with the infected morning glory was suppressed in the transgenic lines as compared with non-transgenic ones. These transgenic lines were shown to be highly resistant not only to primary but also to secondary infection by SPFMV-S. Thus we concluded that the three transgenic lines with the CP gene of SPFMV-S can be used for coat protein-mediated resistance to the virus.

RT-PCR procedures for detection of multiple species of tospovirus from plant tissues were investigated. Downstream primers were designated from the 3' untranslated sequences of the S RNA. An upstream primer was designated from the degenerated sequences of the nucleocapsid protein. Approximately 450 bp DNA fragments were detected when Tomato spotted wilt virus (TSWV)- or Impatiens necrotic spot virus (INSV)- infected tissues were examined. Approximately 350 bp DNA fragments were detected when Watermelon silver mottle virus (WSMoV)- or Melon yellow spot virus (MYSV)-infected tissues were examined. Template RNA was extracted using CF 11 cellulose powder, and nonspecific amplification became unnoticeable when double-stranded RNA was used. The amplified fragments of WSMoV were differentiated from those of MYSV by AluI or TaqI digestion. The amplified fragments of TSWV were differentiated from those of INSV by DraI or HindIII digestion. An alstroemeria plant that was infected with an unidentified tospovirus was also examined, and a 350 bp fragment that was 97% identical to Iris yellow spot virus was detected. This method, therefore, detected at least five distinct Tospovirus species.

Cucumber plants (Cucumis sativus) with necrotic spots, yellowing and mosaic symptoms were found in Kochi Prefecture, Japan in 1995. The causal agent was identified as Melon yellow spot virus (MYSV) on the basis of host reactions, stability in crude sap, particle morphology, transmission by Thrips palmi, serological relationships and the identity of amino acid sequence of the N protein. This is the first report of the occurrence of MYSV on cucumber, and the name “potted wilt”s was proposed for the disease.

In February 1999, cineraria (Senecio×hybridus) plants with necrotic ring spot were found in Okayama Prefecture, Japan. A spherical virus with a diameter of ca. 85∼120nm was isolated. The virus infected seven plant species of four families after mechanical inoculation. In serological tests, the virus was closely related to Impatiens necrotic spot virus (INSV). In RT-PCR with INSV-specific primers, a fragment of about 900bp was amplified. The fragment had a high degree of nucleotide sequence homology with the S RNA of INSV. Based on these results, the virus isolated from cineraria was identified as INSV. This is the first report of INSV on cineraria in Japan.

We investigated the cellular basis of the age-related decline in antibody (Ab) and delayed-type hypersensitivity (DTH) responses to sheep red blood cells (SRBC) in vivo in short-lived senescence-accelerated mouse (SAM) P1. In SAMP1 mice, age-related decreases in CD4+ T cells in the peripheral blood occurred earlier than in control mice and occurred in parallel with the age-related decline in Ab and DTH responses. In addition, the involution of the thymus was faster. The injection of thymic T cells from young mice before sensitization completely restored the Ab responses in aged SAMP1 mice. These data suggest that the age-related decline in Ab response is due to the age-related early loss of helper-T (TH) cells. On the other hand, the local transfer of spleen cells from sensitized aged donors into the footpads of naive syngeneic recipients evoked strong DTH responses, demonstrating the existence of DTH-mediating T (TDTH) cells in the spleens of aged SAMP1 mice. Moreover, the local injection of naive spleen cells from young donors, together with the antigen, into the footpads caused DTH responses in sensitized aged recipients. These findings indicate that TDTH cells were induced and were able to migrate and function as effector cells in aged mice. When naive spleen cells from aged donors were injected locally into the footpad, they restored the DTH response in aged mice, but this effect did not work if the cells were injected intravenously. This demonstrates that the inflammatory cells of the aged mice were able to work at the local site, but could not migrate there. The intravenous injection of naive spleen cells from young donors restored the DTH response in aged mice, suggesting that the endothelial cells of aged mice were not impaired and permitted the inflammatory cells to migrate into the extravascular tissues. Thus, although the age-related decline of the Ab and DTH responses occur in parallel, we found different effects of aging on TH and TDTH cells in SAMP1 mice. Furthermore, our data suggest that the reason for the low DTH response in aged SAMP1 mice is not the loss of TDTH cells, but rather the impaired migration of inflammatory cells into the local site.

