Homologous npdGI Genes in 2,4-Dinitrophenol- and 4-Nitrophenol-Degrading Rhodococcus spp.

Rhodococcus (opacus) erythropolis HL PM-1 grows on 2,4,6-trinitrophenol or 2,4-dinitrophenol (2,4-DNP) as a sole nitrogen source. The NADPH-dependent F₄₂₀ reductase (NDFR; encoded by npdG) and the hydride transferase II (HTII; encoded by npdI) of the strain were previously shown to convert both nitrophenols to their respective hydride Meisenheimer complexes. In the present study, npdG and npdI were amplified from six 2,4-DNP degrading Rhodococcus spp. The genes showed sequence similarities of 86 to 99% to the respective npd genes of strain HL PM-1. Heterologous expression of the npdG and npdI genes showed that they were involved in 2,4-DNP degradation. Sequence analyses of both the NDFRs and the HTIIs revealed conserved domains which may be involved in binding of NADPH or F₄₂₀. Phylogenetic analyses of the NDFRs showed that they represent a new group in the family of F₄₂₀-dependent NADPH reductases. Phylogenetic analyses of the HTIIs revealed that they form an additional group in the family of F₄₂₀-dependent glucose-6-phosphate dehydrogenases and F₄₂₀-dependent N⁵,N¹⁰-methylenetetrahydromethanopterin reductases. Thus, the NDFRs and the HTIIs may each represent a novel group of F₄₂₀-dependent enzymes involved in catabolism.     

Long-lasting adenovirus transgene expression in mice through neonatal intrathymic tolerance induction without the use of immunosuppression.

The major barrier to the clinical application of adenovirus gene therapy for diseases that require stable transgene expression is the immunogenicity of recombinant adenovirus, which ordinarily limits the duration of its effects to a period of about 2 weeks. We postulated that tolerance to adenovirus could be induced and transgene expression could be prolonged if T lymphocytes underwent thymic selection in the presence of adenovirus antigens. Mice were inoculated in the thymus with a recombinant adenovirus containing the lacZ marker gene during the neonatal period at a time before T-cell maturation had occurred. When the virus was administered intravenously to these mice in adulthood, they were found to have an impaired adenovirus-specific cytotoxic T-lymphocyte response which allowed prolonged hepatic lacZ expression, for up to 260 days. The ability to achieve unresponsiveness to a recombinant adenovirus after inoculation of the thymus in neonates extends the paradigm of intrathymic tolerance induction. Furthermore, this model will enable the study of stable adenovirus transgene expression in vivo without the use of immunosuppression and ultimately may have clinical utility.     

Ragweed sensitization alters pulmonary vascular responses to bronchoprovocation in beagle dogs

Theodorou, Andreas, Natalie Weger, Kathleen Kunke, KyooRhee, David Bice, Bruce Muggenberg, and Richard Lemen. Ragweed sensitization alters pulmonary vascular responses to bronchoprovocation in beagle dogs. J. Appl. Physiol.83(3): 912-917, 1997.In ragweed (RW)-sensitized beagle dogs, wetested the hypothesis that reactivity of the pulmonary vasculature wasenhanced with aerosolized histamine (Hist) and RW. Seven dogs wereneonatally sensitized with repeated intraperitoneal RW injections, and12 dogs were controls (Con). The dogs were anesthetizedwith intravenous chloralose, mechanically ventilated, and instrumentedwith femoral arterial and pulmonary artery catheters. Specific lungcompliance(CL_sp),specific lung conductance (G_sp),systemic vascular resistance index, and pulmonary vascular resistanceindex (PVRI) were measured before and after bronchoprovocation withHist and RW. After Hist inhalation (5 breaths of 30 mg/ml), both Conand RW dogs had significant (P < 0.05) decreases inCL_sp(51 ± 4 and 53 ± 5%, respectively) andG_sp (65 ± 5 and69 ± 3%, respectively), but only RW-sensitized dogs had asignificant increase in PVRI (38 ± 10%). After RW inhalation (60 breaths of 0.8 mg/ml), only RW-sensitized dogs had significant increases (62 ± 20%) in PVRI and decreases inG_sp (77 ± 4%) and CL_sp(65 ± 7%). We conclude that, compared with Con,RW-sensitized beagle dogs have increased pulmonary vasoconstrictiveresponses with Hist or RW inhalation.

