Ageing and chronic disease-related changes in the morphometric characteristics of ovarian follicles in cynomolgus monkeys (Macaca fascicularis).

STUDY QUESTION: How is the localisation of ovarian follicles affected by ageing and chronic diseases? SUMMARY ANSWER: Ovarian follicles shift deeper towards the medulla, due to thickening of the tunica albuginea (TA), with ageing and some major common chronic diseases. WHAT IS KNOWN ALREADY: The ovary undergoes morphological and functional changes with ageing. The follicular pool follows these changes with alterations in the amount and distribution of residual follicles. Diseases causing a chronic inflammatory process are associated with morphological changes and impaired ovarian function. STUDY DESIGN, SIZE, DURATION: We conducted a cross-sectional study, examining 90 ovaries from 90 female monkeys. The samples were collected from April 2018 to March 2019 at Tsukuba Primate Research Center in National Institutes of Biomedical Innovation, Health and Nutrition, Japan. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian samples were obtained from cynomolgus monkeys that died from natural causes or were euthanised. Ovarian sections were stained with haematoxylin and eosin (H&E) for histological analyses. In ovarian sections from 64 female macaques aged 0-25 years, a total of 13 743 follicles at different developmental stages (primordial, intermediary, primary, early secondary and late secondary) were assessed to determine the depth of each follicle from the outer surface of the ovarian cortex to the far end of the follicle, by using a digital imaging software. TA thickness was measured as sum of basal membrane and tunica collagen layer for each ovary under H&E staining. To explore the possibility of age-related trends in ovarian morphometric characteristics, samples were divided into four different age groups (0-3 years (pre-menarche), 4-9 years, 10-14 years and 15-20 years). To evaluate the effect of common chronic diseases on ovarian morphometric characteristics, macaques with diabetes mellitus (DM) (n = 10), endometriosis (n = 8) or inflammatory bowel disease (IBD) (n = 8) were compared to age-matched controls without chronic diseases. MAIN RESULTS AND THE ROLE OF CHANCE: Ovarian morphometric analysis revealed that the relative location of follicles became deeper in all age groups according to development of follicles (P < 0.05). Total follicle distance from the ovarian surface was increased with ageing (P < 0.05). In a sub-analysis according to developmental stage, only primordial and intermediary follicles were localised deeper with increasing age (P < 0.05). TA thickness was also increased with ageing (P < 0.05). The localisation of the total number of follicles became deeper in ovaries from monkeys with DM, endometriosis or IBD as compared to the control group (P < 0.05). With DM, analysis of follicles distance at almost each developmental stage was significantly deeper compared to controls (P < 0.05) with the exception of early secondary follicles. With endometriosis, follicles at primary and early and late secondary stages were significantly deeper compared to controls (P < 0.05). Also with IBD, follicles at primary and early and late secondary follicles were significantly deeper compared to controls (P < 0.001). The TA was thicker with DM and endometriosis compared to controls (P < 0.05), but not with IBD (P = 0.16). LARGE SCALE DATA: NA. LIMITATIONS, REASONS FOR CAUTION: Two-dimensional histology was used to assess follicle localisation. The possibility of minimal variations between the measured distance to the actual distance in a spherical structure cannot be excluded. Additionally, the severity of disease was not assessed. WIDER IMPLICATIONS OF THE FINDINGS: This study is the first step towards enhancing our understanding of how ageing and chronic diseases affect the relative localisation of dormant and developing follicles. These observations, combined with possible future human studies, may have managerial implications in the field of fertility preservation and other conditions involving ovarian tissue cryopreservation. STUDY FUNDING/COMPETING INTEREST(S): The present work was supported by the Grant-in-Aid for Scientific Research B (19H03801) (to K.K.), Challenging Exploratory Research (18K19624), Japan Agency for Medical Research and Development, Mochida Memorial Foundation for Medical and Pharmaceutical Research, Takeda Science Foundation and Naito Foundation (to K.K.). All authors have no conflicts of interest directly relevant to the content of this article.

Adipose tissue reduction in mice lacking the translational inhibitor 4E-BP1.