Transposable elements have often been discovered as new insertion sequences in known genes, and minisatellites are often employed as molecular markers in diagnostic and mapping studies. We compared the genes for flower pigmentation in a line of the common morning glory bearing fully colored flowers with those in two anthocyanin flaked mutable lines producing variegated flowers and found RFLPs at the region of the ANS gene for anthocyanin biosynthesis. The DNA rearrangements detected by the RFLPs are due to integration of a novel type of minisatellite, MiniSip1, having a long LTR retrotransposon, RTip1, inserted in the mutable lines. The structural analysis of the rearranged region revealed that the 12.4-kb RTip1 element is flanked by 5-bp target duplications within the MiniSip1 sequence and contains two LTR sequences of about 590 bp, a primer binding site for tRNALys, a typical polypurine tract and another new type of minisatellite, MiniSip2. Since no long open reading frame corresponding to the gag and pol genes was found, RTip1 appears to be a defective Ty3/gypsy-like element. Interestingly, the 269-bp-long MiniSip1 element comprises two alternating motifs of 41 bp and 19 bp, whereas the 962 bp long MiniSip2 element consists of two partially alternating motifs of 86 bp and 90 bp which are partially homologous to each other. Possible evolutionary processes that may have generated the rearranged structure at the ANS gene region are also discussed.

A series of related strains of senescence-accelerated mouse (SAM) shows strain-unique age-related diseases, such as amyloidosis, deficit in learning and memory, osteoporosis, and brain atrophy, while many of these disease-prone mouse (SAMP) strains have impaired immune activity as young adults, and have a short life span, probably not due directly to the diseases. Because the mean life span was prolonged and the time of the disease onset was delayed by a low-calorie dietary condition or a specific pathogen-free environment, both of which ameliorate the impaired immune activity, the enhancement of immune activity may help decrease the deteriorative process of aging, to that seen in ordinary strains of mice. Studies using the SAMP model may help elucidate the role of immunity in the aging process. Herein, we review the cellular and genetic basis of the immune abnormality in SAMP mice, then discuss the relationship between immune abnormality and development of the age-related disease, senile amyloidosis, findings obtained on SAMP hybrid mice and congenic mice for disease-related genes. Activation of the gene(s) for senile amyloid per se shortened the life span, and the early development of the immune dysfunction primarily seems to be both genetically and physiologically independent of amyloidosis, although the disease may be indirectly modified in the aged with depressed immune activity.

Schwann cell basal lamina tubes serve as attractive conduits for regeneration of peripheral nerve axons. In the present study, by using basal lamina tubes prepared by in situ freeze-treatment of rat saphenous nerve, the effects of exogenously applied basic fibroblast growth factor (bFGF) on peripheral nerve regeneration was examined 2 and 5 days after bFGF administration. Regenerating axons were observed by light and electron microscopy using PGP9.5-immunohistochemistry for specific staining of axons. In addition, the localizations of bFGF and its receptor (FGF receptor-1) were examined by immunohistochemistry using anti-bFGF antibody and anti-FGF receptor-1 antibody, respectively. Regenerating axons extended further in the bFGF-administered segment than in the bFGF-untreated control segment. Electron microscopy showed that regenerating axons grew out unaccompanied by Schwann cells. Findings concerning angiogenesis and Schwann cell migration were very similar between the bFGF treated and control nerve segment. bFGF-immunoreactivity was not detected in the control nerve segment. In contrast, bFGF-immunoreactivity was detected on the basal lamina tubes as well as on the plasmalemma of regenerating axons facing the basal lamina in the bFGF treated nerve segment up to 5 days after administration, suggesting that exogenous bFGF can be retained in the basal lamina for several days after administration. FGF receptor was detected on the plasma membrane of regenerating axons where they abutted the basal lamina. These results indicate that bFGF could promote the extension of early regenerating axons by directly influencing the axons, but not via Schwann cells or angiogenesis.

