A steady-state and pre-steady-state kinetics study of the tungstoenzyme formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus

Formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus is a homotetrameric protein with one tungstodipterin and one [4Fe–4S] cubane per 69-kDa subunit. The enzyme kinetics have been studied under steady-state conditions at 80 °C and pre-steady state conditions at 50 °C, in the latter case via monitoring of the relatively weak (ε ≈ 2 mM−1 cm−1) optical spectrum of the tungsten cofactor. The steady-state data are consistent with a substrate substituted-enzyme mechanism for three substrates (formaldehyde plus two ferredoxin molecules). The KM value for free formaldehyde (21 μM) with ferredoxin as an electron acceptor is approximately 3 times lower than the value measured when benzyl viologen is used as an acceptor. The KM of ferredoxin (14 μM) is an order of magnitude less than previously reported values. An explanation for this discrepancy may be the fact that high concentrations of substrate are inhibitory and denaturing to the enzyme. Pre-steady-state difference spectra reveal peak shifts and a lack of isosbestic points, an indication that several processes happen in the first seconds of the reaction. Two fast processes (kobs1 = 4.7 s−1, kobs2 = 1.9 s−1) are interpreted as oxidation of the substrate followed by rearrangement of the active site. Alternatively, these processes could be the entry/binding of the substrate followed by its oxidation. The release of the product and the electron shuffling over the tungsten and iron–sulfur center in the absence of an external electron acceptor are slower (kobs3 = 6.10 × 10−2 s−1, kobs4 = 2.18 × 10−2 s−1). On the basis of these results in combination with results from previous electron paramagnetic resonance studies an activation route plus catalytic redox cycle is proposed.
Introduction
Tungstoenzymes and molybdoenzymes typically catalyze the generic n = 2 reaction: R + H2O ↔ RO + 2[H], where R is either an oxoanion (e.g., sulfite) or a relatively small organic metabolite (e.g., an aldehyde). On the basis of structural (cofactor and protein) and functional properties molybdoenzymes are classified in three families: the sulfite oxidase family, the xanthine oxidase family, and the dimethyl sulfoxide reductase (DMSOR) family [1]. The latter also encompasses a limited number of tungsten-containing formate dehydrogenases. The majority of the tungstoenzymes, however, cannot be classified in any of these three families; they form a separate family of aldehyde oxidoreductases (AORs) together with a few apparently less common AOR molybdoenzyme members [2]. The mechanism of action of molybdoenzymes has been extensively studied for several decades, especially for xanthine oxidase [3]. In contrast, attempts to understand the reaction mechanism of tungstoenzymes, in particular for the members of the AOR family, are more recent and of limited extent, although the available structural information on tungstoenzymes and that on molybdoenzymes are comparable. The present study leads to the first proposal of a complete catalytic cycle of the AOR family member formaldehyde oxidoreductase (FOR).
The cofactor common to the enzymes of all four families consists of a single Mo(VI/IV) or W(VI/IV) ion coordinated by the dithioleno sulfurs of one or two three-ring pterin ligands sometimes with an additional nucleotide attached through a phosphoester bond [4]. All tungsten systems appear to be nonnucleotide dipterin systems with an additional magnesium phosphate coordination between the pterins [5, 6]. The coordination number of the Mo or W is 4–7; extra ligands are provided by an amino acid side chain and/or by O,S of a small, nonprotein ligand. The vast majority of the Mo/W enzymes have additional prosthetic groups for interaction with a second substrate (e.g., NADH) and/or for electron transfer with a protein natural redox partner (e.g., ferredoxin). These heme, flavin, and/or iron–sulfur prosthetic groups are strongly colored to the extent that optical monitoring of the relatively weakly colored (see below) metallopterin has thus far not been tried for mechanistic studies of these complex enzymes. Electron paramagnetic resonance (EPR) spectroscopy is not a competitive alternative, because the only EPR-detectable oxidation state, Mo(V) or W(V), does not appear to be a competent intermediate in the primary, n = 2 reaction.
A small group of molybdoenzymes, exemplified by DMSOR, are exceptional because these proteins contain no other prosthetic groups in addition to the active-site molybdopterin. Furthermore, substitution of W for Mo in Rhodobacter capsulatus DMSOR affords an active enzyme with an unmodified 3D structure. The Mo(VI) and W(VI) metallopterin UV–vis spectra of this enzyme have been reported to exhibit several broad peaks in the visible and near-UV region with extinction coefficients of the order of ε ≈ 2 mM−1 cm−1 [7], and the molybdopterin absorption has been used as monitor in a pre-steady-state kinetics study [8].
Following the DMSOR, the next step up in optical complexity is found in the AOR family of tungstoenzymes, which all contain—in addition to the tungstopterin active site—a single [4Fe–4S](2+;1+) cluster for electron transfer. The visible spectrum of the iron–sulfur cubane consists of a single broad line at approximately 400–430 nm, which is not only a simpler pattern to that found for [2Fe–2S] clusters (e.g., in the xanthine oxidase family of molybdoenzymes), but it also has a significantly smaller extinction coefficient than flavin or heme prosthetic groups, and so chances are more favorable for monitoring the weaker absorptions from the metallopterin cofactor in these complex enzymes. The homotetrameric (4 × 69 kDa) Pyrococcus furiosus FOR is a member of this family, and we describe here a study of this enzyme as the first example of pre-steady-state kinetics research of a complex, group-6 metalloenzyme using the color of the active center as the monitor. Our choice of FOR was also indicated by the availability of a 3D structure, which was the basis of an initial proposal of part of the enzyme’s working mechanism [6], and was further stimulated by the unusual behavior of the enzyme’s substrate in aqueous solution [9, 10].
Materials and methods
P. furiosus (DSM 3638) was grown at 90 °C under anaerobic conditions with starch as a carbon source, as previously described [11]. Cells were broken by osmotic shock, diluting with 5 vol 30 mM tris(hydroxymethyl)aminomethane (Tris)/HCl, pH 8.0, containing, 0.1 mg ml−1 DNase I, 0.1 mg ml−1 RNase, and 1 mM cysteine. A cell-free extract was obtained as the supernatant after 30 min centrifugation at 15,000g.
“High-activity” FOR was purified as reported previously [9, 10]. Samples were anaerobically purified in 20 mM Tris/HCl buffer, pH 8.0. An extra column of (diethylamino)ethyl (2.0 cm × 10 cm) was equilibrated with 20 mM piperazine/diethanesulfonic acid buffer, pH 6.4. A 100-ml gradient was used from 0 to 0.5 M NaCl. Ferredoxin was purified as reported previously [12]. Samples were anaerobically purified in 20 mM Tris/HCl buffer, pH 8.0. Protein concentration was determined using the bicinchoninic acid method using bovine serum albumin as the standard. Subunit molecular weight and degree of purity were determined with sodium dodecyl sulfate polyacrylamide gel electrophoresis using a Phast system (GE Healthcare).
FOR activity was routinely assayed at 80 °C, under anaerobic conditions, with formaldehyde, or glutardialdehyde, as the substrate and 3 mM benzyl viologen as the electron acceptor in 50 mM 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (Epps) buffer, pH 8.4.
Steady-state kinetics of FOR were determined by activity measurements in triplicate in an optical assay with different concentrations of formaldehyde (0.5, 1, 2, 5, 10, 25, 50 mM) as a substrate. Benzyl viologen (ε600 = 10.6 mM−1 cm−1 [13]) or ferredoxin (ε400 = 17.0 mM−1 cm−1 [14]) were used as an electron acceptor at various concentrations in 50 mM Epps buffer, pH 8.4, and  80 °C, where one unit is defined as 2 μmol electron acceptor reduced per minute. The concentrations of benzyl viologen and ferredoxin were, respectively, 50–3,000 and 10–200 μM, and the ferredoxin concentration is per dimer [15].
The UV–vis spectrum was recorded with a Hewlett-Packard 8452A diode-array spectrophotometer. The optimal excitation wavelength for fluorescence measurements was obtained using a Shimadzu RF-5001PC spectrofluorophotometer. Pre-steady-state studies were carried out under anaerobiosis in 50 mM Epps buffer, pH 8.4, at 50 °C with formaldehyde (25 mM) and formaldehyde-d2 (25 mM) as a substrate using an SX.18 MV stopped-flow apparatus equipped with a sequential mixing capability and a diode-array rapid-scan detection system (Applied Photophysics). The UV–vis data were obtained by recording 400 spectra at different time scales (16, 65 and 250 s). A spectrum obtained 2 ms after mixing was taken as the “oxidized” reference spectrum. Fluorescence data were obtained by exciting the samples at 280 nm, and the change in maximum fluorescence emission was measured at 340 nm.
Results
Steady-state kinetics
Steady-state kinetics studies at 80 °C were performed on the combinations FOR/formaldehyde/benzyl viologen and FOR/formaldehyde/ferredoxin. FOR catalyzes a two-electron transfer from formaldehyde to the one-electron acceptor benzyl viologen or ferredoxin. This results in a reaction with three substrates; one formaldehyde molecule and two benzyl viologen or two ferredoxin molecules. The possible mechanisms for this reaction are so-called enzyme-substitution mechanisms: the triple-transfer mechanism or the concerted-substitution mechanism [16].
Triple-transfer mechanism:
Concerted-substitution mechanism: The equation for the reaction rate can be simplified, when substrates B and C (ferredoxin or viologen) are the same. The results have the same form as equations for the reaction rate of an enzyme with two substrates. The triple-transfer mechanism will transform into the substituted-enzyme mechanism for two substrates and the concerted-substitution mechanism will transform into the ternary-complex mechanism for two substrates [16]. The apparent KM values for the second and third substrates are identical, and the measured KM is twice the real KM of the second or third substrate [16, 17]. To predict whether the systems function according to a ternary-complex mechanism or a substituted-enzyme mechanism, one substrate was varied, while the other substrate concentration was held constant.
