Effects of some steroid hormones on head-to-head association in bovine spermatozoa

In the presence of 2 × 10^?6 M Ca²⁺ in Tris-buffered medium 0.5 × 10^?6 M, oestradiol-17β or corticosterone significantly increased the head-to-head association of washed bull spermatozoa; in the same concentration, testosterone and 5α-dihydrotestosterone had no significant effects, whereas progesterone significantly dissociated the associated spermatozoa. At 8 × 10^?6 M Ca²⁺ in the same medium, all five hormones increased the association to about the same level. In Tyrode solution with a Ca²⁺ concentration of 1.4 × 10^?3 M, oestradiol-17β and corticosterone acted as above, whereas progesterone and the two testosterones effected dissociation. In Tyrode solution each of the dissociating hormones was combined with oestradiol-17β. In each case a sum of the effects of the two hormones was obtained without any stimulation or inhibition. All five hormones still produced significant effects at 5 × 10^?7 M in Tyrode solution. A corresponding value for ATP was found at 1 × 10^?5 M.     

Studies on the Assembly Characteristics of Large Subunit Ribosomal Proteins in S. cerevisae

During the assembly process of ribosomal subunits, their structural components, the ribosomal RNAs (rRNAs) and the ribosomal proteins (r-proteins) have to join together in a highly dynamic and defined manner to enable the efficient formation of functional ribosomes. In this work, the assembly of large ribosomal subunit (LSU) r-proteins from the eukaryote S. cerevisiae was systematically investigated. Groups of LSU r-proteins with specific assembly characteristics were detected by comparing the protein composition of affinity purified early, middle, late or mature LSU (precursor) particles by semi-quantitative mass spectrometry. The impact of yeast LSU r-proteins rpL25, rpL2, rpL43, and rpL21 on the composition of intermediate to late nuclear LSU precursors was analyzed in more detail. Effects of these proteins on the assembly states of other r-proteins and on the transient LSU precursor association of several ribosome biogenesis factors, including Nog2, Rsa4 and Nop53, are discussed.     

Detection of oxidases on polyacrylamide gels

Direct transfer of electrons, from the reduced flavin moiety of several oxidases to phenazine methosulfate and then to nitroblue tetrazolium, serves to detect activity of the oxidases of l-amino acids, urate, diamines, hydroxy acids (both long- and short-chain), aldehydes, and sarcosine. It is demonstrated that the method specifically detects oxidases and is more sensitive than other methods available. It is noted that, in the detection of certain dehydrogenases on polyacrylamide gels by the usual NAD-phenazine methosulfate-nitroblue tetazolium technique, one or more of the isozymes may actually represent the corresponding oxidase rather than the dehydrogenase in question.     

Renoprotection by α-mangostin is related to the attenuation in renal oxidative/nitrosative stress induced by cisplatin nephrotoxicity

Cisplatin (CDDP) is a chemotherapeutic agent that produces nephrotoxicity associated with oxidative/nitrosative stress. α-Mangostin (α-M) is a xanthone extracted from mangosteen with antioxidant and anti-inflammatory properties. The purpose of this study was to evaluate the renoprotective effect of α-M on the CDDP-induced nephrotoxicity. α-M was administered (12.5 mg/kg/day, i.g.) for 10 days (7 days before and 3 days after CDDP injection). On day 7, rats were treated with a single injection of CDDP (7.5 mg/Kg, i.p.); 3 days after the rats were killed. α-M attenuated renal dysfunction, structural damage, oxidative/nitrosative stress, decrease in catalase expression and increase in mRNA levels of tumour necrosis factor alpha and transforming growth factor beta. In conclusion the renoprotective effect of α-M on CDDP-induced nephrotoxicity was associated with the attenuation in oxidative/nitrosative stress and inflammatory and fibrotic markers and preservation of catalase activity.     

Changes in apoptotic rate and cell viability in three fish epidermis cultures after exposure to nonylphenol and to a wastewater sample containing low concentrations of nonylphenol

Three types of epidermal cultures of fish were used for toxicological investigations, a primary cell culture and a tissue culture prepared from the rainbow trout Oncorhynchus mykiss Walbaum and the cell line EPC, derived from a skin tumour of the carp Cyprinus carpio L. Two studies were carried out to compare the different culture systems. In the first cultures were incubated with nonylphenol and in the second set of experiments the cell cultures were exposed to a wastewater sample containing low concentrations of nonylphenol (NP). Both cell cultures were similarly sensitive to nonylphenol with respect to the endpoints cell viability (LC₅₀ (24 h) 47.1 μM NP (primary cell culture) and 44.2 μM NP (EPC)) values and apoptotic rate (significantly increased apoptotic rate after exposure to 50 μM NP for 24 h, p < 0.001 (primary cell culture), p = 0.008 (EPC)). The explant culture was slightly less sensitive (increased apoptotic rate after exposure to 50 μM NP for 24 h, but not significant: p = 0.385), which could be due to the capabilities of a differentiated tissue, providing more protective repair mechanisms, compared with single cells. All cultures revealed a concentration–response relationship for the endpoint apoptotic rate after the application of nonylphenol for 24 h. After wastewater exposure, a significant decrease in the apoptotic rate was measured in the primary cell culture (dilution wastewater : medium 1:1:p = 0.018; dilution wastewater : medium 1:2:p = 0.003), whereas the cell line EPC did not reveal any effects. Our results show that the endpoint apoptotic rate is more sensitive than the parameter cell viability for detecting adverse effects of a wastewater sample.     

