[Clinical efficacy of local injection of platelet-rich plasma combined with double-layer artificial dermis in treating wounds with exposed tendon on extremity].

Objective: To investigate the clinical efficacy of local injection of platelet-rich plasma (PRP) combined with double-layer artificial dermis in treating wounds with exposed tendon on extremity. Methods: A retrospective observational study was conducted. From December 2017 to October 2022, 16 patients were admitted to Department of Orthopaedic Trauma of the Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine, and 32 patients were admitted to Department of Burns and Plastic Surgery of Guiyang Steel Factory Staff Hospital. All the patients had wounds with exposed tendon on extremity caused by various reasons and met the inclusion criteria. There were 39 males and 9 females, aged 26 to 58 years. The patients were divided into PRP alone group, artificial dermis alone group, and PRP+artificial dermis group, with 16 patients in each group. The wounds were treated with autologous PRP, double-layer artificial dermis, or thei combination of autologous PRP and double-layer artificial dermis, followed by autologous split-thickness scalp grafting after good growth of granulation tissue. On the 7th day after the secondary surgery, the autograft survival was observed, and the survival rate was calculated. The wound healing time and length of hospital stay of patients were recorded. At 3 and 6 months after wound healing, the Vancouver scar scale (VSS) was used to score the pigmentation, height, vascularity, and pliability of scars, and the total score was calculated. Adverse reactions during the entire treatment process were recorded. Data were statistically analyzed with chi-square test, Fisher's exact probability test, one-way analysis of variance, least significant difference test, Kruskal-Wallis H test, Nemenyi test, and Bonferroni correction. Results: On the 7th day after the secondary surgery, there was no statistically significant difference in the autograft survival rate of patients among PRP alone group, artificial dermis alone group, and PRP+artificial dermis group (P>0.05). The wound healing time and length of hospital stay of patients in PRP+artificial dermis group were (20.1±3.0) and (24±4) d, respectively, which were significantly shorter than (24.4±5.5) and (30±8) d in PRP alone group (P<0.05) and (24.8±4.9) and (32±8) d in artificial dermis alone group (P<0.05). At 3 and 6 months after wound healing, the pliability scores of patients in PRP+artificial dermis group were significantly lower than those in PRP alone group (with Z values of 12.91 and 15.69, respectively, P<0.05) and artificial dermis alone group (with Z values of 12.50 and 12.91, respectively, P<0.05). There were no statistically significant differences in pigmentation, vascularity, height scores, and total score of scar of patients among the three groups (P>0.05). In artificial dermis alone group, one patient experienced partial liquefaction and detachment of the double-layer artificial dermis due to local infection of Staphylococcus epidermidis, which received wound dressing change, second artificial dermis transplantation, and subsequent treatment as before. No adverse reactions occurred in the remaining patients during the whole treatment process. Conclusions: Local injection of PRP combined with double-layer artificial dermis is effective in treating wounds with exposed tendon on extremity, which can not only significantly shorten wound healing time and length of hospital stay, but also improve scar pliability after wound healing to some extent in the long term. It is a clinically valuable treatment technique that is worth promoting and applying.

Effects of taurine on growth performance, antioxidant capacity, and lipid metabolism in broiler chickens.

