Standardization of fine needle aspiration cytology of the breast – comparison of Auto Cyto Fix and conventional smears

In Japan, there are some problems with fine needle aspiration (FNA) cytology of the breast, such as insufficient smeared cells, air-drying artefact and excessive erythrocytes. Liquid-based cytology has been found to solve these problems. Equipment for such preparations has been developed, but can be expensive to purchase and operate. We developed Auto Cyto Fix 1000 (ACF), which is inexpensive and automatically smears and fixes cells. The purpose of this study was to compare the various cytological features of conventional and ACF specimens. We evaluated whether the ACF method would be able to replace the conventional method. Forty-eight FNA specimens of breast were studied. All specimens were prepared by the direct smeared (DS) and ACF methods and evaluated for unsatisfactory cell collection, air-drying artefacts, background findings and epithelial cell findings. Although ACF specimens were prepared using the cells remaining in the needle and syringe after preparing DS specimens, the cellularity of two of the ACF specimens was better than that of the corresponding DS specimens. ACF specimens never showed air-drying artefact. Unlike DS specimens, which have many erythrocytes in the background, erythrocytes were filtered out and the background of ACF specimens was clean. We believe that many problems attributable to conventional FNA specimen preparation have been solved in this study. Preparation using the ACF apparatus can reduce running costs and can be used to prepare FNA specimens of the breast for cytological examination as an alternative to the conventional method.     

Evolution and Divergence of the Genes for Cytoplasmic,Mitochondrial, and Flagellar Creatine Kinases

Creatine kinase (CK) plays a central role in energy homeostasis in cells that display high and variable rates of energy turnover. A number of CK genes exist, each being targeted to particular intracellular compartments. In the vertebrates, two genes code for proteins which form homo- and heterodimers targeted to the cytoplasm, while two additional genes code for primarily octameric proteins targeted to the mitochondrial intermembrane space. Yet another gene is present in certain groups which codes for three fused, complete CK domains and is typically targeted to the flagellar membrane of primitive-type spermatozoa. CK is widely distributed in protochordates and both protostome and deuterostome invertebrate groups. The evolutionary relationships of these CK genes have not been fully elucidated. The present communication reports new cDNA-derived deduced amino acid sequences for four cytoplasmic and three mitochondrial CKs and one flagellar CK from lophotrochozoan, protostome invertebrates as well as a new cytoplasmic CK sequence from a protochordate tunicate. These new sequences, coupled with available sequences in the databases and sequences extracted from genome sequencing projects, provide revealing insights into the evolution and divergence of CK genes. Phylogenetic analyses showed that single cytoplasmic, mitochondrial, and flagellar CK genes were present prior to the divergence of the protostomes and deuterostomes. The flagellar CK gene may have evolved within the cytoplasmic gene clade, although the evidence is somewhat equivocal. The two cytoplasmic genes in the vertebrates, and most likely the two mitochondrial genes, evolved after the divergence of the craniates from the protochordates. Comparison of the structure of the genes for selected cytoplasmic, mitochondrial, and flagellar CKs revealed two identical intron boundaries, further reinforcing the notion of a common evolutionary origin, but also showed patterns of changes in structure consistent with each gene type. These studies show that the cytoplasmic, mitochondrial, and flagellar CK genes are rather ancient and that there has been a systematic pattern of duplication and divergence consistent with changing nature of energy demands and physicochemical environment in the cells where they are expressed.[Reviewing Editor: Martin Kreitman]     

Wnt-3a-dependent cell motility involves RhoA activation and is specifically regulated by dishevelled-2

Wnts stimulate cell migration, although the mechanisms responsible for this effect are not fully understood. To investigate the pathways that mediate Wnt-dependent cell motility, we treated Chinese hamster ovary cells with Wnt-3a-conditioned medium and monitored changes in cell shape and movement. Wnt-3a induced cell spreading, formation of protrusive structures, reorganization of stress fibers and migration. Although Wnt-3a stabilized beta-catenin, two inhibitors of the beta-catenin/canonical pathway, Dickkopf-1 and a dominant-negative T cell factor construct, did not reduce motility. The small GTPase RhoA also was activated by Wnt-3a. In contrast to beta-catenin signaling, inhibition of Rho kinase partially blocked motility. Because Dishevelled (Dvl) proteins are effectors of both canonical and noncanonical Wnt signaling, we used immunofluorescent analysis and small interference RNA technology to evaluate the role of Dvl in cell motility. Specific knock-down of Dvl-2 expression markedly reduced Wnt-3a-dependent changes in cell shape and movement, suggesting that this Dvl isoform had a predominant role in mediating Wnt-3a-dependent motility in Chinese hamster ovary cells.     

