Catalysis by entropic effects: the action of cytidine deaminase on 5,6-dihydrocytidine

In neutral solution, 5,6-dihydrocytidine undergoes spontaneous deamination (k25 approximately 3.2 x 10(-5) s(-1)) much more rapidly than does cytidine (k25 approximately 3.0 x 10(-10) s(-1)), with a more favorable enthalpy of activation (DeltaDeltaH# = -8.7 kcal/mol) compensated by a less favorable entropy of activation (TDeltaDeltaS# = -1.8 kcal/mol at 25 degrees C). E. coli cytidine deaminase enhances the rate of deamination of 5,6-dihydrocytidine (kcat/k(non) = 4.4 x 10(5)) by enhancing the entropy of activation (DeltaDeltaH# = 0 kcal/mol; TDeltaDeltaS# = +7.6 kcal/mol, at 25 degrees C). Binding of the competitive inhibitor 3,4,5,6-tetrahydrouridine (THU), a stable analogue of 5,6-dihydrocytidine in the transition state for its deamination, is accompanied by a release of enthalpy (DeltaH = -7.1 kcal/mol, TDeltaDeltaS = +2.2 kcal/mol) that approaches the estimated enthalpy of binding of the actual substrate in the transition state for deamination of 5,6-dihydrocytidine (DeltaH = -8.1 kcal/mol, TDeltaDeltaS = +6.0 kcal/mol). Thus, the shortcomings of THU in capturing all of the binding affinity expected of an ideal transition-state analogue reflect a less favorable entropy of association. That difference may arise from the analogue's inability to displace a water molecule from the "leaving group site" at which ammonia is generated in the normal reaction. The effect on binding of removing the 4-OH group from the transition-state analogue THU, to form 3,4,5,6-tetrahydrozebularine (THZ) (DeltaDeltaH = -2.1 kcal/mol, TDeltaDeltaS = -4.4 kcal/mol), is mainly entropic, consistent with the inability of THZ to displace water from the "attacking group site". These results are consistent with earlier indications [Snider, M. J., and Wolfenden, R. (2001) Biochemistry 40, 11364] that site-bound water plays a prominent role in substrate activation and inhibitor binding by cytidine deaminase.     

Determination of the affinity of each component of a composite quaternary transition-state analogue complex of creatine kinase

Recombinant rabbit muscle creatine kinase (CK) was titrated with MgADP in 50 mM Bicine and 5 mM Mg(OAc)2, pH 8.3, at 30.0 degrees C by following a decrease in the protein's intrinsic fluorescence. In the presence of 50 mM NaOAc, but in the absence of added creatine or nitrate, MgADP has an apparent K(d) of 135 +/- 7 microM, and the total change in fluorescence on saturation (Delta%F) is 15.3 +/- 0.3%. Acetate was used as the anion in this experiment because it does not promote the formation of a CK.MgADP.anion.creatine transition-state analogue complex (TSAC) [Millner-White and Watts (1971) Biochem. J. 122, 727-740]. In the presence of 80 mM creatine, but no nitrate, the apparent K(d) for MgADP remains essentially unchanged at 132 +/- 10 microM, while Delta%F decreases slightly to 13.2 +/- 0.3%. In the presence of 10 mM nitrate, but no creatine, the apparent K(d) is once again essentially unchanged at 143 +/- 23 microM, but the Delta%F is markedly reduced to 4.2 +/- 0.2%. The presence of both 10 mM nitrate and 80 mM creatine during titration reduces the apparent K(d) for MgADP 10-fold to 13.7 +/- 0.7 microM, and Delta%F increases to 20.6 +/- 0.3%, strongly suggesting that the simultaneous presence of saturating levels of creatine and nitrate increases the affinity of CK for MgADP and promotes the formation of the enzyme*MgADP*nitrate*creatine TSAC. When the fluorescence of CK was titrated with MgADP in the presence of 80 mM creatine and fixed saturating concentrations of various anions, apparent K(d) values for MgADP of 132 +/- 10 microM, 25.2 +/- 1.3 microM, 18.8 +/- 0.9 microM, 13.7 +/- 0.7 microM, and 6.4 +/- 0.7 microM were observed as the anion was changed from acetate to formate to chloride to nitrate to nitrite, respectively. This is the same trend reported by Millner-White and Watts for the effectiveness of various monovalent anions in forming the CK.MgADP.anion.creatine TSAC. On titration of CK with MgADP in the presence of 80 mM creatine and various fixed concentrations of NaNO3, the apparent K(d) for MgADP decreases with increasing fixed concentrations of nitrate. A plot of the apparent K(d) for MgADP vs [NO3-] suggests a K(d) for nitrate from the TSAC of 0.39 +/- 0.07 mM. Similarly, titration with MgADP in the presence of 10 mM NaNO3 and various fixed concentrations of creatine gives a value of 0.9 +/- 0.4 mM for the dissociation of creatine from the TSAC. The data were used to calculate K(TDAC), the dissociation constant of the quaternary TSAC into its individual components, of 3 x 10(-10) M3. To our knowledge this is the first reported dissociation constant for a ternary or quaternary TSAC.     

