First Report of Root-Knot Nematode Meloidogyne arenaria on Atractylodis macrocephalae in China.

Atractylodis macrocephalae is an important Chinese herbal medicine plant and its rhizome is of high medicinal value. In recent years, a severe decline in yield has been observed in Bozhou City (China's largest A. macrocephalae producing area), Anhui Province, China. A survey for plant-parasitic nematodes was conducted in this area from June to September 2011. Stunted plants displayed chlorotic or necrotic lower leaves near the ground part by the growth reduction; examination of the roots of stunted plants revealed the presence of galls typical of infestation by root knot nematode. Root nodules were found on the tap and lateral roots caused the fleshy tap root deformity. The incidence of diseased plants was estimated to be 45%, and yield loss was quantified as 43.5%. Nematodes were extracted from the root samples as previously described (4) and identified by morphology, enzyme analysis, and molecular characterization. Morphology of the female perineal patterns and measurements of the second-stage juveniles (J2s) matched those of the original description of Meloidogyne arenaria. Enzyme analysis of the esterase (Est) phenotype was also typical of the AII phenotype in M. arenaria (2). DNA was extracted according to a modified protocol (1), and the rDNA-internal transcribed spacer (ITS1_5.8S_ITS2) region was amplified with universal primers V5367 (5'-TTGATTACGTCCCTGCCCTTT-3') and 26S (5'-TTTCACTCGCCGTTACTAAGG-3'). PCR yielded a fragment of 764 bp and the purified product was sequenced by Sanger's dideoxy chain termination method (ABI3730). Sequences were identical to that of M. arenaria in GenBank (Accession No. AF387092) (3). Amplification of the D2/D3 fragments of the 28S RNA with universal primers D2A (5'-ACAAGTACCGTGAGGGAAAGTTG-3') and D3B (5'-TCGGAAGGAACCAGCTACTA-3') yielded a PCR fragment of 758 bp. These sequences were also identical to that of M. arenaria in GenBank (Accession No. AF435803). For further confirmation, amplification of the IGS region with universal primers 5S (5'-TTAACTTGCCAGATCGGACG-3') and 18S (5'-TCTAATGAGGGAACCAGCTACTA-3') yielded a PCR fragment of 713 bp. These sequences were 99.64% homologous to that of M. arenaria (GenBank Accession No. MAU42342). To our knowledge, this is the first report of M. arenaria species on A. macrocephalae in China. The fleshy tap root of A. macrocephalae is the main edible part of the plant, and the damage caused by root knot nematode will potentially reduce the yield and quality of this herb. References: (1) J. L. Cenis et al. Phytopathology 83:76, 1993. (2) P. R. Esbenshade and A. C. Triantaphyllou. J. Nematol. 17:6, 1985. (3) T. C. Vrain et al. Appl Nematol. 15:563, 1992. (4) L. F. Wang et al. Forest Res. 14:484, 2001.

Molecular and immunological approaches to mammalian fertilization.

By means of hybridoma technology, we obtained six hydriboma cell lines producing monoclonal antibody (mAb) to porcine zona pellucid (ZP), two of which recognizes the steric structure of common antigens between porcine ZP and humans. Furthermore, we have analyzed all or partial structures of N- and O-linked sugar chains of ZP glycprotein from porcine or murine oocytes. Then, we have clarified that beta-galactose and Le(X) residues on ZP played the binding roles to sperm cells in porcine and murine fertilization. We have also succeeded Sp38 cDNA cloning from cDNA library of porcine testis. We found that Sp38 protein bind to porcine ZP2 and expressed in murine and human sperm cells. Corresponding to the presence of major histocompatibility complex (MHC) class II on murine sperm, CD4 on the murine egg plasma membrane was clearly shown by indirect IIF and immunoprecipitation test. Furthermore, the transcriptional expression of CD4/p56(lck) in eggs was confirmed by RT-PCR method. In addition, the p56(lck) associated with CD4 underneath the plasma membrane of eggs was autophosphorylated after cross-linking of CD4 with anti CD4 mAb. The binding between eggs or Sf9-CD4 cells labeled with anti-CD4 mAb and sperm cells labeled with anti-monomorphic region of class II mAb was completely blocked. Considering these findings together with the fact that an interspecies' heterogeneity is present in CD4 amino acid sequence at the interactive site with class II, we elucidated that one of species' specific intercellular adhesions between two gametes at the fusion step in fertilization is definitely mediated by class II located on the posterior region of sperm head and CD4/p56(lck) complex on the plasma membrane of egg.

