Binding of Campylobacter jejuni FliW Adjacent to the CsrA RNA-Binding Pockets Modulates CsrA Regulatory Activity.

Campylobacter jejuni CsrA is an mRNA-binding, post-transcriptional regulator that controls many metabolic- and virulence-related characteristics of this important pathogen. In contrast to E. coli CsrA, whose activity is modulated by binding to small non-coding RNAs (sRNAs), C. jejuni CsrA activity is controlled by binding to the CsrA antagonist FliW. In this study, we identified the FliW binding site on CsrA. Deletion of the C-terminus of C. jejuni CsrA, which is extended relative to sRNA-binding CsrA proteins, abrogated FliW binding. Bacterial two-hybrid experiments were used to assess the interaction of FliW with wild-type CsrA and mutants thereof, in which every amino acid was individually mutated. Two CsrA mutations (V51A and N55A) resulted in a significant decrease in FliW binding. The V51A and N55A mutants also showed a decrease in CsrA-FliW complex formation, as assessed by size-exclusion chromatography and surface plasmon resonance. These residues were highly conserved in bacterial species containing CsrA orthologs whose activities are predicted to be regulated by FliW. The location of FliW binding was immediately adjacent to the two RNA-binding sites of the CsrA homodimer, suggesting the model that FliW binding to CsrA modulates its ability to bind to its mRNA targets either by steric hindrance, electrostatic repulsion, or by altering the overall structure of the RNA-binding sites.

RNA Binding by the Campylobacter jejuni Post-transcriptional Regulator CsrA.

Campylobacter jejuni is a Gram-negative rod-shaped bacterium that commensally inhabits the intestinal tracts of livestock and birds, and which also persists in surface waters. C. jejuni is a leading cause of foodborne gastroenteritis, and these infections are sometimes associated with the development of post-infection sequelae such as Guillain-Barré Syndrome. Flagella are considered a primary virulence factor in C. jejuni, as these organelles are required for pathogenicity-related phenotypes including motility, biofilm formation, host cell interactions, and host colonization. The post-transcriptional regulator CsrA regulates the expression of the major flagellin FlaA by binding to flaA mRNA and repressing its translation. Additionally, CsrA has previously been shown to regulate 120-150 proteins involved in diverse cellular processes. The amino acid sequence of C. jejuni CsrA is significantly different from that of Escherichia coli CsrA, and no previous research has defined the amino acids of C. jejuni CsrA that are critical for RNA binding. In this study, we used in vitro SELEX to identify the consensus RNA sequence mAwGGAs to which C. jejuni CsrA binds with high affinity. We performed saturating site-directed mutagenesis on C. jejuni CsrA and assessed the regulatory activity of these mutant proteins, using a reporter system encoding the 5' untranslated region (5' UTR) upstream of flaA linked translationally to the C. jejuni astA gene. These assays allowed us to identify 19 amino acids that were involved in RNA binding by CsrA, with many but not all of these amino acids clustered in predicted beta strands that are involved in RNA binding by E. coli CsrA. Decreased flaA mRNA binding by mutant CsrA proteins L2A and A36V was confirmed by electrophoretic mobility shift assays. The majority of the amino acids implicated in RNA binding were conserved among diverse Campylobacter species.

Evaluation of ceftiofur crystalline free acid sterile suspension for control of metritis in high-risk lactating dairy cows.