To investigate the effects of dietary taurine supplementation on growth performance, antioxidant status, and lipid metabolism in broilers, 384 male broilers (Arbor Acres, 1 D of age) were randomly allocated into 4 groups with 8 replicates of 8 birds. Dietary treatments were supplemented with taurine at the level of 0.00, 2.50, 5.00, and 7.50 g/kg of the diet (denoted as CON, TAU1, TAU2, TAU3, respectively). The BW gain from 1 to 21 D and from 22 to 42 D were all increased linearly (linear, P < 0.001) by taurine supplementation. Throughout the trial period, the highest BW gain and favorable gain-to-feed ratio were observed in the TAU2 group. Taurine supplementation increased the antioxidant enzyme activities and decreased (linear, P < 0.001) the content of malondialdehyde in both serum and the liver of broilers and alleviated oxidative damage through enhancing (P < 0.05) the hepatic genes expression of nuclear factor erythroid-2-related factor 2 (NRF2), glutathione peroxidase (GPX), and heme oxygenase-1 (HO-1). Correspondingly, in serum, the activities of hepatic lipase and total lipase were decreased linearly and quadratically (linear and quadratic, P < 0.001) with the increasing inclusion of taurine in the diet. Meanwhile, in serum, the content of triglycerides was significantly decreased (P < 0.05), and except for TAU3, the total cholesterol content was also significantly decreased (P < 0.05) by taurine supplementation. In addition, the hepatic content of triglycerides was significantly decreased (P < 0.05) in the TAU1 and TAU2 groups. Compared with the CON group, the hepatic genes expression of adenosine monophosphate-activated protein kinase alpha (AMPKα), silent 1, (SIRT1) and carnitine palmitoyltransferase 1 (CPT-1) were all increased (P < 0.05), and sterol regulatory element-binding protein-1 (SREBP-1) expression was decreased (P < 0.05) in the TAU2 group. These results indicated that taurine supplementation improved the growth performance, antioxidant capacity, and lipid metabolism of broilers.

The Discriminative Efficacy of Retinal Characteristics on Two Traditional Chinese Syndromes in Association with Ischemic Stroke.

We aimed to investigate the efficacy of an objective method using AI-based retinal characteristic analysis to automatically differentiate between two traditional Chinese syndromes that are associated with ischemic stroke. Inpatient clinical and retinal data were retrospectively retrieved from the archive of our hospital. Patients diagnosed with cerebral infarction in the department of acupuncture and moxibustion between 2014 and 2018 were examined. Of these, the patients with Qi deficiency blood stasis syndrome (QDBS) and phlegm stasis in channels (PSIC) syndrome were selected. Those without retinal photos were excluded. To measure and analyze the patients' retinal vessel characteristics, we applied a patented AI-assisted automated retinal image analysis system developed by the Chinese University of Hong Kong. The demographic, clinical, and retinal information was compared between the QDBS and PSIC patients. The t-test and chi-squared test were used to analyze continuous data and categorical data, respectively. All the selected clinical information and retinal vessel measures were used to develop different discriminative models for QDBS and PSIC using logistic regression. Discriminative efficacy and model performances were evaluated by plotting a receiver operating characteristic curve. As compared to QDBS, the PSIC patients had a lower incidence of insomnia problems (46% versus 29% respectively, p=0.023) and a higher tortuosity index (0.45 ± 0.07 versus 0.47 ± 0.07, p=0.027). Moreover, the area under the curve of the logistic model showed that its discriminative efficacy based on both retinal and clinical characteristics was 86.7%, which was better than the model that employed retinal or clinical characteristics individually. Thus, the discriminative model using AI-assisted retinal characteristic analysis showed statistically significantly better performance in QDBS and PSIC syndrome differentiation among stroke patients. Therefore, we concluded that retinal characteristics added value to the clinical differentiation between QDBS and PSIC.

Dynamic QTL analysis for fruit lycopene content and total soluble solid content in a Solanum lycopersicum x S. pimpinellifolium cross.

Fruit lycopene content and total soluble solid content are important factors determining fruit quality of tomatoes; however, the dynamic quantitative trait loci (QTL) controlling lycopene and soluble solid content have not been well studied. We mapped the chromosomal regions controlling these traits in different periods in F(2:3) families derived from a cross between the domestic and wild tomato species Solanum lycopersicum and S. pimpinellifolium. Fifteen QTLs for lycopene and soluble solid content and other related traits analyzed at three different fruit ripening stages were detected with a composite interval mapping method. These QTLs explained 7-33% of the individual phenotypic variation. QTLs detected in the color-changing period were different from those detected in the other two periods. On chromosome 1, the soluble solid content QTL was located in the same region during the color-changing and full-ripe periods. On chromosome 4, the same QTL for lycopene content was found during the color-changing and full-ripe periods. The QTL for lycopene content on chromosome 4 co-located with the QTL for soluble solid content during the full-ripe period. Co-location of lycopene content QTL and soluble solid content QTLs may be due to pleiotropic effects of a single gene or a cluster of genes via physiological relationships among traits. On chromosome 9, the same two QTLs for lycopene content at two different fruit ripening periods may reflect genes controlling lycopene content that are always expressed in tomato fruit development.

