Stimulation Of DNA Synthesis Research Articles

EXPRESSION OF CONCERN: Neurotensin and EGF induce synergistic stimulation of DNA synthesis by increasing the duration of ERK signaling in ductal pancreatic cancer cells.

EXPRESSION OF CONCERN: Neurotensin and EGF induce synergistic stimulation of DNA synthesis by increasing the duration of ERK signaling in ductal pancreatic cancer cells.

Nutraceuticals known to promote hair growth do not interfere with the inhibitory action of tamoxifen in MCF7, T47D and BT483 breast cancer cell lines.

Hair loss/thinning is a common side effect of tamoxifen in estrogen receptor (ER) positive breast cancer therapy. Some nutraceuticals known to promote hair growth are avoided during breast cancer therapy for fear of phytoestrogenic activity. However, not all botanical ingredients have similarities to estrogens, and in fact, no information exists as to the true interaction of these ingredients with tamoxifen. Therefore, this study sought to ascertain the effect of nutraceuticals (+/- estrogen/tamoxifen), on proliferation of breast cancer cells and the relative expression of ERα/β. Kelp, Astaxanthin, Saw Palmetto, Tocotrienols, Maca, Horsetail, Resveratrol, Curcumin and Ashwagandha were assessed on proliferation of MCF7, T47D and BT483 breast cancer cell lines +/- 17β-estradiol and tamoxifen. Each extract was analysed by high performance liquid chromatography (HPLC) prior to use. Cellular ERα and ERβ expression was assessed by qRT-PCR and western blot. Changes in the cellular localisation of ERα:ERβ and their ratio following incubation with the nutraceuticals was confirmed by immunocytochemistry. Estradiol stimulated DNA synthesis in three different breast cancer cell lines: MCF7, T47D and BT483, which was inhibited by tamoxifen; this was mirrored by a specific ERa agonist in T47D and BT483 cells. Overall, nutraceuticals did not interfere with tamoxifen inhibition of estrogen; some even induced further inhibition when combined with tamoxifen. The ERα:ERβ ratio was higher at mRNA and protein level in all cell lines. However, incubation with nutraceuticals induced a shift to higher ERβ expression and a localization of ERs around the nuclear periphery. As ERα is the key driver of estrogen-dependent breast cancer, if nutraceuticals have a higher affinity for ERβ they may offer a protective effect, particularly if they synergize and augment the actions of tamoxifen. Since ERβ is the predominant ER in the hair follicle, further studies confirming whether nutraceuticals can shift the ratio towards ERβ in hair follicle cells would support a role for them in hair growth. Although more research is needed to assess safety and efficacy, this promising data suggests the potential of nutraceuticals as adjuvant therapy for hair loss in breast cancer patients receiving endocrine therapy.

Saccharomyces cerevisiae DNA polymerase IV overcomes Rad51 inhibition of DNA polymerase δ in Rad52-mediated direct-repeat recombination.

Saccharomyces cerevisiae DNA polymerase IV (Pol4) like its homolog, human DNA polymerase lambda (Polλ), is involved in Non-Homologous End-Joining and Microhomology-Mediated Repair. Using genetic analysis, we identified an additional role of Pol4 also in homology-directed DNA repair, specifically in Rad52-dependent/Rad51-independent direct-repeat recombination. Our results reveal that the requirement for Pol4 in repeat recombination was suppressed by the absence of Rad51, suggesting that Pol4 counteracts the Rad51 inhibition of Rad52-mediated repeat recombination events. Using purified proteins and model substrates, we reconstituted in vitro reactions emulating DNA synthesis during direct-repeat recombination and show that Rad51 directly inhibits Polδ DNA synthesis. Interestingly, although Pol4 was not capable of performing extensive DNA synthesis by itself, it aided Polδ in overcoming the DNA synthesis inhibition by Rad51. In addition, Pol4 dependency and stimulation of Polδ DNA synthesis in the presence of Rad51 occurred in reactions containing Rad52 and RPA where DNA strand-annealing was necessary. Mechanistically, yeast Pol4 displaces Rad51 from ssDNA independent of DNA synthesis. Together our in vitro and in vivo data suggest that Rad51 suppresses Rad52-dependent/Rad51-independent direct-repeat recombination by binding to the primer-template and that Rad51 removal by Pol4 is critical for strand-annealing dependent DNA synthesis.