Transmembrane movement of phosphatidylserine (PS) and various PS analogs at the plasma membrane is thought to occur by an ATP-dependent, protein-mediated process. To isolate mutant CHO cells defective in this activity, we first obtained conditions which inhibited the endocytic, but not the non-endocytic pathway of lipid internalization since PS may enter cells by a combination of these two pathways. We found that acidic treatment of cells, which blocks clathrin-dependent endocytosis, enhanced the energy-dependent uptake of 1-palmitoyl-2-(6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl -sn- glycero-3-phosphoserine (C6-NBD-PS) in CHO cells from donor vesicles (liposomes) by about twofold. Control experiments demonstrated that the enhanced uptake of C6-NBD-PS at acidic pH was not due to: (a) an increase in the capacity of the plasma membrane to incorporate C6-NBD-PS from the donor vesicles; (b) a decrease in the rate of loss of C6-NBD-PS from the cells; or (c) fusion or engulfment of the donor vesicles. When cytosolic acidification (to pH 6.3) was imposed without acidification of the extracellular medium, C6-NBD-PS uptake by intact cells was increased by about 50% compared to control values determined in the absence of acidification. These results suggested that a protein and energy dependent system(s) for transbilayer movement of the fluorescent PS was stimulated by cytosolic acidification. A screening method for mutant cells defective in the non-endocytic uptake of fluorescent PS analogs with replica cell colonies at acidic pH was then devised. After selection of mutagenized CHO-K1 cells by in situ screening, we obtained a mutant cell line in which uptake of fluorescent PS analogs was reduced to about 25% of the wild type level at either pH 6.0 or 7.4. Control experiments demonstrated that the reduced uptake of fluorescent PS analogs in the mutant cells was unrelated to multidrug resistance, and that endocytosis of another plasma membrane lipid marker occurred normally in the mutant cells. These results suggested that a non-endocytic pathway responsible for uptake of fluorescent PS analogs was specifically affected in the mutant cells.

A quasi-spherical virus was isolated from a cultivated Amazon lily plant (Eucharis grandiflora) that could be mechanically transmitted to healthy E. grandiflora plants, subsequently producing mild mosaic or mottle symptoms on the leaves. The purified virus consisted of three quasi-spherical particles about 20 nm wide and 70, 40 and 30 nm in length, containing three segmented genomes of 3,169, 2,507 and 2,530 nucleotides, respectively. Sequence analysis showed that the newly isolated virus is related to pelargonium zonate spot virus, a member of the genus Anulavirus. We propose that the virus should be designated as Amazon lily mild mottle virus (ALiMMV).

Fourteen strains of cucumber mosaic virus (CMV) from Australia have been characterized by their host range and symptomatology. They were classified as subgroup I or II strains by a dot-blot molecular hybridization assay between their total viral RNAs and selected cDNAs. The strains F(NY) and L(NY), both from the USA, were used as the subgroup I- and subgroup II-type strains, respectively. A range of serological tests was used to compare these isolates. Gel immunodiffusion tests, with standard antigens homologous to the antisera prepared against glutaraldehyde-fixed virus of 11 strains, showed that they could be divided into three serogroups on the basis of spur formation in heterologous reactions. Two of the serogroups included either subgroup I or subgroup II isolates, whereas the third serogroup consisted of only one strain (Y(WA)) which was homologous to all the strains tested. Use of heterologous standard antigens in this test failed to show further subgrouping of the antigens. Double-antibody sandwich (DAS)-ELISA using polyclonal antibodies to distinct virus strains also placed the 14 strains in the same three serogroups. When eight different monoclonal antibodies (MAbs) were used in indirect ELISA, one of them distinguished subgroup-I strains and another distinguished subgroup-II strains; the Y(WA) strain fell into subgroup II. Other MAbs showed narrower or broader specificity. Thus both molecular hybridization with total RNA and specific MAbs may be useful for separating isolates of CMV into subgroups I and II. Spur formation using heterologous standard antigens to the antisera, as well as being more difficult to interpret, was not a reliable criterion for classification.

The full nucleotide sequence of the S RNA segment of Melon yellow spot virus (MYSV) was determined to be 3232 nucleotides long and to have every feature of the S RNAs of tospoviruses reported so far. From an amino acid sequence comparison of nonstructural proteins (NSs), MYSV is most closely related to two tospoviruses in serogroup IV. MYSV had nucleotide and amino acid sequence identities over 96% with Physalis severe mottle virus in all regions of S RNA except in an intergenic region and in deduced open reading frames, including that on complementary strand RNA.

We determined the complete or partial nucleotide sequences of eight Sweet potato feathery mottle virus (SPFMV) isolates and compared them with 12 other partial SPFMV sequences. The genome organization of the isolate Bungo (strain group C) was very different from those of isolates in the russet crack, ordinary (O), and east Africa groups. 10-O appeared to be a recombinant of isolates S and O, with a recombination site within the P1 gene. This study will help to provide a better understanding of the taxonomy and biology of SPFMV and how these features relate to virulence.

