Nascent shifts in renal cellular metabolism, structure, and function due to chronic empagliflozin in prediabetic mice.

Sodium-glucose cotransporter, type 2 inhibitors (SGLT2i) are emerging as the gold standard for treatment of type 2 diabetes (T2D) with renal protective benefits independent of glucose lowering. We took a high-level approach to evaluate the effects of the SGLT2i, empagliflozin (EMPA) on renal metabolism and function in a prediabetic model of metabolic syndrome. Male and female 12-wk-old TallyHo (TH) mice, and their closest genetic lean strain (Swiss-Webster, SW) were treated with a high-milk-fat diet (HMFD) plus/minus EMPA (@0.01%) for 12-wk. Kidney weights and glomerular filtration rate were slightly increased by EMPA in the TH mice. Glomerular feature analysis by unsupervised clustering revealed sexually dimorphic clustering, and one unique cluster relating to EMPA. Periodic acid Schiff (PAS) positive areas, reflecting basement membranes and mesangium were slightly reduced by EMPA. Phasor-fluorescent life-time imaging (FLIM) of free-to-protein bound NADH in cortex showed a marginally greater reliance on oxidative phosphorylation with EMPA. Overall, net urine sodium, glucose, and albumin were slightly increased by EMPA. In TH, EMPA reduced the sodium phosphate cotransporter, type 2 (NaPi-2), but increased sodium hydrogen exchanger, type 3 (NHE3). These changes were absent or blunted in SW. EMPA led to changes in urine exosomal microRNA profile including, in females, enhanced levels of miRs 27a-3p, 190a-5p, and 196b-5p. Network analysis revealed "cancer pathways" and "FOXO signaling" as the major regulated pathways. Overall, EMPA treatment to prediabetic mice with limited renal disease resulted in modifications in renal metabolism, structure, and transport, which may preclude and underlie protection against kidney disease with developing T2D.NEW & NOTEWORTHY Renal protection afforded by sodium glucose transporter, type 2 inhibitors (SGLT2i), e.g., empagliflozin (EMPA) involves complex intertwined mechanisms. Using a novel mouse model of obesity with insulin resistance, the TallyHo/Jng (TH) mouse on a high-milk-fat diet (HMFD), we found subtle changes in metabolism including altered regulation of sodium transporters that line the renal tubule. New potential epigenetic determinants of metabolic changes relating to FOXO and cancer signaling pathways were elucidated from an altered urine exosomal microRNA signature.

Investigation of scan delays for CT evaluation of inner wall layering and peak enhancement of the canine stomach and small intestine using a 20 second fixed-injection-duration and bolus tracking technique.

Many gastrointestinal diseases affect the mucosal layer, suggesting that on computed tomography (CT) examination, detection of consistent inner wall layering of the gastrointestinal tract may aid in detection of disease. Changes in wall enhancement can also characterise specific diseases and provide prognostic information. The objectives of this mixed retrospective and prospective analytical study were therefore to identify the scan delays for peak detection of canine stomach and small intestinal inner wall layering and enhancement when using a 20 s fixed-injection-duration and bolus tracking technique. For each patient, 700 mg I/kg iohexol was administered intravenously. Bolus tracking was used to determine aortic arrival. Diagnostic scans were performed after a post-aortic arrival scan delay. Postcontrast CT series were grouped according to post-aortic arrival scan delay: 5 s (n = 17), 10 s (n = 18), 15 s (n = 23), 20 s (n = 10), 25 s (n = 6), 30 s (n = 14), 35 s (n = 17), 40 s (n = 24), and 180 s (n = 60). The stomach and small intestine were assessed for the presence of a contrast-enhancing inner wall layer and wall enhancement. Statistical modeling showed that the scan delays for peak inner wall layering and enhancement were 10 and 15 s for the small intestine, respectively, and 40 s for the stomach. For the injection protocol used in this study, assessment of the canine gastrointestinal tract may use scan delays of 10-15 s and 40 s.

Hepatic computed tomography and cholangiography by use of gadoxetic acid in healthy cats.