Substrate inhibition of one of the substrates is described by Eq. 3, where KsiB is the constant that defines the strength of the inhibition of substrate B with concentration b: Primary plots of the concentration of the inhibitor divided by the reaction rate (b/v) versus the concentration of the inhibitor (b) are parabolic and intersect at a common point on the b/v axis. The primary plots of the concentration of the noninhibiting substrate divided by the reaction rate (a/v) versus the concentration of the noninhibiting substrate (a) are linear and intersect at a positive value of the concentration of noninhibiting substrate [16]. A more complex situation occurs when both substrates show substrate inhibition. At low concentrations of both substrates the shapes of both curves are parabolic. Substrate inhibition by one substrate at low concentrations of the other provides strong positive evidence that the substituted-enzyme mechanism applies [16].
The plots of a/v versus a and b/v versus b, with substrate A is formaldehyde and B is benzyl viologen or ferredoxin, are presented in Figs. 1 and 2. All intersection points are situated within experimental error at positive values of the substrates. The parabolic shape of all the figures indicates, as stated above, substrate inhibition by both substrates. All four graphs in Figs. 1 and 2 are consistent with a substrate substituted-enzyme mechanism. Fig. 1Primary plots for Pyrococcus furiosus formaldehyde oxidoreductase with formaldehyde as a substrate and benzyl viologen as an electron acceptor. Formaldehyde was used in concentrations of 1, 2, 5, 10, 25, and 50 mM. Benzyl viologen was used at concentrations of 50, 100, 500, 1,000, 2,000, and 3,000 μM. Each data point is the average value from three measurementsFig. 2Primary plots for P. furiosus formaldehyde oxidoreductase with formaldehyde as a an substrate and ferredoxin as electron acceptor. Formaldehyde was used at concentrations of 0.5, 2, 5, 10, 25, and 50 mM. Ferredoxin was used at concentrations of 10, 20, 40, 80, 100, and 200 μM. Each data point is the average value from three measurements
One-substrate Michaelis–Menten kinetics was used to fit the data and to calculate KMapp and Vmaxapp at the different concentrations of substrate and acceptor. To calculate the KM and the Vmax of the different substrates the KMapp and vapp data were fitted using the equations for the apparent values of the Michaelis–Menten parameters for the substituted-enzyme mechanism [16]: with equivalent expressions for substrate A.
The KM and Vmax values of formaldehyde measured with excess benzyl viologen as an electron acceptor (Table 1) are in agreement with previous measurements [10, 18]. The KM value for formaldehyde (3.8 mM) with ferredoxin as an electron acceptor is approximately 3 times less than when benzyl viologen is used as the acceptor. The conversion rate of formaldehyde with ferredoxin as the electron acceptor is 6 times less than when benzyl viologen is used as the acceptor. The values for KM are unusually high for a physiological substrate. We have previously provided experimental evidence indicating that only “free” formaldehyde (i.e., the nonhydrated form which is 0.2% at 80 °C) is a substrate for FOR [10]. The corrected KM values for free formaldehyde with acceptor benzyl viologen or ferredoxin are, respectively, 71 and 21 μM. Note that Vmax decreases by a factor of 4 when the temperature is lowered from 80 to 20 °C, and that KM for free formaldehyde is temperature-independent [10].
Table 1
Catalytic properties of formaldehyde oxidoreductase determined at 80 °C with formaldehyde as the substrate and ferredoxin or benzyl viologen as the electron acceptor
Electron acceptor
Benzyl viologen
Ferredoxin
KM total formaldehyde (mM)
13 ± 2
3.8 ± 0.5
KM free formaldehyde (μM)
71 ± 10
21 ± 3
Vmax formaldehyde (s−1)
28 ± 1a
4.5 ± 0.5
KM electron acceptor (μM)
73 ± 19
14 ± 7
Vmax electron acceptor (s−1)
32 ± 2
6.2 ± 3.2
KM glutardialdehyde (mM)
12 ± 2
Vmax glutardialdehyde (s−1)
13 ± 1
The ratio of activities at 80 °C compared with 50 °C is 2.8 under standard conditions (see “Materials and methods”) with 50 mM total formaldehyde
aIn our previous work [10] Vmax = 54 s−1 was reported on a per tungsten atom basis; this is equal to 28 s−1 on a per protein molecule basis
Varying the formaldehyde concentration gives the KM and Vmax values for the electron acceptors. The KM value of benzyl viologen of 73 μM is comparable to values found in literature for other enzymes that react with benzyl viologen as an electron acceptor. The KM value of ferredoxin (14 μM) is, however, uncharacteristically low: previously reported literature values for FOR and AOR are 100 and 200 μM, respectively [9, 19]. An explanation for this discrepancy may be found in the fact that high concentrations of substrate, up to 50 mM of total formaldehyde for FOR, have been used in previous studies to determine KM of ferredoxin. In the present work concentrations from 0 to 10 mM were used to determine KMapp and vapp. When activity was measured with high concentrations of formaldehyde (i.e., resulting in significant substrate inhibition) the apparent KM of ferredoxin increased to approach values found in the literature (data not shown).
In a previous study alternative substrates for FOR were reported with KM values of the order of a few millimoles per liter. The lowest KM value was found for glutardialdehyde (KM = 0.8 mM) with benzyl viologen as an electron acceptor [9]. We have been unable to reproduce these results. In our hands the KM for glutardialdehyde was determined to be 12 mM, i.e., approximately the same as the apparent KM for total formaldehyde.
Pre-steady-state kinetics
Pre-steady-state kinetics studies were performed on FOR (20 μM) and formaldehyde at 50 °C. Formaldehyde was used at a concentration of 25 mM (50 μM free formaldehyde). UV–vis spectra were recorded at 40- and at 160-ms intervals. At least two spectra per time point were averaged for noise reduction. In the spectrum of oxidized FOR the broad feature of the [4Fe–4S]2+ cluster dominates. However, close inspection reveals weak (εmax ≈ 1–2 mM−1 cm−1) absorptions at higher wavelengths similar to those reported for the single-cofactor DMSOR [20]. After subtraction of the spectrum at t = 2 ms, difference spectra were obtained (Fig. 3), at 0.1, 0.5, 1, 2, 5, 10, and 16 s, which reveal peak shifts and a lack of isosbestic points. Fig. 3Difference in absorbance versus wavelength measured in pre-steady-state experiments with formaldehyde as a substrate at times 0.1, 0.5, 1, and 2 s (a) and 2, 5, 10, and 16 s (b). The reference spectrum was taken at t = 2 ms. c UV–vis spectra of the oxidized and the substrate-reduced form (after 65-s incubation time) of P. furiosus formaldehyde oxidoreductase
From the difference spectra five different wavelengths were selected: 335, 390, 435, 595, and 695 nm. At 595 and 695 nm, approximately corresponding absorption bands can be found in the spectrum of oxidized DMSOR from R. capsulatus [8]. Amplitude versus time traces of the 40 and 160 ms spectra at the different wavelengths were combined. After 65 s the time traces for all the wavelengths essentially leveled off (Fig. 4). Data points were taken up to 250 s but no further significant changes were found (not shown). Fig. 4Stopped-flow UV–vis traces obtained for the reduction of P. furiosus formaldehyde oxidoreductase by formaldehyde at 335, 390, 435, 595, and 695 nm
The major changes at the different wavelengths occur in the first seconds. Three phases starting at different times can be identified in Fig. 5a. The first phase starts at or before t ≈ 20 ms; the second and the third phases start at approximately 100 and 300 ms, respectively. Changes in the amplitude versus time traces at 390 and 435 nm start at or before 20 ms; the trace of the wavelength of 595 nm follows the trend of the 335 nm trace, with the difference that all phases start approximately 200 ms later. All data at the different wavelengths were globally simulated with a four-phase exponential least-squares fit (Table 2). Fig. 5Stopped-flow UV–vis traces plus fits on a logarithmic time scale obtained for the reduction of P. furiosus formaldehyde oxidoreductase by a formaldehyde or b deuterated formaldehyde at 335, 390, 435, 595, and 695 nm. c Stopped-flow fluorescence trace plus fit obtained for the reduction of P. furiosus formaldehyde oxidoreductase by formaldehyde at an excitation wavelength of 295 nm and an emission wavelength of 340 nm. d Stopped-flow UV–vis traces plus fits obtained for the binding of formate by P. furiosus formaldehyde oxidoreductase at 335, 390, 435, 595, and 695 nmTable 2Rate constants from pre-steady-state kinetics of formaldehyde oxidoreductase plus formaldehyde or deuterated formaldehyde or formate at 50 °CUV–visFluorescenceCH2OCD2OFormateCH2Ok1 (s−1)4.71.120.514.7k2 (s−1)1.90.942.00 × 10−21.9k3 (s−1)6.10 × 10−26.07 × 10−26.10 × 10−2k4 (s−1)2.18 × 10−22.26 × 10−2
A previous steady-state kinetics study on the substrate kinetic isotope effect of FOR showed a significant primary isotope effect for C–H/D bond breaking. The specific activity of FOR dropped by a factor of 3 when deuterated formaldehyde was used as a substrate [21]. In the present work the pre-steady-state kinetics of FOR with formaldehyde-d2 was determined in the same fashion as for the experiments with formaldehyde. The amplitude versus time traces at selected wavelengths of FOR incubated with formaldehyde-d2 resemble the traces of the different wavelengths when formaldehyde was used as the substrate; the phases are similar, only the duration and the onset of the changes of states are different. Again the data were fitted with a four-phase exponential fit (Fig. 5b; Table 2).
