Anion exchange in human serum transferrin N-lobe: a model study with variant His249Ala

The removal of Fe(III) from human serum transferrin by chelators is thought to proceed through intermediate species in which the chelator becomes associated with the metal center of the protein. The visible spectral shifts associated with the formation of such intermediates in the wild-type (WT) protein are too small for reliable kinetic data to be obtained. Therefore, studies were undertaken with the recombinant N-terminal lobe variant H249A, a variant showing more pronounced spectral changes. The kinetics of the synergistic anion-exchange reaction between nitrilotriacetate (NTA) and carbonate in variant H249A was studied by stopped-flow spectrophotometry as a model for this process in the WT protein. Anion exchange occurs by two pathways at pH 7.4 and 25 degrees C: an NTA-independent dissociative pathway to form a carbonate-free intermediate Fe-H249A (Eq. 1) that subsequently reacts with NTA (Eq. 2):and an NTA-dependent associative pathway (the major pathway) in which a quaternary Fe-H249A-(CO(3))(NTA) intermediate is formed (Eq. 3), which then decays to product (Eq. 4):The reverse reaction, where HCO(3)(-) exchanges for NTA, likewise follows these two pathways. The overall apparent equilibrium constant for formation of Fe-H249A-NTA from Fe-H249A-CO(3) is K'=442 at pH 7.4. The NTA complex is favored over the carbonate complex both kinetically and thermodynamically in the pH range 7.4-8.2.     

Consequences of different forms of conservation for large mammals in Tanzania: preliminary analyses

We examined the effects of protection from human activities and effects of tourist hunting on densities of 21 large mammal species in Tanzania. Aerial censuses revealed that mammal biomass per km² was highest in National Parks. Densities of nine ungulate species were significantly higher in National Parks and Game Reserves than in areas that permitted settlement; these tended to be the larger species favoured by poachers. The presence of tourist hunters had little positive or negative impact on ungulate densities, even for sought-after trophy species; limited ground censuses confirmed these results. Our analyses suggest that prohibition of human activity, backed up by on-site enforcement, maintains ungulate populations at relatively high densities, and challenge the idea that enforcement is only effective when spending is high.     

Recovery of Aging-Related Size Increase of Skin Epithelial Cells: In vivo Mouse and In vitro Human Study

The size increase of skin epithelial cells during aging is well-known. Here we demonstrate that treatment of aging cells with cytochalasin B substantially decreases cell size. This decrease was demonstrated on a mouse model and on human skin cells in vitro. Six nude mice were treated by topical application of cytochalasin B on skin of the dorsal left midsection for 140 days (the right side served as control for placebo treatment). An average decrease in cell size of 56±16% resulted. A reduction of cell size was also observed on primary human skin epithelial cells of different in vitro age (passages from 1 to 8). A cell strain obtained from a pool of 6 human subjects was treated with cytochalasin B in vitro for 12 hours. We observed a decrease in cell size that became statistically significant and reached 20–40% for cells of older passage (6–8 passages) whereas no substantial change was observed for younger cells. These results may be important for understanding the aging processes, and for cosmetic treatment of aging skin.     

Acute toxicity effects of ibuprofen on behaviour and haematological parameters of African catfish Clarias gariepinus (Burchell, 1822)

Indiscriminate discharge of pharmaceutical waste into the aquatic ecosystem may pose serious health challenges to aquatic biota. The effect of acute exposure to ibuprofen was evaluated using changes in behaviour and haematological parameters under static bio-assay method in Clarias gariepinus. Test specimens were exposed to acute concentrations of ibuprofen (0.28, 0.33, 0.38, 0.43 and 0.48 mg l^?1) for 24, 48, 72 and 96 h durations respectively. Behavioural and phenotypic changes were observed in surviving fish. There were significant (p < 0.05) concentration and duration-dependent increases in erythrocyte (RBC), haemoglobin (Hb), pack cell volume (PCV) and leukocytes (WBC) in treated fish compared to the control. Insignificant decreases (p > 0.05) in mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were observed in treated fish compared to the control. Ibuprofen elicited dose and duration- dependent decrease in neutrophil counts with the decreases being significant (p < 0.05) in the higher doses of 0.43 and 0.48 mg l^?1. Ibuprofen did not elicit any significant changes in monocytes, basophils and eosinophils. Changes observed in this study showed that ibuprofen negatively affected the health of the fish and we recommend that discharge of ibuprofen into the aquatic environment should be monitored and controlled.     

Biochemical markers of ongoing joint damage in rheumatoid arthritis - current and future applications, limitations and opportunities

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease associated with potentially debilitating joint inflammation, as well as altered skeletal bone metabolism and co-morbid conditions. Early diagnosis and aggressive treatment to control disease activity offers the highest likelihood of preserving function and preventing disability. Joint inflammation is characterized by synovitis, osteitis, and/or peri-articular osteopenia, often accompanied by development of subchondral bone erosions, as well as progressive joint space narrowing. Biochemical markers of joint cartilage and bone degradation may enable timely detection and assessment of ongoing joint damage, and their use in facilitating treatment strategies is under investigation. Early detection of joint damage may be assisted by the characterization of biochemical markers that identify patients whose joint damage is progressing rapidly and who are thus most in need of aggressive treatment, and that, alone or in combination, identify those individuals who are likely to respond best to a potential treatment, both in terms of limiting joint damage and relieving symptoms. The aims of this review are to describe currently available biochemical markers of joint metabolism in relation to the pathobiology of joint damage and systemic bone loss in RA; to assess the limitations of, and need for additional, novel biochemical markers in RA and other rheumatic diseases, and the strategies used for assay development; and to examine the feasibility of advancement of personalized health care using biochemical markers to select therapeutic agents to which a patient is most likely to respond.     

