Enzyme-linked immunosorbent assay (ELISA) with covalently bound protein on glass tubes: 1. Stable antigenicity and binding of IgG as a model antigen after repeated use

A modification of the ELISA procedure is described. The system is based on the covalent binding of protein to glass tubes. Human IgG was used as model antigen. Optimal conditions were tested for the removal of alkaline phosphatase-labeled antibodies from their antigen. Under such optimal conditions, a regenerable system could be created which exhibited many advantages, as compared with conventional ELISAs with antigens absorbed unspecifically to plastic surfaces. The advantages are: 1. A higher density of IgG as model antigen on the solid phase, i.e., 1 molecule IgG per 94 nm2 with glass tubes as compared with 110 nm2 with polystyrene (PS) or 143 nm2 with polyvinylchloride (PVC) microtiter plates. 2. A much smaller unspecific absorption of less than 1% as compared with 39% with PS-plates or 16% with PVC-plates treated under identical conditions. 3. A higher stability of the binding of the model antigen to the solid phase, i.e., a drastically reduced protein loss of less than 6% during the first ELISA procedure (including the regeneration) and of less than 1% during the subsequent ELISA and regeneration cycles with glass tubes as compared with 46% (PS plates) or 55% (PVC plates). 4. A smaller intraday variation coefficient of the ELISAs of 4.8% with glass tubes as opposed to 9.7% or 7.5% with PS or PVC plates respectively. The system with covalently bound antigens on glass tubes could be used in at least 20 consecutive measuring cycles. In five consecutive cycles, a protein loss of less than 5% was observed and the interday variation coefficient of the ELISA reaction was smaller than 5% using the same tubes repeatedly. Our results indicate that covalent linkage of protein can improve ELISA and lead to repeatedly usable systems as long as the antigen is stable against the regeneration procedure. Such an ELISA system may be helpful with highly purified proteins.     

The Presence of Progressive Motility Sustaining Factor (PMSF) in Human Epididymis

Progressive Motility Sustaining Factor (PMSF) was isolated from human epididymides. PMSF could significantly improve progressive motility of oligozoospermic as well as asthenozoospermic semen. PMSF was found to increase cyclic AMP accumulation by the human spermatozoa indicating its possible mode of action.     

Degradation and resynthesis of cyclic 3', 5' -guanosine monophosphate in truncated rod photoreceptors from bovine retina

Truncated rod photoreceptors containing outer segments with inner segment attachments were prepared from bovine retinas. In the absence of a high-energy donor, the preparation transformed cyclic GMP to 5′-GMP, guanosine and a small amount of cyclic XMP. In the presence of ATP. the preparation formed GDP and GTP from 5′-GMP, and GTP endogenously formed was converted to cyclic GMP. Conditions for optimal activity of the above reactions were defined and a rank ordering of the specific activities of the reactions established. The specific activity of each enzyme was greater in truncated photoreceptors than in homogenates of retina. The efficiency of the metabolic cycle to resynthesize cyclic GMP from 5′-GMP in vivo probably depends upon the energy state of the visual cell and upon the degree of compartmentation of substrates within the inner and outer segment of the photoreceptor.     

Effects of estrogen via estrogen receptors on parvalbumin levels in cardiac myocytes of ovariectomized rats

The study investigated the effects of estrogen on parvalbumin (PV) levels in cardiac myocytes of ovariectomized rats, which is a model system for postmenopausal woman. Parvalbumin acts as a relaxing factor in cardiac myocytes. Adult female Wistar rats, 12 weeks old, were randomly divided into 5 groups of 10: sham-operated (SHAM), ovariectomized (OVX), and OVX receiving estrogen replacement of 10 μg/kg (Es10), 20 μg/kg (Es20) and 40 μg/kg (Es40). After 10 weeks, serum estrogen levels were measured and the α and β estrogen receptors in cardiac myocytes were investigated by immunohistochemistry. PV levels were examined by immunohistochemistry and Western blot analysis. Cardiac myocytes of all animals showed strong staining intensities for α immunoreactive (Es α-ir), but weak staining for β immunoreactive (Es β-ir) estrogen receptors. The Es α-ir was reduced in the cardiac myocytes of the OVX groups, but increased in the Es10, Es20 and Es40 groups. We therefore suggest that estrogen effects are mediated via Es α receptors rather than Es β receptors in female rat hearts. Estrogen and PV immunoreactive (PV-ir) levels and the intensity of the PV band observed in the OVX group were less than those of the SHAM group. In the Es10, Es20 and Es40 groups, the increased intensity of the PV-ir and PV bands correlated with the increased estrogen levels. The low PV levels in cardiac myocytes induced by low estrogen were restored by estrogen replacement therapy. Therefore a reduction of PV may lead to diastolic dysfunction in menopause.     