SPOCK1 Overexpression Suggests Poor Prognosis of Ovarian Cancer

Sparc/osteonectin, cwcv, and kazal-like domains proteoglycan 1 (SPOCK1) has been found in a variety of malignant tumors and is associated with a poor prognosis. We aimed to explore the role of SPOCK1 in ovarian cancer. Ovarian cancer cell lines SKOV3 and SW626 were transfected with SPOCK1 overexpressing or empty vector using electroporation. Cells were studied by immunostaining and an automated Western blotting system. BrdU uptake and wound healing assays assessed cell proliferation and migration. SPOCK1 expression in human ovarian cancer tissues and in blood samples were studied by immunostaining and ELISA. Survival of patients with tumors exhibiting low and high SPOCK1 expression was analyzed using online tools. Both transfected cell lines synthesized different SPOCK1 variants; SKOV3 cells also secreted the proteoglycan. SPOCK1 overexpression stimulated DNA synthesis and cell migration involving p21CIP1. Ovarian cancer patients had increased SPOCK1 serum levels compared to healthy controls. Tumor cells of tissues also displayed abundant SPOCK1. Moreover, SPOCK1 levels were higher in untreated ovarian cancer serum and tissue samples and lower in recipients of chemotherapy. According to in silico analyses, high SPOCK1 expression was correlated with shorter survival. Our findings suggest SPOCK1 may be a viable anti-tumor therapeutic target and could be used for monitoring ovarian cancer.

Potent anticancer effects of vitamin C and vitamin C-rich fruit extract (Citrus sinensis L.) on human lung cancer cells

Introduction: Studies have shown that synthetic agents are connected with some complications. This work was designed to study the effects of vitamin C and Citrus sinensis fruit extract (CSFE) on viability, DNA synthesis, and apoptosis stimulation in human lung cancer cells (COR-L105). Methods: The total contents of the phenolics, flavonoids, and ascorbic acid in CSFE were assessed through the Folin-Ciocalteu, aluminum chloride, and dinitrophenyl hydrazine methods, respectively. The cytotoxicity of vitamin C and CSFE on COR-L105 cells was evaluated by the cell viability assay. Measurement of the DNA synthesis was done through the BrdU solution assay. The expression levels of some apoptosis regulatory genes were also evaluated. Results: The total phenolic, flavonoids, and ascorbic acid contents of CSFE were 94.31 ± 2.27 gallic acid equivalents (mg/g) of dry extract, 63.26 ± 2.86 quercetin equivalents (mg/g) of dry extract, and 59 mg/L, respectively. The 50% cytotoxicity concentration (CC50) values of vitamin C and CSFE on cancer cells were 54.6 and 82.7.6 μg/mL, respectively. Vitamin C and CSFE dose-dependently declined the amount of DNA production in the cancer cells. The expression levels of caspase-3 and Bax genes were markedly (P < 0.001) elevated by vitamin C and CSFE, while they reduced the level of Bcl-2 gene (P < 0.05). Conclusion: The findings showed the potent anticancer effects of vitamin C and CSFE against human lung cancer cell lines. DNA synthesis reduction and apoptosis induction can be considered as possible mechanisms of action. However, further surveys are necessary to clarify the accurate mechanism and their efficacy.

Replication stress by MMS stimulates DNA synthesis in post-replicative G2-phase in S. pombe .

DNA replication is generally limited to S-phase but replication stress can drive cells to undergo DNA synthesis outside of S-phase. Mitotic DNA synthesis pathway is known to be activated to deal with replication stress-induced chromosomal instability. There is also growing evidence that residual DNA synthesis can occur in G2. We demonstrate that fission yeast cells stimulate DNA synthesis in G2-phase but not in M-phase in response to DNA alkylating agent MMS. Auxin-induced degradation of DNA replication helicase Mcm4 during G2, but not during mitosis, inhibits post-replicative DNA synthesis.