Hematology and blood biochemistry in infant baboons (Papio hamadryas)

Although published normative reference standards for hematologic and clinical chemistry measures are available for adult baboons, their applicability to infants has not been addressed. We analyzed these measures in 110 infant baboons (55 females and 55 males) from a large breeding colony at the Southwest Regional Primate Research Center in San Antonio, Texas. The sample consists of olive baboons and olive/yellow baboon hybrids, 1 week to 12 months of age. We produced cross-sectional reference values and examined the effects of age, sex, and subspecies on these variables. Hematology reference ranges for infant baboons are similar to, but wider than, those for adults. Reference ranges for blood biochemistry measures are generally more dissimilar to adults, indicating that for many variables, reference ranges for adult baboons are not adequate for infants. Although sex and subspecies differences are rare, age accounts for more than 10% of the variance in many of the variables.     

Identification and characterization of a potent, selective, and orally active antagonist of the CC chemokine receptor-1

The CC chemokine receptor-1 (CCR1) is a prime therapeutic target for treating autoimmune diseases. Through high capacity screening followed by chemical optimization, we identified a novel non-peptide CCR1 antagonist, R-N-[5-chloro-2-[2-[4-[(4-fluorophenyl)methyl]-2-methyl-1-piperazinyl ]-2-oxoethoxy]phenyl]urea hydrochloric acid salt (BX 471). Competition binding studies revealed that BX 471 was able to displace the CCR1 ligands macrophage inflammatory protein-1alpha (MIP-1alpha), RANTES, and monocyte chemotactic protein-3 (MCP-3) with high affinity (K(i) ranged from 1 nm to 5.5 nm). BX 471 was a potent functional antagonist based on its ability to inhibit a number of CCR1-mediated effects including Ca(2+) mobilization, increase in extracellular acidification rate, CD11b expression, and leukocyte migration. BX 471 demonstrated a greater than 10,000-fold selectivity for CCR1 compared with 28 G-protein-coupled receptors. Pharmacokinetic studies demonstrated that BX 471 was orally active with a bioavailability of 60% in dogs. Furthermore, BX 471 effectively reduces disease in a rat experimental allergic encephalomyelitis model of multiple sclerosis. This study is the first to demonstrate that a non-peptide chemokine receptor antagonist is efficacious in an animal model of an autoimmune disease. In summary, we have identified a potent, selective, and orally available CCR1 antagonist that may be useful in the treatment of chronic inflammatory diseases.     

Psychological Distress, Depression, Anxiety, and Burnout among International Humanitarian Aid Workers: A Longitudinal Study

Background

International humanitarian aid workers providing care in emergencies are subjected to numerous chronic and traumatic stressors.

Objectives

To examine consequences of such experiences on aid workers'' mental health and how the impact is influenced by moderating variables.

Methodology

We conducted a longitudinal study in a sample of international non-governmental organizations. Study outcomes included anxiety, depression, burnout, and life and job satisfaction. We performed bivariate regression analyses at three time points. We fitted generalized estimating equation multivariable regression models for the longitudinal analyses.

