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PubMed2000

Kinetics of UV light-induced cyclobutane pyrimidine dimers in human skin in vivo: an immunohistochemical analysis of both epidermis and dermis.

It is well known that UV exposure of human skin induces DNA damage, and the cumulative effect of such repeated damage is an important contributor to the development of skin cancer. Here, we demonstrate UV dose- and time-dependent induction of DNA damage in the form of cyclobutane pyrimidine dimers (CPD) in skin cells following a single exposure of human skin to UV radiation. CPD+ cells were identified by an immunohistochemical technique using monoclonal antibodies to thymine dimers. The percentage of CPD+ cells was UV dose-dependent, even a suberythemal (0.5 minimal erythemal dose [MED]) dose resulted in detectable level of cells that contained pyrimidine dimers. Forty-eight hours after irradiation the percent of total epidermal cells positive for CPD ranged from 19 +/- 8, 36 +/- 10, 57 +/- 12 and 80 +/- 10, and total percent dermal cells positive for CPD ranged from 1 +/- 1, 7 +/- 3, 16 +/- 3 and 20 +/- 5, respectively, following 0.5, 1.0, 2.0 and 4.0 MED. CPD were also observed in deeper reticular dermis, which suggest the penetrating ability of UV radiation into the skin. The change in CPD+ cells from 0.5 to 240 h post-UV exposure in both epidermal and dermal compartments of the skin was also quantitated. CPD+ cells were observed in skin biopsies at early time points after UV exposure which remained elevated for 48 h, then declined significantly by 3 days post-UV. A close examination of the skin at and after 3 days following UV exposure indicates the significant removal of DNA damaged cells from the epidermis. Ten days after UV exposure the levels of CPD+ cells in both epidermis and dermis were not significantly different from that in unirradiated skin.

Immunohistochemistry↗
PubMed2002

Ultraviolet-B-induced cyclobutane-pyrimidine dimer formation and repair in Arctic marine macrophytes.

The significance of ultraviolet-B radiation (UVBR: 280-315 nm)-induced DNA damage as a stress factor for Arctic marine macrophytes was examined in the Kongsfjord (Spitsbergen, 78 degrees 55.5'N, 11 degrees 56.0'E) in summer. UVBR penetration in the water column was monitored as accumulation of cyclobutane-pyrimidine dimers (CPD) in bare DNA. This showed that UVBR transparency of the fjord was variable, with 1% depths ranging between 4 and 8 m. In addition, induction and repair kinetics of CPD were studied in several subtidal macrophytes obtained from the Kongsfjord (5-15 m). Surface exposure experiments demonstrated CPD accumulation in Palmaria palmata, Devaleraea ramentacea, Phycodrys rubens, Coccotylus truncatus and Odonthalia dentata. In artificial light, field collected material of P. palmata, D. ramentacea, P. rubens and Laminaria saccharina showed efficient CPD repair, with only 10% of the artificially induced CPD remaining after 5 h. No significant differences in repair rate were observed among these species. CPD repair was slower or absent in O. dentata, C. truncatus and Monostroma arcticum, indicating that fast repair mechanisms such as photolyase were not continuously expressed in these species. CPD repair rates were not directly related to the vertical distribution of algae in the water column and to the reported UV sensitivity of the examined species. Dosimeter incubations showed that maximal exposure to DNA damaging wavelengths was low for all examined species. Furthermore, most species collected below the 1% depth for DNA damage displayed efficient CPD repair, suggesting that UVBR-induced CPD currently impose a minor threat for mature stages of these species growing in the Kongsfjord, Spitsbergen.

Arctic Regions↗
PubMed2000

Blunt chest trauma in the elderly patient: how cardiopulmonary disease affects outcome.