Conservation of the insert-2 motif confers Rev1 from different species with an ability to disrupt G-quadruplexes and stimulate translesion DNA synthesis.

In some organisms, the replication of G-quadruplex (G4) structures is supported by the Rev1 DNA polymerase. We previously showed that residues in the insert-2 motif of human Rev1 (hRev1) increased the affinity of the enzyme for G4 DNA and mediated suppression of mutagenic replication near G4 motifs. We have now investigated the conservation of G4-selective properties in Rev1 from other species. We compared Rev1 from Danio rerio (zRev1), Saccharomyces cerevisiae (yRev1), and Leishmania donovani (lRev1) with hRev1, including an insert-2 mutant form of hRev1 (E466A/Y470A or EY). We found that zRev1 retained all of the G4-selective prowess of the human enzyme, but there was a marked attenuation of G4 binding affinity for the EY hRev1 mutant and the two Rev1 proteins lacking insert-2 (yRev1 and lRev1). Perhaps most strikingly, we found that insert-2 was important for disruption of the G4 structure and optimal stimulation of processive DNA synthesis across the guanine-rich motif by DNA polymerase kappa (pol κ). Our findings have implications for how Rev1 might contribute to G4 replication in different species spanning the evolutionary tree - signaling the importance of selection for enzymes with robust G4-selective properties in organisms where these non-B DNA structures may fulfill taxa-specific physiological functions.

Multiple DNA repair pathways contribute to MMS-induced post-replicative DNA synthesis in S. pombe .

Replication stress can induce DNA synthesis outside of replicative S-phase. We have previously demonstrated that fission yeast cells stimulate DNA synthesis in G2-phase but not in M-phase in response to DNA alkylating agent MMS. In this study, we show that various DNA repair pathways, including translesion synthesis and break-induced replication contribute to post-replicative DNA synthesis. Checkpoint kinases, various repair and resection proteins, and multiple polymerases are also involved.

Endothelial senescence mediates hypoxia-induced vascular remodeling by modulating PDGFB expression.

Uncontrolled accumulation of pulmonary artery smooth muscle cells (PASMCs) to the distal pulmonary arterioles (PAs) is one of the major characteristics of pulmonary hypertension (PH). Cellular senescence contributes to aging and lung diseases associated with PH and links to PH progression. However, the mechanism by which cellular senescence controls vascular remodeling in PH is not fully understood. The levels of senescence marker, p16INK4A and senescence-associated β-galactosidase (SA-β-gal) activity are higher in PA endothelial cells (ECs) isolated from idiopathic pulmonary arterial hypertension (IPAH) patients compared to those from healthy individuals. Hypoxia-induced accumulation of α-smooth muscle actin (αSMA)-positive cells to the PAs is attenuated in p16fl/fl-Cdh5(PAC)-CreERT2 (p16iΔEC) mice after tamoxifen induction. We have reported that endothelial TWIST1 mediates hypoxia-induced vascular remodeling by increasing platelet-derived growth factor (PDGFB) expression. Transcriptomic analyses of IPAH patient lungs or hypoxia-induced mouse lung ECs reveal the alteration of senescence-related gene expression and their interaction with TWIST1. Knockdown of p16INK4A attenuates the expression of PDGFB and TWIST1 in IPAH patient PAECs or hypoxia-treated mouse lungs and suppresses accumulation of αSMA–positive cells to the supplemented ECs in the gel implanted on the mouse lungs. Hypoxia-treated mouse lung EC-derived exosomes stimulate DNA synthesis and migration of PASMCs in vitro and in the gel implanted on the mouse lungs, while p16iΔEC mouse lung EC-derived exosomes inhibit the effects. These results suggest that endothelial senescence modulates TWIST1-PDGFB signaling and controls vascular remodeling in PH.

