Sex specific response of cultured human bone cells to ERα and ERβ specific agonists by modulation of cell proliferation and creatine kinase specific activity

We have previously reported that human cultured bone cells (hObs) respond to estradiol-17β (E2) by stimulating DNA synthesis, creatine kinase BB specific activity (CK) and other parameters sex-specifically. We now investigate the sex specificity of the response of these hObs to estrogen receptor (ER) α and ERβ specific agonists. Real time PCR revealed that all cells express mRNA for both ERs. ERα mRNA but not ERβ mRNA was stimulated by all estrogenic compounds in both pre- and post-menopausal hObs with no effect in male hObs. Cells treated with E2, 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERβ specific agonist) and 4,4',4″-[4-propyl-(1H)-pyrazol-1,3,5-triyl] tris-phenol (PPT; ERα specific agonist) showed increased DNA synthesis and CK in all female but not male hObs. Raloxifene (Ral), a specific ERα antagonist, inhibited the stimulation of DNA synthesis and CK by E2 or PPT, but not by DPN. DPN and PPT like E2 modulated the expression of both 12 and 15 lipooxygenase (LO) mRNA in both female but not male hObs. 12 and 15 HETE production was modulated only by DPN and PPT in these cells. The LO inhibitor baicaleine inhibited only E2 and PPT but not DPN effects in both female hObs. In conclusion, we provide herein evidence for the separation of age- and sex-dependent mediation via both ERα and ERβ pathways in the effects of estrogens on hObs, with a yet unknown mechanism.     

Heightened stress response in primary fibroblasts expressing mutant eIF2B genes from CACH/VWM leukodystrophy patients

Childhood ataxia with central nervous system hypomyelination (CACH), also called vanishing white matter (VWM) leukoencephalopathy, is a fatal genetic disease caused by mutations in eukaryotic initiation factor 2B (eIF2B) genes. The five subunits eIF2B factor is critical for translation initiation under normal conditions and regulates protein synthesis in response to cellular stresses. Primary fibroblasts from CACH/VWM patients and normal individuals were used to measure basal eIF2B activity as well as global protein synthesis and ATF4 induction in response to stress in the endoplasmic reticulum. We show that although the cells expressing mutant eIF2B genes respond normally to stress conditions by reduced global translation rates, they exhibit significantly greater increase in ATF4 induction compared to normal controls despite equal levels of stress and activity of the upstream eIF2α kinase. This heightened stress response observed in primary fibroblasts that suffer from minor loss of basal eIF2B activity may be employed as an initial screening tool for CACH/VWM leukodystrophy.     

Gangliosides of organ-confined versus metastatic androgen-receptor-negative prostate cancer

Prior development of a unique androgen-receptor (AR)-negative cell line (HH870) from organ-confined (T2b) human prostate cancer (CaP) enabled comparison of the gangliosides associated with normal and neoplastic prostate epithelial cells, organ-confined versus metastatic (DU 145, PC-3), and AR-negative versus AR-positive CaP cell lines. Resorcinol-HCl and specific monoclonal antibodies were used to characterize gangliosides on 2D-chromatograms, and to visualize them on the cell surface with confocal-fluorescence microscopy. AR-negative cells expressed GM1b, GM2, GD2, GD1a, and GM3. GM1a, GD1b, and GT1b were undetectable. GM1b and GD1a were more prominent in AR-negative than in AR-positive cells. PC-3 and HH870 cells were unique in the expression of O-acetylGD2 (O-AcGD2) and two alpha2,3-sialidase-resistant, alkali-susceptible GMR17-reactive gangliosides. Expression of GD1a, GM1b, doublets of GD3, GD2, and O-AcGD2, and the presence of an additional alkali-labile-14.G2a-reactive ganglioside, two alkali-susceptible, and three alkali-resistant GMR17-reactive gangliosides makes HH870 a potential component of a polyvalent-vaccine for active-specific immunotherapy of CaP.     