Two studies were conducted to determine if clinical metritis could be prevented or decreased in at-risk lactating dairy cows by a single treatment with Excede Sterile Suspension (ceftiofur crystalline free acid sterile suspension [CCFA-SS]) administered within 24 hours after an abnormal calving. Study 1 was a preliminary study and study 2 was a clinical trial (designed to confirm the results of study 1). In both studies, abnormal calving was defined as cows that had dystocia (required assistance), twins, abortion, retained fetal membranes for 12 hours or more, or any combination thereof. A randomized block design with cows blocked on order-of-entry within dairy without regard to parity was used in both studies. In study 1, cows that had abnormal calving from six commercial dairies were randomly assigned to either untreated control (N = 122) or 6.6 mg ceftiofur equivalents/kg of body weight sc in the base of the ear (CCFA-SS, N = 121), within 24 hours after calving. Cows with normal calving during the enrollment period received no treatment and were included for observational purposes (N = 122). Health observations and rectal temperatures were recorded daily, and physical examinations were conducted on Days 1 ± 1, 7 ± 2, 14 ± 2, and 21 ± 2, and uterine swabs (for bacterial culture) were collected from a subsample of cows on Days 3 or 4, 7 ± 2, 14 ± 2, and 21 ± 2. These observations were made by treatment-blinded personnel. In study 2, cows with abnormal calving from 12 commercial dairies were assigned to receive either saline (control, N = 247) or CCFA-SS (N = 247) within 24 hours after calving. Health observations and rectal temperatures were recorded daily, and physical examinations were conducted on Days 0 to 2, 7 ± 1, and 14. In study 1, the incidence of metritis on Day 14 ± 2 was 20.2% versus 36.8% for CCFA-SS and control, respectively, with an odds ratio of 2.30 (P < 0.05). In study 2, incidences of metritis on Day 14 were 28.7% versus 43.5% for CCFA-SS and saline, respectively, with an odds ratio of 1.92 (P < 0.05). Rectal temperatures on Days 1 and 2 and the average for the first 6 days were lower (P < 0.05) for CCFA-SS compared with control cows for both studies. Treatment of cows with a single dose of CCFA-SS within 24 hours after abnormal calving reduced the incidence of subsequent metritis in lactating dairy cows.

Biofilm in milking equipment on a dairy farm as a potential source of bulk tank milk contamination with Listeria monocytogenes.

The objective of this study was to assess the presence of a Listeria monocytogenes-containing biofilm in milking equipment as a potential source of bulk tank milk contamination on a dairy farm where milk contamination had been previously documented. Samples were collected from milking equipment and milking parlor premises on 4 occasions and analyzed for the presence of L. monocytogenes. Pulsed-field gel electrophoresis (PFGE) typing was conducted on L. monocytogenes isolates from the milking equipment, parlor and storage room floors, bulk tank milk, and in-line milk filters. Pieces from milk meters and rubber liners were obtained to visually assess the presence of a biofilm using scanning electron microscopy. A total of 6 (15%), 4 (25%), and 1 (6%) samples were culture-positive for L. monocytogenes in the first, second, and third sample collection, respectively. Two samples were L. monocytogenes hly PCR-positive but were culture-negative in the fourth sample collection. Combined AscI and ApaI restriction analysis yielded 6 PFGE types for 15 L. monocytogenes isolates obtained from milking equipment, parlor, bulk tank milk, and milk filters. A predominant and persistent PFGE type (PFGE type T) was observed among these L. monocytogenes isolates (9/15 isolates). Scanning electron microscopy of samples from the bottom cover of 2 milk meters showed the presence of individual and clusters of bacteria, mainly associated with surface scratches. The presence of a bacterial biofilm was observed on the bottom covers of the 2 milk meters. Prevention of the establishment of biofilms in milking equipment is a crucial step in fulfilling the requirement of safe, high-quality milk.

First Report of Limber Pine Dwarf Mistletoe (Arceuthobium cyanocarpum) on Sugar Pine (Pinus lambertiana) from California.