Tryptophan residues are excellent probes for protein conformational changes. In FOR a single tryptophan (Trp441) can be found in the active site [6]. A fluorescence stopped-flow experiment was performed using 280 nm as the excitation wavelength and 340 nm as the emission wavelength. The concentrations of FOR and formaldehyde used were the same as in the previous UV–vis stopped-flow experiments. Traces were measured on different time scales: 1, 10, and 100 s, and were combined (Fig. 5c). The data can be fitted to a three-phase exponential model. The noise levels however are high. The fluorescence data were fitted simultaneously with the UV–vis data of Fig. 5a, affording a consistent set of parameters (Table 2).
Formaldehyde is oxidized to formate by FOR with reduction of the W(VI) center. We checked whether FOR can oxidize formate to carbon dioxide. Activity measurements in an optical assay were done at 80 °C using formate as the substrate and methyl viologen as the electron acceptor. FOR was unable to oxidize formate. Pre-steady-state binding experiments with oxidized FOR using formate as the nonreducible substrate were done at the same time intervals as for the experiments with formaldehyde (Fig. 5d). Although formate is not converted to carbon dioxide by FOR, absorption changes are found in the time traces at the different wavelengths. The first phase starts at t ≈ 20 ms; the second and the third phases start at approximately 100 and 300 ms, respectively. The time trace at 335 nm resembles the trace obtained with formaldehyde. The time traces at 390 and 435 nm are not similar to each other in contrast to when formaldehyde was used as the substrate. The time traces at 595 and 695 nm are essentially unaffected by the formate. All data at the different wavelengths were globally fitted with a two-phase exponential least-squares model (Table 2).
Discussion
Steady-state kinetics
An initial proposal for part of the working mechanism of P. furiosus FOR was deduced by Hu et al. [6] from the enzyme’s 3D structure: two electrons are transferred from the substrate to the tungsten center. The pterin acts as a noninnocent ligand and transfers the electrons via the N8 atom one by one through Cys491 to the iron–sulfur cluster of FOR. The iron–sulfur cluster of ferredoxin accepts an electron through its Asp14 [6]. The electron is then transferred from the iron–sulfur cluster of ferredoxin to a membrane-bound hydrogenase [22]. After reduction of the iron–sulfur cluster of ferredoxin, the electron transfer comes to a halt. Only one electron at a time can pass through this route. Two ferredoxin molecules are necessary to transport both electrons; alternatively, a ferredoxin dimer [15] can twice in a row bind to FOR and accept an electron. This means that a two-substrate mechanism is insufficient to describe this three-substrate system. The second and the third substrates are both ferredoxin. This results in steady-state Michaelis–Menten equations that have the same form as equations for the reaction rate of an enzyme with two substrates; however, the measured KM for ferredoxin will be twice the “real” KM.
The triple-transfer mechanism for three substrates with substrates two and three (ferredoxin) the same is consistent with the experiments.
Both substrates, either formaldehyde and ferredoxin or formaldehyde and benzyl viologen, inhibit FOR. The KM values of 20–70 μM for free formaldehyde are in agreement with our previous studies [10]. When a nonphysiological electron acceptor such as benzyl viologen is used then the KM for formaldehyde is 3–4 times higher. It is possible that when ferredoxin binds to FOR the enzyme experiences a slight conformational change, resulting in a lower KM for formaldehyde.
In these studies a KM of 14 μM was determined for ferredoxin, which is 5 times lower than the KM of benzyl viologen. The high KM reported in the literature for ferredoxin was determined with high concentrations of formaldehyde. An explanation for this high value is that at high concentrations (50 mM) formaldehyde inhibits and partially denatures FOR.
Pre-steady-state kinetics
Pre-steady-state kinetics studies were performed at 50 °C. This relatively low temperature was dictated by technical limitations of the stopped-flow apparatus. FOR activity at 50 °C is 2.8 times less than at 80 °C, which affords an increased time resolution of enzyme intermediates.
Difference spectra from the pre-steady-state data revealed peak shifts and a lack of isosbestic points. These features are an indication that several processes are happening in the first seconds of the reaction. The visible spectrum of the iron–sulfur cubane consists of a single broad line at approximately 400–430 nm; the relatively weakly colored metallopterin is found at different wavelengths.
A minimal first-order exponential fit model was used to fit the pre-steady-state data. The UV–vis data could be fitted globally with a three-phase exponential fit. Two fast processes and one slow process were found. The fluorescence data were initially modeled with a two-phase exponential fit, resulting in one fast and one slow process. The fast and slow processes deduced from the fluorescence and from the UV–vis data were clearly different. A four-phase exponential model afforded a significantly improved fit to the UV-data (two fast and two slow processes), and the fluorescence data were fitted more accurately with a three-phase exponential model (two fast processes and one slow process). The complete set of fluorescence and UV–vis data obtained with the same enzyme and substrate were then fitted in a global analysis with a three-phase and a four-phase exponential model, respectively. The same four-phase model was used to fit the pre-steady-state data obtained using formaldehyde-d2 as the substrate. The fit is comparable with the fit of formaldehyde but the rate constants of the first and second phases are lower, as expected from the steady-state studies. The first phase is 4 times slower (k1 = 1.12 s−1) than with formaldehyde as the substrate. The rate constant of the second phase (k2 = 0.94 s−1) is lower than when formaldehyde is used as the substrate, consistent with a previously reported steady-state kinetics experiment [21]. The rates for the first two phases with CD2O as the substrate are not significantly different within experimental error. The rate constant for intramolecular electron shuffling appears to be unaffected by the use of formaldehyde-d2.
Residuals of the individual fits plotted versus time were essentially randomly distributed around the x-axis, thus attesting to the quality of the global fit. Several intermediate species are formed during the first seconds of the reaction. These results are in contrast with previous studies on molybdenum–DMSOR, where in stopped-flow experiments clear isosbestic points were found, and analysis of the time course produced a simple exponential, indicating a simple concerted two-electron reduction of the enzyme with no intermediate species [23].
Two alternative interpretations can be envisioned for the three phases in pre-steady-state kinetics (Fig. 6). Fig. 6Proposed alternative models for the pre-steady-state phases of fully oxidized P. furiosus formaldehyde oxidoreductase reacting with formaldehyde. Rate constants with capital letters as a subscript involve binding, oxidizing the substrate, releasing the product, and rearranging electrons between the tungsten center and the iron–sulfur cluster. Alternatively, rate constants with numbered subscripts involve oxidation of substrate, a conformational change leading to weakened product binding, release of product, and electron rearrangement
FOR is represented in Fig. 6 as a rectangle with two redox groups, a tungstopterin (W) with oxidation states 6+, 5+, or 4+, and a [4Fe–4S] cubane cluster with oxidation states 2+ or 1+. On the left of W is an empty binding pocket which can be occupied by substrate (A), by product (P), or by ready-to-leave deactivated product (P*). Occupation by A means that the enzyme has acquired a molecule of substrate in the previous reaction. Similarly, a W capped by a double-bonded O means that the enzyme has previously acquired an oxygen (protonation undefined) from the solvent. The protein is initially in the fully oxidized form; the tungsten center is 6+ and has an oxygen atom bound that was previously acquired from a water molecule, and the iron–sulfur cluster is 2+.
In the first model the initial step (kA = 4.7 s−1) observed in the optical monitoring is taken to be the overall binding process of formaldehyde to the enzyme. No changes to the formal oxidation state of the tungsten center and iron–sulfur cluster are assumed to occur in this stage. Subsequently, in what appears to be a single event, the oxygen atom is transferred, the substrate is oxidized to the product (kB = 1.9 s−1), and the tungsten center receives two electrons from the substrate and is reduced to 4+. The product then leaves the protein (kC = 6.10 × 10−2 s−1). In this step there are no changes in the oxidation states of the tungsten center and the iron–sulfur cluster. The active site of FOR has been proposed by Hu et al. [6] to be closed off from the solvent by a lid provided by the protein itself. It is possible that the substrate, free formaldehyde, is so small that it will not experience significant resistance in reaching the active site. The bigger and charged product may have more difficulty getting free of the enzyme, resulting in a low rate. The protein will then, in the absence of an external electron acceptor, redistribute (to a minor extent; see below) its electrons over the tungsten center and the iron–sulfur cluster (kD = 2.18 × 10−2 s−1). The tungsten center will become 5+ and the iron–sulfur cluster 1+, consistent with previously determined redox properties for “high-activity” enzyme [10]. An isotope effect of 4.2 is found for the rate constant of the first step (Table 1), which seems to be unrealistically large for a noncovalent binding event, and would rather seem to point toward an event involving the breaking and making of covalent chemical bonds. Therefore, the following would appear to be a rather more likely interpretation of the stopped-flow data.
Alternatively, if the entry and binding of the substrate does not lead to a detectable change in the optical absorption, then the first step (k1 = 4.7 s−1) can be the two-electron reduction of the tungsten. In this model the second step (k2 = 1.9 s−1) is a conformational change leading to an “open” state with decreased affinity for the product formed. The third step (k3 = 6.10 × 10−2 s−1) then is the ready release of this now weakly bound formate. The pre-steady-state phase ends with the two-electron-reduced intermediate which, in the absence of an external electron acceptor, slowly, and only partially converts, by electron shuffling, to the EPR-detectable, paramagnetic side product. Previous EPR studies on FOR revealed this slow electron shuffling at 20–60 °C between the tungsten center and the iron–sulfur cluster upon reduction with formaldehyde or with dithionite [10]. Only after minutes the W(V) signal appeared in the EPR measurements. This observation is in qualitative agreement with the low rate constants found here for the decay of the fourth state.