Blunt trauma patients with rib fractures have significant risk of morbidity and mortality. The risk of complications increases with age and cardiopulmonary disease. We reviewed our experience at a community hospital Level II trauma center over a 5-year period. A review of the trauma registry revealed 62 patients over the age of 65 with multiple rib fractures and no associated injuries. Thirty-one patients with cardiopulmonary disease (CPD+) were compared with 31 patients without cardiopulmonary disease (CPD-). Charts were reviewed for morbidity, mortality, the need to upgrade level of care (readmission to the hospital or intensive care unit), and length of hospitalization. Complications occurred in 17 of 31 CPD+ patients and in four of 31 CPD- patients (P < 0.001). The only three deaths were in CPD+ patients. Ten CPD+ patients and four CPD- patients required an upgrade in the level of care (P < 0.05). The CPD+ patients had longer hospitalization than the CPD- patients: 8.5 versus 4.3 days (P < 0.05). We conclude that elderly patients with multiple rib fractures and cardiopulmonary disease are at significant risk for complications that result in readmission to the hospital and intensive care unit and prolonged length of hospitalization. Admission to the intensive care unit with attention to cardiac and pulmonary status upon transfer to the ward is warranted.

Age Factors↗
PubMed1992

Restoration of antioxidants in liver by methionine feeding in experimental rat urolithiasis.

The effect of methionine or citrate on antioxidant defense system has been studied in urolithic rat. Liver weight and its protein concentration did not change in the rats fed with calculi producing diet (CPD) when compared to normal diet fed rats. Feeding rats along with citrate (c-CPD) or methionine (m-CPD) improved their body weight gain. Liver microsomes and mitochondria fractions of CPD and c-CPD fed groups showed increased susceptibility for lipid peroxidation in presence of ascorbate and t-butyl hydroperoxide when compared to either control or m-CPD fed groups. Increased superoxide dismutase and xanthine oxidase activities, decreased catalase, glutathione peroxidase and glucose-6-phosphate dehydrogenase activities, decreased concentrations of reduced glutathione, total thiols, ascorbic acid and vitamin-E and increased formation of hydroxyl radical, hydroperoxides and diene conjugates were observed in the liver of both CPD fed group as well as c-CPD fed group. Except SOD and xanthine oxidase, all other parameters were normalized in m-CPD fed group. This suggested that feeding methionine reduced the susceptibility for lipid peroxidation by restoration of the level of free radical scavengers.

Animals↗
PubMed1981

Transplantation of cadaveric paediatric donor kidneys into adult recipients.

To assess the justification for the use of cadaveric paediatric donor (CPD) kidneys in adult recipients, the results of transplantation of 50 CPD kidneys into adult recipients (CPD group) were compared to the outcome of 250 transplantations of adult cadaveric donor kidneys into adult recipients (ACD group). No differences in graft and patient survival were observed between both groups. The functional capacity of the kidneys and the incidence of surgical and urological complications was not different in both groups. Hypertension was observed more frequently in recipients of CPD kidneys compared to recipients of ACD kidneys (p less than 0.025). The occurrence of hypertension was related to the surgical technique used for the arterial anastomosis. When no arterial patches were available hypertension occurred more frequently in the CPD group (9/9 cases) compared to the ACD group (31/70 cases) (p less than 0.01). In recipients of CPD kidneys the incidence of hypertension was lower then arterial patches were used (11/19 cases) compared to recipients of CPD kidneys where no patches were used (9/9) (p less than 0.05). The results indicate that the use of CPD kidneys in adult recipients is justified. However, to diminish the occurrence of hypertension, donor nephrectomy of CPD kidneys should take place according to the so-called en bloc technique, so that arterial patches can be obtained.

Adolescent↗
PubMed1994

Analysis of repair of cyclobutane pyrimidine dimers and pyrimidine 6-4 pyrimidone photoproducts in transcriptionally active and inactive genes in Chinese hamster cells.

Irradiation of cells with short wave ultraviolet light (UV-C) induces both cyclobutane pyrimidine dimers (CPD) as well as pyrimidine 6-4 pyrimidone photoproducts (6-4 PP). We have focused on the removal of both types of DNA photolesions from the transcriptionally active adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HPRT) genes and the inactive c-mos gene. Induction levels of both CPD and 6-4 PP were similar for all three genes analyzed, with the induction of 6-4 PP being about 3-fold lower than of CPD. Repair of CPD was analyzed using the CPD-specific enzyme T4 endonuclease V; repair of 6-4 PP was examined employing Escherichia coli UvrABC excinuclease. Unlike the HPRT gene, in which CPD were removed selectively from the transcribed strand, both strands of the 16-kilobase fragment encompassing the 2.6-kilobase APRT gene were repaired efficiently. This suggests the existence of multiple transcription units in the APRT region including transcription units running in the opposite direction of the APRT gene. Only a marginal part of the CPD was removed from the inactive c-mos gene after 24 h. In all three genes investigated, 6-4 PP were repaired more rapidly than CPD and, as demonstrated for the HPRT and APRT genes, without strand specificity. The difference in the repair phenotype of CPD between the HPRT gene and the APRT gene coincides with differences between both genes with regard to the DNA strand distribution of previously published UV-induced mutations.