Selectively Imprinted β‐cyclodextrin Polymer for Colorimetric Assay of Lysophosphatidic Acid for Point of Care Detection of Ovarian Cancer

AbstractIn the present study, a simple molecular imprinting technique for the quantitative measurement of lysophosphatidic acid (LPA) is reported for the first time through colorimetry. LPA is a phospholipid bioactive involved in many physiological and pathological processes like stimulating DNA synthesis, proliferating ovarian cancer and inducing cell migration. It is reported that the concentration of LPA in patients with gynecological cancers like ovarian cancer is considerably higher than in the control. The early diagnosis of ovarian cancer may exceed the survival rate of patients. There is a need for an analytical tool for point of care detection of ovarian cancer at an early stage. Herein, molecular imprinting technique is used to develop an imprinted β‐Cyclodextrin (β‐CD) polymer using LPA as a template. This MIP prepared was imprinted with an LPA cavity, which has been extended for colorimetry assay. The colorimetric LPA assay was conducted in the spiked blood serum sample. The detection limit of LPA in spiked serum samples is calculated as 0.078 μmol/L.

Hydrostatic Pressure Controls Angiogenesis through Endothelial YAP1 during Lung Regeneration

Pulmonary artery (PA) pressure increases during lung growth after unilateral pneumonectomy (PNX). The objective of this study is to understand the mechanism by which increases in PA pressure control angiogenesis during lung regeneration. Mechanosensitive transcriptional co‐activator, yes‐associated protein (YAP1), in endothelial cells (ECs) is necessary for angiogenesis during post‐PNX lung growth. We investigate whether increases in PA pressure following PNX controls angiogenesis through YAP1. When hydrostatic pressure is applied to human pulmonary arterial ECs (HPAECs), the expression of YAP1, transcription factor TEAD1, and angiogenic factor receptor Tie2 increases, while these effects are inhibited when HPAECs are treated with YAP1 siRNA or YAP1S94A mutant that fails to bind to TEAD1. Hydrostatic pressure also stimulates DNA synthesis and cell migration in HPAECs, while YAP1 knockdown or YAP1S94A mutant inhibits the effects. Gene enrichment analysis reveals that the levels of extracellular matrix or cell adhesion molecules are altered in post‐PNX mouse lung ECs, which interact with YAP1. These results suggest that increases in PA pressure stimulate angiogenesis through YAP1 during regenerative lung growth.

Effects of Dexamethasone on DNA Synthesis in Lens Epithelial Cells Are Dependent on Cell Type and Growth Factor

Purpose To determine the factors that influence the ability of dexamethasone (dex) to inhibit or stimulate the growth of lens epithelial cells. Method Different growth factors with or without dex (10−6 M) were added to quiescent cultures of two clones of Nakano mouse lens epithelial cells (NK11) in serum-free medium. DNA synthesis was then measured after 8–12 hours by the incorporation of tritiated thymidine. Results Dex was found to both stimulate and inhibit mitogen-induced 3H-thymidine incorporation into the DNA of cultured mouse lens epithelial cells. Enhancement or repression by dex was found to depend on the growth factor used to stimulate the quiescent cell. EGF and insulin were consistently inhibited with dex. Basic fibroblast growth factor (bFGF) and retinoblastoma-derived growth factor (RbDGF) were both enhanced and inhibited by dex, depending on the growth factor concentration and the cell clone used for the experiment. Additionally, RbDGF protects against the dex inhibition of insulin stimulation, but not the inhibition of EGF stimulation. Progesterone, an inhibitor of the activation of the glucocorticoid receptor, blocks the dex inhibitory effect on the EGF and insulin stimulation of DNA synthesis. The ability of progesterone to affect the dex inhibition is consistent with the dex receptor modulating DNA synthesis. The dex effect on DNA synthesis, either stimulatory or inhibitory, was still seen if dex was added as late as 10 hours after the growth factor. Conclusions The study demonstrated that dex reduces the overall growth and activity of lens epithelial cells in vitro. This result provides insight into the risk of developing posterior subcapsular cataracts (PSC) in patients on oral glucocorticoid therapy. Understanding the basic mechanisms by which steroids mediate lens cell growth may provide the ability to more accurately predict who will develop PSC. The present studies show the difference in the effect of dex from lens cell to lens cell, but, more importantly, suggest a pattern of dependent variables that might prove useful in such predictions.