OBJECTIVE: To evaluate 3 doses of gadoxetic acid (Gd-EOB-DPTA) for hepatic CT and cholangiography in cats and to determine optimal timing for hepatobiliary image acquisition and evaluation of the contrast-enhanced hepatobiliary anatomy. ANIMALS: 6 healthy cats. PROCEDURES: Cats were anesthetized; sequential CT scans were performed 0, 5, 25, 45, 65, and 85 minutes after IV administration of Gd-EOB-DTPA at low (0.0125 mmol/kg), medium (0.1 mmol/kg), and high (0.3 mmol/kg) doses. Hepatobiliary enhancement for each dose was objectively assessed over time and by use of a subjective semiquantitative visual assessment score. RESULTS: No contrast-related adverse effects were detected. Each increase in dose of contrast medium resulted in a significant increase in HU across the hepatobiliary system. The liver had a significantly higher number of HU at 45 minutes, with homogenous enhancement at all doses of contrast medium. Contrast-enhanced cystic and bile duct HU were significantly higher and maximal at 65 minutes. Contrast-enhanced gallbladder HU did not plateau by 85 minutes. At a high dose of contrast medium, 12 of 60 (20%) biliary tract scores indicated no enhancement, 34 (57%) indicated poor enhancement, and 14 (23%) indicated moderate enhancement. No cat had excellent enhancement of the biliary tract at any dose. CONCLUSIONS AND CLINICAL RELEVANCE: Gd-EOB-DTPA-enhanced hepatic CT and cholangiography in cats were safely performed and provided good hepatic enhancement but poor to moderate enhancement of the biliary tract. This technique may be useful for assessing the liver parenchyma in cats, but its value for assessing the biliary tract is questionable.

Vascular conspicuity differs among injection protocols and scanner types for canine multiphasic abdominal computed tomographic angiography.

Multiphasic multidetector computed tomographic angiography is a standard diagnostic test for canine abdominal vascular disorders. Three imaging protocols have been previously described. The test-bolus protocol allows precise timing but can be time consuming to perform. Bolus-tracking software is fast and easy to use but can be problematic for exact timing of vascular phases. A recently described fixed-injection-duration protocol is not influenced by body weight and provides a wider temporal window for arterial acquisitions. Objectives of this retrospective and prospective, multicentric, method comparison study were to determine which of the three multidetector computed tomographic angiography protocols allows best vascular conspicuity of the canine abdomen and to assess the influence of different multidetector computed tomography (CT) scanners on study quality. Triple-phase multidetector computed tomographic angiography canine abdominal studies from 30 dogs were retrospectively retrieved from three different institutions. Each institution performed one of the three computed tomographic angiography protocols (4-row and 16-row multidetector CT). Prospectively, the three protocols were also acquired with similar conditions on a 64-row MDCT in 21 dogs. Main abdominal vessels were scored by blinded readers for each phase. The fixed-injection-duration protocol had the best combined arterial and portal vascular conspicuity on scanners of limited speed, while the test-bolus protocol provided the best overall vascular conspicuity on 64-row multidetector CT scanner. The quality of arterial studies performed on 64-row MDCT scanner was improved compared to the ones performed on four- to 16-row multidetector CT scanners. Findings supported the fixed-injection-duration protocol as the best compromise between an ideal portal vascular enhancement and an easily reproducible protocol on scanners with low and high number of detector rows.

Breathing new life into West Nile virus therapeutics; discovery and study of zafirlukast as an NS2B-NS3 protease inhibitor.