All nuclear-encoded mRNAs contain a 5' cap structure (m7GpppN, where N is any nucleotide), which is recognized by the eukaryotic translation initiation factor 4E (eIF4E) subunit of the eIF4F complex. The eIF4E-binding proteins constitute a family of three polypeptides that reversibly repress cap-dependent translation by binding to eIF4E, thus preventing the formation of the eIF4F complex. We investigated the biological function of 4E-BP1 by disrupting its gene (Eif4ebp1) in the mouse. Eif4ebp1-/- mice manifest markedly smaller white fat pads than wild-type animals, and knockout males display an increase in metabolic rate. The males' white adipose tissue contains cells that exhibit the distinctive multilocular appearance of brown adipocytes, and expresses the uncoupling protein 1 (UCP1), a specific marker of brown fat. Consistent with these observations, translation of the peroxisome proliferator-activated receptor-gamma co-activator 1 (PGC1), a transcriptional co-activator implicated in mitochondrial biogenesis and adaptive thermogenesis, is increased in white adipose tissue of Eif4ebp1-/- mice. These findings demonstrate that 4E-BP1 is a novel regulator of adipogenesis and metabolism in mammals.

Effects of reproductive stage, GH, and 11-ketotestosterone on expression of growth differentiation factor-9 in the ovary of the eel, Anguilla australis.

In order to study the regulation of the growth differentiation factor-9 (gdf9) gene in a primitive teleost with semelparous life history, we cloned a cDNA encoding shortfinned eel Gdf9, expressed a partial peptide in Escherichia coli, and raised an antiserum to evaluate changes in Gdf9 expression during its pituitary homogenate-induced reproductive cycle. The effects of in vivo and in vitro exposure to the androgen 11-ketotestosterone (11-KT), known to affect previtellogenic (PV) oocyte growth, were also determined. Furthermore, we investigated whether Gdf9 expression was metabolically gated by treating PV fish with recombinant GH in vivo. Immunoreactive proteins of ca. 52 and 55 kDa were identified by western blot analysis. Gdf9 message and protein were most abundant in PV oocytes, and peaked slightly earlier for mRNA than for protein. Captivity resulted in reduced gdf9 mRNA levels, which were restored following pituitary homogenate treatment. As oocytes progressed through induced oogenesis, Gdf9 expression decreased. Neither 11-KT nor GH treatment affected gdf9 mRNA levels in PV fish, although GH could partially restore handling- or captivity-induced decreases in gdf9 mRNA levels. Semelparous eels thus show an expression pattern of Gdf9 during oogenesis that is similar to that seen in other vertebrates, that appears responsive to handling or captivity stress, and whose control remains to be elucidated.

Identification of SARS-COV spike protein-derived and HLA-A2-restricted human CTL epitopes by using a new muramyl dipeptidederivative adjuvant.

Severe acute respiratory syndrome (SARS) spread during the winter of 2003, and attempts have been made to develop vaccines against SARS corona virus (SARS-CoV). The present study provides a strategy to rapidly identify SARS-CoV-derived antigenic peptides recognized by HLA-A2-restricted cytotoxic T lymphocytes (CTLs). Forty-three candidate peptides having HLA-A2-binding motifs were selected in silico and HLA-A2/Db chimeric MHC class I-transgenic mice were immunized with these peptides and a new derivative of muramyl dipeptide that can induce upregulation of HLA-DR, CD80, CD86, and CD40 in human CD14+ antigen presenting cells, was administered as an adjuvant. Six HLAA2-restricted mouse CTL epitopes were identified, including two new epitopes which have never been reported before. One of the novel peptides was naturally processed and successfully induced HLAA2-restricted specific CTLs in both HLA transgenic mice and healthy donors. The method was useful, convenient and efficient for rapid identification of CTL epitopes derived from SARS-CoV proteins and will be possibly applicable for other pathogens to develop a peptide-based vaccine.

Antigenic variation and resistance to neutralization in poliovirus type 1.

Mutations have been identified in variants of poliovirus, type 1 (Mahoney) on the basis of their resistance to neutralization by individual monoclonal antibodies. The phenotypes of these variants were defined in terms of antibody binding; the pattern of epitopes expressed or able to be exploited for neutralization were complex. Single amino acid changes can have distant (in terms of linear sequence) and generalized effects on the antigenic structure of poliovirus and similarly constituted virions.

