BTN3A2 expression in epithelial ovarian cancer is associated with higher tumor infiltrating T cells and a better prognosis

BTN3A2/BT3.2 butyrophilin mRNA expression by tumoral cells was previously identified as a prognostic factor in a small cohort of high grade serous epithelial ovarian cancer (HG-EOC). Here, we evaluated the prognostic value of BT3.2 at the protein level in specimen from 199 HG-EOC patients. As the only known role of butyrophilin proteins is in immune regulation, we evaluated the association between BT3.2 expression and intratumoral infiltration of immune cells by immunohistochemistry with specific antibodies against BT3.2, CD3, CD4, CD8, CD20, CD68 and CD206. Epithelial BT3.2 expression was significantly associated with longer overall survival and lower risk of disease progression (HR?=?0.651, p?=?0.006 and HR?=?0.642, p?=?0.002, respectively) and significantly associated with a higher density of infiltrating T cells, particularly CD4+ cells (0.272, p<0.001). We also observed a strong association between the relative density of CD206+ cells, as evaluated by the ratio of intratumoral CD206+/CD68+ expression, and risk of disease progression (HR?=?1.355 p?=?0.044, respectively). In conclusion, BT3.2 protein is a potential prognostic biomarker for the identification of HG-EOC patients with better outcome. In contrast, high CD206+/CD68+ expression is associated with high risk of disease progression. While the role of BT3.2 is still unknown, our result suggest that BT3.2 expression by epithelial cells may modulates the intratumoral infiltration of immune cells.     

Conserved central domains control the quaternary structure of type I and type II Hsp40 molecular chaperones

Heat shock protein (Hsp)40s play an essential role in protein metabolism by regulating the polypeptide binding and release cycle of Hsp70. The Hsp40 family is large, and specialized family members direct Hsp70 to perform highly specific tasks. Type I and Type II Hsp40s, such as yeast Ydj1 and Sis1, are homodimers that dictate functions of cytosolic Hsp70, but how they do so is unclear. Type I Hsp40s contain a conserved, centrally located cysteine-rich domain that is replaced by a glycine- and methionine-rich region in Type II Hsp40s, but the mechanism by which these unique domains influence Hsp40 structure and function is unknown. This is the case because high-resolution structures of full-length forms of these Hsp40s have not been solved. To fill this void, we built low-resolution models of the quaternary structure of Ydj1 and Sis1 with information obtained from biophysical measurements of protein shape, small-angle X-ray scattering, and ab initio protein modeling. Low-resolution models were also calculated for the chimeric Hsp40s YSY and SYS, in which the central domains of Ydj1 and Sis1 were exchanged. Similar to their human homologs, Ydj1 and Sis1 each has a unique shape with major structural differences apparently being the orientation of the J domains relative to the long axis of the dimers. Central domain swapping in YSY and SYS correlates with the switched ability of YSY and SYS to perform unique functions of Sis1 and Ydj1, respectively. Models for the mechanism by which the conserved cysteine-rich domain and glycine- and methionine-rich region confer structural and functional specificity to Type I and Type II Hsp40s are discussed.     

Alterations in the testis of hormone sensitive lipase-deficient mice is associated with decreased sperm counts, sperm motility, and fertility

Hormone-sensitive lipase (HSL, Lipe, E.C.3.1.1.3) functions as a triglyceride and cholesteryl esterase, supplying fatty acids, and cholesterol to cells. Gene-targeted HSL-deficient (HSL(-/-)) mice reveal abnormal spermatids and are infertile at 24 weeks after birth. The purpose of this study was to follow the evolution of spermatid abnormalities as HSL(-/-) mice age, characterize sperm motility in older HSL(-/-) mice, and determine if mice expressing a human testicular HSL transgene (HSL(-/-)ttg) produce normal motile sperm. In situ hybridization indicated that HSL is expressed exclusively in steps 5-16 spermatids, but not in Sertoli cells. In HSL(-/-) mice, abnormalities were evident in step 16 spermatids at 5 weeks after birth, with defects progressively increasing in spermatids with age. The defects included multinucleation of spermatids, abnormal shapes and a reduction of elongating spermatids. In older HSL(-/-) mice, sperm counts appeared reduced by 42%, but this value was lower because samples were compromised by the presence of small degenerating germ cells in addition to sperm, both of which appeared of similar size and density. Sperm motility was dramatically reduced with only 11% classified as motile in HSL(-/-) mice compared to 76-78% of sperm in wild-type and HSL(-/-)ttg mice. Sperm morphology, counts, and motility were normal in HSL(-/-)ttg mice, as was their fertility. Collectively, the data indicate that HSL deficiency results in abnormal spermatid development with defects arising at 5 weeks of age and progressively increasing at later ages. HSL(-/-) mice also show a dramatic reduction in sperm counts and motility and are infertile.     