The West Nile virus (WNV) has spread throughout the world causing neuroinvasive diseases with no treatments available. The viral NS2B-NS3 protease is essential for WNV survival and replication in host cells and is a promising drug target. Through an enzymatic screen of the National Institute of Health clinical compound library, we report the discovery of zafirlukast, an FDA approved treatment for asthma, as an inhibitor for the WNV NS2B-NS3 protease. Zafirlukast was determined to inhibit the protease through a mixed mode mechanism with an IC50 value of 32 µM. A structure activity relationship study of zafirlukast revealed the cyclopentyl carbamate and N-aryl sulfonamide as structural elements crucial for NS2B-NS3 protease inhibition. Replacing the cyclopentyl with a phenyl improved inhibition, resulting in an IC50 of 22 µM. Experimental and computational docking analysis support the inhibition model of zafirlukast and analogs binding at an allosteric site on the NS3 protein, thereby disrupting the NS2B cofactor from binding, resulting in protease inhibition.

Use of gadoxetic acid for computed tomographic cholangiography in healthy dogs.

OBJECTIVE To evaluate the effect of gadoxetic acid (contrast) dose on biliary tract enhancement, determine the optimal time after contrast injection for CT image acquisition, and assess the feasibility of CT cholangiography in sedated dogs. ANIMALS 8 healthy dogs. PROCEDURES The study had 2 parts. In part 1, 4 dogs were anesthetized and underwent CT cholangiography twice. Gadoxetic acid was administered IV at a low dose (0.025 mmol/kg) for the first procedure and high dose (0.3 mmol/kg) for the second procedure. Serial CT scans were obtained at predetermined times after contrast injection. In part 2, 4 dogs were sedated and underwent CT angiography 85 minutes after IV administration of the high contrast dose. Contrast enhancement of the biliary tract on all scans was objectively assessed by measurement of CT attenuation and qualitatively assessed by use of a subjective 4-point scoring system by 3 independent reviewers. All measurements were compared over time and between contrast doses for the dogs of part 1. Subjective measurements were compared between the sedated dogs of part 2 and anesthetized dogs of part 1. RESULTS Enhancement of the biliary tract was positively associated with contrast dose and time after contrast injection. Optimal enhancement was achieved 65 minutes after contrast injection. Subjective visualization of most biliary structures did not differ significantly between sedated and anesthetized dogs. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated CT cholangiography with gadoxetic acid was feasible in sedated dogs. The high contrast dose provided better visualization of biliary structures than the low dose; CT scans should be obtained 65 minutes after contrast injection.

Estimation of time to peak contrast enhancement of the aorta and liver for dual-phase computed tomography on the basis of contrast medium arrival time, injection duration, and injection technique in dogs.

OBJECTIVE To evaluate the accuracy of estimating time to peak enhancement (TPE) of the aorta and liver parenchyma on the basis of contrast medium arrival time in the aorta, injection duration, and injection technique in dogs. ANIMALS 18 dogs of specific body weight categories (≥ 2 dogs/category) with no liver abnormalities detected via CT. PROCEDURES Dogs were randomly assigned within weight categories to receive contrast medium IV at a fixed injection rate (5 mL/s) or fixed injection duration (20 seconds). Time-contrast attenuation curves were generated from dynamic CT scans acquired at the hepatic hilus. Data collected for contrast medium arrival time and injection duration were used to estimate TPEs of the aorta and liver, and results were compared with the observed TPEs for the aorta and liver. RESULTS Contrast medium arrival time, injection duration, and injection technique were significantly associated with observed values for aortic TPE and explained 96.1% of variation in TPE. For the fixed rate technique, the regression equation for estimating aortic TPE was 0.8 × (injection duration + contrast medium arrival time) + 1.6. For the fixed duration technique, the regression equation changed by only the constant (-2.6). However, the hepatic TPE estimated from the 3 predictor variables was not significantly different from the mean of observed TPEs. CONCLUSIONS AND CLINICAL RELEVANCE Aortic TPE could be accurately estimated from contrast medium arrival time, injection duration, and injection technique in dogs with apparently healthy livers. The regression equations derived from this relationship can be used to improve the efficiency of dual-phase CT of the liver in dogs.

CT findings in two cats with broncholithiasis.