Adenine Phosphoribosyltransferase↗
PubMed1994

Stimulation of growth by recombinant human growth hormone in children undergoing peritoneal or hemodialysis treatment. German Study Group for Growth Hormone Treatment in Chronic Renal Failure.

Fourteen patients on hemodialysis (HD) and 17 patients on continuous peritoneal dialysis (CPD) were treated with 28-30 IU/m2/week of recombinant human growth hormone (rhGH) for at least 12 months. The HD and CPD patient groups were comparable with regard to age, bone age, height standard deviation scores (SDS), and height velocity at start of treatment. During the first year of rhGH treatment, height velocity increased from 2.9 +/- 1.9 to 5.5 +/- 1.7 cm/year in the HD group and from 3.0 +/- 2.2 to 7.2 +/- 3.2 cm/year in the CPD group. The increase in growth rate was significant in both groups (p < 0.001), and tended to be significantly greater in the CPD than in the HD group (p = 0.09). The marked acceleration of growth under rhGH treatment resulted in an increase in relative height by 0.37 +/- 0.38 SDS in the HD group and by 0.47 +/- 0.69 SDS in the CPD group during the first treatment year. Seven HD and 7 CPD patients completed a second year of rhGH treatment. In these patients, height velocity dropped to 3.6 +/- 2.7 cm/year (HD) and 3.6 +/- 2.3 cm/year (CPD), respectively during the second treatment year. We conclude that rhGH treatment accelerates growth in children both on HD and CPD. The treatment response tends to be greater in CPD compared to HD patients. The efficacy of treatment decreases considerably during the second treatment year.

Adolescent↗
PubMed2002

Transient and sustained ERK phosphorylation and nuclear translocation in growth control.

Growth stimulation and inhibition are both associated with tyrosine phosphorylation. We examined the effects of epidermal growth factor (EGF), a growth stimulant, and compound 5 (Cpd 5), a protein-tyrosine phosphatase (PTPase) inhibitor, which inhibits the growth of the same Hep3B hepatoma cells. We found that both EGF and Cpd 5 induced tyrosine phosphorylation of EGF receptor (EGFR) and ERK. However, the phosphorylation caused by EGF was transient and that caused by Cpd 5 was prolonged. Furthermore, Cpd 5 action caused a strong nuclear phospho-ERK signal and induced phospho-Elk-1, a nuclear target of ERK activation, in contrast to the weak effects of EGF. An ERK kinase assay demonstrated that ERK activated by Cpd 5 could phosphorylate its physiological substrate, Elk-1. The MEK inhibitors PD098056 and U0126 abrogated both the induction by Cpd 5 of phospho-ERK, its nuclear translocation and phospho-Elk-1 and also antagonized its growth inhibitory effects. Furthermore, phospho-ERK phosphatase and phospho-Elk-1 activities were lost from nuclear extracts from Cpd 5 treated, but not EGF treated cells. In conclusion, the data show that Cpd 5 causes growth inhibition as a consequence of prolonged ERK and Elk-1 phosphorylation, likely a result of inhibition of multiple PTPases, including those acting on phospho-EGFR, on phospho-ERK, and on phospho-Elk-1, in contrast to the kinase driven transient activation resulting from EGF.

Active Transport, Cell Nucleus↗
PubMed2004

On prediction of the cesarean delivery risk in a large private practice.