Synthesis, 18F-labeling, and in vitro and in vivo studies of bombesin peptides modified with silicon-based building blocks

The gastrin-releasing peptide receptor (GRPr) is overexpressed on various human tumors. The goal of our study was the synthesis of new 18F-labeled bombesin analogues for the PET imaging of GRPr expression in prostate tumor using a silicon-based one-step n. c. a. radiolabeling method. The silicon-containing building blocks were efficiently coupled to the N-terminus of the peptides via solid-phase synthesis. Radiolabeling of the obtained peptide precursors proceeded smoothly under acidic conditions (34-85% conversion). Using the di-tert-butyl silyl building block as labeling moiety, products containing a hydrolytically stable 18F-label were obtained. In in vitro receptor binding experiments 2-(4-(di-tert-butylfluorosilyl)phenyl)acetyl-Arg-Ava-Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH 2 ( 4b, IC50 = 22.9 nM) displayed a 12-fold higher binding affinity than 2-(4-(di-tert-butylfluorosilyl)phenyl)acetyl-Arg-Ava-Gln-Trp-Ala-Val-Gly-His(3Me)-Sta-Leu-NH2 ( 3b, IC50 = 276.6 nM), and 4b was therefore chosen for further evaluation. In vitro and ex vivo metabolite studies of [18F]4b showed no significant degradation. In biodistribution experiments, tumor uptake of [18F]4b was low and unspecific, whereas the GRPr-rich pancreas revealed a high and specific accumulation of the radiotracer. This study demonstrates the applicability of our silicon-based one-step n. c. a. radiolabeling method for the synthesis of new 18F-labeled bombesin derivatives. This innovative approach represents a general, straightforward access to radiolabeled peptides as PET imaging probes.     

Hereditary cancer-associated missense mutations in hMSH6 uncouple ATP hydrolysis from DNA mismatch binding

Hereditary nonpolyposis colorectal cancer is caused by germline mutations in DNA mismatch repair genes. The majority of cases are associated with mutations in hMSH2 or hMLH1; however, about 12% of cases are associated with alterations in hMSH6. The hMSH6 protein forms a heterodimer with hMSH2 that is capable of recognizing a DNA mismatch. The heterodimer then utilizes its adenosine nucleotide processing ability in an, as of yet, unclear mechanism to facilitate communication between the mismatch and a distant strand discrimination site. The majority of reported mutations in hMSH6 are deletions or truncations that entirely eliminate the function of the protein; however, nearly a third of the reported variations are missense mutations whose functional significance is unclear. We analyzed seven cancer-associated single amino acid alterations in hMSH6 distributed throughout the functional domains of the protein to determine their effect on the biochemical activity of the hMSH2-hMSH6 heterodimer. Five alterations affect mismatch-stimulated ATP hydrolysis activity providing functional evidence that missense variants of hMSH6 can disrupt mismatch repair function and may contribute to disease. Of the five mutants that affect mismatch-stimulated ATP hydrolysis, only two (R976H and H1248D) affect mismatch recognition. Thus, three of the mutants (G566R, V878A, and D803G) appear to uncouple the mismatch binding and ATP hydrolysis activities of the heterodimer. We also demonstrate that these three mutations alter ATP-dependent conformation changes of hMSH2-hMSH6, suggesting that cancer-associated mutations in hMSH6 can disrupt the intramolecular signaling that coordinates mismatch binding with adenosine nucleotide processing.     