The pre-steady-state data from the experiments with formate and FOR were fitted with a two-phase exponential model: there is one fast process and one slow process. Formate is not oxidized or reduced by FOR; therefore, the fast process (kobs1 = 0.51 s−1) could be the entry and the binding of formate to the active site of FOR. As in the FOR/formaldehyde system the slow process (kobs2 = 2.00 × 10−2 s−1) is probably a nonphysiological process: the protein reaches a dead end.
Excluding the very slow electron redistribution step in the absence of external electron acceptor (the last step in Fig. 6) a complete cycle could now be written in which the enzyme shuttles between fully oxidized, one-electron-reduced, and two-electron-reduced, by inclusion of the reoxidation steps of the two-electron-reduced intermediate through serial reactions with two molecules of oxidized ferredoxin. However, such a scheme would be inconsistent with steady-state turnover rates, because the rate constant (k3 or kC) of the product-release step (the penultimate step in Fig. 6) is more than an order of magnitude less than kcat. On the other hand, it is conceivable, that the one-electron-reduced intermediate reacts with a second substrate molecule to become three-electron-reduced (with respect to the resting enzyme). This would lead to the cycle presented in Fig. 7 in which the enzyme shuttles between one-electron-reduced, two-electron-reduced, and three-electron-reduced. Fig. 7Proposed model for the catalytic redox cycle of P. furiosus formaldehyde oxidoreductase. A is the substrate formaldehyde; P is the product formate. The cube-enclosing circle represents the natural redox partner, ferredoxin. Pre-steady-state kinetics measures either the steps with rate constants kA through kD or, alternatively, those with constants k1 through k4. The fully oxidized enzyme is reductively activated by the reaction with formaldehyde to enter a redox cycle in which the enzyme shuttles between one--electron-reduced, two--electron-reduced, and three-electron-reduced states. See the text for additional explanation
Under this model the steps observed in the pre-steady-state experiments would actually constitute an activation process. Note that once the steady state of this cycle has been reached, the enzyme reacts sequentially with one formaldehyde and two ferredoxin molecules consistent with the “A+2B” Michaelis–Menten steady-state analysis.
The proposed reaction cycle of Fig. 7 is based on a combination of steady state-kinetics, pre-steady-state kinetics, and EPR studies in the absence of an electron acceptor and available structural data. Future pre-steady-state kinetics studies in the presence of excess natural electron acceptor ferredoxin may put the model to a rigorous, be it experimentally challenging, test.

pre-steady-state kinetics
steady-state kinetics
pyrococcus furiosus
tungsten
formaldehyde oxidoreductase


Practical recommendations on the use of lenalidomide in the management of myelodysplastic syndromes

Lenalidomide, an oral immunomodulatory agent, has received approval in the USA from the Food and Drug Administration (FDA) for the management of myelodysplastic syndromes (MDS) classified by the International Prognostic Scoring System (IPSS) as low risk or intermediate-1 risk and with a deletion 5q (del(5q)) cytogenetic abnormality. Although some patients with del(5q) have a relatively good prognosis, all del(5q) patients will become transfusion-dependent at some point during the course of their disease. The results of two clinical trials in more than 160 patients with MDS have demonstrated clear therapeutic benefits of lenalidomide, with >60% of patients achieving independence from transfusion during therapy, irrespective of age, prior therapy, sex, or disease-risk assessment. The recommendations presented in this review will aid the safe administration of lenalidomide for the treatment of patients with low-risk or intermediate-1-risk MDS and a del(5q) cytogenetic abnormality, and they will help physicians avoid unnecessary dose reduction or interruption, thus assuring the best efficacy for patients.
Introduction
Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis and leading to peripheral cytopenias and a genetic instability with enhanced risk of disease transformation to acute myeloid leukemia (AML).
In the general population, MDS affects approximately five in every 100,000 individuals and is regarded as a relatively rare disease [1]. However, its incidence is highest in people aged >70 years, and in this age group, MDS is the most frequently occurring malignant hematological disorder: twice as frequent as AML, more common than chronic lymphocytic leukemia, and more common than all other malignant lymphomas combined [2]. All patients diagnosed with MDS will eventually die from their disease, and in about 30%, the disease will progress to AML [3].
Between 40% and 60% of patients with primary MDS and about 90% of patients with secondary MDS have chromosomal abnormalities at the time of diagnosis [4]. Chromosomal abnormalities may occur as a single abnormality or as part of a complex karyotype; disease severity increases with the number of abnormalities. The most common chromosomal abnormalities are deletions of chromosome 5 and chromosome 7 and trisomy of chromosome 8 [5]. In a subgroup of patients who have an isolated deletion 5q (del(5q)), termed 5q-syndrome, the clinical profile is distinct [6]. Typically, this clinical profile includes macrocytic anemia, a normal to increased platelet count, mild leukopenia, hypolobulated megakaryocytes in the bone marrow, a medullary blast count <5%, and an isolated del(5q) abnormality including a common deleted region between 5q31 and 5q33 [4, 7]. Consistent with the observation that disease severity is related to the number of chromosomal abnormalities, patients with del(5q) plus additional cytogenetic abnormalities have a worse prognosis than those with an isolated del(5q) abnormality [3, 5].
On the basis of the results of an international phase II trial in 148 patients [8], the oral immunomodulatory, antiangiogenic, and antineoplastic agent lenalidomide (Revlimid®; Celgene Corporation, NJ, USA) was approved in December 2005 by the US Food and Drug Administration (FDA) for the treatment of patients with transfusion-dependent International Prognostic Scoring System (IPSS) classified low-risk to intermediate-1-risk MDS with a del(5q) cytogenetic abnormality, with or without additional cytogenetic abnormalities. The first 46 patients recruited received lenalidomide 10mg/day for 21days every 28-day cycle, for up to 24weeks. After a protocol amendment, the remaining 102 patients received lenalidomide 10mg/day on a continuous dosing schedule [8]. Overall, 67% of patients who were initially transfusion-dependent achieved transfusion independence, and another 9% achieved a decrease (≥50%) in the total number of transfusions required during lenalidomide therapy [8]. Forty-five percent of the 85 evaluable patients in this study achieved a complete cytogenetic response, irrespective of chromosomal complexity [8].
In January 2007, an international group of MDS specialists met to discuss the practical management of lenalidomide in patients with transfusion-dependent, IPSS-classified low-risk or intermediate-1-risk MDS and a del(5q) cytogenetic abnormality. The recommendations presented here are based on the clinical data derived from the current literature [8, 9], from the known clinical profile of lenalidomide in other disorders, and the clinical experience of the panel members in the practical use of this agent. Although lenalidomide has been used in IPSS-classified intermediate-2-risk and high-risk patients and in patients without an associated del(5q) abnormality, the recommendations presented here focus on the current approved indication of the drug in the USA.
Selection of patients for lenalidomide therapy
The FDA approved lenalidomide for patients with transfusion-dependent anemia due to low- or intermediate-1-risk MDS associated with a del(5q) cytogenetic abnormality, with or without additional cytogenetic abnormalities. This is an important limitation, as patients with a complex karyotype and del(5q) are eligible for treatment only if they have no more than one cytopenia (i.e., anemia, neutropenia, or thrombocytopenia) and their medullary bone marrow blast count is <5%. Furthermore, patients with a bone marrow blast count up to 10% receive a score of 0.5 points in the IPSS classification (Table 1) [3]. Therefore, in order to remain in the IPSS intermediate-1-risk category, one additional chromosomal abnormality (not involving chromosome 7) or two or more peripheral cytopenias are allowed, but not both. Although teratogenicity has not been reported with lenalidomide, as it is an analogue of thalidomide, women of childbearing potential should have two negative pregnancy tests performed within 14days prior to lenalidomide intake.
Table 1International prognostic scoring system for myelodysplastic syndromes: survival and evolution of acute myeloid leukemia Score valuePrognostic variable00.51.01.52.0Bone marrow blasts, %<55–10–11–2021–30KaryotypeaGoodIntermediatePoor  Cytopeniasb0/12/3   Scores for risk groups are as follows: Low, 0; Int-1, 0.5–1.0; Int-2, 1.5–2.0; and High, ≥2.5aGood: normal, −Y, isolated del(5q), isolated del(20q); poor: ≥3 abnormalities or chromosome 7 anomalies; intermediate: other abnormalitiesbNeutrophils <1800 × 106/l, hemoglobin <10 g/dl, platelets <100 × 109/lTable reproduced with permission from [3] © The American Society of Hematology
Lenalidomide therapy is effective regardless of prior erythropoietin [8, 9] or prior thalidomide therapy [9], and those previous therapies should not be regarded as contraindications for its use, although concurrent therapy with erythropoietin is not recommended, owing to concerns regarding venous thromboembolism.
Clinical data have shown that the frequency of response to lenalidomide is similar across all age groups evaluated [8]. Importantly, the overall adverse-event burden did not increase in older patients, but serious adverse events were more frequent in patients aged over 65years compared with younger patients (54% vs. 33%, respectively) [10].
Although the impact of renal impairment on lenalidomide efficacy or toxicity in patients with MDS has not been evaluated, the risk of toxicity is expected to be greater in patients with impaired renal function because lenalidomide is renally excreted [11]. In patients with known renal impairment, the expert panel support the dosing schedule proposed by Chen et al., which is based on creatinine clearance (Table 2) [11]. As advancing age is associated with declining renal function, it is recommended that patients older than 65years who are prescribed lenalidomide are monitored carefully throughout their treatment (Table 3).