The effects of aging on AV nodal recovery properties.

The purpose of this study was to assess the changes of AV nodal recovery properties with aging. Although in children and young adults it was found that there were age dependent changes in their AV nodal recovery properties, in the older population this information was not available. In 92 subjects (aged 16-92 years) without AV nodal disease or dual AV nodal pathway physiology, their AV nodal recovery curves were studied by delivering premature atrial extrastimuli coupled to basic atrial beats during cardiac electrophysiological study. Data were analyzed using linear regression and curve-fitting techniques. Patients were grouped by age, group I < 40 years (n = 33), group II 40-59 years (n = 26), and group III > 60 years (n = 33). The results showed that the AV nodal recovery curve did not change significantly in the aging process except that the AV nodal effective refractory period had a positive correlation with increasing age. The latter was significantly increased in group III when compared to group I or group II. For this parameter, when patients whose AV nodal refractory period was limited by the atrial refractory period were excluded, there was still a statistically significant increase in group III compared to group II (P < 0.05): group I (n = 27): 202+/-42 ms; group II (n = 17): 197+/-26 ms; and group III (n = 17): 224+/-46 ms. The results suggest that the AV nodal recovery curve remains unchanged once it reaches adulthood, with the exception that the nodal effective refractory period becomes slightly longer after age 60.

[Study on the preparation of baicalin complex with phospholipid].

OBJECTIVE: To choose the optimization conditions for the formation of baicalin complex with phospholipid. METHOD: A reasonable criterion of accurate evaluation was established, by which such factors as temperature, the ratio of baicalin to phospholipid and the concentration of reactants were examined and the orthographic design was used. RESULT: The best conditions were obtained through statistical analysis and proof test. CONCLUSION: The formation of baicalin complex with phospholipid was greatly influenced by the solvent, concentration and reactant ratio.

Cell growth and gene modulatory activities of Yunzhi (Windsor Wunxi) from mushroom Trametes versicolor in androgen-dependent and androgen-insensitive human prostate cancer cells.

The incidence of prostate cancer varies greatly throughout the world; it is highest in African-Americans and lowest in the Asian populations of China, India, and Japan. Geographical differences in both prevalence of latent prostate cancer and mortality have been postulated to be influenced by diverse tumor-promoting and protective factors, both environmental and dietary. Prostate cancer is a tumor with an extremely long latency; the pattern of prostate tumorigenesis, in terms of the display and sequence of appearance of particular molecular or biochemical features, or morphological changes, characterizing different stages of the carcinogenic process, is expected to be heterogeneous. Some insights into tumor heterogeneity and progression can be obtained from studies using cell lines, particularly those derived from different anatomical sites. The present study aims to investigate whether hormone-responsive LNCaP and androgen-refractory JCA-1, PC-3, and DU-145 prostate cancer cells are responsive to Yunzhi (YZ), a proprietary dietary supplement prepared from extracts of Trametes versicolor, also known as Coriolus versicolor (a mushroom consumed by Chinese for its purported health benefits), and to elucidate its mechanism of action. Ethanolic extracts (70%) of YZ significantly reduced LNCaP cell growth, down-regulated the levels of secreted PSA, but had less effects on the expression of intracellular PSA and did not affect levels of the androgen receptor. In androgen-unresponsive prostate cancer cells, YZ had a much less pronounced suppressive effect on proliferation of PC-3 and DU-145 cells, compared to LNCaP, and was inactive against JCA-1 cells. Western blot analyses show that the expression of Rb, a key regulatory protein in G1/S transition, and PCNA, integrally involved in mammalian cell DNA replication, were significantly reduced by treatment with YZ in PC-3 and DU-145 cells, respectively. In contradiction, none of these biochemical parameters were affected in JCA-1 cells under identical treatment conditions. Further analysis shows that YZ increased the levels of signal transducer and activator family of transcription factors STAT 1 and STAT 3 in JCA-1 and not LNCaP cells. The greater sensitivity of LNCaP cells to this polysaccharopeptide raises the possibility that YZ may be considered as an adjuvant therapy in the treatment of hormone responsive prostate cancer; additionally, it may have chemopreventive potential to restrict prostate tumorigenic progression from the hormone-dependent to the hormone-refractory state.

