Metabolism of Round Spermatids: A Possible Regulation of Hexose Transport

Round spermatids (steps 1–8) were isolated from rat testes and glucose transport into the cells was examined. The exposure of spermatids to glucose resulted in an extremely low level of ATP. In contrast, the level of ATP remained constant in the presence of pyruvate. Transport of a glucose analogue, 2-deoxy-D-[³H]glucose ([³H]dGlc) into spermatids was correlated with intracellular levels of ATP and was much greater in cells with higher rather than lower levels of ATP. [³H]dGlc transport into spermatids with low levels of ATP was partially reversed when the cells were incubated with pyruvate. Inhibition of [³H]dGlc transport was exerted on Vmax and not on Km. Moreover, glucose acted as a competitive inhibitor of [³H]dGlc uptake (Km increased; Vmax unaltered). These results suggest that glucose transport into spermatids is active in vitro and probably regulated by the intracellular level of ATP.     

The Effect of Hydrocortisone on the Epidermal Proliferation in an Organ Culture of Chick Embryonic Skin

Hydrocortisone is regarded as an initiator of keratinization in embryonic skin. The present investigation dealt with the effect of hydrocortisone on the proliferation of epidermal cells during early development: Cell kinetic analyses using ³H-thymidine autoradiography were applied to a skin organ culture prepared from a 13-day chick embryo.
Hydrocortisone at a concentration between 0.01 and 1.0 μg/ml was effective in initiating a morphological change leading to the epidermal keratinization in vitro and caused a marked decrease in the mitotic and labeling indices of epidermal basal cells, the decrease being maximum at 2 days of culture previous to the morphological change.
During continuous labeling with ³H-thymidine, the number of labeled basal cells reached 100% within 2 days in the control and 4 days in the culture treated with hydrocortisone. This confirmed that the growth fraction of epidermal basal cells was 1.0 even after the administration of hydrocortisone.
The duration of each cell cycle phase at 2 days of culture was determined by percent labeled mitoses and double-labeling analyses. It was concluded that hydrocortisone extended the generation time of epidermal basal cells at this time point about three fold over the control. This extension was mainly due to the elongation of the G ₁ phase.     

Pathway of mannitol formation during photosynthesis in brown algae

Eisenia bicyclis, Arame, was allowed to photosynthesize in seawatercontaining H¹⁴CO₃^–, and ¹⁴C-mannitol was isolated fromits fronds. The ratio of ¹⁴C-total/¹⁴C₁ + ¹⁴C₆ in the ¹⁴C-mannitolwas found to be about 8.0 at 1 min-illumination, but graduallydecreased with time to 3.0, showing uniform radioactivity distribution.Mannitol therefore seems to be formed in brown algae throughthree carbon compounds. Enzymes which may be involved in the possible biosynthetic pathwayof mannitol, i.e. aldolase, hexose diphosphatase, mannitol-1-phosphataseand glucosephosphate isomerase were present in extracts fromseveral brown algae. Some of their properties are discussed. ¹Contribution from the Shimoda Marine Biological Station ofTokyo Kyoiku University, No. 187. ²Present address: Reseach Institute, Seikagaku Kogyo Co., Ltd.,Yamato-machi, Kitatama-gun, Tokyo, Japan. (Received December 13, 1968; )     

EFFECTS OF THYROXINE AND PROLACTIN ON THE RATES OF PROTEIN SYNTHESIS IN THE THIGH BONES OF THE TADPOLE OF RANA CATESBEIANA

In thigh bones isolated from a Rana catesbeiana tadpole which has been kept in a 5 × 10⁻⁸ M thyroxine solution for several days, the rate of ¹⁴C-leucine incorporation into protein becomes higher than that in the thigh bones of control animals. Intraperitoneal injection of prolactin also results in an increase in the rate of ¹⁴C-leucine incorporation into protein in the thigh bones at a rate very similar to that in thyroxine-treated animals. In the thigh bones of the thyroxine-treated tadpoles, the rate of ¹⁴C-proline incorporation into protein is markedly higher than that of control animals. Prolactin treatment of the tadpoles also causes an increase in the rate of ¹⁴C-proline incorporation, but the rate is lower than that found in thyroxine-treated animals. The injection of prolactin into thyroxine-treated tadpoles fails to cause further increase in the rates of incorporation of these amino acids into protein. In the thigh bones of tadpoles at the climax of metamorphosis, prolactin injection does not cause any increase in the rates of ¹⁴C-labeled proline and leucine incorporation, whereas both rates become slightly higher in the thigh bones of thyroxine-treated tadpoles at this stage. The thigh bones probably become insensitive to prolactin when they are exposed to thyroxine.     