Assessment of Testicular Corticosterone Biosynthesis in Adult Male Rats

Corticosterone is synthesized in the adrenal glands and is circulated throughout the body to perform regulatory functions in various tissues. The testis is known to synthesize and secrete testosterone and other androgens. We developed an accurate method to measure steroid content using liquid chromatography-mass spectrometry analysis. In the present study, significant levels of the precursor compounds of testosterone and corticosterone synthesis could be detected in rat testis using this method. After adrenalectomy, corticosterone remained in the blood and testicular tissue at approximately 1% of the amount present in the control testis. When the excised testicular tissue was washed and incubated with NADH, NADPH and progesterone, not only testosterone and its precursors but also 11-deoxycorticosterone and corticosterone were produced; the levels of 11-deoxycorticosterone and corticosterone increased with incubation time. The production rate of 11-deoxycorticosterone from progesterone was estimated to be approximately 1/20 that of 17-hydroxyprogesterone, and the corticosterone level was approximately 1/10 that of testosterone. These ratios coincided with those in the testicular tissue of the adrenalectomized rats, indicating that corticosterone was synthesized in the testis and not in the blood. A primary finding of this study was that corticosterone and testosterone were synthesized in a 1/10-20 ratio in the testis. It is concluded that corticosterone, which has various functions, such as the regulation of glycolysis and mediating spermatogenesis, is produced locally in the testis and that this the local production is convenient and functional to respond to local needs.     

Reciprocal conversion of Gtr1 and Gtr2 nucleotide-binding states by Npr2-Npr3 inactivates TORC1 and induces autophagy

Autophagy is an intracellular degradation process that delivers cytosolic material to lysosomes and vacuoles. To investigate the mechanisms that regulate autophagy, we performed a genome-wide screen using a yeast deletion-mutant collection, and found that Npr2 and Npr3 mutants were defective in autophagy. Their mammalian homologs, NPRL2 and NPRL3, were also involved in regulation of autophagy. Npr2-Npr3 function upstream of Gtr1-Gtr2, homologs of the mammalian RRAG GTPase complex, which is crucial for TORC1 regulation. Both npr2∆ mutants and a GTP-bound Gtr1 mutant suppressed autophagy and increased Tor1 vacuole localization. Furthermore, Gtr2 binds to the TORC1 subunit Kog1. A GDP-bound Gtr1 mutant induced autophagy even under nutrient-rich conditions, and this effect was dependent on the direct binding of Gtr2 to Kog1. These results revealed that 2 molecular mechanisms, Npr2-Npr3-dependent GTP hydrolysis of Gtr1 and direct binding of Gtr2 to Kog1, are involved in TORC1 inactivation and autophagic induction.     

Loss of heterochromatin protein 1 gamma reduces the number of primordial germ cells via impaired cell cycle progression in mice