     

Serotonin sensitive adenylate cyclase activity in monkey anterior limbic cortex: antagonism by molindone and other antipsychotic drugs.

Serotonin (5-HT) sensitive adenylate cyclase in monkey anterior limbic cortex homogenates was further characterized and the effects of antipsychotic drugs and 5-HT anatagonists investigated. Differences in time course for stimulation by agonists and in responsiveness to receptor anatagonists of 5-HT-and dopamine (DA)-stimulated activities, were observed. Also there was an additivity of 5-HT and DA at maximally effective concentrations. Classical 5-HT antagonists blocked the 5-HT sensitive adenylate cyclase with a rank order of potency: methiothepin > cyproheptadine > methysergide. These 5-HT antagonists also effectively inhibited DA sensitive adenylate cyclase. Most antipsychotic drugs tested antagonized 5-HT stimulated activity although these drugs exhibited greater efficacies in blocking DA stimulated activity. Exceptions were molindone which failed to antagonize DA sensitive adenylate cyclase but effectively blocked 5-HT sensitive cyclase and pipamperone which was inactive in both cyclase systems. Haloperidol was a more selective antagonist of the DA sensitive adenylate cyclase than were the other antipsychotic drugs tested.     

3H-Spiroperidol binding and dopamine-stimulated adenylate cyclase: evidence for multiple classes of receptors in primate brain regions.

Studies of displacement by agonist and antagonist drugs of ³H-spiroperidol binding in brain regions of $\text{Cebus}$ and rhesus monkeys revealed one type of receptor in caudate nucleus and a second type of receptor in both frontal and anterior limbic cortex. Compared with caudate, the cortical regions were more sensitive to clozapine and loxapine, equally sensitive to fluphenazine and relatively less sensitive to haloperidol. Also, the cortical regions were insensitive to molindone. Parallel studies using the dopamine-stimulated adenylate cyclase have demonstrated three types of receptors, one in caudate, a second in frontal cortex, and a third in anterior limbic cortex. In each region studied, relative sensitivities to drug using these two methods differed, suggesting that in each of these regions only a relatively small portion of ³H-spiroperidol receptors are coupled to adenylate cyclase.     

Restriction of PGK-B to spermatogenic cells: Evidence from experimentally cryptorchid mice

The hypothesis that PGK-B, like LDH-C₄, is restricted to spermatogenic cells was explored by examining isozyme patterns in testes from mice depleted of germinal cells by surgical cryptorchism. In experimentally cryptorchized C57BL/10Sn males, decline in PGK-B activity paralleled decline in LDH-C₄ activity and was correlated with degeneration of spermatocytes, spermatids, and spermatozoa. Trace amounts of these sperm isozymes found in cryptorchid testes after the depletion of maturing germ cells probably came from degenerated spermatids and spermatocytes and not from somatic testicular cells.     

Division of labor within the DNA damage tolerance system reveals non-epistatic and clinically actionable targets for precision cancer medicine

Crosslink repair depends on the Fanconi anemia pathway and translesion synthesis polymerases that replicate over unhooked crosslinks. Translesion synthesis is regulated via ubiquitination of PCNA, and independently via translesion synthesis polymerase REV1. The division of labor between PCNA-ubiquitination and REV1 in interstrand crosslink repair is unclear. Inhibition of either of these pathways has been proposed as a strategy to increase cytotoxicity of platinating agents in cancer treatment. Here, we defined the importance of PCNA-ubiquitination and REV1 for DNA in mammalian ICL repair. In mice, loss of PCNA-ubiquitination, but not REV1, resulted in germ cell defects and hypersensitivity to cisplatin. Loss of PCNA-ubiquitination, but not REV1 sensitized mammalian cancer cell lines to cisplatin. We identify polymerase Kappa as essential in tolerating DNA damage-induced lesions, in particular cisplatin lesions. Polk-deficient tumors were controlled by cisplatin treatment and it significantly delayed tumor outgrowth and increased overall survival of tumor bearing mice. Our results indicate that PCNA-ubiquitination and REV1 play distinct roles in DNA damage tolerance. Moreover, our results highlight POLK as a critical TLS polymerase in tolerating multiple genotoxic lesions, including cisplatin lesions. The relative frequent loss of Polk in cancers indicates an exploitable vulnerability for precision cancer medicine.     