Hydrostatic Pressure Controls Angiogenesis Through Endothelial YAP1 During Lung Regeneration.

Pulmonary artery (PA) pressure increases during lung growth after unilateral pneumonectomy (PNX). Mechanosensitive transcriptional co-activator, yes-associated protein (YAP1), in endothelial cells (ECs) is necessary for angiogenesis during post-PNX lung growth. We investigate whether increases in PA pressure following PNX control-angiogenesis through YAP1. When hydrostatic pressure is applied to human pulmonary arterial ECs (HPAECs), the expression of YAP1, transcription factor TEAD1, and angiogenic factor receptor Tie2 increases, while these effects are inhibited when HPAECs are treated with YAP1 siRNA or YAP1S94A mutant that fails to bind to TEAD1. Hydrostatic pressure also stimulates DNA synthesis, cell migration, and EC sprouting in HPAECs, while YAP1 knockdown or YAP1S94A mutant inhibits the effects. Gene enrichment analysis reveals that the levels of genes involved in extracellular matrix (ECM), cell adhesion, regeneration, or angiogenesis are altered in post-PNX mouse lung ECs, which interact with YAP1. Exosomes are known to promote tissue regeneration. Proteomics analysis reveals that exosomes isolated from conditioned media of post-PNX mouse lung ECs contain the higher levels of ECM and cell-adhesion proteins compared to those from sham-operated mouse lung ECs. Recruitment of host lung ECs and blood vessel formation are stimulated in the fibrin gel containing exosomes isolated from post-PNX mouse lung ECs or pressurized ECs, while YAP1 knockdown inhibits the effects. These results suggest that increases in PA pressure stimulate angiogenesis through YAP1 during regenerative lung growth.

SV40 T antigen helicase domain regions responsible for oligomerisation regulate Okazaki fragment synthesis initiation.

The initiation of Okazaki fragment synthesis during cellular DNA replication is a crucial step for lagging strand synthesis, which is carried out by the primase function of DNA polymerase α‐primase (Pol‐prim). Since cellular replication protein A (RPA) prevents primase from starting RNA synthesis on single‐stranded DNA (ssDNA), primase requires auxiliary factors, such as the simian virus 40 (SV40) T antigen (Tag), for the initiation reaction on RPA‐bound ssDNA. Here, we investigated the ability of Tag variants and Tag protein complexes to bind to ssDNA and their resulting effects on the stimulation of Pol‐prim on free and RPA‐bound ssDNA. Atomic force microscopy imaging showed that while Tag131‐627(V350E/P417D) and Tag131‐627(L286D/R567E) (abbreviated as M1 and M2, respectively) could bind to ssDNA as monomers, these monomeric Tags could come together and bind to ssDNA as dimers as well. In a model assay for the initiation of Okazaki fragment synthesis, full‐length Tag SV40 Tag1‐708 and monomeric M2 stimulated DNA synthesis of Pol‐prim on ssDNA and on RPA‐bound ssDNA. In contrast, neither monomeric M1 nor M1‐M2 dimers could stimulate Pol‐prim, on ssDNA or on RPA‐bound ssDNA. Overall, we show that a lack of stimulatory activity of monomeric M1 and M1‐M2 dimers suggests that residues V350 and P417 are not only important for interactions between Tag molecules but also for protein–protein interactions within Okazaki fragment initiation complexes. Thus, we highlight that mutations in M1 are dominant negative with regard to Okazaki fragment initiation.

Transient Receptor Potential Melastatin 8, a sensor of cold temperatures mediates expression of cyclin-dependent kinase inhibitor, p21/Cip1, a regulator of epidermal cell proliferation.