Expression of Fas-Fas ligand in murine testis.

PROBLEM: During spermatogenesis, it has been suggested that the number of germ cells to be matured is regulated and restricted through the apoptotic mechanism. In the present study, we investigated the expression and apoptotic role of Fas and Fas ligand (L) in the murine testis. METHOD OF STUDY: The expression of Fas-FasL in the murine testis was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR)-Southern blot hybridization, in situ hybridization, and Western blot methods. The terminal deoxynucleotide transferase mediated dUTP-nick end label (TUNEL) and DNA fragmentation methods were applied to detect the generation of apoptosis in germ cells. RESULTS: By means of RT-PCR-Southern blot hybridization, we demonstrated the positive expression of Fas in testicular germ cells, and of FasL in testicular cells. supporting the findings with in situ hybridization that Fas was localized in germ cells, whereas FasL was localized in Sertoli cells of murine testis. A specific band at 45 kDa was obtained in the lysates from testis and germ cells with Western blot analysis. Then, the co-incubation of germ cells with Spodoptera frugiperda (Sf9)-FasL cells in vitro resulted in the induction of apoptosis in germ cells detected by the TUNEL method. Furthermore, DNA fragmented ladders were also demonstrated in germ cells co-incubated with Sf9-FasL cells. CONCLUSION: Fas-FasL system seemed to play an apoptotic role in spermatogenesis by the molecular interaction between FasL on Sertoli cells and Fas on germ cells.

Isolation and identification of apoptosis inducing nucleosides from CD57(+)HLA-DRbright natural suppressor cell line.

The CD57(+)HLA-DRbright natural suppressor (57.DR-NS) cell line derived from human decidual tissue generated the apoptosis in K562/Molt4 target cells mediated by soluble factors released into the culture supernatant. The factors in the culture fluid of the 57. DR-NS cell line were isolated by the physicochemical procedures as follows, first by a preparative octadecyl sorbent column, further by thin-layer-chromatography (TLC), finally by reverse-phase high-performance liquid chromatography (HPLC). The six major components (P1-P6) obtained by HPLC demonstrated the generation of apoptotic cell death of target cells. The physicochemical characters of six active components were strongly suggested to be nucleosides and their modified forms in nature. Then, the physicochemical structures of the six active components were finally determined by fast atom bombardment-mass spectrometry (FAB-MS) and nuclear magnetic resonance (1H NMR) spectrometry as follows: P1, 2'-deoxyuridine; P2, ribothymidine; P3, 2'-O-methyluridine; P4, thymidine; P5, 2'-O-methylinosine; P6, 2'-O-methylguanosine. Thus, we collectively named them "apoptosis inducing nucleosides (AINs)." Then, we demonstrated that the induction of apoptosis in target cells by the 57.DR-NS cell line was mediated by a series of AINs.

Receptive field properties of visual neurons in the avian nucleus lentiformis mesencephali.

The receptive field (RF) properties of visual neurons extracellularly recorded from the nucleus lentiformis mesencephali (nLM) in pigeons (Columba livia) were quantitatively analyzed using a workstation computer. These cells were actively spontaneous, and direction-and velocity-selective. Using spatial gratings as visual stimuli, these cells could be divided into three groups: uni- (74%), bi- (17%), and omnidirectional (9%) cells in terms of their directionality. On the basis of their velocity selectivity, they could be named slow cells (84%), preferring low velocity (0.1-11 degrees/s), and fast cells (14%), preferring rapid motion (34-67 degrees/s), with one cell (2%) responding maximally to an intermediate velocity of 18 degrees/ s. These two properties were correlated in the way that all unidirectionals were slow cells, omnidirectionals were fast cells, and bidirectionals were either slow or fast cells including the intermediate cell. Using small targets as visual stimuli, it was found that the majority of cells examined had RFs that each consisted of an excitatory RF (ERF) and an inhibitory RF (IRF) that overlapped. The unidirectionals were mainly of this type of RF structure, whereas the omnidirectionals apparently had ERFs alone. The direction preference of ERF was opposite to that of IRF for unidirectional cells tested, whereas they were perpendicular to each other for one bidirectional cell. The overall responses of these cells resulted from interaction between excitation and inhibition induced by directionally different motion. Under certain conditions, visual responses of a particular cells to a small target moving through its ERF were equal in responsive strength to those to whole-field gratings swept over the screen. It was suggested that optokinetic nystagmus produced by wholefield gratings results from population activity of large group(s) of neurons in some optokinetic nuclei, at least one of which is nLM.