Table 2Recommended dose adjustments for patients with impaired renal functionaRenal impairmentDoseMild10 mg (full dose) every 24 h (80 > CLcr ≥ 50 ml/min)Moderate5 mg every 24 h (30 ≤ CLcr < 50 ml/min)Severe5 mg every 48 h (CLcr < 30 ml/min, not requiring dialysis)End-stage renal disease5 mg three times a week after each dialysis (CLcr < 30 ml/min, requiring dialysis)aRecommendations based on a pharmacokinetic study by Chen et al. [11]CLcr creatinine clearanceTable adapted from [11] © 2007 SAGE Publications. Adapted by permission of SAGE Publications, Inc.Table 3Recommendations for laboratory monitoring during treatmentFunctionTestRecommendation(s)Kidney functionCreatinineEvery 4 weeks in patients aged 65 years and olderBloodFBCWeekly monitoring of full blood count mandatory for the first 2 months (it may be continued for 5 months).Biweekly or monthly monitoring should be considered thereafter, depending on hematological status.If treatment is interrupted in patients who had a previous episode of neutropenia or thrombocytopenia while on lenalidomide treatment, the same monitoring guidelines apply at re-initiationThyroid functionTSH, T4Monitor every month during the course of treatmentIn case of loss of response during lenalidomide treatmentGonadal functionTestosteroneIn case of loss of response during lenalidomide treatmentDigoxinDigoxinIn patients concomitantly taking Digoxin, the plasma level should be monitored periodicallyPregnancy testUrine testDay −14 and day 0 at initiation of therapy, monthly thereafter (in women of childbearing potential)Bone marrowBM aspiration and cytogenetic testing; Trephine biopsy optionalAt commencement of therapy.In case of loss of response to rule out progressive disease or cytogenetic evolutionBM Bone marrow, FBC full blood count, T4 thyroxine, TSH thyroid-stimulating hormone
Likewise, the safety, efficacy, and pharmacokinetics of lenalidomide therapy have not been fully investigated in patients with evidence of hepatic impairment, or those with frank hepatic dysfunction. Clinical trials conducted to date have excluded patients with inadequate hepatic function, with individual trials using different serum transaminase measures as exclusion criteria; most trials excluded patients with serum transaminase levels greater than three times the upper limit of normal.
Pharmacokinetic studies have revealed that the co-administration of lenalidomide with digoxin may increase the maximal digoxin concentration, although the area under the concentration–time curve remained unchanged [10]. Importantly, lenalidomide does not interact with the cytochrome P450 system, and so, can be confidently co-administered with other medications metabolized by this route (Celgene data on file) [10]. This latter finding is particularly relevant for older patients, who may be more likely to be receiving concomitant medications for co-morbidities. The expert panel concurred with the current prescribing information for lenalidomide and recommended that patients taking digoxin should have their digoxin plasma levels monitored periodically (Table 3).
Treatment with lenalidomide in del(5q) MDS
The time-to-response during lenalidomide therapy was evaluated in the larger of the two pivotal clinical trials [8]. For most patients, the median time to initiation of the transfusion-independent period was 4.6weeks, but responses have been noted to take up to 12months [8]. Therefore, the expert panel recommends a treatment duration of at least 4months in order to obtain an initial response (Table 4). Patients were followed for a median of 104weeks, but as more than half of them remained transfusion-free at this assessment point, the median duration of transfusion independence could not be calculated. Encouragingly, among the pooled patients with MDS with a del(5q) abnormality from the two studies [8, 9], transfusion independence and major erythroid response were maintained for a median duration of at least 2years [12]. Even though there are anecdotal reports of long-lasting erythroid remissions in some patients with hematological complete response who discontinued lenalidomide, the panel recommends continuation of lenalidomide treatment in responders for as long as it is tolerated. In patients where the dose has initially been reduced for mild adverse events, dose escalations may be considered (Table 4). In responders with high ferritin levels due to long transfusion histories who achieve normal hemoglobin levels, lenalidomide treatment should be continued and therapeutic phlebotomy be considered to reduce the secondary iron overload. In responders who discontinue due to adverse events, the panel recommends checking for response continuation before initiating another treatment (Table 4). Preliminary data show that prolonged treatment with lenalidomide does not increase the risk of transition to acute myeloid leukemia [13]. Of note, there are reports of patients acquiring additional cytogenetic abnormalities while on lenalidomide [8], but at this time the relevance of this phenomenon is not entirely clear. Until there is data to the contrary, patients with continuous hematological response should remain on the drug. Patients losing their response and developing a complex karyotype should be approached as they would be in the absence of lenalidomide, and alternative therapies, including transplantation, should be considered.
Table 4Recommendations for treatment duration Recommendation(s)Initial treatmentTreatment should be continued for at least 4 months in order to obtain an initial responsePatients who Have a complete hematological responseContinue lenalidomide therapy for as long as it continues to be well tolerated to avoid relapse (both erythroid and cytogenetic) Have a partial responseContinue lenalidomide therapy, and consider an escalation of the lenalidomide dose to a maximum of 10 mg per day, if tolerable Discontinue treatment because of adverse eventsPatients should not begin another therapy immediately; it is recommended to wait 8–12 weeks to determine whether the response continues
Tolerability of lenalidomide in del(5q) MDS
Lenalidomide has been shown to be well tolerated in the two clinical trials reported to date [8, 9]. The most commonly reported adverse events of grade 3 or 4 severity were neutropenia (55%), thrombocytopenia (44%), anemia (7%), leukopenia (6%), rash (6%), diarrhea (3%), pruritus (3%), pneumonia (3%), and fatigue (3%) [8].
Management of hematological adverse events
Hematological adverse events, including neutropenia and thrombocytopenia, were the most common adverse events with lenalidomide therapy and the most frequent reasons for dose adjustment [9]. As such, thrombocytopenia and neutropenia should be expected to occur in most patients—in fact, they appear to be associated with a higher likelihood of a response to therapy [14]. In general, regular monitoring of blood cell counts is recommended (Table 3).
The majority of hematological adverse events (62%) occurred early in the course of treatment (within the first 8weeks) [8]. Grade 3 or 4 neutropenia was reported in 55% of patients [8]. Monitoring and, in some cases, additional treatment with granulocyte colony-stimulating factor (G-CSF) is recommended (Tables 3 and 5, and Fig. 1). Neutropenic sepsis was the only reported cause of death (occurring in three patients) that was regarded as possibly related to the study drug [8], therefore, patients should receive clear guidance on how to react in the event of febrile neutropenia (Table 5). Grade 3 or 4 thrombocytopenia was observed in 44% of patients [8]. In some thrombocytopenic patients, interruption of lenalidomide treatment may be necessary (Table 5 and Fig. 1). Lenalidomide has not been tested in patients with a baseline neutrophil count <500 × 106/l or baseline platelet count <50 × 109/l.
Fig. 1Recommendations for the management of a neutropenia and b thrombocytopenia. aNo consensus was reached, recommendation is based on the opinion of some of the panel members G-CSF granulocyte colony-stimulating factorTable 5Recommendations for the management of hematological adverse events Recommendation(s)NeutropeniaFor treatment recommendations for neutropenia see Fig. 1Febrile neutropeniaProvide patients with clear guidance on how to react in the event of febrile neutropenia (patient education, specialized hematological care at all times, and broad-spectrum antibiotics within 3 h of fever onset)ThrombocytopeniaFor treatment recommendations for thrombocytopenia see Fig. 1VTEVTE prophylaxis is not generally recommended in patients with MDS. Combining lenalidomide with erythropoietin is also not recommended.If erythropoietin is used, be aware of a potentially increased risk of VTE. Patients should be informed about the risk of VTE and monitored for symptomsIf VTE does occur, interrupt lenalidomide treatment, treat the VTE, and carefully re-introduce lenalidomide once stable anticoagulation has been establishedPolycythemiaLenalidomide should be continued and phlebotomy considered, depending on ferritin levels. Although polycythemia is usually transient, treatment interruption may be necessary if additional risk factors for VTE are presentMDS myelodysplastic syndromes, VTE venous thromboembolism
In the opinion of the panel, patients presenting with a neutrophil count <1,000 × 106/l prior to the start of lenalidomide therapy can still be treated with lenalidomide if G-CSF is co-administered. When neutropenia occurs during lenalidomide therapy, interruption of the lenalidomide treatment schedule might be necessary (Table 5 and Fig. 1). When thrombocytopenia (platelet count <25 × 109/l) is diagnosed at presentation, based on the rationale that once a response is achieved thrombocytopenia is likely to resolve, some experts would consider administering lenalidomide with platelet support (Table 5 and Fig. 1). However, other experts advise against the use of lenalidomide in patients with a platelet count <50 × 109/l because there is a risk of sustained deterioration of thrombocytopenia, despite an erythroid response. In some patients responding to lenalidomide, polycythemia may occur [8]; if appropriate, therapeutic phlebotomy or interruption of lenalidomide therapy may be considered (Table 5).
Grade 3 or 4 venous thromboembolism (VTE) was observed in 3% of patients [8]. Generally, the risk of VTE is increased if there is a history of superficial vein thrombosis, or previous VTE [15, 16]. It is unknown if this applies to patients treated with lenalidomide as well. Nevertheless, these patients should be carefully monitored. In patients with a history of previous VTE, low-molecular-weight heparin should be used to prevent recurrent thrombosis. Although aspirin is effective in the prevention of thrombosis in multiple myeloma, aspirin cannot be recommended alongside lenalidomide as lenalidomide frequently leads to grade 3 or 4 thrombocytopenia in patients with MDS with del(5q). There are insufficient data on low-dose coumadin treatment for the prophylaxis of VTE, and therefore the panel did not recommend its use. Of particular relevance to patients with MDS, VTE risk is increased by concomitant erythropoietin use [17]. Since the incidence of VTE in patients with lenalidomide-treated MDS is generally low [8], prophylaxis in patients without antecedents of VTE is not recommended. If VTE does occur, it should be treated according to standard protocols. Treatment with lenalidomide should be interrupted until stable anticoagulation is achieved and then carefully reintroduced (Table 5). Once VTE has occurred during lenalidomide therapy, patients must remain on anticoagulation therapy as lenalidomide treatment continues.