Limber pine dwarf mistletoe (Arceuthobium cyanocarpum (A. Nelson ex Rydberg) Coulter & Nelson; Viscaceae) severely parasitizes limber pine (Pinus flexilis James) and several other white pines, including western white pine (P. monticola Dougl. ex D. Don) and whitebark pine (P. albicaulis Engelm.), over an extensive geographic range in the western United States (1). However, limber pine dwarf mistletoe has not been previously reported on sugar pine (P. lambertiana Dougl.), another white pine found within the range of limber pine dwarf mistletoe (1). In August 2009, we found a sugar pine infected with limber pine dwarf mistletoe approximately 0.8 km northeast of Tahquitz Peak in the San Jacinto Mountains, California (33°45'24''N, 116°40'24''W; elevation 2,640 m). The infected sugar pine was 13 inches (33 cm) in diameter and had 13 infections on five of its lower branches. Ten of the infections were producing mature male and female mistletoe plants with open flowers or developing fruits, respectively. Two of the infected branches were forming witches' brooms in response to infection by limber pine dwarf mistletoe. The infected sugar pine was growing within 3 m of four limber pines severely infected with limber pine dwarf mistletoe. The male and female plants produced on the infected sugar pine were morphologically identical to those growing on the infected limber pines. Limber pine dwarf mistletoe can be distinguished from sugar pine dwarf mistletoe (A. californicum Hawksw. & Wiens), the principal dwarf mistletoe parasitizing sugar pine in California, by its smaller plants (mean height 3 cm versus 8 cm) and flowering period (August to September versus June to July). In an attempt to determine the relative susceptibility of sugar pine to limber pine dwarf mistletoe, we conducted a survey of the infested limber pine stand. Because there were no additional sugar pines growing in the area, it was impossible to assess the general susceptibility of sugar pine to limber pine dwarf mistletoe, but the production of many mature plants from 10 of the infections on the sugar pine suggests this tree species may be highly susceptible. However, this is currently the only known location where sugar pine co-occurs with limber pine dwarf mistletoe (1), so assessing the susceptibility of sugar pine to this dwarf mistletoe will depend on locating additional sites where they co-occur. It should be noted also that previous surveys in the San Jacinto Mountains failed to detect infection by limber pine dwarf mistletoe on sugar pine (1). Specimens of limber pine dwarf mistletoe on sugar pine were collected and deposited at the Deaver Herbarium (ASC), Northern Arizona University, Flagstaff (Accession No. 92697). To our knowledge, this is the first report of limber pine dwarf mistletoe parasitizing sugar pine. References: (1) F. G. Hawksworth and D. Wiens. USDA For. Serv. Agric. Handb. 709, 1996.

First Report of Mountain Hemlock Dwarf Mistletoe (Arceuthobium tsugense subsp. mertensianae) on Sugar Pine (Pinus lambertiana) from Oregon.

Mountain hemlock dwarf mistletoe (Arceuthobium tsugense (Rosendahl) G.N. Jones subsp. mertensianae Hawksw. & Nickrent, Viscaceae) commonly parasitizes mountain hemlock (Tsuga mertensiana (Bong.) Carr.) from the central Sierra Nevada Mountains to the southern Cascades in Oregon (1,3). It has also been reported to commonly parasitize whitebark pine (Pinus albicaulis Engelm.) and occasionally western white pine (P. monticola Dougl. ex D. Don) (1,4). In September of 2008, we found mountain hemlock dwarf mistletoe infecting two sugar pines (P. lambertiana Dougl.) 4.5 km north of Windigo Pass, Oregon (42°24'40″N, 123°35'26″W, elevation 2,710 m). One of the sugar pines was 63.5 cm (25 inches) in diameter and had three infections. The other tree was 22.4 cm (9 inches) in diameter, but had 17 infections, many with mistletoe plants that allowed identification of the mistletoe using published descriptions (1,2). Mountain hemlock dwarf mistletoe can be distinguished from sugar pine dwarf mistletoe (A. californicum Hawksw. & Wiens) by its smaller plant size (mean height 5 cm versus 8 cm) and plant color (green-brown versus green to yellow) (1,2). An area (site) of approximately 1 ha around the infected sugar pines was examined and none of the other sugar pines we observed (33 trees) were found to be infected. Because mountain hemlock dwarf mistletoe also occurs in the principal range of sugar pine in the Sierra Nevada Mountains, it may also parasitize this tree there. However, our observations in several mountain hemlock stands infested with mountain hemlock dwarf mistletoe in California have failed to uncover infection of sugar pine by this mistletoe. Therefore, we would tentatively classify sugar pine as a rare host of mountain hemlock dwarf mistletoe (1). Specimens of mountain hemlock dwarf mistletoe on sugar pine were collected and deposited at the Deaver Herbarium (ASC), Northern Arizona University, Flagstaff (Accession No. 87122). To our knowledge, this is the first report of mountain hemlock dwarf mistletoe parasitizing sugar pine. References: (1) F. G. Hawksworth and D. Wiens. USDA For. Serv. Agric. Handb. 709, 1996. (2) F. G. Hawksworth et al. Novon 2:204, 1992. (3) R. L. Mathiasen and C. M. Daugherty. Novon 17:222, 2007. (4) R. L. Mathiasen and F. G. Hawksworth. For. Sci. 34:429, 1988.