CASE SERIES SUMMARY: Chronic inflammatory airway disease with secondary broncholithiasis was diagnosed in two cats from CT and bronchoalveolar lavage cytological findings. In one cat with progressively worsening lower respiratory tract signs, more than 80 discrete, highly attenuating endobronchial opacities were detected on thoracic CT. The broncholiths were distributed throughout the right middle, and left and right caudal lung lobes, and the caudal part of the left cranial and accessory lobes. In the other cat broncholithiasis was an incidental finding on thoracic radiographs taken during diagnostic investigation of inappetence. On thoracic CT, 25 calcified endobronchial opacities were detected in the left caudal lung lobe in secondary and tertiary bronchi. CT features of chronic inflammatory airway disease were present in both cases, including bronchiectasis, atelectasis, flattening of the diaphragm and bronchial wall thickening. RELEVANCE AND NOVEL INFORMATION: This is the first report to document CT features of broncholithiasis in cats. Feline broncholithiasis should be considered as a differential diagnosis in any case where calcified endobronchial material is evident on thoracic radiographs or CT.

EFFECTS OF TWO CONTRAST INJECTION PROTOCOLS ON FELINE AORTIC AND HEPATIC ENHANCEMENT USING DYNAMIC COMPUTED TOMOGRAPHY.

This prospective study compared aortic and hepatic enhancement achieved using a contrast injection protocol with a fixed rate of 5 ml/s vs. that achieved using a protocol with fixed injection duration of 20 s in eight cats. Cats were assigned into two groups (Group 1, rate 5 ml/s; Group 2, duration 20 s). The dose of contrast was the same in both groups (740 mgI/kg). Regions of interest (ROI) were drawn in the aorta and liver for transverse scans acquired at the hepatic hilus. Time to peak aortic enhancement occurred significantly earlier in Group 1 (M = 11s, SD = 1.63) than in Group 2 (M = 25.5 s, SD = 2.51). Peak aortic enhancement was significantly higher in Group 1 (M = 1906.51 HU, SD = 368.64) than in Group 2 (M = 745.08 HU, SD = 201.84). Duration of aortic enhancement equal to or above 300 HU was statistically longer in Group 2 (M = 24.5 s, SD = 8.39) than in Group 1 (M = 10 s, SD = 1.63). There were no significant differences in time to peak liver enhancement, peak liver enhancement, or duration of hepatic arterial phase between groups. Findings supported the hypothesis that longer injection duration results in a broader bolus geometry with a longer time to peak and a lower peak aortic enhancement in cat. This strong influence of injection duration on timing of aortic enhancement may help future users optimize protocols for CT angiography of the aorta and multiphasic evaluation of the liver, pancreas, and small intestine.

Malaria parasite infection compromises control of concurrent systemic non-typhoidal Salmonella infection via IL-10-mediated alteration of myeloid cell function.

Non-typhoidal Salmonella serotypes (NTS) cause a self-limited gastroenteritis in immunocompetent individuals, while children with severe Plasmodium falciparum malaria can develop a life-threatening disseminated infection. This co-infection is a major source of child mortality in sub-Saharan Africa. However, the mechanisms by which malaria contributes to increased risk of NTS bacteremia are incompletely understood. Here, we report that in a mouse co-infection model, malaria parasite infection blunts inflammatory responses to NTS, leading to decreased inflammatory pathology and increased systemic bacterial colonization. Blunting of NTS-induced inflammatory responses required induction of IL-10 by the parasites. In the absence of malaria parasite infection, administration of recombinant IL-10 together with induction of anemia had an additive effect on systemic bacterial colonization. Mice that were conditionally deficient for either myeloid cell IL-10 production or myeloid cell expression of IL-10 receptor were better able to control systemic Salmonella infection, suggesting that phagocytic cells are both producers and targets of malaria parasite-induced IL-10. Thus, IL-10 produced during the immune response to malaria increases susceptibility to disseminated NTS infection by suppressing the ability of myeloid cells, most likely macrophages, to control bacterial infection.

Structure-activity relationships of diamine inhibitors of cytochrome P450 (CYP) 3A as novel pharmacoenhancers. Part II: P2/P3 region and discovery of cobicistat (GS-9350).