Management of non-hematological adverse events
Unlike thalidomide, lenalidomide does not lead to dose-dependent peripheral neuropathy or somnolence [18], but other non-hematological adverse events (NHAEs) may occur. The most common NHAEs (all grades) associated with lenalidomide therapy are diarrhea (49%), pruritus (42%), rash (36%), and fatigue (31%) [10]. The most commonly reported grade ≥3 NHAEs were rash (6%), fatigue (3%), diarrhea (3%), and pruritus (3%) [8]. In general, should NHAEs arise during lenalidomide therapy, alternative causes should be ruled out and symptomatic treatment initiated (Table 6). Dry skin and pruritus occur regularly, and itching of the scalp is a characteristic adverse event during the first few weeks of therapy. These NHAEs are usually self-limiting and seldom need treatment. Unselective antihistamines (e.g., clemastine) are usually helpful. In severe cases, a short course of systemic corticosteroids (10mg of prednisone or equivalent), or local steroid application may be helpful. Rash often resolves spontaneously with time [19], and the panel do not recommend discontinuation of lenalidomide treatment should rash arise; however, in patients with severe or persistent rash, the temporary interruption of lenalidomide may be necessary (Table 6).
Table 6Recommendations for management of non-hematological adverse events (NHAEs)NHAERecommendation(s)RashUsually resolves within 2–3 weeks, no interruption of lenalidomide treatment neededIf required treat with unselective antihistamines (e.g. clemastine), topical steroids, or a short course (14 days) of oral 10 mg prednisoneIf rash is severe or persists after treatment, lenalidomide should be interrupted until rash resolves. In the experience of the panel, lenalidomide can be restarted thereafter without recurrence of rashDiarrheaTreat symptomatically after ruling out other underlying causesHypothyroidismIn case of hypothyroidism, thyroid replacement therapy must be initiatedOther NHAEsTreat symptomatically after ruling out other underlying causes such as anemia or autoimmune disorders
Diarrhea is a frequent problem and may impact on a patient’s quality of life. Patients with known lactose intolerance should add lactase to their diet, as lenalidomide capsules contain small amounts of lactose. Diverse symptomatic therapies have been reported to be effective in those patients, including, but not limited to, loperamide, pipaverium bromide, uzara root extract, and tinctura opii. Muscle cramps have been reported by a number of patients after a variable time of lenalidomide intake. Magnesium dietary supplements may be tried, but are usually of limited value. Quinine sulphate (up to 200mg thrice-daily) works well, however, physicians should check for other cytochrome-P450-interacting drugs (e.g., sotalol, terfenadine, astemizole, voriconazole, erythromycin, rifampicin, and cisapride) that might prolong QT time in these patients. Patients who experience fatigue will be likely to benefit from counselling [20], and in cases of severe fatigue, dose reduction may be needed. Hypothyroidism has been reported in approximately 7% of patients [10]. It is almost exclusively of autoimmune cause. Patients should be screened for hypothyroidism every other month during the course of treatment (Table 3). If hypothyroidism is confirmed, hormone replacement therapy is indicated (Table 6). Patients losing their response to lenalidomide during treatment may have developed either hypothyroidism or hypogonadism, and should be screened for both conditions.
Conclusions
The approval of lenalidomide for the treatment of MDS patients with low-risk or intermediate-1-risk disease with a deletion of chromosome 5q represents a significant step forward for this underserved group of patients. The key goal of the strategies presented here is to avoid unnecessary dose delays and reductions, and to maximize probability of response. Patient selection is straightforward, as efficacy does not appear to be influenced appreciably by relevant prognostic factors including age, prior erythropoietin therapy, sex, French–American–British type, or IPSS score. Treatment should be continued for a minimum of 4months to ensure treatment response. Neutropenia and thrombocytopenia are the most common adverse events during lenalidomide therapy but can be managed by the introduction of G-CSF and dose interruption. Other side-effects are generally manageable, and a range of simple (prophylactic) interventions are recommended to address the more common and more serious associated adverse events.

lenalidomide
myelodysplastic syndromes
mds
treatment guidelines


Perceived Conflicts Between Pastoralism and Conservation of the Kiang Equus kiang in the Ladakh Trans-Himalaya, India

An emerging conflict with Trans-Himalayan pastoral communities in Ladakh’s Changthang Plateau threatens the conservation prospects of the kiang (Equus kiang) in India. It is locally believed that Changthang’s rangelands are overstocked with kiang, resulting in forage competition with livestock. Here, we provide a review and preliminary data on the causes of this conflict. Erosion of people’s tolerance of the kiang can be attributed to factors such as the loss of traditional pastures during an Indo-Chinese war fought in 1962, immigration of refugees from Tibet, doubling of the livestock population in about 20 years, and increasing commercialization of cashmere (pashmina) production. The perception of kiang overstocking appears misplaced, because our range-wide density estimate of 0.24 kiang km−2 (± 0.44, 95% CL) is comparable to kiang densities reported from Tibet. A catastrophic decline during the war and subsequent recovery of the kiang population apparently led to the overstocking perception in Ladakh. In the Hanle Valley, an important area for the kiang, its density was higher (0.56 km−2) although even here, we estimated the total forage consumed by kiang to be only 3–4% compared to 96–97% consumed by the large livestock population (78 km−2). Our analysis nevertheless suggests that at a localized scale, some herders do face serious forage competition from kiang in key areas such as moist sedge meadows, and thus management strategies also need to be devised at this scale. In-depth socioeconomic surveys are needed to understand the full extent of the conflicts, and herder-centered participatory resolution needs to be facilitated to ensure that a sustainable solution for livelihoods and kiang conservation is achieved.
One of the seven equid species in the world, the kiang Equus kiang, occurs in parts of China and India, with small populations also reported from Pakistan and Nepal. Unlike the Asian wild ass E. hemionus, of which the population has declined drastically over the last century, kiang continues to have a wide distribution with fairly large populations (Schaller 1998). Within India, Ladakh (approximately 75° 50′ to 75° 80′ E; 32° 30′ N to 32° 37′ N) remains a stronghold for the kiang (Fox and others 1991; Shah 1996), where local Buddhist communities have been fairly tolerant of the species, and large herds of kiang can be relatively easily seen in eastern Ladakh. Kiang is classed as a Least Concern species under the IUCN Red List category; however the Western Kiang, E. k. kiang, which occurs in Ladakh, is classed as “data deficient” (Shah 2002).
Yet, with growing integration of the local economy of Ladakh with better developed cash markets, pastoral communities are fast losing their tolerance towards the kiang and it is increasingly seen as a competitor of livestock (Fox and others 1991). Ironically, its relatively large population and conspicuousness are becoming concerns for the continued conservation of the species. Although the species is not persecuted (except for driving them away from pastures by herders on horseback) in Ladakh largely due to the Buddhist beliefs of the local pastoral communities, this perceived conflict is currently one of the most serious issues being faced by the local district administration and the wildlife department, with vehement demands for compensation. Indeed, a decreasing tolerance for the species may lead in future to a worsening willingness to preserve it. There is a belief among the administration that Ladakh’s rangelands are presently overstocked by kiang, and that they are degrading the pastures (Anon 2003). Over the last decade, as a measure to protect forage from kiang and make it available to livestock, the local government and some nongovernmental organizations have started fencing the most productive pastures, the sedge meadows near rivers and streams, thereby increasing forage availability for livestock and reducing the available habitat for the kiang (Richard 1999; Bhatnagar and Wangchuk 2001).
The purpose of this article is to document this intensifying conflict between pastoralism and conservation of the kiang in Ladakh. The kiang subspecies in question is categorized as “data-deficient” by the IUCN (Moehlman 2002). We examine the primary ecological, socioeconomic, and political aspects of kiang–human conflict, and summarize the results of our rangewide surveys of the kiang population in Ladakh. We also discuss the growing linkages between a traditional way of life and the global cashmere market, and their consequences for conservation of the kiang.
Methods
Study Area
Administratively, the Ladakh region in the state of Jammu and Kashmir (India) is divided into two districts: the western Kargil district and the eastern Leh district. Leh district is a high-altitude cold desert spread over approximately 45,000 km2. This rain-shadow region is semiarid to arid, with winter temperatures dropping to below −25°C. Western and central Leh are mostly rugged, relatively lower in elevation, and inhabited by agropastoralists. The eastern part of the district, called Changthang, on the other hand, comprises high plateaus and rolling hills (usually >4000 m) interspersed with lake and river basins that have moist patches of relatively dense graminoid vegetation. Most of the remaining arid region is dominated by medium to sparse steppe vegetation. Changthang covers approximately 20,000 km2 and is inhabited by the nomadic Changpa community and Tibetan refugees, both of whom primarily rear goat and sheep. According to the human census in 2001 conducted by the government, the population of the Leh district was 117,637, with the majority (85%) being in the western region. The population is sparse in the Changthang (13,444) with a density of approximately 0.7 km−2. Ladakh is home to a diverse assemblage of wild carnivores, ungulates, and birds (Pfister 2004).