Aberrant expression and dysfunction of Fas antigen in MRL/MpJ-lpr/lpr murine ovary.

In lpr mice the insertion of an early transposable element (ETn) into intron 2 of the Fas gene, which mediates apoptosis, causes the development of massive lymphadenopathy, splenomegaly and autoimmune disease. In the present study we investigated the influence of this mutation on ovarian development of lpr mice. By means of in situ hybridisation, the expression of Fas mRNA was detected at the same levels in the ovarian cells of MRL/MpJ-lpr/lpr (MRL/lpr) mice as in those of MRL/MpJ-(+)/+ (MRL/+) mice. However, indirect immunofluorescence (IIF) staining with anti-Fas monoclonal antibody (mAb) on the membrane of follicle and egg of MRL/lpr mice was significantly weaker than that of MRL/+ mice. Furthermore, the expression level of Fas protein at the 45 kDa band from ovarian cell lysates of MRL/lpr mice was much lower than that of MRL/+ mice. The co-incubation of Spodoptera frugiperda (Sf9)-Fas lig- and (L) cells with eggs of MRL/+ mice resulted in apoptosis of eggs, as detected by the terminal deoxynucleotide transferase mediated dUPT-nick end labelled (TUNEL) method. In contrast the co-incubation of Sf9-FasL cells with eggs of MRL/lpr mice did not generate apoptosis in eggs. Following intraperitoneal administration of anti-Fas mAb into both types of mice, most oocytes, a proportion of granulosa cells in the ovary and hepatocytes in liver of MRL/+ mice were positively stained by the TUNEL method, corresponding to the appearance of DNA fragmented ladders by DNA fragmentation assay, while negative signals were obtained in those cells of MRL/lpr mice. As the mice aged, the ovarian size of MRL/lpr mice was found to be much larger than that of MRL/+ mice due to the increased number of ovarian follicles. Therefore, the ovarian adenopathy in MRL/lpr mice was strongly suggested to be caused by the dysfunction of Fas antigen in the ovary.

Expression of Fas ligand in murine ovary.

Corresponding to the expression of Fas in the ovarian oocytes as previously reported (Guo et al., Biochem Biophys Res Commun 1994; 203:1438-1446; Mori et al., JSIR 1995; 9:49-50), the expression of Fas ligand (FasL) in the ovarian follicle was found to be restricted in the area of granulosa cells by the indirect immunofluorescence (IIF) test. Reverse transcriptase/polymerase chain reaction (RT/PCR) technique coupled with Southern blot hybridization analysis showed that the highest level of FasL mRNA was demonstrated in murine ovaries and granulosa cells 1 day after the administration of pregnant mare's serum gonadotropin (PMSG), while the level of FasL mRNA became very weak on the day 5, respectively. The observed gradual decrease in FasL mRNA could not be attributed to a generalized degradation of cellular RNA during atresia, as evidenced by the presence of constitutive expression of elongation factor 1 alpha (EF-1 alpha) mRNA in murine ovaries and granulosa cells treated with PMSG. Furthermore, in situ hybridization analysis with a FasL-specific probe confirmed that FasL was specifically localized in the granulosa cells of most follicles and its expression was regulated by PMSG administration. FasL localized in granulosa cells might possibly play an important role in the formation of the ovarian atretic follicles, most likely depending on PMSG administration.

Expression of Fas-Fas ligand system associated with atresia through apoptosis in murine ovary.

We examined the contribution of Fas and its ligand (FasL) in the process of follicular atresia using murine intraovarian follicles and pregnant mare's serum gonadotropin (PMSG)-hyperovulated eggs. Reverse transcriptase/polymerase chain reaction-Southern blot hybridization demonstrated positive expression of Fas in both intraovarian oocytes and hyperovulated eggs. In contrast, expression of FasL was only detected in granulosa cells. These findings were histologically confirmed by in situ hybridization using Fas- and FasL-specific probes. A time-course study showed that Fas mRNA was positive in atretic follicles through day 0 and day 2 of PMSG stimulation and negative thereafter. Levels of FasL mRNA were the highest on day 1 and tapered off toward day 5 of PMSG stimulation. Levels of elongation factor 1 alpha mRNA, a constitutive element, were constantly maintained throughout the experimental period. Coculture of ovulated eggs, intact and zona-free, and granulosa cells demonstrated positive TUNEL staining only in zona-free eggs. These findings indicate that follicular atresia is caused by apoptosis, and the apoptosis associated with internucleosomal DNA fragmentation is directly regulated by the Fas/FasL system.