OBJECTIVE: This study was undertaken to develop an easily usable integrated formula for predicting the probability of cephalopelvic disproportion/failure to progress (CPD) and cesarean delivery (CS) as a function of demographic factors in a middle-class private practice, and to evaluate risk factors for CS in a low-risk primiparous population. METHODS: We studied 3355 primiparous women who delivered singleton births between February 1993 and July 2001 in a large private practice. We calculated body mass index (BMI) and weight gain during pregnancy by using clinical data from a comprehensive clinical database. Multivariable logistic regression analysis was used to estimate the relationship between the probability of CS and CS carried out for CPD and 6 demographic factors: maternal age, maternal height, initial pregnancy BMI, pregnancy weight gain, gestational age, and birth weight. Three methods were used to assess the accuracy of the model: the Hosmer-Lemeshow goodness-of-fit statistic, association of predicted probabilities, and direct comparison of the formula to the actual data. Odds ratios with a 95% CI are also calculated for each of these factors. RESULTS: The overall rate of primiparous CS for the practice is 21.7%, with 11.7% carried out for CPD. Formulas were developed to predict the probability of CS and the probability of CS caused by CPD. Our analysis shows that both the risk of CS and the risk of CS caused by CPD are significantly associated with all 6 demographic factors. We also develop an easily usable Web page-based calculator to instantly estimate any woman's probability of a CS or CPD at the beginning or at the end of her pregnancy. CONCLUSION: The probability of CS and CS performed for CPD is higher for shorter, older, more obese women with large pregnancy weight gains, larger fetal birth weights, and longer gestation ages. Accurate formulas that predict the probabilities of CS and CS performed for CPD in this large private practice have been developed.

Adult↗
PubMed2004

Backward masking and the central performance drop.

Kehrer [Spatial Vision 2 (1987) 247] found that texture discrimination performance sometimes peaks in the parafovea rather than at the fovea, and he referred to this phenomenon as the central performance drop (CPD). Kehrer used a backward mask to limit performance and Morikawa [Vision Res. 40 (2000) 3517] argued that in some cases the temporal aspects of the backward mask may be critical to the emergence of the CPD. In one experiment Morikawa showed that the CPD does not emerge when a simultaneous noise-mask (different from the mask used by Kehrer) is used to limit performance. In another experiment Morikawa showed that unmasked texture displays comprising short lines do not elicit the CPD. In both cases, changes in the temporal aspects of the texture displays were accompanied by changes in the spatial structure of the mask or stimulus. For the spatio-temporal theory of the CPD to be sustained one would have to show that noise masks elicit a CPD when used as backward masks and that the short-line textures elicit a CPD when followed by backward masks. Our evidence provides little if any support for either of these predictions. Furthermore, an analysis of a simple filter-rectify-filter model of texture segmentation shows that a greatly attenuated CPD is to be expected when a noise mask is used as a source of spatial noise.

Adult↗
PubMed2003

Differential effects of two growth inhibitory K vitamin analogs on cell cycle regulating proteins in human hepatoma cells.

A comparison was made between two K vitamin analogs. Growth in vitro of Hep G2 hepatoma cells was inhibited both by Compound 5 (Cpd 5), a recently synthesized thioalkyl analog of vitamin K or 2-(2-mercaptoethanol)-3-methyl-1, 4-naphthoquinone, as well as by synthetic vitamin K3 (menadione). Using synchronized Hep G2 hepatoma cells, the actions of both Cpd 5 and vitamin K3 on cell cycle regulating proteins were examined. Cpd 5 decreased the levels of cyclin D1, Cdk4, p16, p21 and cyclin B1. By contrast, VK3 only decreased the level of cyclin D1, but had no effect on the levels of Cdk4, p16 or p21. Interestingly, both VK3 and VK2 increased the levels of p21. The naturally occurring K vitamins had little effect on cell growth and none on the cyclins or Cdks. Amounts and activity of the G1/S phase controlling Cdc25A were measured. We found that Cpd 5 directly inhibited both Cdc25A activity and its protein expression, whereas VK3 did not. Thus, the main effects of Cpd 5 were on G1 and S phase proteins, especially Cdk4 and Cdc25A amounts in contrast to VK3. Computer docking studies of Cpd 5 and VK3 to Cdc25A phosphatase showed three binding sites. In the best conformation, Cpd 5 was found to be closer to the enzyme active site than VK3. These findings show that Cpd 5 represents a new class of anticancer agent, being a protein tyrosine phosphatase (PTP) antagonist, that binds to Cdc25A with suppression of its activity. Tumors expressing high levels of oncogenic Cdc25A phosphatase may thus be susceptible to the growth inhibitory activities of this class of compound.