Diffuse large B-cell lymphoma showing an interfollicular pattern of proliferation: a study of the Osaka Lymphoma Study Group.

AIMS: Diffuse large B-cell lymphoma (DLBCL) usually proliferates effacing lymph follicles. In occasional cases, tumour cells show an interfollicular pattern of proliferation preserving lymph follicles. The aim was to analyse clinicopathological findings in DLBCL showing an interfollicular pattern of proliferation to determine whether this type of lymphoma is a distinct entity of DLBCL. METHODS AND RESULTS: Clinicopathological findings in 12 cases of DLBCL showing an interfollicular pattern of proliferation [interfollicular group (IF)] were examined and compared with those in 30 cases of DLBCL with ordinary morphology [control group (CG)]. IF showed a significantly lower lactate dehydrogenase level and International Prognostic Index scores than CG (P = 0.023 and P < 0.01, respectively). The frequency of localized disease, clinical stage 1 and 2, in IF was higher than that in CG (P = 0.016). A morphologically polymorphous pattern of proliferation was found in seven of 12 cases (58.3%) in IF, which was higher than that in CG, five (16.7%) of 30 cases (P < 0.01). Clonality analysis with the polymerase chain reaction method revealed that all 11 IF cases examined showed a monoclonal pattern. Immunohistochemically, the majority (11 of 12) of IF cases showed a non-germinal centre B-cell phenotype and the frequency was higher than that in CG (P = 0.021). CONCLUSION: Diffuse large B-cell lymphoma with an interfollicular pattern of proliferation shows distinct clinical and pathological findings from ordinary DLBCL.

Comparative aspects on the role of polypyrimidine tract-binding protein in internal initiation of hepatitis C virus and picornavirus RNAs.

We compared the effects of polypyrimidine tract-binding protein (PTB) on hepatitis C virus (HCV genotype IIa), encephalomyocarditis virus (EMCV) and poliovirus internal ribosome entry site (IRES) activities in vitro. It bound strongly to EMCV IRES, but weakly to PV and HCV RNAs. PV IRES showed the strongest dependency to PTB and it showed less than one-tenth of IRES activity after the immuno-depletion of PTB from HeLa S10 lysate with pre-coated anti-PTB IgG beads, comparing to the normal IgG beads-treated S10 lysate. EMCV IRES activity was approximately 40% of that of normal control after PTB depletion. Especially, HCV IRES activity was approximately 95%, and most weekly affected by the depletion of PTB. Repletion of PTB to depleted S10 lysate restored activities of PV and EMCV IRESs. The data suggest that PTB plays an important role in picornaviral IRESs, but not in HCV IRES.

Complete nucleotide sequences of all three poliovirus serotype genomes. Implication for genetic relationship, gene function and antigenic determinants.

The complete nucleotide sequences of the genomes of the type 2 ( P712 , Ch, 2ab ) and type 3 (Leon 12a1b ) poliovirus vaccine strains were determined. Comparison of the sequences with the previously established genome sequence of type 1 (LS-c, 2ab ) poliovirus vaccine strain revealed that 71% of the nucleotides in the genome RNAs were common, that the 5' and 3' termini of the genomes were highly homologous, and that more than 80% of the nucleotide differences in the coding region occurred in the third letter position of in-phase codons, resulting in a low frequency of amino acid difference. These results strongly suggested that the serotypes of poliovirus derived from a common prototype. A comparison of the amino acid sequences predicted from the genome sequences showed highest variation in the capsid protein region, whereas non-structural proteins are highly conserved. Initiation of polyprotein synthesis occurs in all three strains more than 740 nucleotides downstream from the 5' end. An analysis of the non-coding region suggests that small peptides that could potentially originate from this region are conserved. The amino acid sequences immediately surrounding the cleavage signals, however, show a higher than average degree of variation. The analysis of the amino acid sequences of the capsid protein VP1 of all serotypes has led to the prediction of potential antigenic sites on the virion involved in neutralization.

Complete genomic sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120.