Signals from extraembryonic tissues in mice determine which proximal epiblast cells become primordial germ cells (PGCs). After their specification, approximately 40 PGCs appear at the base of the allantoic bud and migrate to the genital ridges, where they expand to about 25?000 cells by Embryonic Day (E)13.5. The heterochromatin protein 1 (HP1) family members HP1alpha, HP1beta, and HP1gamma (CBX5, CBX1, and CBX3, respectively) are thought to induce heterochromatin structure and to regulate gene expression by binding methylated histone H3 lysine 9. We found a dramatic loss of germ cells before meiosis in HP1gamma mutant (HP1gamma(-/-)) mice that we generated previously. The reduction in PGCs in HP1gamma(-/-) embryos was detectable from the early bud stage (E7.25), and the number of HP1gamma(-/-) PGCs was gradually reduced thereafter. Bromodeoxyuridine incorporation into PGCs was significantly reduced in E7.25 and E12.5 HP1gamma(-/-) embryos. Furthermore, a lower proportion of HP1gamma(-/-) PGCs than wild-type PGCs was in S phase, and a higher proportion, respectively, was in G1 phase at E12.5. Moreover, the proportion of p21 (Cip, official symbol CDKN1A)-positive HP1gamma(-/-) PGCs was increased, suggesting that the G1/S phase transition was inhibited. However, no differences were detected between fate determination, migration, apoptosis, or histone modification of PGCs of control embryos and those of HP1gamma(-/-) embryos. Therefore, the reduction in PGCs in HP1gamma(-/-) embryos could be caused by impaired cell cycle in PGCs. These results suggest that HP1gamma plays an important role in keeping enough germ cells by regulating the PGC cell cycle.     

Serum Interleukin-18 Levels Are Associated with Physical Activity in Japanese Men

Objective

To investigate the link between serum interleukin-18 (IL-18) levels and physical activity in Japanese men.

Methods

A total of 81 men (45.7±17.6 years old) was enrolled in this cross-sectional investigation study. We assessed anthropometric and body composition parameters. Serum IL-18 levels, physical activity by uniaxial accelerometers, peak oxygen uptake and metabolic risk parameters were also evaluated.

Results

Serum IL-18 levels were 179.4±84.7 pg/mL. Physical activity evaluated by Σ[metabolic equivalents × h per week (METs⋅h/w)]was significantly and negatively correlated with serum IL-18 levels (r = −0.252, p = 0.0235). These associations remained even after adjusting for age, peak oxygen uptake and other confounding factors.

Conclusion

Serum IL-18 levels were closely associated with physical activity independent of peak oxygen uptake in Japanese men.     

Purification and Properties of a Lytic Enzyme from Streptomyces griseus H-402

A lytic enzyme which was capable of lysing cells of Streptococcus mutans was purified from the culture filtrate of Streplomyces griseus H–402 by Amberlite CG–50 treatment, CM-cellulose and hydroxylapatite column chromatographies, and Sephadex G–150 gelfiltration. The lytic enzyme was obtained in a crystalline form which was homogeneous in polyacrylamide gel electrophoresis. The molecular weight was estimated to be 2×10⁴ by the thin-layer gel-filtration method on Sephadex G–75, and 2.3 × 10⁴ by the method of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was found to be a N-acetylmuramidase whose activity was lost by N-bromosuccinimide as an inhibitor.     

Sexual reproduction is the default mode in apomictic Hieracium subgenus Pilosella, in which two dominant loci function to enable apomixis

Asexual seed formation, or apomixis, in the Hieracium subgenus Pilosella is controlled by two dominant independent genetic loci, LOSS OF APOMEIOSIS (LOA) and LOSS OF PARTHENOGENESIS (LOP). We examined apomixis mutants that had lost function in one or both loci to establish their developmental roles during seed formation. In apomicts, sexual reproduction is initiated first. Somatic aposporous initial (AI) cells differentiate near meiotic cells, and the sexual pathway is terminated as AI cells undergo mitotic embryo sac formation. Seed initiation is fertilization-independent. Using a partially penetrant cytotoxic reporter to inhibit meioisis, we showed that developmental events leading to the completion of meiotic tetrad formation are required for AI cell formation. Sexual initiation may therefore stimulate activity of the LOA locus, which was found to be required for AI cell formation and subsequent suppression of the sexual pathway. AI cells undergo nuclear division to form embryo sacs, in which LOP functions gametophytically to stimulate fertilization-independent embryo and endosperm formation. Loss of function in either locus results in partial reversion to sexual reproduction, and loss of function in both loci results in total reversion to sexual reproduction. Therefore, in these apomicts, sexual reproduction is the default reproductive mode upon which apomixis is superimposed. These loci are unlikely to encode genes essential for sexual reproduction, but may function to recruit the sexual machinery at specific time points to enable apomixis.