High Content Screening as High Quality Assay for Biological Evaluation of Photosensitizers In Vitro

A novel single step assay approach to screen a library of photdynamic therapy (PDT) compounds was developed. Utilizing high content analysis (HCA) technologies several robust cellular parameters were identified, which can be used to determine the phototoxic effects of porphyrin compounds which have been developed as potential anticancer agents directed against esophageal carcinoma. To demonstrate the proof of principle of this approach a small detailed study on five porphyrin based compounds was performed utilizing two relevant esophageal cancer cell lines (OE21 and SKGT-4). The measurable outputs from these early studies were then evaluated by performing a pilot screen using a set of 22 compounds. These data were evaluated and validated by performing comparative studies using a traditional colorimetric assay (MTT). The studies demonstrated that the HCS assay offers significant advantages over and above the currently used methods (directly related to the intracellular presence of the compounds by analysis of their integrated intensity and area within the cells). A high correlation was found between the high content screening (HCS) and MTT data. However, the HCS approach provides additional information that allows a better understanding of the behavior of these compounds when interacting at the cellular level. This is the first step towards an automated high-throughput screening of photosensitizer drug candidates and the beginnings of an integrated and comprehensive quantitative structure action relationship (QSAR) study for photosensitizer libraries.     

Local Mechanical Stimuli Regulate Bone Formation and Resorption in Mice at the Tissue Level

Bone is able to react to changing mechanical demands by adapting its internal microstructure through bone forming and resorbing cells. This process is called bone modeling and remodeling. It is evident that changes in mechanical demands at the organ level must be interpreted at the tissue level where bone (re)modeling takes place. Although assumed for a long time, the relationship between the locations of bone formation and resorption and the local mechanical environment is still under debate. The lack of suitable imaging modalities for measuring bone formation and resorption in vivo has made it difficult to assess the mechanoregulation of bone three-dimensionally by experiment. Using in vivo micro-computed tomography and high resolution finite element analysis in living mice, we show that bone formation most likely occurs at sites of high local mechanical strain (p<0.0001) and resorption at sites of low local mechanical strain (p<0.0001). Furthermore, the probability of bone resorption decreases exponentially with increasing mechanical stimulus (R² = 0.99) whereas the probability of bone formation follows an exponential growth function to a maximum value (R² = 0.99). Moreover, resorption is more strictly controlled than formation in loaded animals, and ovariectomy increases the amount of non-targeted resorption. Our experimental assessment of mechanoregulation at the tissue level does not show any evidence of a lazy zone and suggests that around 80% of all (re)modeling can be linked to the mechanical micro-environment. These findings disclose how mechanical stimuli at the tissue level contribute to the regulation of bone adaptation at the organ level.     

Cell-to-Cell Transmission Can Overcome Multiple Donor and Target Cell Barriers Imposed on Cell-Free HIV

Virus transmission can occur either by a cell-free mode through the extracellular space or by cell-to-cell transmission involving direct cell-to-cell contact. The factors that determine whether a virus spreads by either pathway are poorly understood. Here, we assessed the relative contribution of cell-free and cell-to-cell transmission to the spreading of the human immunodeficiency virus (HIV). We demonstrate that HIV can spread by a cell-free pathway if all the steps of the viral replication cycle are efficiently supported in highly permissive cells. However, when the cell-free path was systematically hindered at various steps, HIV transmission became contact-dependent. Cell-to-cell transmission overcame barriers introduced in the donor cell at the level of gene expression and surface retention by the restriction factor tetherin. Moreover, neutralizing antibodies that efficiently inhibit cell-free HIV were less effective against cell-to-cell transmitted virus. HIV cell-to-cell transmission also efficiently infected target T cells that were relatively poorly susceptible to cell-free HIV. Importantly, we demonstrate that the donor and target cell types influence critically the extent by which cell-to-cell transmission can overcome each barrier. Mechanistically, cell-to-cell transmission promoted HIV spread to more cells and infected target cells with a higher proviral content than observed for cell-free virus. Our data demonstrate that the frequently observed contact-dependent spread of HIV is the result of specific features in donor and target cell types, thus offering an explanation for conflicting reports on the extent of cell-to-cell transmission of HIV.