The nucleotide sequence of the entire genome of a filamentous cyanobacterium, Anabaena sp. strain PCC 7120, was determined. The genome of Anabaena consisted of a single chromosome (6,413,771 bp) and six plasmids, designated pCC7120alpha (408,101 bp), pCC7120beta (186,614 bp), pCC7120gamma (101,965 bp), pCC7120delta (55,414 bp), pCC7120epsilon (40,340 bp), and pCC7120zeta (5,584 bp). The chromosome bears 5368 potential protein-encoding genes, four sets of rRNA genes, 48 tRNA genes representing 42 tRNA species, and 4 genes for small structural RNAs. The predicted products of 45% of the potential protein-encoding genes showed sequence similarity to known and predicted proteins of known function, and 27% to translated products of hypothetical genes. The remaining 28% lacked significant similarity to genes for known and predicted proteins in the public DNA databases. More than 60 genes involved in various processes of heterocyst formation and nitrogen fixation were assigned to the chromosome based on their similarity to the reported genes. One hundred and ninety-five genes coding for components of two-component signal transduction systems, nearly 2.5 times as many as those in Synechocystis sp. PCC 6803, were identified on the chromosome. Only 37% of the Anabaena genes showed significant sequence similarity to those of Synechocystis, indicating a high degree of divergence of the gene information between the two cyanobacterial strains.

Complete genome structure of the nitrogen-fixing symbiotic bacterium Mesorhizobium loti.

The complete nucleotide sequence of the genome of a symbiotic bacterium Mesorhizobium loti strain MAFF303099 was determined. The genome of M. loti consisted of a single chromosome (7,036,071 bp) and two plasmids, designated as pMLa (351,911 bp) and pMLb (208, 315 bp). The chromosome comprises 6752 potential protein-coding genes, two sets of rRNA genes and 50 tRNA genes representing 47 tRNA species. Fifty-four percent of the potential protein genes showed sequence similarity to genes of known function, 21% to hypothetical genes, and the remaining 25% had no apparent similarity to reported genes. A 611-kb DNA segment, a highly probable candidate of a symbiotic island, was identified, and 30 genes for nitrogen fixation and 24 genes for nodulation were assigned in this region. Codon usage analysis suggested that the symbiotic island as well as the plasmids originated and were transmitted from other genetic systems. The genomes of two plasmids, pMLa and pMLb, contained 320 and 209 potential protein-coding genes, respectively, for a variety of biological functions. These include genes for the ABC-transporter system, phosphate assimilation, two-component system, DNA replication and conjugation, but only one gene for nodulation was identified.

Cloned infectious complementary DNA of the poliovirus Sabin 1 genome: biochemical and biological properties of the recovered virus.

A complete cDNA copy of the genome of the attenuated type 1 poliovirus vaccine (Sabin 1) strain was constructed and inserted into the EcoRI site of the plasmid pBR325. When cultured mammalian cells were transfected with this recombinant plasmid, 20 to 50 poliovirus plaques per 10 micrograms plasmid DNA were observed. Fingerprints of the RNA of the recovered virus showed no changes when compared with those of the parental virus genome, an observation indicating that the primary structure of the cloned cDNA is a reflection of authentic poliovirus RNA. The recovered virus had the same properties as those of the Sabin 1 strain in regard to antigenicity, sensitivity to temperature, and dependency on bicarbonate concentration. These results suggest that the virus obtained by DNA transfection is indistinguishable from the Sabin 1 strain. The recombinant plasmid could therefore be used as a stable repository of the virus and as inoculum for the oral polio live vaccine.

Purification of enzymatically active poliovirus proteinase 3C produced in Escherichia coli.

A viral protein 3C of the poliovirus (PV) Sabin 2 strain, a possible core region of the viral proteinase, was expressed in Escherichia coli using a recombinant DNA technology. The protein was recovered as a soluble protein from the insoluble protein fraction of the bacterial lysate, and was purified by a simple procedure with column chromatography. The viral capsid precursor P1 (1ABCD) of the PV Sabin 3 strain, which had been similarly produced in E. coli, was mixed with the purified or crude recombinant 3C. Immunoblotting assay with monoclonal antibodies specific to capsid proteins 1C (VP3) and 1D (VP1) of the PV Sabin 3 strain revealed that the in vitro reaction products contained 1C (VP3), 1D (VP1) and 1ABC (VP0-VP3). The data indicated that processing of the polyprotein P1 by the recombinant 3C proceeded properly in vitro, although an undigested product, 1ABC, is always detected in the reaction mixture. The results strongly suggest that, in addition to a protein 3CD, the 3C protein itself is also catalytically active in the processing of the viral capsid precursor polyprotein P1.