The HIV protease inhibitor (PI) ritonavir (RTV) has been widely used as a pharmacoenhancer for other PIs, which are substrates of cytochrome P450 3A (CYP3A). However the potent anti-HIV activity of ritonavir may limit its use as a pharmacoenhancer with other classes of anti-HIV agents. Ritonavir is also associated with limitations such as poor physicochemical properties. To address these issues a series of compounds with replacements at the P2 and/or P3 region was designed and evaluated as novel CYP3A inhibitors. Through these efforts, a potent and selective inhibitor of CYP3A, GS-9350 (cobicistat) with improved physiochemical properties was discovered.

Effects of stifle flexion angle and scan plane on visibility of the normal canine cranial cruciate ligament using low-field magnetic resonance imaging.

Low-field magnetic resonance imaging (MRI) is commonly used to evaluate dogs with suspected cranial cruciate ligament injury; however, effects of stifle positioning and scan plane on visualization of the ligament are incompletely understood. Six stifle joints (one pilot, five test) were collected from dogs that were scheduled for euthanasia due to reasons unrelated to the stifle joint. Each stifle joint was scanned in three angles of flexion (90°, 135°, 145°) and eight scan planes (three dorsal, three axial, two sagittal), using the same low-field MRI scanner and T2-weighted fast spin echo scan protocol. Two experienced observers who were unaware of scan technique independently scored visualization of the cranial cruciate ligament in each scan using a scale of 0-3. Visualization score rank sums were higher when the stifle was flexed at 90° compared to 145°, regardless of the scan plane. Visualization scores for the cranial cruciate ligament in the dorsal (H (2) = 19.620, P = 0.000), axial (H (2) = 14.633, P = 0.001), and sagittal (H (2) = 8.143, P = 0.017) planes were significantly affected by the angle of stifle flexion. Post hoc analysis showed that the ligament was best visualized at 90° compared to 145° in the dorsal (Z = -3.906, P = 0.000), axial (Z = -3.398, P = 0.001), and sagittal (Z = -2.530, P = 0.011) planes. Findings supported the use of a 90° flexed stifle position for maximizing visualization of the cranial cruciate ligament using low-field MRI in dogs.

Structure-activity relationships of diamine inhibitors of cytochrome P450 (CYP) 3A as novel pharmacoenhancers, part I: core region.

Ritonavir (RTV), an HIV-1 protease inhibitor (PI), is also a potent mechanism-based inhibitor of human cytochrome P450 3A (CYP3A) and has been widely prescribed as a pharmacoenhancer. As a boosting agent for marketed PIs, it reduces pill burden, and improves compliance. Removal of the hydroxyl group from RTV reduces, but does not eliminate HIV PI activity and does not affect CYP3A inhibition. Herein we report the discovery of a novel series of CYP3A inhibitors that are devoid of antiviral activity. The synthesis and evaluation of analogs with extensive modifications of the 1,4-diamine core along with the structure activity relationships with respect to anti-HIV activity, CYP3A inhibitory activity, selectivity against other CYP enzymes and the human pregnane X receptor (PXR) will be discussed.

Malaria-associated L-arginine deficiency induces mast cell-associated disruption to intestinal barrier defenses against nontyphoidal Salmonella bacteremia.

Coinfection with malaria and nontyphoidal Salmonella serotypes (NTS) can cause life-threatening bacteremia in humans. Coinfection with malaria is a recognized risk factor for invasive NTS, suggesting that malaria impairs intestinal barrier function. Here, we investigated mechanisms and strategies for prevention of coinfection pathology in a mouse model. Our findings reveal that malarial-parasite-infected mice, like humans, develop L-arginine deficiency, which is associated with intestinal mastocytosis, elevated levels of histamine, and enhanced intestinal permeability. Prevention or reversal of L-arginine deficiency blunts mastocytosis in ileal villi as well as bacterial translocation, measured as numbers of mesenteric lymph node CFU of noninvasive Escherichia coli Nissle and Salmonella enterica serotype Typhimurium, the latter of which is naturally invasive in mice. Dietary supplementation of malarial-parasite-infected mice with L-arginine or L-citrulline reduced levels of ileal transcripts encoding interleukin-4 (IL-4), a key mediator of intestinal mastocytosis and macromolecular permeability. Supplementation with L-citrulline also enhanced epithelial adherens and tight junctions in the ilea of coinfected mice. These data suggest that increasing L-arginine bioavailability via oral supplementation can ameliorate malaria-induced intestinal pathology, providing a basis for testing nutritional interventions to reduce malaria-associated mortality in humans.