Transient Receptor Potential Melastatin 8 (TRPM8) is a calcium-permeable, non-selective cation channel of the transient receptor potential superfamily, required for the transduction of moderate cold temperatures. TRPM8 is also known to regulate proliferation of prostate, pancreatic, breast, and melanoma carcinoma cells. Here, we examined a key factor in the regulation of TRPM8-mediated proliferation of epidermal cells, which are directly affected by cold temperatures. Experiments involving knockdown and ectopic expression of TRPM8 in normal keratinocyte HaCaT and squamous carcinoma SAS cells suggest that TRPM8 inhibits cell proliferation by upregulating the expression of cyclin-dependent inhibitor p21/Cip1. Whereas these findings were observed in the absence of an endogenous agonists, additions of the synthetic TRPM8 agonist icilin reduced DNA synthesis in HaCaT cells but stimulated that in SAS cells by altering p21/Cip1 levels in a TRPM8-independent manner, indicating that icilin poses a risk of stimulating carcinoma cell proliferation. Unexpectedly, the TRPM8 blocker, used for the treatment of overactive bladder and bladder pain, N-(3-aminopropyl)-2-{[(3-methylphenyl) methyl] oxy}-N-(2-thienylmethyl) benzamide hydrochloride salt (AMTB) reduced DNA synthesis by upregulating p21/Cip1 expression. However, another TRPM8 blocker, N-(4-Tertiarybutylphenyl)-4-(3-chloropyridin- 2-yl) tetrahydropyrazine-1 (2H)-carbox-amide (BCTC), stimulated DNA synthesis by downregulating p21/Cip1 expression, indicating that it may pose a risk of carcinogenesis associated with dysregulated cell cycles when used to treat overactive bladder and bladder pain.

Aged and young mice differentially respond to tape-stripping in epidermal gene expression.

Disruption of epidermal permeability barrier induces an increase in proinflammatory cytokine expression and release, stimulation of epidermal lipid and DNA synthesis, and expression of antimicrobial peptides. Although alterations in epidermal function in the aged skin are known, whether the epidermal transcriptomic responses to barrier disruption differ between aged and young mice remains unknown. Here, we performed RNA sequencing of the epidermis in 2-month- vs. 20-month-old mice following barrier disruption with repeated tape-stripping. At baseline condition, the epidermis of 20-month-old mice displayed an upregulation of inflammation-associated genes and down-regulation of epidermal structure- and development-related genes in comparison to 2-month-old mice. Barrier disruption upregulated expression levels of 327genes and downregulated 209genes in 2-month-old mice. In 20-month-old mice, the numbers of upregulated and down-regulated genes were 537 and 299, respectively. In comparison to young mice, the prominently upregulated genes in the 20-month-old mice were associated with the IL-17signalling pathway, while downregulated genes were mainly involved in the cytokine-cytokine receptor interaction pathway. These results indicate that inflammation-associated signalling pathways are upregulated, while epidermal structure- and development-related genes are downregulated in the epidermis of aged mice, with further aggravation following barrier disruption, suggesting the importance of improving epidermal function in the elderly.

Growth Hormone Signaling Pathway Leading to the Induction of DNA Synthesis and Proliferation in Primary Cultured Hepatocytes of Adult Rats.