Results

Study participants from 19 NGOs were assessed at three time points: 212 participated at pre-deployment; 169 (80%) post-deployment; and 154 (73%) within 3–6 months after deployment. Prior to deployment, 12 (3.8%) participants reported anxiety symptoms, compared to 20 (11.8%) at post-deployment (p = 0·0027); 22 (10.4%) reported depression symptoms, compared to 33 (19.5%) at post-deployment (p = 0·0117) and 31 (20.1%) at follow-up (p = .00083). History of mental illness (adjusted odds ratio [AOR] 4.2; 95% confidence interval [CI] 1·45–12·50) contributed to an increased risk for anxiety. The experience of extraordinary stress was a contributor to increased risk for burnout depersonalization (AOR 1.5; 95% CI 1.17–1.83). Higher levels of chronic stress exposure during deployment were contributors to an increased risk for depression (AOR 1·1; 95% CI 1·02–1.20) comparing post- versus pre-deployment, and increased risk for burnout emotional exhaustion (AOR 1.1; 95% CI 1.04–1.19). Social support was associated with lower levels of depression (AOR 0·9; 95% CI 0·84–0·95), psychological distress (AOR = 0.9; [CI] 0.85–0.97), burnout lack of personal accomplishment (AOR 0·95; 95% CI 0·91–0·98), and greater life satisfaction (p = 0.0213).

Conclusions

When recruiting and preparing aid workers for deployment, organizations should consider history of mental illness and take steps to decrease chronic stressors, and strengthen social support networks.     

Early intestinal Th1 inflammation and mucosal T cell recruitment during acute graft-versus-host reaction

Little is understood about the earliest cytokine responses and the role(s) of donor CD4 T cells in the intestine during the induced graft-vs-host reaction (GVHR). We investigated the activation and mucosal homing phenotype of the donor CD4 cells and the kinetics of cytokine responses within the intestine and associated lymphoid tissues during early GVHR. Significant frequencies of donor CD4 cells accumulated within recipient Peyer's patches (PP), mesenteric lymph nodes (MLN), lamina propria (LP), and spleen (SP), during the first 9 days of GVHR. Many donor CD4 cells in SP, MLN, and LP expressed CD44 and also expressed de novo the mucosal homing integrin alpha(4)beta(7) (LPAM-1). A large IFN-gamma response occurred by day 3 in cells from PP and MLN, but much later (day 9) in SP and LP cells. IL-10 production by SP and MLN cells was elevated initially but declined substantially by day 9. IL-4 production by SP, MLN, and PP cells was low on day 3 and showed gradual decline in LP by day 9. IL-5 production by LP cells gradually increased in direct contrast to IL-5 production by MLN cells. The MLN CD4 cells showed the most dynamic changes, with high numbers of activated/effector donor CD4 cells and altered cytokine production consistent with a developing Th1 response. The IFN-gamma responses in PP and MLN preceded that of the SP, suggesting an intestinal origin for some Th1 effector cells in GVHR. Donor CD4 T cells apparently acquire the ability to home to the LP during early GVHR.     

UK-73,093: A non-peptide neurotensin receptor antagonist

UK-73,093 was identified in a screening program as a compound able to displace [³H]-neurotensin from its bovine brain receptor. We describe the discovery of this compound, species differences in receptor affinity and its characterization as a functional neurotensin antogonist in vitro and in vivo.     

Genotypic differences in thermotolerance are dependent upon prestress capacity for antioxidant protection of the photosynthetic apparatus in Gossypium hirsutum

Numerous studies have illustrated the need for antioxidant enzymes in acquired photosynthetic thermotolerance, but information on their possible role in promoting innate thermotolerance is lacking. We investigated the hypothesis that genotypic differences in source leaf photosynthetic thermostability would be dependent upon prestress capacity for antioxidant protection of the photosynthetic apparatus in Gossypium hirsutum. To test this hypothesis, thermosensitive (cv. ST4554) and reportedly thermotolerant (cv. VH260) G. hirsutum plants were exposed to control (30/20°C) or high‐day temperature (38/20°C) conditions during flowering and source leaf gas exchange, chlorophyll content and maximum photochemical efficiency (F_v/F_m) were measured for each treatment. The relationship between source leaf thermostability and prestress antioxidant capacity was quantified by monitoring the actual quantum yield response of photosystem II (PSII) (Φ_PSII) to a range of temperatures for both cultivars grown under the control temperature regime and measuring antioxidant enzyme activity for those same leaves. VH260 was more thermotolerant than ST4554 as evidenced by photosynthesis and F_v/F_m being significantly lower under high temperature for ST4554 but not VH260. Under identical growth conditions, VH260 had significantly higher optimal and threshold temperatures for Φ_PSII and glutathione reductase (GR; EC 1.8.1.7) activity than ST4554, and innate threshold temperature was dependent upon endogenous GR and superoxide dismutase (SOD; EC 1.15.1.1) activity. We conclude that maintaining a sufficient antioxidant enzyme pool prior to heat stress is an innate mechanism for coping with rapid leaf temperature increases that commonly occur under field conditions.