Multivariate analysis of pulmonary fibrosis after electron beam irradiation for postmastectomy chest wall and regional lymphatics: evidence for non-dosimetric factors.

BACKGROUND AND PURPOSE: To evaluate the factors associated with pulmonary fibrosis after postmastectomy electron beam irradiation of chest wall and regional lymphatics in patients with breast cancer. MATERIALS AND METHODS: From July 1987 through July 1994, 109 women with stage II and III breast cancer receiving modified radical mastectomies were managed by postoperative electron beam irradiation. Doses of 46 to 50.4 Gy were delivered to the chest wall covered with bolus, internal mammary nodes, supraclavicular nodes and axillary lymph nodes via 12 or 15 MeV single portal electron beam. Seventeen patients received additional 10-16 Gy surgical scar boost via 9 MeV electron beam. Comparison of pre-treatment and post-treatment chest X-ray films were used to monitor the development of pulmonary fibrosis. RESULTS: Only Grade 1 radiation-induced late pulmonary toxicity was noted in 33 patients (29%). Twenty-six patients (24%) developed pulmonary fibrosis under unbolused chest wall. Lung fibrosis under bolused chest wall was noted in 11 patients (10%). Statistical difference (P<0.01) was noted between the incidence of fibrosis in these two sites. In multivariate analysis of lung fibrosis under unbolus-covered chest wall, the independent prognostic factors are low body mass index (BMI) (P<0.01), tamoxifen taking (P=0.03), and no treatment interruption (P=0.03). No independent factor was associated with lung fibrosis under bolus-covered chest wall in multivariate analysis. CONCLUSIONS: In the analysis of pulmonary fibrosis induced by unbolused electron beam, BMI rather than body weight and body height is a strong prognostic factor. Tamoxifen and short overall time can predispose the development of lung fibrosis.

Chinese herbal mixture PC SPES in treatment of prostate cancer (review).

The Chinese herbal preparation denoted PC SPES, a mixture consisting of extracts from eight herbs, is being used with increasing frequency by prostate cancer patients worldwide. Evidence has emerged that PC SPES is an effective modality that alleviates some symptoms in advanced prostate cancer in a significant proportion of patients including the cases that failed conventional therapy. This evidence as well as published data that show the effects of PC SPES in suppressing growth of prostate cancer in animal model studies, is reviewed together with the available data on the possible side effects of PC SPES. The review also covers in vitro studies that reveal the cell cycle specificity, induction of apoptosis, effects on androgen receptor and other molecular and metabolic changes induced by PC SPES that may explain its anticancer activity. Individual chemical components that are present in herbs of which PC SPES is composed, known to have anti-proliferative, antitumor, antimutagenic, analgesic and/or differentiation inducing activity, are listed and discussed. The effectiveness of PC SPES in prostate cancer is explained as due to its complex composition which may target many signal transduction and metabolic pathways simultaneously thereby eliminating the back-up or redundant mechanisms that otherwise promote cell survival when single-target agents are used.