We investigated the signal transduction pathway associated with growth hormone (GH)-stimulated DNA synthesis and proliferation in primary cultured hepatocytes. Adult rat hepatocytes were isolated from normal livers by two-step in situ collagenase perfusion to facilitate disaggregation of the adult rat liver. Then hepatocytes were cultured in serum-free Williams' medium E supplemented with GH (1-100 ng/ml) in the presence or absence of test reagents. GH-induced hepatocyte DNA synthesis and proliferation were determined, and the phosphorylation activities of Janus kinase (JAK) 2 (JAK2) (p125 kDa), p95-kDa RTK, and ERK1/2 were measured by western blotting. Hepatocytes grown in serum-free defined medium proliferated within 5 h of culture in the presence of GH (100 ng/ml) in a concentration- and time-dependent manner (EC50 75 ng/ml). These proliferative effects of GH were almost completely blocked by an anti-GH receptor monoclonal antibody (85 ng/ml) and an anti-insulin-like growth factor (IGF)-I receptor monoclonal antibody. In addition, the proliferative effects of GH were significantly blocked by a JAK2 inhibitor (TG101209, 10-6 M), as well as specific signal-transducing inhibitors of phospholipase C (PLC; U-73122, 10-6 M), RTK (AG538, 10-6 M), phosphoinositide 3-kinase (PI3K; LY294002, 10-6 M), mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK; PD98059, 10-6 M), and mammalian target of rapamycin (mTOR; rapamycin, 10 ng/ml). GH significantly induced the phosphorylations of JAK2 (p125 kDa), p95-kDa IGF-I receptor tyrosine kinase (RTK), and ERK2 in this order according to western blotting analysis. The proliferative action of GH is mediated by two main signaling pathways. One includes activation of the GH receptor/JAK2/PLC/Ca2+ pathway, and the other involves activation of the p95-kDa IGF-I RTK/PI3K/ERK2/mTOR pathway in primary cultures of adult rat hepatocytes.

The Use of Low Intensity Laser in Wound Healing in Dog

Background: Low-intensity laser is effective in cellular metabolism, analgesia, and tissue repair. The bioelectric, bioenergetic, and biochemical effects of laser therapy stimulate local circulation, collagen formation, and epithelization. The objective of this study was to report the use of gallium-aluminum arsenide (GaAlAs) laser (830 nm) in healing two wounds caused by necrosis in a dog.Case: An 8-year-old bitch, a rescued victim of animal abuse, had tibiotarsal dislocation in the left posterior limb. Orthopedic surgery was performed with the placement of an external fixation device with six pins in the distal third of the left posterior limb. A radiographic examination performed 42 days after the surgery revealed the loss of the tibiotarsal ratio, decreased radiopacity of the carpal bones, edema, inflammation, muscle atrophy, and the rotation of the anatomical axis of the metatarsals and phalanges. It was then decided to perform a second orthopedic surgery, in which a bone graft and arthrodesis with an 8-screw titanium plate were performed. Four of the plate screws were placed proximally to the tibia and four distally to the tarsus in addition to a screw and a pin transfixed through the distal metaphyseal region of the tibia and calcaneus. Traction was felt during suturing in the dermis and epidermis of the limb, suggesting the possibility of dehiscence of the stitches. After 3 h of surgery, the limb was cold and edematous. After 5 days, the tissue of the medial region of the tarsus developed necrosis, exposing a wound 6 cm long, 1.5 cm wide, and 2 mm deep (wound 1). Another necrotic lesion was observed in the dorsal region of the tarsos - 6.5 cm long, 2 cm wide, 3 mm deep, and exposing 5 cm of the titanium plate (wound 2). Therapy with GaAlAs laser was then performed over the entire length of the wounds. In each therapeutic session, the laser was first used in punctual mode at 10 J/cm², 830 nm, 20 s at each point of the length of the lesion and then in scan mode at 10 J/cm², 830 nm, 1000 Hz, and 40 s continuously. Laser therapy sessions were conducted at 2-4 day intervals, with a 12-day interval between the eighth and ninth sessions. During the entire treatment, cleansing and debridement of the wounds were performed every 48 h with saline and chlorhexidine digluconate, using a compression bandage, as described earlier. Wound 1 healed completely after two laser therapy sessions. In wound 2, tissue repair stagnated after the ninth and last laser session, leaving 4 cm of the titanium plate still exposed. The limb could not support the body weight of the patient, and radiographic examination revealed that the anatomical axis of the metatarsals and the phalanges was rotated and bone conformation was poor. Given the anatomical conditions associated with the patient's clinical picture, it was decided to amputate the limb between the femur and tibia.Discussion: Therapeutic lasers act on mitochondrial respiration, increasing respiratory metabolism and stimulating DNA synthesis and tissue proliferation. In the present case, there was a decrease in inflammatory cells, edema, and the size of the wounds. The pathological conditions of the affected site, influence of trauma, and degree of tissue damage affected the results of the laser therapy. Despite the size difference between wounds 1 and 2, the exposed titanium plate was a determining factor for the partial healing of wound 2. Keywords: laser, laser therapy, scar, necrosis, orthopedics, physical therapy, tissue repair. Título: A utilização do laser de baixa intensidade na cicatrização de ferida em cão.Descritores: laser, laserterapia, cicatriz, necrose, ortopedia, fisioterapia, reparo tecidual.