Within Ladakh, the kiang occurs in the eastern Changthang part of the Leh district. Our survey covered most of this region, from the Pangong Tso in the North, along the eastern border with Tibet, to Hanle in the south (Figure 1). We divided the entire region into five blocks based on their location and overall similarity in terms of topography and vegetation, and surveyed each block (Table 1). Our repeated counts of kiang were conducted in the Hanle Valley. This valley (79° 0′ 45″ E, 32° 37′ 29″ N; approximately 2800 km2) extends from the Zanskar mountains in the south to the Indus Valley in the north into which the Hanle River drains at Loma. The higher reaches are primarily rolling mountains and plateaus, while the mid and lower portion is a wide valley fringed by a high range exceeding 5000 m on the east, and a lower, more gradual ridge on the west.
Fig. 1The five blocks surveyed for kiang in eastern Ladakh during 2000 to 2003. The estimated “viewscape” is based on modeling for areas visible within 3 km on either side of the road (see text for details). Distribution of kiang in Ladakh is also shown (based on Chundawat and Qureshi 1999). We also surveyed some additional adjacent areas with similar habitat.Table 1Density of kiang in the surveyed blocks of eastern Ladakh, IndiaSurvey blockTransect length (km)Area (km2)No. KiangKiang density (km−2)Loma-Demchok (Indus)822882490.86Hanle Valley66347780.23Chushul-Loma63299190.06Parma Valley7436090.03Pongong Tso80359100.03Total36516533650.24 (95% CL ± 0.44)
Data Collection and Analysis
Through archival research and field observations, we documented the intensifying human–kiang conflict in Ladakh. We describe the perceptions of the Ladakhi administration regarding this conflict, and the efforts they have undertaken to address it. Through archival research, we also examine recent land-use changes in eastern Ladakh, describe the growing pashmina or cashmere industry, and its fallouts for the kiang–human conflict. Literature was also surveyed for obtaining information on the past status and distribution of the kiang. We also obtained data on livestock populations from the Sheep Husbandry Departments in Nyoma and Leh, to estimate the livestock density in eastern Ladakh. We had informal conversations with over 50 herders to learn about their perceptions regarding conflicts.
Our data on the kiang population in Ladakh come from rangewide surveys conducted in the year 2000. The study area was surveyed from a slow-moving vehicle (traveling at 10–12 km/hour) and areas on either side were periodically scanned using an 8× binocular and/or 20× spotting scope. On every sighting, animals were counted and classified, and habitat variables such as topographic feature, elevation, slope, aspect, dominant plant species, and approximate plant cover were recorded. Classification of kiang by sex was not possible in most instances due to the similarity between the sexes, especially when seen from a distance. A total distance of 365 km was covered in the Changthang, traveling on an average 60–70 km per day. We also covered approximately 50 km on foot in the upper catchment of the Hanle Valley. Subsequently, in the spring of 2001, 2003, and 2004, we conducted repeated vehicle counts of kiang along an approximately 100-km stretch in the Hanle Valley, which had emerged as an area with moderate to high kiang density during the 2000 survey.
Density for kiang and livestock was calculated by estimating the area covered in each segment using Geographical Information System (GIS) tools. The area surveyed along the roads was estimated by creating a “viewscape” in ArcInfo, which effectively created polygons of all area visible from the road. The base map used for this estimation was the National Imagery and Mapping Agency’s Vector Map Level 0 (Digital Chart of the World) Edition: 5. This was integrated with the Digital Elevation Model (resolution 90 m or 3 arc seconds) Shuttle Radar Topography Mission (SRTM) data from the Space Shuttle mission (for the year 2000) obtained from ftp://e0mss21u.ecs.nasa.gov/srtm/ to generate maps of the study area with numerous layers of information that included elevation, aspect, and drainage. After creating the viewscape, we used a cutoff distance of 3 km on either side of the road, within which all visible areas were calculated. This was generally our maximum sighting distance for the kiang (most Rangefinders do not work at this distance). We estimated the extent of the surveyed areas (through viewscapes) for both the rangewide surveys as well as the region of our repeated kiang counts in Hanle Valley.
As a preliminary exercise to understand the extent of forage consumption by kiang vis-à-vis livestock, we estimated total daily forage consumption by kiang and livestock through established relationships between body mass and daily food intake (as percent of body mass) among herbivores (Foose 1982). In the absence of population structure data for both kiang and livestock, we used adult body masses (averaged between sexes) from Mishra and others (2002). We used the established relationships for foregut fermenters (goat, sheep, cattle, yak, cattle-yak hybrids) and hindgut fermenters (kiang, horses, donkeys), as appropriate. Forage quality in the highly seasonal cold desert steppe of Ladakh is expected to vary strongly between seasons, with high-protein, low-fiber forage being available in summer, and low-protein, high-fiber forage in winter. We therefore estimated forage consumption by each species separately for winter and summer, using the relationships for low-protein, high-fiber grass hay diet for winter (4.5–7.4% crude protein and 65–70% cell wall) and high-protein, low-fiber legume hay diet for summer (17.4–22.2% crude protein and 31–56% cell wall) (Foose 1982). For hindgut fermenters, daily forage intake as a percent of body mass (W) in summer was estimated as 13.8W−0.315, and 6.95W−0.236 in winter. Similarly, for the ruminants (goat, sheep, cow, dzo/dzomo, yak), it was estimated as 7.31W−0.231 in summer and 4.04 W−0.184 in winter (Foose 1982).
Results and Discussion
Genesis of the Conflict: Kiang Population Changes in Recent History
Kiang inhabits the open plains and rolling mountains of the Changthang plateau in eastern Ladakh at altitudes between 4000 m and 5500 m. It has a total estimated range of approximately 7400 km2 in Ladakh (Chundawat and Qureshi 1999; Figure 1). The species was common in Ladakh during the earlier part of the 20th century (Stockley 1936), but is believed to have declined substantially with the buildup of armed forces in Ladakh during the war between India and China in 1962 (Fox and others 1991). There are no population estimates available for the period before or immediately following this war, but it is believed that the population had recovered by the 1980s. The only published estimate of their population comes from the late 1980s when approximately 1500 animals were estimated to occur in Ladakh (Fox and others 1991; Shah 2002).
It thus appears that for at least a decade after the war, the kiang population remained relatively small in Ladakh. The genesis of the present conflict can be traced back to the early 1980s, by which time their population had apparently recovered (Fox and others 1994). The prevalent belief in Ladakh during that time, and one that continues until today, is that the kiang population has drastically increased in eastern Ladakh because many animals have moved from Tibet to the Indian side of the border to escape persecution (Fox and others 1991; Anon. 2003). Even in the 1980s, there were complaints by herders regarding the extent of forage consumption by kiang and consequent competition with livestock, and demands that the government should “drive the kiang back into Tibet” (Fox and others 1991).
Intensification of the Conflict: Changing Face of Pastoralism in Ladakh
The Tibetan plateau was inhabited by nomadic hunting people 30,000 years ago, and nomadic pastoralists have been herding livestock on the plateau for at least two millennia (Schaller 1998). The nomads, who became Buddhists presumably in the last millennium, live in tents (ribos) in seasonal camps. In the past, the few nomadic groups inhabiting eastern Ladakh belonging to the Changpa community traveled distances exceeding 100 km during their seasonal migrations (Phuntsok 2000; Hagalia 2004). This has recently changed, and most seasonal movements are now restricted to within 40–50 km (Hagalia 2004).
After the 1960s, there has been a steady increase in the population of the Changpa and their livestock, presumably facilitated by access to better health care, provisions, and subsidies, both from the government as well as the army (Chaudhuri 2000; Phuntsok 2001; Bhatnagar and Wangchuk 2001). Supplemental forage provided by the government in the event of severe winters now offsets most of the starvation-related livestock mortality, which in the past was perhaps an important factor limiting the livestock population. The 1962 war between India and China also led to the curtailment of movement of the Changpa herders from eastern Ladakh into parts of Tibet, considerably reducing the availability of grazing land. Many of their pastures such as Skagzung, the primary winter pasture of the Rupshu Changpas (Figure 1), became unavailable, effectively escalating the stocking density in eastern Ladakh. Furthermore, a large number of Tibetan refugees have moved into eastern Ladakh with their livestock herds since the late 1950s, thus contributing to a substantial increase in the number of herding families and the livestock population (Bhatnagar and Wangchuk, 2001; Hagalia 2004). As per the government records, the livestock population in Ladakh (including Changthang) has doubled in about two decades, from approximately 212,500 in 1977 to 487,000 in 1999 (Richard 1999; Bhatnagar and Wangchuk 2001). In 1999, Changthang constituted 44% of Ladakh’s livestock population, most of which (94%) were sheep and goats.
One of the most important livestock products from the region is the pashmina or cashmere, a fine luxury fiber derived from the underwool of the local Changra goat. Traditionally, more than 95% of cashmere from Ladakh has been used for barter with or sale to traders from the neighboring Kashmir region under a treaty with the erstwhile kingdom of Kashmir dating from 1684 (Rizvi 1999). Over the last two decades, however, cashmere production in Ladakh has been promoted by the government in a substantial way. This has included setting up of goat breeding farms, efforts at improving yield, and provisioning of veterinary services to nomads that has brought down the kid mortality rates to 2–3% from an estimated 30–40% (Jina 1995). However, the efforts at increasing the per animal cashmere yield have apparently had very limited success (Jina 1995), suggesting that increasing cashmere production necessitates an increase in the goat population. More recently, with part support from the central government of India, the International Fund for Agricultural Development and the UNDP, the Ladakh administration has set up a cashmere de-hairing plant, expected to result in considerable added value to the cashmere from Ladakh (Joshi and Morup 2003; Mital 2004). Presently, Ladakh contributes merely 30,000 kg (0.37%) to the global cashmere production and the administration is eager to increase Ladakh’s contribution in global trade.