Individual and combined effects of DNA methylation and copy number alterations on miRNA expression in breast tumors

Background

The global effect of copy number and epigenetic alterations on miRNA expression in cancer is poorly understood. In the present study, we integrate genome-wide DNA methylation, copy number and miRNA expression and identify genetic mechanisms underlying miRNA dysregulation in breast cancer.

Results

We identify 70 miRNAs whose expression was associated with alterations in copy number or methylation, or both. Among these, five miRNA families are represented. Interestingly, the members of these families are encoded on different chromosomes and are complementarily altered by gain or hypomethylation across the patients. In an independent breast cancer cohort of 123 patients, 41 of the 70 miRNAs were confirmed with respect to aberration pattern and association to expression. In vitro functional experiments were performed in breast cancer cell lines with miRNA mimics to evaluate the phenotype of the replicated miRNAs. let-7e-3p, which in tumors is found associated with hypermethylation, is shown to induce apoptosis and reduce cell viability, and low let-7e-3p expression is associated with poorer prognosis. The overexpression of three other miRNAs associated with copy number gain, miR-21-3p, miR-148b-3p and miR-151a-5p, increases proliferation of breast cancer cell lines. In addition, miR-151a-5p enhances the levels of phosphorylated AKT protein.

Conclusions

Our data provide novel evidence of the mechanisms behind miRNA dysregulation in breast cancer. The study contributes to the understanding of how methylation and copy number alterations influence miRNA expression, emphasizing miRNA functionality through redundant encoding, and suggests novel miRNAs important in breast cancer.     

Tyrosine phosphorylation directs TACE into extracellular vesicles via unconventional secretion

When studying how HIV‐1 Nef can promote packaging of the proinflammatory transmembrane protease TACE (tumor necrosis factor‐α converting enzyme) into extracellular vesicles (EVs) we have revealed a novel tyrosine kinase‐regulated unconventional protein secretion (UPS) pathway for TACE. When TACE was expressed without its trafficking cofactor iRhom allosteric Hck activation by Nef triggered translocation of TACE into EVs. This process was insensitive to blocking of classical secretion by inhibiting endoplasmic reticulum (ER) to Golgi transport, and involved a distinct form of TACE devoid of normal glycosylation and incompletely processed for prodomain removal. Like most other examples of UPS this process was Golgi reassembly stacking protein (GRASP)‐dependent but was not associated with ER stress. These data indicate that Hck‐activated UPS provides an alternative pathway for TACE secretion that can bypass iRhom‐dependent ER to Golgi transfer, and suggest that tyrosine phosphorylation might have a more general role in regulating UPS.      

Development of microsatellite markers for the Manila clam Ruditapes philippinarum

We isolated nine polymorphic microsatellites from the Manila clam Ruditapes philippinarum. These loci provide one class of highly variable genetic marker as the number of alleles ranged from six to 22, and the observed and expected heterozygosity ranged from 0.136 to 0.909 and from 0.553 to 0.954, respectively. We consider that these loci are potentially useful for detailing the genetic structure and gene flow among R. philippinarum populations.     

THIS ARTICLE HAS BEEN RETRACTEDDevelopment of microsatellite markers in the noxious red tide‐causing dinoflagellate Heterocapsa circularisquama (Dinophyceae)

We isolated 15 polymorphic microsatellites from one of the most noxious red tide‐causing dinoflagellate species, Heterocapsa circularisquama. These loci provide one class of highly variable genetic markers, as the number of alleles ranged from two to six, and the estimate of gene diversity was from 0.205 to 0.684 across the 15 microsatellites. These loci have the potential to reveal genetic structure and gene flow among H. circularisquama populations.     

Development of compound microsatellite markers in the harmful dinoflagellate Cochlodinium polykrikoides (Dinophyceae)

We isolated 15 polymorphic microsatellites from Cochlodinium polykrikoides. These loci provide a class of highly variable genetic markers, as the number of alleles ranged from two to 15, and the estimate of gene diversity was from 0.083 to 0.880 across the 15 microsatellites. We consider that these loci have a potential to reveal the genetic structure and gene flow among C. polykrikoides populations.