Blotting, Western↗
PubMed2002

Induction of apoptosis via mitogen-activated protein kinase pathway by a K vitamin analog in rat hepatocytes.

BACKGROUND/AIMS: Compound 5 (Cpd 5), a vitamin K analog, inhibits rat hepatocyte DNA synthesis and hepatoma cell growth. The aim of this study was to determine if the inhibitory effect of Cpd 5 on cell growth was related to apoptosis. METHODS: Isolated rat hepatocytes were cultured with Cpd 5, and mitogen-activated signaling pathway and apoptosis pathway were investigated using Western blot analysis. RESULTS: When rat hepatocytes were cultured with Cpd 5 for 48 h, apoptosis was evident, which included characteristic morphological changes, DNA fragmentation, and the activation of caspase 3 (CPP 32)-like protease. Examination of upstream events of apoptosis pathway showed that the expression of Bax was induced and bcl-2 was inhibited by Cpd 5 treatment. Concomitant with the induction of apoptosis, Cpd 5 activated the extracellular signal-regulated kinase (ERK) signaling pathway. PD 98059, a mitogen-activated protein kinase kinase inhibitor, and glutathione, an anti-thiol-oxidant, not only blocked Cpd 5-induced ERK phosphorylation, but also antagonized the activation of CPP-32, the altered Bcl-2/Bax expression, and DNA fragmentation. CONCLUSIONS: The data suggest that the ERK signaling pathway may be involved in the regulation of rat hepatocyte apoptosis induced by Cpd 5.

Animals↗
PubMed2000

Patient assessment of quality of care in a chronic peritoneal dialysis facility.

The percentage of patients with end-stage renal disease (ESRD) maintained on chronic peritoneal dialysis (CPD) in the United States remains well less than the percentage in several other countries. Furthermore, there has recently been a decline in the percentage of patients with ESRD in the United States undergoing CPD. The reasons for this decline are uncertain, and investigators have implicated problems with the kinetics of peritoneal dialysis, peritonitis and exit-site infections, and psychosocial stresses imposed by the therapy. Few studies, however, have considered the role of the dialysis facility itself and patient perceptions of the facility as contributing to problems with the long-term acceptance of CPD. This study is designed to examine patients' perceptions of the organization and structure of the peritoneal dialysis facility and their interactions with the facility, focusing attention on areas of patient satisfaction and dissatisfaction with the facility. The study was conducted in a large, freestanding peritoneal dialysis program in an urban area that currently treats 140 patients undergoing CPD. Thirty patients were randomly selected to participate in the present study. A structured interview that included open-ended questions was administered and tape-recorded by a trained interviewer not affiliated with the dialysis unit. Patient responses were then reviewed by two investigators, and a taxonomy of patient satisfaction and dissatisfaction was developed, using a modification of the classification proposed by Concato and Feinstein. Patient responses were then categorized according to the taxonomy. The most frequently cited areas of patient satisfaction included the amount of information and instruction provided by the staff (n = 30), personal atmosphere of the facility (n = 30), efficiency of delivery of the dialysis supplies (n = 23), and availability of the primary nurse (n = 18). The importance of the nurse-patient interaction was emphasized by all 30 patients, whereas the physician-patient interaction was cited by only 14 patients. The most frequently cited area of dissatisfaction noted by all 30 patients concerned the dialysis regimen itself. The present study focuses attention on patient perceptions of their CPD facility, identifying areas of satisfaction and dissatisfaction. The analysis is important not only in providing a framework for CPD facilities with which to review their own interactions with CPD patients, but also for identifying those areas that require attention to maintain the long-term viability of CPD therapy.

Adult↗
PubMed1999

Selection of virulent isolates of entomopathogenic hyphomycetes against Clavigralla tomentosicollis Stål. and evaluation in cage experiment using three cowpea varieties.