Autocrine secretion of insulin-like growth factor-I mediates growth hormone-stimulated DNA synthesis and proliferation in primary cultures of adult rat hepatocytes

The intracellular signaling pathway of growth hormone (GH)-stimulated DNA synthesis and proliferation was investigated in primary cultures of adult rat hepatocytes. DNA synthesis and cell proliferation were detected in hepatocyte parenchymal cells grown in serum-free, defined medium containing GH (100 ng/ml). GH-stimulated hepatocyte DNA synthesis and proliferation were almost completely blocked by TG101209 (10−6 M), a selective Janus kinase (JAK)2 inhibitor, U-73122 (10−6 M), a selective phospholipase C (PLC) inhibitor, and a monoclonal antibody to insulin-like growth factor-I (IGF-I) receptor (100 ng/ml) or anti-secretion agents such as somatostatin (10−6 M) and BAPTA/AM (10−7 M). In addition, blocking monoclonal antibodies to IGF-I, but not transforming growth factor-α, completely inhibited GH-induced hepatocyte DNA synthesis and proliferation. IGF-I levels in the culture medium increased rapidly versus baseline levels within 5 min in response to GH (100 ng/ml), and the maximum IGF-I level (100 pg/ml) was reached 20 min after GH stimulation. Autocrine secretion of IGF-I into the culture medium was inhibited by a growth-inhibitory dose of TG101209, U-73122, somatostatin, or BAPTA/AM. These data indicate that the proliferative mechanism of action of GH is mediated mainly through a GH receptor/JAK2/PLC-stimulated increase in the autocrine secretion of IGF-I by primary cultured hepatocytes, followed by stimulation of the 95 kDa IGF-I receptor tyrosine kinase signaling pathway.

Pif1, RPA, and FEN1 modulate the ability of DNA polymerase δ to overcome protein barriers during DNA synthesis

Successful DNA replication requires carefully regulated mechanisms to overcome numerous obstacles that naturally occur throughout chromosomal DNA. Scattered across the genome are tightly bound proteins, such as transcription factors and nucleosomes, that are necessary for cell function, but that also have the potential to impede timely DNA replication. Using biochemically reconstituted systems, we show that two transcription factors, yeast Reb1 and Tbf1, and a tightly positioned nucleosome, are strong blocks to the strand displacement DNA synthesis activity of DNA polymerase δ. Although the block imparted by Tbf1 can be overcome by the DNA-binding activity of the single-stranded DNA-binding protein RPA, efficient DNA replication through either a Reb1 or a nucleosome block occurs only in the presence of the 5'-3' DNA helicase Pif1. The Pif1-dependent stimulation of DNA synthesis across strong protein barriers may be beneficial during break-induced replication where barriers are expected to pose a problem to efficient DNA bubble migration. However, in the context of lagging strand DNA synthesis, the efficient disruption of a nucleosome barrier by Pif1 could lead to the futile re-replication of newly synthetized DNA. In the presence of FEN1 endonuclease, the major driver of nick translation during lagging strand replication, Pif1-dependent stimulation of DNA synthesis through a nucleosome or Reb1 barrier is prevented. By cleaving the short 5' tails generated during strand displacement, FEN1 eliminates the entry point for Pif1. We propose that this activity would protect the cell from potential DNA re-replication caused by unwarranted Pif1 interference during lagging strand replication.