Protective Role of miR-34c in Hypoxia by Activating Autophagy through BCL2 Repression

Hypoxia leads to significant cellular stress that has diverse pathological consequences such as cardiovascular diseases and cancers. MicroRNAs (miRNAs) are one of regulators of the adaptive pathway in hypoxia. We identified a hypoxia-induced miRNA, miR-34c, that was significantly upregulated in hypoxic human umbilical cord vein endothelial cells (HUVECs) and in murine blood vessels on day 3 of hindlimb ischemia (HLI). miR-34c directly inhibited BCL2 expression, acting as a toggle switch between apoptosis and autophagy in vitro and in vivo. BCL2 repression by miR-34c activated autophagy, which was evaluated by the expression of LC3-II. Overexpression of miR-34c inhibited apoptosis in HUVEC as well as in a murine model of HLI, and increased cell viability in HUVEC. Importantly, the number of viable cells in the blood vessels following HLI was increased by miR-34c overexpression. Collectively, our findings show that miR-34c plays a protective role in hypoxia, suggesting a novel therapeutic target for hypoxic and ischemic diseases in the blood vessels.     

Does Isolated Unilateral Hip or Knee Osteoarthritis Lead to Adverse Changes in Extremity Composition?

BackgroundWhile muscle atrophy is a function of normal aging, loss of muscle in the setting of hip and knee osteoarthritis (OA) has been observed using radiographic studies. There is limited data available regarding changes in extremity composition using bioimpedance (BIA). The purpose of this study was to assess the changes in extremity composition in patients with isolated, unilateral hip or knee OA using BIA.MethodsPatients presenting to our institution’s adult reconstruction clinic from February 2020 to April 2021 were retrospectively reviewed to identify those with isolated, unilateral hip and knee OA. The InBody 770 Body Composition Analyzer (InBody USA, Cerritos, California) was used to perform a complete body composition assessment, per protocol. Lean extremity mass (LEM), fat mass (FM), intracellular water (ICW), extremity body water (EBW = ICW + extracellular water (ECW)) and phase angle (PA) were determined. Differences between the affected (OA) and unaffected (no OA) extremities were compared using t-tests.Results38 patients had isolated hip OA. The mean age was 60.8 (±11.7) years, mean BMI was 31.7 (±6.8) kg/m2, and 39.5% were female. LEM, FM, EBW, ICW, and PA were significantly decreased in the hip OA extremity (LEM: 20.0 vs. 20.4 kg, p=0.0008, FM: 8.8 vs. 8.9 kg, p=0.0049, EBW: 15.7 vs 16.0, p=0.0011, ICW: 9.5 vs. 9.7 L, p=0.0004, PA: 4.5 vs 4.9º, p<0.0001). There were 25 patients with isolated knee OA. Mean age was 62.8 (±11.3) years, mean BMI was 33.6 (±6.9) kg/m2, and 52.0% were female. FM and PA were significantly lower in the knee OA extremity (11.3 vs 11.4 kg, p=0.0291, 4.5 vs 4.9º, p<0.0001). There were no significant differences in LEM, EBW, and ICW between the knee OA extremity and the unaffected extremity.ConclusionPatients with isolated, unilateral hip OA had decreased LEM, FM, EBW, and ICW in the affected extremity. Both unilateral hip and knee OA was associated with decreased PA, suggestive of greater underlying dysfunction in muscle or cellular performance. Further study is needed to better define when these abnormalities develop, how they progress over time, and the impact of targeted interventions in reversing these changes. Level of Evidence: III