The structural basis of the thermostability of SP1, a novel plant (Populus tremula) boiling stable protein

We previously reported on a new boiling stable protein isolated from aspen plants (Populus tremula), which we named SP1. SP1 is a stress-related protein with no significant sequence homology to other stress-related proteins. It is a 108-amino-acid hydrophilic polypeptide with a molecular mass of 12.4 kDa (Wang, W. X., Pelah, D., Alergand, T., Shoseyov, O., and Altman, A. (2002) Plant Physiol. 130, 865-875) and is found in an oligomeric form. Preliminary electron microscopy studies and matrix-assisted laser desorption ionization time-of-flight mass spectrometry experiments showed that SP1 is a dodecamer composed of two stacking hexamers. We performed a SDS-PAGE analysis, a differential scanning calorimetric study, and crystal structure determination to further characterize SP1. SDS-PAGE indicated a spontaneous assembly of SP1 to one stable oligomeric form, a dodecamer. Differential scanning calorimetric showed that SP1 has high thermostability i.e. Tm of 107 degrees C (at pH 7.8). The crystal structure of SP1 was initially determined to 2.4 A resolution by multi-wavelength anomalous dispersion method from a crystal belonging to the space group I422. The phases were extended to 1.8 A resolution using data from a different crystal form (P21). The final refined molecule includes 106 of the 108 residues and 132 water molecules (on average for each chain). The R-free is 20.1%. The crystal structure indicated that the SP1 molecule has a ferredoxin-like fold. Strong interactions between each two molecules create a stable dimer. Six dimers associate to form a ring-like-shaped dodecamer strongly resembling the particle visualized in the electron microscopy studies. No structural similarity was found between the crystal structure of SP1 and the crystal structure of other stress-related proteins such as small heat shock proteins, whose structure has been already determined. This structural study further supports our previous report that SP1 may represent a new family of stress-related proteins with high thermostability and oligomerization.     

Physical stresses at the air-wall interface of the human nasal cavity during breathing.

The nose is the front line defender of the respiratory system and is rich with mechanoreceptors, thermoreceptors, and nerve endings. A time-dependent computational model of transport through nasal models of a healthy human has been used to analyze the fields of physical stresses that may develop at the air-wall interface of the nasal mucosa. Simulations during quiet breathing revealed wall shear stresses as high as 0.3 Pa in the noselike model and 1.5 Pa in the anatomical model. These values are of the same order of those known to exist in uniform large arteries. The distribution of temperature near the nasal wall at peak inspiration is similar to that of wall shear stresses. The lowest temperatures occur in the vicinity of high stresses due to the narrow passageway in these locations. Time and spatial gradients of these stresses may have functional effects on nasal sensation of airflow and may play a role in the well-being of nasal breathing.     

The cross-generation transmission of oxytocin in humans

Animal studies demonstrated that the neuropeptide oxytocin (OT), implicated in bond formation across mammalian species, is transmitted from mother to young through mechanisms of early social experiences; however, no research has addressed the cross-generation transmission of OT in humans. Fifty-five parents (36 mothers and 19 fathers) engaged in a 15-min interaction with their infants. Baseline plasma OT was sampled from parents and salivary OT was sampled from parents and infants before and after play and analyzed with ELISA methods. Interactions were micro-coded for parent and child's socio-affective behavior. Parent and infant's salivary OT was individually stable across assessments and showed an increase from pre- to post-interaction. Significant correlations emerged between parental and infant OT at both assessments and higher OT levels in parent and child were related to greater affect synchrony and infant social engagement. Parent-infant affect synchrony moderated the relations between parental and infant OT and the associations between OT in parent and child were stronger under conditions of high affect synchrony. Results demonstrate consistency in the neuroendocrine system supporting bond formation in humans and other mammals and underscore the role of early experience in shaping the cross-generation transmission of social affiliation in humans.