Evidence for an increased risk of transmission of simian immunodeficiency virus and malaria in a rhesus macaque coinfection model.

In sub-Saharan Africa, HIV-1 infection frequently occurs in the context of other coinfecting pathogens, most importantly, Mycobacterium tuberculosis and malaria parasites. The consequences are often devastating, resulting in enhanced morbidity and mortality. Due to the large number of confounding factors influencing pathogenesis in coinfected people, we sought to develop a nonhuman primate model of simian immunodeficiency virus (SIV)-malaria coinfection. In sub-Saharan Africa, Plasmodium falciparum is the most common malaria parasite and is responsible for most malaria-induced deaths. The simian malaria parasite Plasmodium fragile can induce clinical symptoms, including cerebral malaria in rhesus macaques, that resemble those of P. falciparum infection in humans. Thus, based on the well-characterized rhesus macaque model of SIV infection, this study reports the development of a novel rhesus macaque SIV-P. fragile coinfection model to study human HIV-P. falciparum coinfection. Using this model, we show that coinfection is associated with an increased, although transient, risk of both HIV and malaria transmission. Specifically, SIV-P. fragile coinfected macaques experienced an increase in SIV viremia that was temporarily associated with an increase in potential SIV target cells and systemic immune activation during acute parasitemia. Conversely, primary parasitemia in SIV-P. fragile coinfected animals resulted in higher gametocytemia that subsequently translated into higher oocyst development in mosquitoes. To our knowledge, this is the first animal model able to recapitulate the increased transmission risk of both HIV and malaria in coinfected humans. Therefore, this model could serve as an essential tool to elucidate distinct immunological, virological, and/or parasitological parameters underlying disease exacerbation in HIV-malaria coinfected people.

Inter-professional Education in the Acute-Care Setting: The Clinical Instructor's Point of View.

PURPOSE: The purpose of this study was to gain an understanding of the opportunities and challenges involved in providing clinical inter-professional education (IPE) to physical therapy (PT) students in the acute-care setting from the perspective of PT clinical instructors (CIs). METHODS: Focus groups were conducted in four acute-care hospitals in Toronto. Participants were recruited using a purposive and convenience sampling approach in order to enhance our understanding of the perceptions of acute-care PT CIs. Eighteen full-time PT CIs with an average of 11 years in practice participated. A constant comparative process was employed to identify recurrent issues and themes within and between groups. RESULTS: Three main themes emerged from the focus groups: (1) Clinical IPE happens when inter-professional collaboration (IPC) occurs; however, IPC differs according to setting, access to other professions, time, support, and structure. (2) IPE is a lifelong learning process that applies to both CIs and students. (3) Student preparedness is a prerequisite for clinical IPE. CONCLUSIONS: IPC is an integral part of clinical IPE that requires ongoing commitment and reflection by CIs to ensure that they are ready to instruct students who have some preparation in formal IPE. More knowledge about providing clinical IPE in a structured manner, through academic and health institutions, will allow CIs to become role models for future generations of PT students.

Both hemolytic anemia and malaria parasite-specific factors increase susceptibility to Nontyphoidal Salmonella enterica serovar typhimurium infection in mice.