Three types of apolipoprotein A-II (apoA-II) proteins (A, B and C) were predicted from the nucleotide sequence of apoA-II cDNA. Substitution of amino acid residues was noted at four positions (type A: Pro-5, Asp-20, Met-26, Ala-38; B: Pro-5, Glu-20, Val-26, Val-38; C: Gln-5, Glu-20, Val-26, Ala-38). Each type was identifiable by digestion of amplified apoA-II DNA by PCR, using restriction-fragment-length polymorphism of the apoA-II gene for restriction enzymes Cfr13I and MspI. The molecular type of apoA-II was determined among 23 strains of mice including nine of the senescence accelerated mouse series developed in our laboratory. Examination of types of apoA-II and amyloid deposition in the F2 and F3 hybrid mice showed that apoA-II amyloid deposition was present only in the mice homozygous for type C apoA-II and which were 12-17 months of age. The molecular type of apoA-II may be a factor involved in the development of senile amyloidosis in mice.

The effects of age and dietary restriction on immune response were investigated using an animal model of accelerated senescence (senescence accelerated mouse, SAM). The experimental groups consisted of control (ad libitum fed) and restricted groups (fed 60% of energy intake of the controls). Spleen weight and total number of splenic cells were significantly lower in the food-restricted group at 8 mo of age. Percentages of T (Thy-1.1+) and B (surface Ig+) cells in the splenic cells were not significantly different between the two groups. The number of direct hemolytic plaque-forming cells per 10(6) spleen cells 4 d following immunization with sheep red blood cells and dinitrophenyl-Ficoll was significantly greater in the 8-mo-old mice in the food-restricted group than in the control group. In the latter group, antibody responses Progressively decreased with age. Mitogen responses to concanavalin A and lipopolysaccharide were maintained in the food-restricted group but were depressed in the control group at 8 mo. In addition, though autoantibody to single-stranded DNA increased in the control group with advancing age, there was a steady decrease in the food-restricted group until 8 mo. Serum immunoglobulin (IgA and IgM) concentrations were significantly lower in the food-restricted group than in controls at 8 mo of age. Therefore, our results suggest that when senescence accelerated mice are subjected to food restriction, there may be a modulatory effect on the immune dysfunction associated with advancing age.

The complete nucleotide sequences of the S RNA of two Japanese isolates of Watermelon silver mottle virus were determined. One was isolated from naturally infected watermelon and causes malformation on upper leaves of Tetragonia expansa. The other was isolated from melon and causes characteristic yellow necrotic lesions on upper leaves of T. expansa. The total nucleotide sequences of the S RNA of WS-O and WS-Y were 3553 nt and 3558 nt long, respectively. Both the nucleotide sequences and the deduced amino acid sequences of WS-O and WS-Y were quite similar even though the symptoms on T. expansa are quite different. They were also significantly similar to those of the Taiwanese isolates, Topso-W and Tospo-To. These results suggested that the Japanese isolates and the Taiwanese isolates of WSMoV were classified as one group not only serologically but also genetically. Within the S RNA sequences, the most variable region was the intergenic region between the N gene and the NSs gene. This was due to a 20 nt insertion between the Japanese isolates and the Taiwanese isolates.

The re-innervation process in the periodontal ligament of replanted canine teeth was examined by immunohistochemistry for protein gene product 9.5 (PGP 9.5), a general marker for neurons, and by electron microscopy. Within 1 week of replantation, the periodontal fibers had regenerated, filling the narrow spaces between the alveolar bone and the root surface around the cervical and apical regions. Near the root apex, however, no PGP 9.5-immunopositive nerve fibers were found in the regenerated periodontal ligament except for those in the alveolar half of the ligament. At 2 weeks after replantation, many nerve fibers positive for PGP 9.5 had ascended the periodontal ligament from the thick nerve bundles located near the root apex. Fine nerve endings showing complicated ramification were also present in the apical region. By 3 or 4 weeks after replantation, the vascular network was regenerated and principal periodontal fibers were re-established throughout the entire length of the periodontal ligament. The extensively ramified PGP 9.5-immunopositive structures had increased in thickness and density and showed characteristic tree-like profiles by 3 weeks. Electron microscopy confirmed that most of these structures were Ruffini-like endings, and demonstrated that such nerve terminals were almost regenerated by 4 weeks post-replantation. These results indicated that, in the periodontal ligament of replanted canine teeth, the regeneration of the nerve fibers including mechanosensory receptors first showed signs of regeneration by 2 weeks following tooth replantation and proceeded rapidly thereafter. Regeneration of the periodontal ligament including fiber architecture as well as vascular and neural elements was almost complete by 4 weeks after replantation.