Increased serum mitogenic activity for arterial smooth muscle cells associated with relaxation and low educational level in human subjects with high but not low hostility traits: implications for atherogenesis.

Proliferation of arterial smooth muscle cells (aSMC) is a key component of atherogenesis. A sample of 225 volunteers, aged 21-65 years, was exposed to "frustration," "harassment," or "relaxation," after completing the 50-item Hostility subscale of the Minnesota Multiphasic Personality Inventory (MMPI). Whole blood was measured before and after exposure for platelet-derived growth factor (PDGF), and sera were evaluated for total and HDL cholesterol concentrations and PDGF-independent mitogenic activity (SMA). Blood pressure and pulse rate were also evaluated. Analyses of SMA (i.e., serum independent of PDGF) revealed an increase in mitogenic effect for cultured human aSMC when hostility was treated as a dichotomous modifier. Among high-hostility subjects, surprisingly, those in the relaxed group and those with a lower educational level were found to have a significant mitogen response; no significant effects were observed for the low-hostility groups. The data suggest that endogenous stresses may occur in high-hostility individuals when "relaxed," to influence proliferation of arterial smooth muscle cells, as a contribution to atherogenesis. In individuals with lower educational levels and higher hostility scores, lifestyle changes may play a role.

A case-control study of the relationship between dietary factors and risk of lung cancer in women of Shenyang, China.

A 1:1 case-control study of lung cancer incidence among women in Shenyang was conducted to explore the relationship between diet and the risk of lung cancer, with emphasis on the potential effects of a list of specific dietary constituents on modifying lung cancer risks. Dietary information on 290 cases and population-matched controls was obtained by personal interviews. Multiple logistic regression models were used for the statistical analysis and summarization of the data. A significant difference was found between cases and controls with respect to intake of beta-carotene, vitamin C and fibers, all of which reduced the risk for lung cancer in a dose-dependent manner; yielding calculated odds ratio (OR) of 0.84, 0.75, and 0.46, respectively. The apparent effects of these nutrients persisted after adjusting for cigarette smoking;suggesting that they may function as protective factors to reduce the risk for lung cancer in Chinese women.

Induction of apoptosis and down-regulation of bcl-6 in mutu I cells treated with ethanolic extracts of the Chinese herbal supplement PC-SPES.

PC-SPES, an HPLC-standardized 8-herb dietary supplement prepared by proprietary extraction/mixing technologies, appears to have a number of health benefits when given to cancer patients. These include reduction of serum PSA (prostate specific antigen) in individuals diagnosed with advanced prostate carcinoma, and overall improvement of morbidity and immune status in terminal cancer cases. Since the expression of bcl-6 in T and B lymphocytes has been reported to be significantly down regulated by mitogens, we reason that the immune boosting effects of PC-SPES could involve the modulation of bcl-6 expression. Such a hypothesis was tested in the bcl-6 abundant Mutu I cells. Specifically, we investigated the effects of PC-SPES in regulating cell growth, induction of apoptosis, effecting changes in the retinoblastoma gene RB and the modulation of expression of the bcl-6. Herein we report that proliferation of Mutu I cells was inhibited by a 3-7 day incubation with ethanolic extracts of PC-SPES, with concurrent induction of apoptosis. In addition, a dose-dependent reduction of bcl-6 was observed, with no concomitant change in either the phosphorylated or the unphosphorylated forms of RB. These data raise the possibility that PC-SPES may enhance immune functions in vivo by down-regulating bcl-6 expression. Alternatively, decrease in bcl-6 could serve as a biomarker for testing the efficaciousness of PC-SPES in vivo.

Regulation of androgen receptor (AR) and prostate specific antigen (PSA) expression in the androgen-responsive human prostate LNCaP cells by ethanolic extracts of the Chinese herbal preparation, PC-SPES.