Poly(U) binding activity of hepatitis C virus NS3 protein, a putative RNA helicase.

A non-structural protein of the hepatitis C virus (HCV), NS3, contains amino acid sequence motifs characteristic of serine-proteinases and RNA helicases. RNA binding activity of the NS3 protein with an apparent dissociation constant of 2 x 10(-7) M was detected using a poly(U)-Sepharose resin. Competitive RNA binding analysis suggested that the NS3 protein binds preferentially to the poly(U) sequence, which is located at the 3' end of HCV RNA. Mutational analysis of NS3 protein revealed the possibility that both the RNA helicase region and the serine-proteinase region were necessary for full RNA binding activity.

Detection of hepatitis C virus specific core protein in serum of patients by a sensitive fluorescence enzyme immunoassay (FEIA).

A protein-capture fluorescence enzyme immunoassay (FEIA) was developed using monoclonal antibodies (mAbs) against recombinant hepatitis C virus (HCV) core protein. Four hybridoma cell lines (5E3, 5F11, 515S, 1080S) were established and characterized. These monoclonal antibodies (mAbs) each had IgG1 and OgG2 isotypes, and recognized major B cell epitopes within the immunodominant nucleoprotein amino terminal subregion. Using mAb 5F11 as the first antibody to the solid phase and beta-D-galactosidase-conjugated mAb 5E3 as the second antibody to the protein, we established a specific HCV core protein capturing FEIA capable of detecting as little as 20 pg/ml of recombinant HCV core protein. HCV core protein in serum was detectable after treatment with 4.0% polyethyleneglycol, 0.5 NaOH, and 5% Triton X-100. The results of a peptide inhibition assay indicated that this FEIA is specific for HCV RNA positive sera. The quantity of HCV core protein detected in serum was significantly correlated to the level of HCV RNA. The detection limit for HCV core proteins was an HCV RNA per titer of approximately 10(4)/ml. Using this FEIA system, the detection ratio of HCV core protein in patients with chronic HCV infection was 92.3% (70/76).

Hepatitis C virus replication and pathogenesis.

The mechanism involved in the development of persistent hepatitis C virus (HCV) infection and the pathogenesis remain unclear. The present review is an accumulation of evidence gathered to date. In addition, it discusses the system developed to characterize HCV.

Effects of cyclosporin A on hepatitis C virus infection in bone marrow transplant patients. Bone Marrow Transplantation Team.

The hepatitis C virus (HCV) infection has, in general, been considered not to affect liver function severely during the course of bone marrow transplantation (BMT) except for late hepatitis which coincided with a decrease in immunosuppressive therapy. We examined serial sera of two patients with positive HCV antibody who underwent allogeneic BMT and found that while the dose of cyclosporin A tapered off, the serum concentration of HCV core protein increased before the occurrence of hepatitis. This suggests that viral reactivation and growth might be one of the important mechanisms of hepatitis after BMT in patients with positive HCV antibody.

Expression of poliovirus complementary DNA coding for viral antigenic determinants in Escherichia coli.

DNA sequences coding for the immunogenic capsid protein VP1 and/or VP3 of poliovirus strain LSc-2ab (Sabin 1) were prepared by digesting the cloned complementary DNA with restriction endonuclease PstI. The DNA fragments were inserted into the unique PstI site of Escherichia coli plasmid vectors pBR322, pKT 280 and/or pKT 287 that lay in the region expressed under control of the penicillinase promoter system. In the expression vectors, poliovirus sequences were designed to be read in phase and therefore to be expressed as fusion proteins with the bacterial peptides. In addition, the Escherichia coli tryptophan operon promoter-operator system was inserted upstream of the penicillinase system to obtain stronger expression of the poliovirus sequences. Escherichia coli transformed with these plasmids appeared to produce the antigenic polypeptides, which were detected by immunoprecipitation with antibodies to capsid proteins VP1 and/or VP3 followed by SDS-polyacrylamide gel electrophoresis.