Fine-scale genetic structure of a long-lived reptile reflects recent habitat modification.

Anthropogenic habitat fragmentation--ubiquitous in modern ecosystems--has strong impacts on gene flow and genetic population structure. Reptiles may be particularly susceptible to the effects of fragmentation because of their extreme sensitivity to environmental conditions and limited dispersal. We investigate fine-scale spatial genetic structure, individual relatedness, and sex-biased dispersal in a large population of a long-lived reptile (tuatara, Sphenodon punctatus) on a recently fragmented island. We genotyped individuals from remnant forest, regenerating forest, and grassland pasture sites at seven microsatellite loci and found significant genetic structuring (R(ST)=0.012) across small distances (<500 m). Isolation by distance was not evident, but rather, genetic distance was weakly correlated with habitat similarity. Only individuals in forest fragments were correctly assignable to their site of origin, and individual pairwise relatedness in one fragment was significantly higher than expected. We did not detect sex-biased dispersal, but natural dispersal patterns may be confounded by fragmentation. Assignment tests showed that reforestation appears to have provided refuges for tuatara from disturbed areas. Our results suggest that fine-scale genetic structuring is driven by recent habitat modification and compounded by the sedentary lifestyle of these long-lived reptiles. Extreme longevity, large population size, simple social structure and random dispersal are not strong enough to counteract the genetic structure caused by a sedentary lifestyle. We suspect that fine-scale spatial genetic structuring could occur in any sedentary species with limited dispersal, making them more susceptible to the effects of fragmentation.

Outcomes of patients with AML and MDS who relapse or progress after reduced intensity allogeneic hematopoietic cell transplantation.

We describe treatment, outcomes and prognostic factors for patients who relapse following transplantation with a reduced intensity conditioning regimen. Seventy consecutive patients with high-risk myeloid malignancies underwent transplant and 25 (36%) relapsed, a median of 120 days later. The median percentage of bone marrow blasts at relapse was 24, the median donor chimerism was 73% and new karyotypic abnormalities occurred in 8 out of 20 (40%) evaluable patients. Twenty-one patients (84%) received aggressive treatment for relapse, including chemotherapy (60%), second hematopoietic cell transplantation (HCT; 52%) and/or donor lymphocyte infusion (DLI; 12%). Thirteen achieved a complete response (CR) and four remain in CR. Median overall survival (OS) after relapse was 6 months (95% confidence interval=2.7-9.9 months), and actuarial 1 year OS was 24%. Most deaths were due to disease progression (17/20, 85%). We did not observe an advantage for cellular therapy (DLI or second transplant) compared to chemotherapy. Salvage therapy for relapse after reduced intensity HCT is feasible, associated with low treatment-related mortality, and may result in prolonged survival in select patients. Studies exploring the optimal treatment for relapse following reduced intensity HCT are warranted.

Species and cultural conservation in New Zealand: maori traditional ecological knowledge of tuatara.

Traditional ecological knowledge can be highly informative and integrated with complementary scientific knowledge to improve species management. This is especially true for abundant species with which indigenous peoples have frequent interactions (e.g., through harvest), but has been studied less frequently in isolated or declining species. We examined Maori traditional ecological knowledge of tuatara (Sphenodon spp., reptiles that resemble lizards but are the last living representatives of the order Sphenodontia) through semidirected interviews of elders of Te Atiawa, Ngati Koata, and Ngati Wai Iwi (similar to tribes), the guardians of several islands currently inhabited by tuatara. Maori are indigenous to New Zealand, having settled 800 to 1000 years ago. Tuatara are endemic to New Zealand, have declined in numbers since human settlement, and are now restricted to 37 offshore islands. The detail and volume of tuatara traditional ecological knowledge were less than that recorded in studies of more abundant or accessible species. In addition, traditional knowledge of the cultural significance of tuatara was more common and detailed among the elders than traditional knowledge of tuatara biology or ecology. The traditional knowledge collected, however, provided the first evidence of seven former sites of tuatara occupation, suggested five additional sites tuatara may currently occupy, contained novel hypotheses for scientific testing, and described tuatara cultural roles that have not been reported previously. We conclude that, in at least some cases, traditional ecological knowledge may persist as species decline and may serve as a valuable source of ecological information for conservation.