Severe pediatric malaria is an important risk factor for developing disseminated infections with nontyphoidal Salmonella serotypes (NTS). While recent animal studies on this subject are lacking, early work suggests that an increased risk for developing systemic NTS infection during malaria is caused by hemolytic anemia, which leads to reduced macrophage microbicidal activity. Here we established a model for oral Salmonella enterica serotype Typhimurium challenge in mice infected with Plasmodium yoelii nigeriensis. Initial characterization of this model showed that 5 days after coinoculation, P. yoelii nigeriensis infection increased the recovery of S. Typhimurium from liver and spleen by approximately 1,000-fold. The increased bacterial burden could be only partially recapitulated by antibody-mediated hemolysis, which increased the recovery of S. Typhimurium from liver and spleen by 10-fold. These data suggested that both hemolysis and P. yoelii nigeriensis-specific factors contributed to the increased susceptibility to S. Typhimurium. The mechanism by which hemolysis impaired resistance to S. Typhimurium was further investigated. In vitro, S. Typhimurium was recovered 24 h after infection of hemophagocytic macrophages in 2-fold-higher numbers than after infection of mock-treated macrophages, making it unlikely that reduced macrophage microbicidal activity was solely responsible for hemolysis-induced immunosuppression during malaria. Infection with P. yoelii nigeriensis, but not antibody-mediated hemolysis, reduced serum levels of interleukin-12p70 (IL-12p70) in response to S. Typhimurium challenge. Collectively, studies establishing a mouse model for this coinfection suggest that multiple distinct malaria-induced immune defects contribute to increased susceptibility to S. Typhimurium.

An increase of cytochrome C oxidase mediated disruption of gemcitabine incorporation into DNA in a resistant KB clone.

Mechanistic aberrations leading to Gemcitabine (2',2'-dFdCyd,2,2-difluorodeoxycytidine, Gem) resistance may include alteration in its transport, metabolism and incorporation into DNA. To explore the mechanism of Gem resistance, the restriction fragment differential display PCR (RFDD-PCR) was employed to compare the mRNA expression patterns of KBGem (Gem resistant), KBHURs (hydroxyurea resistant) and KBwt (parental KB cell). Nine gene fragments were overexpressed specifically in the KBGem clone. Sequencing and BLAST results showed that three fragments represent cytochrome C oxidase (CCOX, respiration complex IV) subunit III (CCOX3). The cDNA microarray confirmed that the mRNAs of CCOX and ATP synthase subunits were upregulated in KBGem as compared to KBwt and KBHURs. The increase in CCOX1 protein and activity led to the increase of free ATP concentration, which is consistent with the gene expression profile of KBGem. Furthermore, the sensitivity to Gem could be reversed by sodium azide, a CCOX inhibitor. Following the treatment of sodium azide, the cellular accumulation of [3H]-Gem increased in a dose (of azide)-dependent manner, which is associated with increase of [3H]-Gem incorporation into DNA in KBGem. In summary, an increase of CCOX activity and free ATP level may reduce the transport, metabolism and DNA incorporation of Gem, resulting in Gem resistance.

Ribonucleotide reductase small subunit p53R2 facilitates p21 induction of G1 arrest under UV irradiation.

p53R2, which is one of the two known ribonucleotide reductase small subunits (the other being M2), is suggested to play an important role in supplying deoxynucleotide triphosphates (dNTP) for DNA repair during the G(1) or G(2) phase of the cell cycle. The ability of p53R2 to supply dNTPs for repairing DNA damages requires the presence of a functional p53 tumor suppressor. Here, we report in vivo physical interaction and colocalization of p53R2 and p21 before DNA damage. Mammalian two-hybrid assay further indicates that the amino acids 1 to 113 of p53R2 are critical for interacting with the NH(2)-terminal region (amino acids 1-93) of p21. The binding between p21 and p53R2 decreases inside the nucleus in response to UV, the time point of which corresponds to the increased binding of p21 with cyclin-dependent kinase-2 (Cdk2), and the decreased Cdk2 activity in the nucleus at G(1). Interestingly, p53R2 dissociates from p21 but facilitates the accumulation of p21 in the nucleus in response to UV. On the other hand, the ribonucleotide reductase activity increases at the corresponding time in response to UV. These data suggest a new function of p53R2 of cooperating with p21 during DNA repair at G(1) arrest.