The virulence of 8 isolates of entomopathogenic hyphomycetes against adult and 5th instar nymph of Clavigralla tomentosicollis was evaluated in the laboratory at 4 different concentrations of inoculum. At all concentrations, Beauveria bassiana CPD 9 and Metarhizium anisopliae CPD 5 caused the highest mortality in adult bug ranging from 58 to 97% and 53 to 100%, respectively at 7 days post inoculation. The same isolates had the shortest LT50 (3.5 and 4.1 days, respectively) and the lowest LC50 (1.8 x 10(5) and 9.8 x 10(4) conidia ml-1) values in adult insects. In nymphs, M. anisopliae CPD 5 was the most virulent isolate causing mortality of between 43 to 92% with the shortest LT50 of 2.7 days and the lowest LC50 of 4.6 x 10(5) conidia ml-1 which however did not differ significant from LC50 observed in B. bassiana CPD 9 isolate at 5 days post inoculation. A significant reduction in feeding in both developmental stages treated with fungi was observed at 2 days after treatment with the greatest reduction occurring in insects treated with B. bassiana CPD 9 and M. anisoplia CPD 5. In adult insects treated with these isolates, some bugs ceased feeding 24 h before death. When these two isolates were compared in caged experiment with an untreated control using a susceptible, tolerant and moderately resistant variety of cowpea, percentage pod and seed damage were significantly lower in fungal treated cages than in the control cages on all varieties tested. Grain yield per plant was also significantly higher in fungal treated cages than in the control cages on all varieties. The performance of M. anisopliae CPD 5 was however superior to B. bassiana CPD 9. Application of the fungi on moderately resistant variety of cowpea was found to enhance the performance of the pathogen.

Heteroptera↗
PubMed2003

Activation and proliferation signals in primary human T lymphocytes inhibited by ergosterol peroxide isolated from Cordyceps cicadae.

Effects of ergosterol peroxide (C28H44O3; Cpd 6A) from Cordyceps cicadae on phytohemagglutinin (PHA)-stimulated cell proliferation were studied in primary human T cells. The results showed that Cpd 6A suppressed T-cell proliferation for about 24 h after stimulation with PHA. Cell cycle analysis indicated that Cpd 6A arrested the cell cycle progression of activated T cells from the G1 transition to the S phase. To localize the point in the cell cycle where arrest occurred, a set of key regulatory events leading to the G1/S boundary, including the expression of cyclins D2, E, A1, and B1, interleukin (IL)-2, IL-4, interferon-gamma (IFN-gamma), and activating protein-1 (AP-1), was examined. Cpd 6A suppressed, in activated T lymphocytes, the production and mRNA expression of cyclin E, IL-2, IL-4, IL-10, and IFN-gamma in a dose-dependent manner. Expression of AP-1 proteins, consisting of c-Fos and c-Jun, in activated T lymphocytes was decreased by Cpd 6A. The kinetic study indicated that the inhibitory effects of Cpd 6A on IL-2 mRNA expressed in T cells might be related to blocking c-Fos protein synthesis. T-cell proliferation after Cpd 6A treatment was partially restored by addition of IL-2, IL-4, and IFN-gamma. These suppressant effects of Cpd 6A on T-cell proliferation, activated by PHA, appeared to be mediated, at least in part, through the inhibition of early gene transcripts, especially those of cyclin E, IFN-gamma, IL-2, and IL-4, and by arresting cell cycle progression in the cells.

Adult↗
PubMed2024

Human parainfluenza virus 3 vaccine candidates attenuated by codon-pair deoptimization are immunogenic and protective in hamsters.

Human parainfluenza virus type 3 (HPIV3) is a major pediatric respiratory pathogen lacking available vaccines or antiviral drugs. We generated live-attenuated HPIV3 vaccine candidates by codon-pair deoptimization (CPD). HPIV3 open reading frames (ORFs) encoding the nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and polymerase (L) were modified singly or in combination to generate 12 viruses designated Min-N, Min-P, Min-M, Min-FHN, Min-L, Min-NP, Min-NPM, Min-NPL, Min-PM, Min-PFHN, Min-MFHN, and Min-PMFHN. CPD of N or L severely reduced growth in vitro and was not further evaluated. CPD of P or M was associated with increased and decreased interferon (IFN) response in vitro, respectively, but had little effect on virus replication. In Vero cells, CPD of F and HN delayed virus replication, but final titers were comparable to wild-type (wt) HPIV3. In human lung epithelial A549 cells, CPD F and HN induced a stronger IFN response, viral titers were reduced 100-fold, and the expression of F and HN proteins was significantly reduced without affecting N or P or the relative packaging of proteins into virions. Following intranasal infection in hamsters, replication in the nasal turbinates and lungs tended to be the most reduced for viruses bearing CPD F and HN, with maximum reductions of approximately 10-fold. Despite decreased in vivo replication (and lower expression of CPD F and HN in vitro), all viruses induced titers of serum HPIV3-neutralizing antibodies similar to wt and provided complete protection against HPIV3 challenge. In summary, CPD of HPIV3 yielded promising vaccine candidates suitable for further development.