Expression of Fas-Fas ligand system associated with atresia in murine ovary.

We detected that Fas receptor (Fas) was expressed at transcriptional levels in oocytes/eggs and some granulosa cells of murine ovary, whereas, Fas ligand (FasL) was found to be strongly expressed in granulosa cells by means of in situ hybridisation. These results were supported by an indirect immunofluorescence (IIF) test with anti-Fas monoclonal antibody (mAb)/FasL Ab. The lysates from granulosa cells were precipitated by anti-FasL Ab, exhibiting a specific band at 40 kDa. When zona pellucida (ZP)-free eggs were incubated with granulosa cells in vitro, apoptosis (DNA fragmentation) was induced in the eggs, as detected by the terminal deoxynucleotide transferase mediated dUTP-nick end labelling (TUNEL) method. To examine whether FasL-bearing cells can induce apoptosis in eggs expressing Fas through molecular interaction between FasL and Fas, we employed a baculovirus expression system to generate FasL on the surface of Spodoptera frugiperda (Sf9) cells. The co-incubation of eggs with Autographa californica nuclear polyhedrosis virus (AcNPV) and FasL transfected Sf9 (Sf9-FasL) cells in vitro was performed and resulted in the induction of apoptosis in eggs as detected by the TUNEL method. Apoptosis was absent in eggs co-incubated with AcNPV-1393 transfected Sf9 (Sf9-1393) cells. Thus, ovarian atresia was suggested to be induced by molecular interaction between FasL on granulosa cells and Fas on oocytes/eggs during oogenesis.

Molecular structure and function of CD4 on murine egg plasma membrane.

In the present study, the expression of the CD4 molecule on murine egg plasma membrane was confirmed by the indirect immunofluorescence (IIF) method. The full-length CD4 cDNA from murine eggs was synthesised by the reverse transcriptase-polymerase chain reaction (RT-PCR) method and its authenticity verified by Southern blot hybridisation using an end-labelled internal oligonucleotide. The results of DNA sequencing showed that the nucleotide sequence of the cDNA of CD4 from murine egg mRNA was identical to that of immune T cells. To demonstrate the direct interaction of CD4 from murine egg with murine sperm cells bearing MHC (major histocompatibility complex) class II molecule, we employed a baculovirus expression system to generate CD4 on the surface of Spodoptera frugiperda (Sf9) cells. Expression of CD4 on Sf9 cells infected with Autographa californica nuclear polyhedrosis virus (AcNPV)-CD4 was demonstrated by IIF and immunoblotting. The CD4-expressing Sf9 cells adhered to MHC class II-bearing sperm cells since the adhesion was specifically blocked by anti-CD4 monoclonal antibody (mAb) or anti-monomorphic region of MHC class II mAb. Taking our previous and present experimental results together, they strongly suggest that intercellular membrane adhesion between two gametes at the fusion step in fertilisation is mediated by the MHC class II molecule located on the posterior region of the sperm head and the CD4 molecule on egg plasma membrane.

Identification of Fas antigen associated with apoptotic cell death in murine ovary.

The majority of ovarian follicles including oocytes undergo atresia through a mechanism involving apoptotic cell death. The mechanisms underlying atresia remain to be clarified. In the present study, we detected the expression of the Fas antigen (Fas), which is a cell-surface protein to modulate apoptosis, in murine ovarian oocytes and hyperovulated eggs as well as in several control tissues. Substantial decline in Fas mRNA was found in atretic follicles which were injected with pregnant mare's serum gonadotropin (PMSG) on day 3. The observed decreases in mRNA of Fas could not be attributed to a generalized degradation of cellular RNA during atresia, as evidenced by the presence of intact 18S and 28S ribosomal RNA as well as constitutive expression of EF-1 alpha mRNA in atretic follicles. The data obtained indicate that apoptotic cell death of oocytes seemed to be associated with internucleosomal DNA fragmentation regulated by Fas molecule expressed in atretic ovarian follicles.