The complete nucleotide sequence of a sweet potato feathery mottle virus severe strain (SPFMV-S) genomic RNA was determined from overlapping cDNA clones and by directly sequencing viral RNA. The viral RNA genome is 10 820 nucleotides long, excluding the poly(A) tail and contains one open reading frame (ORF) starting at nucleotide 118 and ending at 10 599, potentially encoding a polyprotein of 3 493 amino acids (Mr 393 800). The ORF was followed by a 3′ untranslated region of 221 nucleotides. The deduced polyprotein includes P1 (74K), HC-Pro (52K), P3 (46K), 6K1, CI (72K), 6K2, NIa-VPg (22K), NIa-Pro (28K), NIb (60K) and coat (35K) proteins, after an analysis of protein cleavage sites analogous to other potyvirus polyproteins. The polyprotein had a high level of amino acid identity with those of other potyviruses, except in the regions of P1 and P3. The P1 of SPFMV-S RNA has 664 amino acid residues, and is the largest and least similar to those of other potyviruses. HC-Pro and CI show high identity with those of other potyviruses. P3 has relatively low identity, however, the length of P3 was within the range of variability among other potyviruses. The 6K1 protein between P3 and C1 is also highly similar to those of other potyviruses. This is the first report on the complete nucleotide sequence of the sweet potato-infecting virus.

The effect of basic fibroblast growth factor (bFGF) applied locally into the bone under physiological conditions was investigated. An aqueous solution containing 0 microgram (vehicle), 100 micrograms or 400 micrograms recombinant human bFGF was percutaneously applied through a needle into the right ilium in rabbit, and the ilia were harvested 4 weeks after the application. Compared with vehicle-treated animals, bone mineral density measured by dualenergy X-ray increased in the 400 micrograms bFGF group. The width of trabeculae in the bFGF-treated groups was greater than in the vehicle group. These results showed that bFGF applied locally into the bone under physiological conditions affected bone formation, and suggested that such an application might have potential for increasing bone.

Recombinant DNA molecules containing cDNA of a sweet potato feathery mottle virus severe strain (SPFMV-S) RNA genome were constructed and the partial nucleotide sequences were determined for three DNA inserts, which cover 4-2 kb from the 3'-terminus excluding the poly (A) tail. This region of the genome consists of an open reading frame of 1340 amino acids (a.a.) and a 3'-non-translated region of 224 nucleotides. The protein products expected were 6K2 (53 a.a.), NIa (435 a.a.), NIb (521 a.a.) and CP (315 a.a.). Among NIa, NIb and coat proteins, the NIb protein was found to be the most conserved (59-68%) when compared to the corresponding proteins of other distinct potyviruses.

The apoA-II gene was amplified by polymerase chain reaction (PCR) from chromosomal DNA of nine inbred strains of mice. Sequence analysis of the PCR products indicated the presence of three types of apoA-II genes. Three types of apoA-II proteins (A, B and C) were predicted from the nucleotide sequence of apoA-II cDNA. Substitution of amino acid residues was noted at 4 positions. Each type was identifiable by digestion of PCR amplified apoA-II DNA, using restriction enzyme Cfrl3l and Mspl. The mouse strains, SAM-P/1, SAM-P/2, SJL/J, A/J had type A apoA-II. These strains generally exhibit amyloid deposition from relatively younger age. Examination of types of apoA-II and amyloid deposition in the F2 hybrid mice showed that apoA-II amyloid deposition was present only in the homozygous mice for type A apoA-II. We postulate that the molecular structure of apoA-II may be an important factor involved in the development of senile amyloidosis in mice.