Animals↗
PubMed1999

Biosynthesis and packaging of carboxypeptidase D into nascent secretory vesicles in pituitary cell lines.

Metallocarboxypeptidase D (CPD) is a membrane-bound trans-Golgi network (TGN) protein. In AtT-20 cells, CPD is initially produced as a 170-kDa endoglycosidase H-sensitive glycoprotein. Within 30 min of chase, the CPD increases to 180 kDa and is resistant to endoglycosidase H as a result of carbohydrate maturation. CPD also undergoes an activation step required for binding to a substrate affinity resin. Blocking the protein exit from the endoplasmic reticulum inhibits the increase in molecular mass but not the step required for affinity column binding, suggesting that enzyme activation precedes carbohydrate maturation and that these reactions occur in distinct intracellular compartments. Only the higher molecular weight mature CPD enters nascent secretory vesicles, which bud from the TGN of permeabilized AtT-20 and GH3 cells. The budding efficiency of CPD into vesicles is 2-3-fold lower than that of endogenous proopiomelanocortin in AtT-20 cells or prolactin in GH3 cells. In contrast, the packaging of a truncated form of CPD, which lacks the cytoplasmic tail and transmembrane domain, was similar to that of proopiomelanocortin. Taken together, the results support the proposal that CPD functions in the TGN in the processing of proteins that transit the secretory pathway and that the C-terminal region plays a major role in TGN retention.

Animals↗
PubMed2003

Persistent ERK phosphorylation negatively regulates cAMP response element-binding protein (CREB) activity via recruitment of CREB-binding protein to pp90RSK.

Compound 5 (Cpd 5) or 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone, is an inhibitor of protein phosphatase Cdc25A and causes persistent activation of extracellular signal-regulated kinase (ERK) and cell growth inhibition. To study the mechanism(s) by which persistent ERK phosphorylation might induce cell growth inhibition, we used Cpd 5 as a tool to examine its effects on the activity of CREB (cAMP response element-binding protein) transcription factor in Hep3B human hepatoma cells. We found that CREB activity, including its DNA binding ability and phosphorylation on residue Ser-133, was strongly inhibited by Cpd 5, followed by suppression of CRE-mediated transcription of cyclin D1 and Bcl-2 genes. Cpd 5-mediated suppression of CREB phosphorylation and transcriptional activity was antagonized by mitogen-activated protein kinase kinase inhibitors PD 98059 and U-0126, implying that this inhibition of CREB activity was regulated at least in part by the ERK pathway. The phosphorylation of ribosomal S6 kinase (pp90(RSK)), a CREB kinase in response to mitogen stimulation, was also found to be inhibited by Cpd 5 action. This inhibition of pp90(RSK) phosphorylation is likely the result of its increased association with CREB-binding protein (CBP), which subsequently caused inhibition of CREB phosphorylation and activity. To support the hypothesis that Cpd 5 effects on Cdc25A inhibition with subsequent ERK activation could cause CREB inhibition, we examined the effects of Cdc25A inhibition without the use of Cpd 5. Hep3B cells were transfected with C430S Cdc25A mutant, and ERK was found to be phosphorylated in a constitutively activated manner, which was accompanied by decreased CREB phosphorylation and increased recruitment of CBP to pp90(RSK). These data provide evidence that CBP.RSK complex formation in response to persistent ERK phosphorylation by Cpd 5 down-regulates CREB activity, leading to inhibition of both cAMP response element-mediated gene expression and cell growth.

CREB-Binding Protein↗