First evidence of prothymosin alpha localization in the acrosome of mammalian male gametes

Prothymosin α (PTMA) is a highly acidic intrinsically unstructured protein. Its expression in male gonads is evolutionary conserved; in rat testis it is specifically localized in the cytoplasm of post‐meiotic germ cells, in association with the developing acrosome system. In the present paper we investigated on PTMA localization inside the head of mammalian spermatozoa (SPZ). We chose a confocal approach to ascertain whether PTMA is expressed in the acrosome or in the perinuclear theca, two regions that are tightly linked and partially overlapped in the mature haploid cells. The obtained results showed that PTMA is specifically localized in the acrosome of rat epididymal SPZ; the same experimental approach evidenced, for the first time, PTMA presence in human ejaculated SPZ. A Western blot analysis on protein extracts from human sperm head fractions confirmed the confocal data and demonstrated that the peptide is specifically associated with the inner acrosomal membrane fraction. Finally, when the acrosome reaction was induced in vitro by progesterone treatment on both rat and human sperm, PTMA signal was retained in the apical region of reacted SPZ. In conclusion, this study confirms the conservation of PTMA distribution in vertebrate male gametes and strongly supports a role for this polypeptide in their physiology. J. Cell. Physiol. 228: 1629–1637, 2013. © 2013 Wiley Periodicals, Inc.     

Triacylglycerol accumulation activates the mitochondrial apoptosis pathway in macrophages

Programmed cell death of lipid-laden macrophages is a prominent feature of atherosclerotic lesions and mostly ascribed to accumulation of excess intracellular cholesterol. The present in vitro study investigated whether intracellular triacylglycerol (TG) accumulation could activate a similar apoptotic response in macrophages. To address this question, we utilized peritoneal macrophages isolated from mice lacking adipose triglyceride lipase (ATGL), the major enzyme responsible for TG hydrolysis in multiple tissues. In Atgl(-/-) macrophages, we observed elevated levels of cytosolic Ca(2+) and reactive oxygen species, stimulated cytochrome c release, and nuclear localization of apoptosis-inducing factor. Fragmented mitochondria prior to cell death were indicative of the mitochondrial apoptosis pathway being triggered as a consequence of defective lipolysis. Other typical markers of apoptosis, such as externalization of phosphatidylserine in the plasma membrane, caspase 3 and poly(ADP-ribose) polymerase cleavage, were increased in Atgl(-/-) macrophages. An artificial increase of cellular TG levels by incubating wild-type macrophages with very low density lipoprotein closely mimicked the apoptotic phenotype observed in Atgl(-/-) macrophages. Results obtained during the present study define a novel pathway linking intracellular TG accumulation to mitochondrial dysfunction and programmed cell death in macrophages.     

The mutagenic potential of madder root in dyeing processes in the textile industry

The roots of Rubia tinctorum L. have a long tradition of being used in dyeing processes of textiles from centuries ago until the present time. The colouring principles belong to the class of hydroxyanthraquinones. Concern arose because several of these compounds were recognised as mutagenic in vitro and even carcinogenic in rodents. To assess the possible risk to humans caused by coloured textiles, mutagenicity was investigated with two madder root samples of different origin (Iran and Bhutan) along the entire dyeing process from root extracts to the dyed wool. The Salmonella/microsome test (Ames assay) with the strains TA98, TA100 and TA1537 was used. Significant mutagenic effects could be detected in madder root extracts and also in the final product, the dyed wool. Madder root from Iran showed considerably higher mutagenic responses than samples from Bhutan. Analytical investigations of the extracts by HPLC showed the presence of a spectrum of anthraquinones typical for madder root. Three mutagenic compounds, lucidine, rubiadine and purpuroxanthine, together with the non-mutagenic alizarine could be detected. The mutagenic response of the different samples was positively correlated with the concentration of the mutagenic anthraquinones, and with lucidine in particular. Based on these investigations a risk to dye-house workers and users of textiles dyed with R. tinctorum must be anticipated.     