As part of the study on the potential use of natural product-based combination therapy for treating prostate cancer, we have investigated the effects of a "HPLC standardized" herbal preparation, PC-SPES, on the prostate LNCaP cell line. Proliferation of the LNCaP cells was inhibited by a 4-6 day incubation with ethanolic extracts of PC-SPES. Decrease of cell growth was accompanied by a 60-70% down-regulation of the proliferating cell nuclear antigen (PCNA) and level of secreted PSA. A smaller and more variable decrease (20-40%) in the level of intracellular PSA was also observed. The PC-SPES-modulated PSA changes occurred concurrently with the decrease of AR expression, based on Western blot analysis and binding to the radioactive ligand [3H]R1881. A 60% decrease in R1881 binding occurred after a 24 h incubation with PC-SPES. These results suggest that PC-SPES negatively affects cell growth in part through its ability to modulate changes in PCNA, and may decrease PSA levels indirectly by suppressing AR expression.

Discovery of high affinity bombesin receptor subtype 3 agonists.

Human bombesin receptor subtype 3 (BRS-3) was cloned based on its homology to the human gastrin-releasing peptide (GRP) receptor and neuromedin B (NMB) receptor. Some bombesin-like peptides were shown to activate BRS-3 expressed in Xenopus laevis oocytes, but only at relatively high concentrations, which suggests that BRS-3 is an orphan receptor. To study the pharmacology of BRS-3 in the context of a mammalian cell, we used BR2 cells, which are Balb/3T3 fibroblasts transfected with BRS-3 cDNA. A number of bombesin-like peptides found in mammals and amphibians stimulated calcium mobilization in BR2 cells but exhibited no effect on nontransfected parental Balb/3T3 cells. Of these peptides, NMB (EC50 approximately 1-10 microM) was the most active for stimulation of calcium mobilization. Testing of a series of NMB analogs truncated at the amino terminus and carboxyl terminus indicated that the minimal size of NMB required for retention of full activity was Ac-NMB(3-10). Systematically replacing each residue with alanine, or changing its chirality, demonstrated that the carboxyl-terminal residues His8, Phe9, and Met10 of NMB are important for optimal activity. We also tested whether a number of bombesin (BN) analogs that are potent pure or partial antagonists of the GRP receptor can activate BRS-3 in BR2 cells. One such analog, D-Phe6-BN(6-13) propyl amide, activated BRS-3-mediated calcium mobilization with an EC50 level of 84 nM. Through additional synthesis, we generated a significantly more potent analog, D-Phe6-Phe13-BN(6-13) propyl amide, which displayed an EC50 level of 5 nM for activation of BRS-3. Taken together, our data show that the core portions of bombesin-like peptides required for activation of BRS-3 are similar to those necessary for activation of the GRP and NMB receptors and thus provide pharmacological evidence that BRS-3 is in the BN receptor family. Furthermore, we have identified an agonist of BRS-3, namely D-Phe6-Phe13-BN(6-13) propyl amide, which is roughly 1000-fold more potent than BRS-3 agonists described previously.

Changes in cell growth, cyclin/kinase, endogenous phosphoproteins and nm23 gene expression in human prostatic JCA-1 cells treated with modified citrus pectin.

Modified citrus pectin (MCP) added to the media of cultured androgen-independent human prostatic JCA-1 cells reduced cell growth and correspondingly [3H]thymidine incorporation into DNA, which was correlated with the down-regulation of cyclin B and p34cdc2 MCP also induced distinct increases in specific endogenous phosphoproteins, including a cAMP-stimulated 52,000 (52-kDa) protein. Since metastatis has been inversely correlated with nm23 gene expression in some cancer cells and was reportedly inhibited by MCP in a rat prostate model, we investigated steady state level changes in the nm23 protein in JCA-1 cells and found it to be unexpectedly suppressed as a result of exposure to MCP.

Growth retardation in senescent arterial smooth muscle cells and its reversal following brain stimulation: implications for atherogenesis.