Fetal heart rate recorder for long-duration use in active full-term pregnant women.

We sought to assess the signal consistency and accuracy of a long-duration fetal heart rate (FHR) recorder (a 1-MHz ultrasonic Doppler transducer composed of six miniprobes and an autocorrelation technique) in active, full-term pregnant women. The FHR data of 15 normal full-term fetuses were obtained every 250 milliseconds in various maternal positions using the new Doppler system or a direct scalp electrocardiographic (ECG) transducer. Differences between simultaneous Doppler and ECG measures were small and within acceptable limits except for short-term variability. Signal loss, assessed in 15 subjects in various positions, was less than 10% except during sitting or walking. The mean (+/- standard deviation) percentages of FHR signals lost over 2 hours in bed, 1 hour out of bed, and 24 hours in and out of bed were 1.2 +/- 2.1, 12.9 +/- 16.2, and 3.8 +/- 4.4, respectively. This system for recording FHR is suitable for clinical research and routine FHR monitoring, and provides data comparable to Holter ECG monitoring in patients with heart disease.

Simple quantitative assay of alpha-fetoprotein mRNA in liver tissue using the real-time detection polymerase chain reaction assay - its application for clinical use.

Alpha-feto protein (AFP) mRNA levels increase in hepatocellular carcinoma (HCC) cells as compared with non-neoplastic tissue. Therefore, detection of AFP mRNA in blood nuclear cells is useful for the evaluation of treatment efficacy and prognosis of HCC. In this study, simple and reproducible methods were developed to quantify AFP mRNA using the real-time RT-PCR assay (Taq Man assay). By using in vitro synthesized AFP and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA, the sensitivity and dynamic range of the RT-PCR assay were established. AFP mRNA in both HCC and non-neoplastic tissue, as well as in cell lines, were measured using this assay system. The expression of the AFP mRNA level was normalized using the GAPDH house keeping gene product as an endogenous reference. AFP and GAPDH mRNA can be quantified in the range of 10-10(8) copies when using this quantitative assay. Among HCC cell lines, Huh 7 and HepG2 cells, respectively, represented 1.5x10(6) and 6.0x10(5) AFP mRNA/10(6) GAPDH mRNA, in contrast to 6, 23 and 230 AFP mRNA/10(6) GAPDH mRNA for HLE, HLF and PLC/PRF/5 cells, respectively. Other cell lines derived from stomach, pancreas, and colon cancers have 10 AFP mRNA copies/10(6) GAPDH mRNA. In liver tissue from patients with chronic hepatitis, and the non-neoplastic portion of the liver from HCC patients, AFP mRNA distributes from 2.5x10(3) to 5.8x10(4)/10(6) GAPDH transcripts. In contrast, AFP mRNA in tumor cells were more than 100-fold higher than that found in corresponding non-neoplastic portions in two patients who had a high level of AFP in serum. The establishment of the TaqMan quantifying system for AFP mRNA may have important clinical implications.

Hepatitis C virus core particle detected by immunoelectron microscopy and optical rotation technique.

Hepatitis C Virus (HCV) are 55-65 nm spherical particles, but the internal structure of the virion remains to be clarified. To clarify the morphology of HCV core particles, we performed an immune electron microscopy (IEM) using plasma samples from two blood donors with high HCV RNA titers and a detergent-treated anti-HCV core antibody-free plasma sample with high HCV RNA titer (1.5x10(8) copies/ml). Spherical particles, with 33-40 nm in diameter (an average diameter of 37 nm) were found in 1.22-1.25 g/ml fractions after sucrose density gradient centrifugation by conventional electron microscopy (EM). IEM using rabbit polyclonal antibody (RR8) specific to the putative HCV core protein and goat anti-rabbit IgG colloidal gold particles revealed that these spherical particles specifically reacted with RR8. This finding indicates that the spherical particles are naked HCV core particles. Some of the HCV core particles had an icosahedron-like structure. Optical rotation technique showed that the HCV core particle exhibits six-fold symmetry and that the length of regular hexagon side is approximately 20 nm. These findings showed that HCV core particles are spherical particles of 33-40 nm in diameter and that HCV core particles may possess an icosahedron-like structure and a buoyant density of 1.22-1.25 g/ml.