Antigen-specific CD8+ T cells and the development of central memory during Mycobacterium tuberculosis infection

Whether true memory T cells develop in the face of chronic infection such as tuberculosis remains controversial. To address this question, we studied CD8+ T cells specific for the Mycobacterium tuberculosis ESAT6-related Ags TB10.3 and TB10.4. The shared epitope TB10.3/10.4(20-28) is presented by H-2 K(d), and 20-30% of the CD8+ T cells in the lungs of chronically infected mice are specific for this Ag following respiratory infection with M. tuberculosis. These TB10.3/10.4(20-28)-specific CD8+ T cells produce IFN-gamma and TNF and express CD107 on their cell surface, which indicates their likely role as CTL in vivo. Nearly all of the Ag-specific CD8+ T cells in the lungs of chronically infected mice had a T effector cell phenotype based on their low expression of CD62L and CD45RB. In contrast, a population of TB10.3/10.4(20-28)-specific CD8+ T cells was identified in the lymphoid organs that express high levels of CD62L and CD45RB. Antibiotic treatment to resolve the infection led to a contraction of the Ag-specific CD8+ T cell population and was accompanied by an increase in the proportion of CD8+ T cells with a central memory phenotype. Finally, challenge of memory-immune mice with M. tuberculosis was accompanied by significant expansion of TB10.3/10.4(20-28)-specific CD8+ T cells, which suggests that these cells are in fact functional memory T cells.     

Development of restoration techniques for Hawaiian thrushes: Collection of wild eggs,artificial incubation,hand‐rearing,captive‐breeding,and re‐introduction to the wild

From 1995 to 1999, two species of endemic Hawaiian thrushes, `Oma`o (Myadestes obscurus) and Puaiohi (M. palmeri), were captive‐reared and re‐introduced into their historic range in Hawai`i by The Peregrine Fund, in collaboration with the U.S. Geological Survey–Biological Resources Division (BRD) and the Hawai`i State Department of Land and Natural Resources. This paper describes the management techniques that were developed (collection of wild eggs, artificial incubation, hand‐rearing, captive propagation, and release) with the non‐endangered surrogate species, the `Oma`o; techniques that are now being used for recovery of the endangered Puaiohi. In 1995 and 1996, 29 viable `Oma`o eggs were collected from the wild. Of 27 chicks hatched, 25 were hand‐reared and released into Pu`u Wa`awa`a Wildlife Reserve. Using the techniques developed for the `Oma`o, a captive propagation and release program was initiated in 1996 to aid the recovery of the endangered Puaiohi. Fifteen viable Puaiohi eggs were collected from the wild (1996–1997) to establish a captive breeding flock to produce birds for re‐introduction. These Puaiohi reproduced for the first time in captivity in 1998 (total Puaiohi chicks reared in captivity 1996–1998 = 41). In 1999, 14 captive‐bred Puaiohi were re‐introduced into the Alaka`i Swamp, Kaua`i. These captive‐bred birds reproduced and fledged seven chicks in the wild after release. This is the first endangered passerine recovery program using this broad spectrum of management techniques (collection of wild eggs, artificial incubation, hand‐rearing, captive‐breeding, and release) in which re‐introduced birds survived and bred in the wild. Long‐term population monitoring will be published separately [BRD, in preparation]. Zoo Biol 19:263–277, 2000. © 2000 Wiley‐Liss, Inc.     

Mutations at the histidine 249 ligand profoundly alter the spectral and iron-binding properties of human serum transferrin N-lobe

Human serum transferrin is an iron-binding and -transport protein which carries iron from the blood stream into various cells. Iron is held in two deep clefts located in the N- and C-lobes by coordinating to four amino acid ligands, Asp 63, Tyr 95, Tyr 188, and His 249 (N-lobe numbering), and to two oxygens from carbonate. We have previously reported the effect on the iron-binding properties of the N-lobe following mutation of the ligands Asp 63, Tyr 95, and Tyr 188. Here we report the profound functional changes which result from mutating His 249 to Ala, Glu, or Gln. The results are consistent with studies done in lactoferrin which showed that the histidine ligand is critical for the stability of the iron-binding site [H. Nicholson, B. F. Anderson, T. Bland, S. C. Shewry, J. W. Tweedie, and E. N. Baker (1997) Biochemistry 36, 341-346]. In the mutant H249A, the histidine ligand is disabled, resulting in a dramatic reduction in the kinetic stability of the protein toward loss of iron. The H249E mutant releases iron three times faster than wild-type protein but shows significant changes in both EPR spectra and the binding of anion. This appears to be the net effect of the metal ligand substitution from a neutral histidine residue to a negative glutamate residue and the disruption of the "dilysine trigger" [MacGillivray, R. T. A., Bewley, M. C., Smith, C. A., He, Q.-Y., Mason, A. B., Woodworth, R. C., and Baker, E. N. (2000) Biochemistry 39, 1211-1216]. In the H249Q mutant, Gln 249 appears not to directly contact the iron, given the similarity in the spectroscopic properties and the lability of iron release of this mutant to the H249A mutant. Further evidence for this idea is provided by the preference of both the H249A and H249Q mutants for nitrilotriacetate rather than carbonate in binding iron, probably because NTA is able to provide a third ligation partner. An intermediate species has been identified during the kinetic interconversion between the NTA and carbonate complexes of the H249A mutant. Thus, mutation of the His 249 residue does not abolish iron binding to the transferrin N-lobe but leads to the appearance of novel iron-binding sites of varying structure and stability.