Advancing age and psychosocial stress are each associated with a rising incidence of atherosclerosis. In this investigation we attempted to answer the question of whether they are independent of each other or not. Since a key feature of atherosclerosis is the proliferation of arterial smooth muscle cells (ASMC), we transplanted aortic tissue from aged rats, half of which had received hypothalamic stimulation, as a model for stress, to growth supporting medium, immediately after stimulation and observed their growth behavior for a period of 4 months. Similar observations were carried out on young animals for comparison. Although there was little difference in outgrowth frequency of explants from young animals between stimulated and non-stimulated subjects, in the case of the older rats, explants from animals which were not stimulated were considerably retarded in their growth, whereas those from subjects which had received HS, grew as well as those of the younger ones. These results show that HS can reverse the growth decline in aortic tissues explanted from senescent rats. They also suggest that age per se is not atherogenic in terms of proliferative behavior of ASMC, whereas when interacted with a stressful condition, this may be the case. Since in the elderly there is a decreased tolerance to stress, the 'atherogenic' effects of age in these individuals may be mediated through the stress response.

Age differences in the proliferative response of cultured arterial smooth muscle cells induced by sera of hypothalamically-stimulated rats.

In an earlier report, it was shown that arterial smooth muscle cells (ASMC) cultured from normal rat aorta, proliferated in response to homologous sera from young rats which had received hypothalamic stimulation (HS), in contrast to the effect of sera from non-stimulated age-matched controls (sham-operated). In the present study, this proliferative response was compared in young and old rats. Isolated target cells were subcultured from primary explants of aortic tissue obtained from young, male, Fischer 344 rats, which were electrode-implanted in the hypothalamus but not stimulated. After an initial quiescence period (growth arrest), target cells were exposed to plasma derived serum (PDS) from 4 experimental groups: young stimulated rats; young sham-operated controls; aged stimulated rats and aged sham-operated controls, at PDS concentrations of 2.5% and 5.0% and counted at days 2 and 5. Proliferative responses of ASMC were found to be influenced by concentration of the PDS, age of the donor animal contributing the serum and the presence of HS. The greatest responses were observed in relation to sera derived from aged stimulated rats, especially at the higher concentration, suggesting an interaction of HS with advanced age. These results are discussed in reference to their possible bearing on the pathogenesis of atherosclerosis.

Some properties of 2-5A binding/nucleolytic activities in gel filtered rabbit reticulocyte lysates.

Rabbit reticulocyte lysates, gel filtered on Sephadex G-25 with or without ATP (or its analogs), were preincubated at 37 degrees C and their subsequent binding to p3A4,3'-[32P]pCp was studied. Lysates filtered without ATP or in the presence of 0.1 mM 8-bromo-ATP, 1,N6-etheno-ATP, or ITP showed a time-dependent decrease in binding activity. This decrease was completely prevented when lysates were filtered with 0.1 mM ATP, 2'-deoxy-ATP, beta-gamma-methylene-ATP, or ATP-gamma-S. The stability of binding provided by ATP or 2'-deoxy-ATP analogs corresponds to a more active 2-5A dependent endonucleolytic (RNAase L) activity based on studies using [3H] viral mRNA. Chromatography on heparin-agarose showed that ATP-supplemented gel-filtered reticulocyte lysates had a different p3A4,3'-[32P]pCp binding activity elution-profile than lysates gel-filtered in the absence of ATP. Covalent cross-linking of periodate-oxidized p3A4,3'-[32P]pC to gel-filtered lysates, preincubated at 0 degree C or 37 degrees C for 30 min, showed the following results: all lysates gave a major cross-linking of the radioactive ligand to an 80 000 dalton polypeptide, regardless of the temperature of preincubation, lysates gel-filtered without ATP, with 0.1 mM ITP, or beta-gamma-methylene-ATP, showed a significant reduction in the cross-linking of the 80 000 dalton protein, after preincubation at 37 degrees C for 30 min. This decrease was accompanied by an increase in the labeling of two smaller polypeptides.