Functional divergence of the duplicated AtKIN14a and AtKIN14b genes: critical roles in Arabidopsis meiosis and gametophyte development

Gene duplication is important for gene family evolution, allowing for functional divergence and innovation. In flowering plants, duplicated genes are widely observed, and functional redundancy of closely related duplicates has been reported, but few cases of functional divergence of close duplicates have been described. Here, we show that the Arabidopsis AtKIN14a and AtKIN14b genes encoding highly similar kinesins are two of the most closely related Arabidopsis paralogs, which were formed by a duplication event that occurred after the split of Arabidopsis and poplar. In addition, AtKIN14a and AtKIN14b exhibit varying degrees of coding sequence divergence. Further genetic studies of plants carrying atkin14a and/or atkin14b mutations indicate that, although these two genes have similar functions, there is clear evidence for functional divergence. Although both genes are important for male and female meiosis, AtKIN14a plays a more critical role in male meiosis than AtKIN14b . Moreover, either one of these two genes is necessary and sufficient for gametophyte development, indicating that they are redundant for this function. Therefore, AtKIN14a and AtKIN14b together play important roles in controlling plant reproductive development. Our results suggest that the AtKIN14a and AtKIN14b genes have retained similar functions in gametophyte development and female meiosis, but have evolved partially distinct functions in male meiosis, with AtKIN14a playing a more substantive role.     

Energy‐Dense Snack Food Intake in Adolescence: Longitudinal Relationship to Weight and Fatness

Objective: The longitudinal relationship between the consumption of energy‐dense snack (EDS) foods and relative weight change during adolescence is uncertain. Using data from the Massachusetts Institute of Technology Growth and Development Study, the current analysis was undertaken to examine the longitudinal relationship of EDS food intake with relative weight status and percentage body fat and to examine how EDS food consumption is related to television viewing. Research Methods and Procedures: One hundred ninety‐six nonobese premenarcheal girls 8 to 12 years old were enrolled between 1990 and 1993 and followed until 4 years after menarche. At each annual follow‐up visit, data were collected on percentage body fat (%BF), BMI z score, and dietary intake. Categories of EDS foods considered were baked goods, ice cream, chips, sugar‐sweetened soda, and candy. Results: At study entry, girls had a mean ± SD BMI z score of ?0.27 ± 0.89, consumed 2.3 ± 1.7 servings of EDS foods per day, and consumed 15.7 ± 8.1% of daily calories from EDS foods. Linear mixed effects modeling indicated no relationship between BMI z score or %BF and total EDS food consumption. Soda was the only EDS food that was significantly related to BMI z score over the 10‐year study period, but it was not related to %BF. In addition, a significant, positive relationship was observed between EDS food consumption and television viewing. Discussion: In this cohort of initially nonobese girls, overall EDS food consumption does not seem to influence weight status or fatness change over the adolescent period.     

Effects on temperature on spontaneous and thyroxine-stimulated locomotor activity of Atlantic cod

Spontaneous locomotor activity of cod Gadus morhua maintained at 6° C tripled from February to May. In contrast, locomotor activity of cod held at 2° C was significantly lower than at 6° C (between 25 and 65% lower) and the seasonal increase was smaller. Plasma levels of both thyroxine (T₄) and triiodothyronine (T₃) did not differ between 2 and 6° C. T₄ injection increased locomotor activity by 10% for both temperature regimes. These data indicate that low water temperature reduces locomotor activity associated with migration in cod and that thyroid hormones are not involved in this decrease. This study provides a possible mechanism through which cold waters may affects migration and distribution of cod via its Effects on locomotor activity and swimming speed.     

Inhibition of pyruvate carboxylase by sequestration of coenzyme A with sodium benzoate

Pyruvate-dependent CO2 fixation by isolated mitochondria was strongly inhibited by sodium benzoate. Pyruvate carboxylase was identified as a site of inhibition by limiting flux measurements to assays of pyruvate carboxylase coupled with malate dehydrogenase. Benzoate reduced pyruvate-dependent incorporation of [14C]KHCO3 into malate and pyruvate-dependent malate accumulation by 74 and 72%, respectively. Aspartate-dependent malate accumulation was insensitive to benzoate, ruling out malate dehydrogenase as a site of action. Inhibition by benzoate was antagonized by glycine, which sharply accelerated conversion of benzoate to hippurate. Assays of coenzyme A and its acyl derivatives revealed inhibition to correlate with depletion of acetyl CoA and accumulation of benzoyl CoA. Depletion of acetyl CoA was sufficient to account for greater than 50% reduction in pyruvate carboxylase activity. Competition between acetyl CoA and benzoyl CoA for the activator site on pyruvate carboxylase was insignificant. Results support the interpretation that the observed inhibition of pyruvate carboxylase occurred primarily by depletion of the activator, acetyl CoA, through sequestration of coenzyme A during benzoate metabolism.