SUMMARY Transmission of different nepoviruses through chickweed (Stellaria media) seed was differently affected by ambient temperature during seed production. Raspberry ringspot and tomato black ring (Scottish isolate) viruses were similarly and frequently transmitted at14, 18 and 22 oC, whereas arabis mosaic virus was transmitted most frequently at 14 oC, and strawberry latent ringspot and tomato black ring (German isolate) viruses at 22 oC. When infected by seed-borne nepoviruses, seedlings of S. media and other species were symptomless at 15–25 oC, and the viruses were therefore detected by inoculating sap to Chenopodium quinoa indicator plants. However, typical symptoms of arabis mosaic and tomato black ring viruses were induced by growing Nicotiana tabacum, N. clevelandii and C. quinoa seedlings infected with seed-borne virus at 33–37 oC during the third and fourth weeks after sowing, preceded and followed by periods at 15–25 oC. The proportion of N. tabacum seedlings developing symptoms was the same as that of untreated seedlings yielding sap-transmissible virus. Seed transmissibility of pseudo-recombinant isolates of raspberry ringspot and tomato black ring viruses, containing RNA-i from one virus strain and RNA-2 from another strain, depended greatly on the transmissibility of the strain contributing RNA-i. The source of RNA-2 had an additional but smaller influence. The satellite RNA (RNA-3) of tomato black ring virus was seed-transmitted in S. media and its occurrence in cultures did not affect the frequency of transmission of the virus. Results of testing the infectivity of extracts of seed from infected mother plants suggested that failure of seed transmission reflected failure to become established in the seed, not subsequent inactivation. Whereas seed transmissibility of raspberry ringspot virus is primarily dependent on information carried in RNA-i, transmissibility by nematode vectors, another property of major ecological importance, is determined by RNA-2. In the field, selection pressures presumably can act independently on the two parts of the genome but evidence was also obtained of selection for mutual compatibility of RNA-i and RNA-2.

A possibly new HL-A specificity, associated with W22, was found in the Japanese. Population and family studies indicate that the gene Sa 1 (frequency 0.0771) behaves as a ‘new’ allele at FOUR sublocus. A case of crossing over between the LA and FOUR loci of the HL-A system was also described. The HL-A system of leukocyte antigens is the major histocompatibility system in man, and exhibits a remarkable degree of allelic polymorphism [6, 8, 9]. The 5th Histocompatibility Workshop was devoted to an anthropological survey of HL-A throughout the world, and 54 different populations were studied by the participating laboratories [7]. These studies have shown that each ethnic group possesses its own characteristic HL-A profile. Also remarkable is the higher frequency of the unknown HL-A antigens of the FOUR series in Orientals [1, 10, 12, 13]. Screening for antibodies with cells of Japanese, therefore, will offer a chance of detecting sera against unknown factors. The present study reports the presence of antibody against a possibly new HL-A specificity, Sa 1, associated with W22, found in the sera of some Japanese multiparous women. In addition, a case of crossing over between the LA and FOUR loci of the HL-A system is presented.

Summary Chenopodium quinoa plants were inoculated with pairs of pseudo-recombinant isolates of raspberry ringspot virus that shared one part of their genome but not the other. Results of typing progeny virus obtained from systemically infected leaves indicated that RNA-1 from different virus strains differed in competitiveness, and also in ability to allow the expression of differences in competitiveness between RNA-2 from different strains. Ability of genotypes to dominate was related to the rapidity with which they induced systemic symptoms in singly infected plants.

Synthesis of circular (RNA 2) and linear (RNA 3) molecules of velvet tobacco mottle virus (VTMoV) satellite RNA (sat RNA) has been studied by incubating strips of tissues excised from systemically infected Nicotiana clevelandii in solutions of [14C]uridine. After a short lag, RNA and virus synthesis proceeded at a constant rate for at least 24 hr, during which time most of the synthesis was directed to the production of RNAs 2 and 3. The kinetics of [14C]uridine incorporation into the sat RNA molecules after increasing times of incubation and during pulses of [14C]uridine followed by chase incubation with excess [12C]uridine suggest that RNA 3 is a percursor of RNA 2. However, not all the RNA 3 synthesized was shown to end up as RNA 2, even after 72 hr of incubation. Several lines of evidence are presented supporting the conclusion that VTMoV-infected cells contain large pools of unencapsidated sat RNA. It is suggested that the sat RNA may have a greater affinity for the VTMoV replicase than the helper viral RNA which results in copious production of the sat RNA.

This paper describes the electron microscopic structure of dyskeratotic cells observed in cutaneous lesions of 3 cases with acquired zinc deficiency. Double-zoned cytoplasm consisted of amorphous cell organelles and irregular tonofilaments occasionally containing lipid droplets and melanosomes. Small nuclei were seen in many dyskeratotic cells around the blister. Various degrees of alteration were noted in cells in erythematous lesions following the disappearance of all desmosomes. The halo formation of dyskeratotic cells with double-zoned cytoplasm indicates an alteration in keratinocytes, which may be caused by superoxide radical anions resulting from inactivity of superoxide dismutase, a zinc-dependent enzyme.