The recent intensification of human–kiang conflict needs to be viewed against these socioeconomic and political developments. The human and livestock populations in kiang habitat have significantly increased in the last four decades due to factors ranging from better healthcare to the influx of refugees. Furthermore, promotion of production and value of cashmere has meant that livestock rearing is fast becoming a cash-based enterprise that increasingly caters directly to the lucrative global cashmere market. Herders are realizing the importance of every bit of pastureland in maximizing cashmere production and see kiang as a direct threat to pasture production. Against this background, it is perhaps not surprising that people are fast losing their tolerance of the kiang.
The Ecological Context: Current Kiang Population and Relationships with Livestock
In our rangewide survey of the kiang, we surveyed an estimated area of 1653 km2, which is 22% of the total range of the kiang in Ladakh (7400 km2; Chundawat and Qureshi 1999). We recorded a total of 181 distinct kiang groups totaling 365 animals. The average density in the surveyed blocks was 0.24 (95% CL ± 0.44) kiang km−2, with the highest abundance along the upper Indus from Loma to Demchok followed by the Hanle Valley (Table 1, Figure 1). With an overall livestock population of 210,000 (as per unpublished records from the Nyoma office of the Sheep Husbandry Department (2004)) in the Nyoma and Durbuk blocks that comprise the Changthang region of Ladakh, the livestock density in the region was 11 km−2. Thus, in terms of density, kiang represents only 2% of the total herbivore population (kiang and livestock together) in the Changthang, with the bulk being contributed by goats (57%).
The group sizes of kiang observed during the rangewide survey varied from 1 to 74. The mean group size was 2.8 (95% CL ± 0.8), with most of the sightings (55%) being of solitary animals. Most kiang were seen in open wide valleys (79%, n = 202), followed by stony slopes (19%) and very rarely in broken areas (2%). There were no sightings on slopes steeper than 30°. We observed kiang between elevations of 4100 and 5500 m, with most sightings between 4200 and 4300 m. Most kiang groups (73%) were seen on sedge (Carex spp. and Kobresia spp.) and grass (Stipa spp.) meadows in the valley bottom, which were occasionally interspersed with bushes of Caragana sp. These meadows appear to be critical habitat for kiang during summer and winter as also reported by Schaller (1998), but are limited in extent to narrow discontinuous strips in moist areas along valleys. The remaining sightings were in areas that had sparse to dense Artemisia steppe (25%; with Tanacitum sp. and Sedum sp. being the other dominant plants) and high altitude forb meadows (2%; Saussurea spp., Salsola sp., and Polygonum sp. as the dominant plants).
At a landscape level covering entire eastern Ladakh, of the total daily forage consumption by large herbivores, we estimated that kiang consume 10–11% and livestock 89–90% (Table 2). Among livestock, goats were estimated to consume the maximum forage (41% to 43% of the total forage consumed), followed by sheep (26% to 27%).
Table 2Herbivore density and estimated forage consumption by kiang vis-à-vis livestock in ChangthangSpeciesBody weight(kg)Density(animals km−2)Total forage consumption in summer (kg km−2)Percent forage consumption in summerTotal forage consumption in winter (kg km−2)Percent forage consumption in winterKiang2750.241.6101.211Livestock  Donkey900.040.110.11  Horse2480.150.950.76  Cattle1910.160.740.54  Yak2980.261.591.110  Yak cattle hybrids2270.010.03<10.02<1  Sheep343.984.4272.926  Goat336.446.9434.541Livestock total112111.0314.5909.789Total11.2716.0810.96Body weights are taken from Mishra (2001) and have been averaged between sexes. Note that this will result in an overestimate of the offtake by kiang because we assumed that all kiang were adult. Livestock density was calculated only for adult animals.
Our subsequent estimate of the kiang population in the Hanle Valley (the region between Rongo and Kalangtartar) based on repeated counts between 2001 and 2004 yielded a density of 0.56 kiang/km2 (95% CL ± 0.08). We estimated that on an average, kiang remove only 3% to 4% of the total forage consumed in the Hanle Valley, with the greater part being consumed by goats (45% to 47%) followed by yaks (20% to 21%) and sheep (20%) (Table 3).
Table 3Herbivore density and estimated forage consumption by kiang vis-à-vis livestock in the Hanle ValleySpeciesBody weight (kg)Density(animals km−2)Total forage consumption in summer (kg km−2)Percent forage consumption in summerTotal forage consumption in winter (kg km−2)Percent forage consumption in winterKiang2750.563.632.84Livestock  Donkey9000000  Horse2481.579.587.49  Cattle1910.662.721.92  Yak2983.9122.82016.521  Yak cattle hybrids22700000  Sheep3421.5223.72015.519  Goat3350.6954.54735.545Livestock total112178.34113.29776.796Total78.9032.479.6
Synthesis of the Conflict: Is It Real or Perceived?
Most wars in the last five decades have resulted in detrimental effects on wildlife (Blom 2000; Dudley and others 2002; Mishra and Fitzherbert 2004), and the war between India and China in 1962 was no exception, particularly from the viewpoint of kiang conservation. Our review suggests that the kiang population in Ladakh declined drastically in the years during and after the war between India and China in the 1960s, possibly due to hunting, land mines, and disturbances associated with the war. Thus, for two decades during and after the war, a generation of herders presumably saw and became used to a very low density of kiang in Ladakh’s rangelands. Not surprisingly, the genesis of kiang–human conflict in Ladakh can be traced back to the 1980s, the period by which the kiang population had recovered. It seems that the recovery of the kiang from a very low density to the current estimated density of 0.24/km2 has led to the perception to a generation of nomads that Ladakh’s rangelands are now overstocked with kiang. Available estimates from three regions in Tibet place the density of kiang between 0.15 and 0.39 kiang km−2 (Schaller 1998), suggesting that the kiang density in Ladakh is comparable with those in Tibet. Furthermore, our results show that the share of forage consumed by the kiang vis-à-vis livestock is relatively small (10–11% in the whole of eastern Ladakh, and 3–4% in Hanle Valley). Thus, at a landscape level, the perception of the overabundance of kiang in Ladakh, and of kiang compromising cashmere production, is clearly unfounded.
Nevertheless, there is variation in kiang density locally (0.03 to 0.86 kiang km−2 recorded in our surveys). Kiang occasionally congregate in large herds and can together remove considerable forage from the low-productivity Trans-Himalayan pastures. Using the data in Table 2, a group of 70 kiang (the largest group seen during the survey was 74) can be estimated to potentially consume 199–254 kg dry forage in a single day. The peak standing biomass in sedge meadows, which seem to be important kiang habitats, has been estimated in the adjoining region of Spiti at 857 kg ha−1 (Mishra 2001). These habitats seem critical for both kiang and livestock, but cover a small fraction of the landscape, with an estimated 150 km2 in the entire approximately 20,000 km2 of Changthang. Illustratively, this means that in a single day a large group of 70 kiang can potentially consume more than a quarter of the standing biomass from 1 ha of a key resource area. This consumption is also very similar to the estimated daily forage consumption by a herd of 200 goats (approximately 215 kg). Because of the long winters (November to April) and short summers (July–August), the potential for regrowth during summer is low, aggravating the impact. Considering the potential of large kiang herds to remove significant amounts of forage, together with the fact that grazing areas have been reduced and the livestock population in Ladakh more than doubled in the last 30 years, it is not difficult to see that at a localized scale of the valued sedge meadows, some herders must be facing serious forage competition from the kiang. Given the intensifying commercialization of cashmere production in Ladakh and its integration with global markets, this localized forage competition is, perhaps understandably, no longer tolerated, and the conflict needs to be addressed immediately. However, it must be borne in mind that the problem seems to be evident at local scales, and will need to be addressed at that scale.
In the absence of any scientific understanding of the conflict so far, or efforts to document it, this local conflict has already begun to take on a serious political dimension. The politicians and the district administration in Ladakh are blaming the kiang for compromising cashmere production, and the wildlife department is being looked upon to provide a solution. The only on-ground reaction of the district administration has been to fence large productive sedge and grass meadows from the kiang. In the Hanle Valley alone, an estimated 120 ha of meadows have been fenced already. Three fourths of our total kiang sightings during the rangewide surveys were in such meadows, suggesting that this is an important habitat for the species, and continued fencing of this habitat may cause a decline in the kiang population of Ladakh.
How can this conflict be managed? Although our review of the recent history of political and socioeconomic developments in kiang habitat and our estimates of density and forage consumption of kiang and livestock bring to light interesting dimensions of the kiang–human conflict, the current level of understanding of the kiang’s ecology and its relationships with livestock is still very preliminary. Intensive research needs to be promoted to assist in better management of the species, and its conflicts with humans. Monitoring programs need to assess kiang and livestock density, as well as the land use changes in kiang habitat. Our article shows that the problem of kiang–human conflict is locally concentrated in the key resource areas that contain the sedge meadows, and management strategies need to be devised at that local scale. Surveys are needed to identify all high-conflict areas, and adequate compensatory mechanisms need to be worked out for herders who face genuine forage competition from the kiang. It needs to be examined whether the fact that herders share the forage with kiang could actually be used for adding value to the cashmere produced in Ladakh. The kiang is a striking and conspicuous wild animal that may provide a potential for ecotourism. Finally, after taking up the above interventions, it is important that fences on the sedge meadows to exclude kiang be removed and the practice of fencing discontinued because they lock out key resource areas from the kiangs’ range. Without access to key resource areas, kiang and other wild herbivores, such as the Tibetan gazelle, Procapra picticaudata, may face severe population declines in Ladakh.

conflicts
pastoralism
equus kiang
ladakh
livestock
nomads