Morphological alterations and distribution of occludin in rat testes after bilateral vasectomy

The aim of study was to investigate the fate and the morphology of the cells which constitute the spermatogenic line, and to determine the distribution of occludin in the testis in adult vasectomized Wistar rats. The rats were divided into two groups: control group (sham-operated) and vasectomized group. One, 3 and 6 months after sham and vasectomy operations, testis samples were examined. The weight of the testes was found to be reduced 3 and 6 months after vasectomy. There was vacuolization in the seminiferous tubules one month after vasectomy. The tubules showed severe atrophy 3 and 6 months after vasectomy. The occludin immunolabeling in the 3- and 6-month groups was weak and diffuse, and the density of the protein was found to be decreased. The increase in the number of apoptotic cells was accompanied by a time-dependent decrease in the number of haploid, diploid and tetraploid cells. This study demonstrated that vasectomy causes degeneration in the seminiferous tubules with alterations in occludin distribution with a decrease in the number of spermatogenic cells. Moreover, these alterations increase in a time-dependent manner.     

Identification and functional analysis of genes which confer resistance to cisplatin in tumor cells

The efficacy of cisplatin during cancer chemotherapy is often impaired by the emergence of cancer cells which become resistant to chemotherapeutic agents. While various mechanisms have been proposed to explain resistance to cisplatin, the genes involved in this process still remain unclear. By using DNA microarrays, we performed a genome-wide analysis of cisplatin-resistant HeLa cells in order to identify genes involved in resistance. We identified nine genes (NAPA, CITED2, CABIN1, ADM, HIST1H1A, EHD1, MARK2, PTPN21, and MVD), which were consistently upregulated in two cisplatin-resistant HeLa cell lines. The upregulated genes, here referred to as cisplatin resistance genes (CPR), were further analyzed for their ability to modify the response of HEK293 cells to cisplatin. Short-hairpin RNA (shRNA) knockdown of CPR genes, individually or in combination, was shown to sensitize HEK293 cells to cisplatin, but not to vincristine or taxol, suggesting that CPR genes may be involved specifically in cisplatin resistance. Among the treatments performed, shRNA knockdown of NAPA was the most efficient treatment able to sensitize cells to cisplatin. Furthermore, shRNA knockdown of a single CPR gene was sufficient to partially reverse acquired cisplatin resistance in HeLa cells. Sensitization to cisplatin following knockdown of CPR genes was also observed in the tumorigenic cell lines Sk-ov-3, H1155, and CG-1. Based on these results, we propose that the CPR genes identified here may represent potential candidates for novel target therapies aimed at preventing resistance to cisplatin during chemotherapy.     

Control of cardiac myofilament activation and PKC-betaII signaling through the actin capping protein, CapZ

Actin capping protein (CapZ) anchors the barbed ends of sarcomeric actin to the Z-disc. Myofilaments from transgenic mice (TG-CapZ) expressing a reduced amount of CapZ demonstrate altered function and protein kinase C (PKC) signaling [Pyle WG, Hart MC, Cooper JA, Sumandea MP, de Tombe PP, and Solaro RJ., Circ. Res. 90 (2002) 1299-306]. The aims of the current study were to determine the direct effects of CapZ on myofilament function and on PKC signaling to the myofilaments. Our studies compared mechanical properties of single myocytes from TG-CapZ mouse hearts to wild-type myocytes from which CapZ was extracted using PIP(2). We found that myofilaments from CapZ-deficient transgenic myocardium exhibited increased Ca(2+) sensitivity and maximum isometric tension. The extraction of CapZ from wild-type myofilaments replicated the increase in maximum isometric tension, but had no effect on myofilament Ca(2+) sensitivity. Immunoblot analysis revealed that the extraction of CapZ was associated with a reduction in myofilament-associated PKC-beta(II) and that CapZ-deficient transgenic myofilaments also lacked PKC-beta(II). Treatment of wild-type myofilaments with recombinant PKC-beta(II) reduced myofilament Ca(2+) sensitivity, whereas this effect was attenuated in myofilaments from TG-CapZ mice. Our results indicate that cardiac CapZ directly controls maximum isometric tension generation, and establish CapZ as an important component in anchoring PKC-beta(II) at the myofilaments, and for mediating the effects of PKC-beta(II) on myofilament function.     

PYDDT,a novel phase 2 enzymes inducer,activates Keap1–Nrf2 pathway via depleting the cellular level of glutathione

Keap1–Nrf2 pathway has emerged as a regulator for the endogenous antioxidant response, which is critical in defending cells against carcinogenesis. Herein, we demonstrated that depleting the cellular level of glutathione (GSH) by a novel electrophilic agent 2-(pro-1-ynyl)-5-(5,6-dihydroxypenta-1,3-diynyl) thiophene (PYDDT) could activate Keap1–Nrf2 pathway. In above process, it was found that Keap1 was modified by S-glutathionylation, an important post-translational modification of protein cysteines with critical roles in oxidative stress and signal transduction. We concluded from our findings that conjugation with intracellular GSH by PYDDT might lead to Keap1 S-glutathionylation and was a key event involved in its Nrf2 inducing activity.     

Exposure of human skin to benzo[a]pyrene: Role of CYP1A1 and aryl hydrocarbon receptor in oxidative stress generation

Exposure to benzo[a]pyrene (BaP) can induce inflammatory skin diseases and skin cancer, which are both associated to oxidative stress. BaP is known to bind with high specificity to the aryl hydrocarbon receptor (AhR), modifying the expression of CYP1A1, involved both in cancer and inflammation.