The Laboratory Rat as an Animal Model for Osteoporosis Research

Osteoporosis is an important systemic disorder, affecting mainly Caucasian women, with a diverse and multifactorial etiology. A large variety of animal species, including rodents, rabbits, dogs, and primates, have been used as animal models in osteoporosis research. Among these, the laboratory rat is the preferred animal for most researchers. Its skeleton has been studied extensively, and although there are several limitations to its similarity to the human condition, these can be overcome through detailed knowledge of its specific traits or with certain techniques. The rat has been used in many experimental protocols leading to bone loss, including hormonal interventions (ovariectomy, orchidectomy, hypophysectomy, parathyroidectomy), immobilization, and dietary manipulations. The aim of the current review is not only to present the ovariectomized rat and its advantages as an appropriate model for the research of osteoporosis, but also to provide information about the most relevant age and bone site selection according to the goals of each experimental protocol. In addition, several methods of bone mass evaluation are assessed, such as biochemical markers, densitometry, histomorphometry, and bone mechanical testing, that are used for monitoring and evaluation of this animal model in preventive or therapeutic strategies for osteoporosis.Abbreviations: BMD, bone mineral density; DEXA, dual-energy X-ray absorptiometry; μCT, microcomputerized tomography; pQCT, peripheral quantitative computerized tomographyOsteoporosis is a multifactorial skeletal disease, characterized by reduction in bone mass and disruption of the microarchitectural structure of bone tissue, resulting in loss of mechanical strength and increased risk of fracture.² The disorder can be localized or involve the entire skeleton. Generalized osteoporosis can be primary (postmenopausal and senile) or secondary. In the European Union, osteoporosis is a leading cause of mortality and morbidity in the elderly and a key factor in the high cost of medical care.³⁴Although osteoporosis usually makes its appearance late in life, and age is a major risk factor, its roots can be tracked back into adolescence. Particularly during periods of rapid bone growth, dietary calcium levels are of high importance.³⁴ Other factors that contribute to the pathogenesis of osteoporosis are lifestyle and genetic and hormonal attributes.^13,71 Reduced physical activity increases the rate of bone loss, and muscle contraction is the prevailing source of skeletal loading. Regarding hormonal factors, women, especially in the decade after menopause, can show a severe reduction of bone mass, thus explaining the high incidence of osteoporotic fractures in women compared with men.³⁴The multiple factors implicated in osteoporosis, its obscure pathogenesis, the dramatic decline in quality of life, high incidence of the disorder (especially in postmenopausal women), financial cost, and high mortality, make the need for further experimentation in animal models imperative. Experimental research can improve our understanding of pathogenesis and of the activity of pharmaceutical agents in the prevention or treatment of the disease. Although many aspects of the disorder have been revealed, others remain unclear, including the mechanisms involved in calcium homeostasis in the extracellular space and its effect on bone physiology and disease⁶⁵ and the cell and molecular pathways triggered after mechanical loading to orchestrate bone renewal.⁵³ Current research is focused on new therapeutic possibilities targeting the osteolytic enzymes of the osteoclast and the mechanisms activating bone progenitor cells and those controlling apoptosis as new potential treatments.^63,64Many therapeutic advances in the management of osteoporosis were studied first in diverse animal models and then entered clinical practice.^31,67,69 All of these models should fulfill similar basic criteria: they must comply with national and local ethical and legislative considerations, be accessible to experimental centers, be easy and safe to handle, have a low cost of acquisition, require little maintenance, reliably reproduce the disease and the biological material to be examined should be readily available. Laboratory rats meet most of these criteria. In addition, the availability of detailed knowledge of the rat skeleton and protocols for rapid induction of osteopenia, have increased this model''s popularity. Here we review the advantages and limitations of the use of the laboratory rat in osteoporosis research.     

The role of vitamin D receptor gene polymorphisms in the bone mineral density of Greek postmenopausal women with low calcium intake

The aim of this study was to investigate the effect of common vitamin D receptor (VDR) gene polymorphisms on the bone mineral density (BMD) of Greek postmenopausal women. Healthy postmenopausal women (n=578) were recruited for the study. The BMD of the lumbar spine and hip was measured using dual-energy X-ray absorptiometry with the Lunar DPX-MD device. Assessment of dietary calcium intake was performed with multiple 24-h recalls. Genotyping was performed for the BsmI, TaqI and Cdx-2 polymorphisms of the VDR gene. The selected polymorphisms were not associated with BMD, osteoporosis or osteoporotic fractures. Stratification by calcium intake revealed that in the low calcium intake group (<680 mg/day), all polymorphisms were associated with the BMD of the lumbar spine (P<.05). After adjustment for potential covariates, BsmI and TaqI polymorphisms were associated with the presence of osteoporosis (P<.05), while the presence of the minor A allele of Cdx-2 polymorphism was associated with a lower spine BMD (P=.025). In the higher calcium intake group (>680 mg/day), no significant differences were observed within the genotypes for all polymorphisms. The VDR gene is shown to affect BMD in women with low calcium intake, while its effect is masked in women with higher calcium intake. This result underlines the significance of adequate calcium intake in postmenopausal women, given that it exerts a positive effect on BMD even in the presence of negative genetic predisposition.     

Oncogenic Role of the Ec Peptide of the IGF-1Ec Isoform in Prostate Cancer

IGF-1 is one of the key molecules in cancer biology; however, little is known about the role of the preferential expression of the premature IGF-1 isoforms in prostate cancer. We have examined the role of the cleaved COO– terminal peptide (PEc) of the third IGF-1 isoform, IGF-1Ec, in prostate cancer. Our evidence suggests that endogenously produced PEc induces cellular proliferation in the human prostate cancer cells (PC-3) in vitro and in vivo, by activating the ERK1/2 pathway in an autocrine/paracrine manner. PEc overexpressing cells and tumors presented evidence of epithelial to mesenchymal transition, whereas the orthotopic injection of PEc-overexpressing, normal prostate epithelium cells (HPrEC) in SCID mice was associated with increased metastatic rate. In humans, the IGF-1Ec expression was detected in prostate cancer biopsies, where its expression correlates with tumor stage. Our data describes the action of PEc in prostate cancer biology and defines its potential role in tumor growth, progression and metastasis.     

Intrauterine growth restriction affects bone mineral density of the mandible and the condyle in growing rats

Objectives:To investigate in growing rats the effect of intrauterine growth restriction (IUGR) on the bone mineral density of the mandible and tibia, as well as the quality of the mandibular and condylar bone.Methods:Twelve male rats were born IUGR by mothers sustaining 50% food restriction during pregnancy. Twelve control male rats were born by mothers fed ad libitum. Dual-energy X-ray absorptiometry (DEXA) of the tibia, proximal tibial metaphysis and the mandible, biochemical markers, histology and histomorphometrical analysis on the mandibular and subchondral bone of the condyle were performed.Results:IUGR significantly affected bone mineral density (BMD) of both tibial and mandibular bones. IUGR rats had significantly lower osteocalcin values (p=0.021) and phosphorus (p=0.028), but not 25-OH vitamin D (p=0.352). Bone area percentage in the mandible was significantly lower (51.21±5.54) in IUGR compared to controls (66.00±15.49), and for subchondral bone of the condyle for IUGR (47.01±6.82) compared to controls (68.27±13.37). IUGR had a significant reduction in the fibrous layer, but not the proliferating layer, with the hypertrophic layer significantly increased.Conclusion:Maternal restricted nutrition during gestation can affect BMD of the mandible and the tibia of the offspring animals.