Low cost of locomotion in lizards that are active at low temperatures.

The nocturnality hypothesis of K. Autumn and coworkers states that nocturnal geckos have evolved a low energetic cost of locomotion (C(min)). A low C(min) increases maximum aerobic speed and partially offsets the decrease in maximum oxygen consumption caused by activity at low nocturnal temperatures. We tested whether a low C(min) is unique to nocturnal geckos or represents a more general pattern of convergent evolution among lizards that enables nocturnality and/or cold-temperature activity. We measured C(min) in four carefully selected lizard species from New Zealand (two nocturnal and two diurnal; n=5-9 individuals per species), including a nocturnal and diurnal gecko (a low C(min) is a gecko trait and is not related to nocturnality), a nocturnal skink (a low C(min) is related to being nocturnal), and a diurnal skink active at low temperatures (a low C(min) is related to being active at low body temperatures). The C(min) values of the four species measured in this study (range=0.21-2.00 mL O(2) g(-1) km(-1)) are lower than those of diurnal lizards from elsewhere, and the values are within or below the 95% confidence limits previously published for nocturnal geckos. A low C(min) increases the range of locomotor speeds possible at low temperatures and provides an advantage for lizards active at these temperatures. We accepted the hypothesis that nocturnal lizards in general have a low C(min) and provide evidence for a low C(min) in lizards from cool-temperate environments. The low C(min) in lizards living at high latitudes may enable extension of their latitudinal range into otherwise thermally suboptimal habitats.

Total lactate dehydrogenase activity of tail muscle is not cold-adapted in nocturnal lizards from cool-temperate habitats.

The dependence of metabolic processes on temperature constrains the behavior, physiology and ecology of many ectothermic animals. The evolution of nocturnality in lizards, especially in temperate regions, requires adaptations for activity at low temperatures when optimal body temperatures are unlikely to be obtained. We examined whether nocturnal lizards have cold-adapted lactate dehydrogenase (LDH). LDH was chosen as a representative metabolic enzyme. We measured LDH activity of tail muscle in six lizard species (n=123: three nocturnal, two diurnal and one crepuscular) between 5 and 35 degrees C and found no differences in LDH-specific activity or thermal sensitivity among the species. Similarly, the specific activity and thermal sensitivity of LDH were similar between skinks and geckos. Similar enzyme activities among nocturnal and diurnal lizards indicate that there is no selection of temperature specific LDH enzyme activity at any temperature. As many nocturnal lizards actively thermoregulate during the day, LDH may be adapted for a broad range of temperatures rather than adapted specifically for the low temperatures encountered when the animals are active. The total activity of LDH in tropical and temperate lizards is not cold-adapted. More data are required on biochemical adaptations and whole animal thermal preferences before trends can be established.

Fludarabine, melphalan, and alemtuzumab conditioning in adults with standard-risk advanced acute myeloid leukemia and myelodysplastic syndrome.

PURPOSE: This prospective phase II study evaluated toxicity, relapse rate, progression-free survival, and overall survival after allogeneic transplantation and conditioning with fludarabine, melphalan, and alemtuzumab in patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). PATIENTS AND METHODS: Fifty-two consecutive adults with AML and MDS were enrolled onto the study. Median age was 52 years (range, 17 to 71 years) and the majority of patients had high-risk disease, comorbidities, and/or modest reduction in performance status. Fifty-six percent of patients had unrelated or mismatched related donors. RESULTS: After a median follow-up of 18 months (range, 2 to 34 months), 1-year survival was 48% (95% CI, 34% to 61%), progression-free survival was 38% (95% CI, 25% to 52%), relapse rate was 27% (95% CI, 15% to 40%), and treatment-related mortality was 33% (95% CI, 20% to 46%). The cumulative probability of extensive chronic graft-versus-host disease (GVHD) was only 18% (95% CI, 8% to 40%); extensive chronic GVHD was only observed in recipients of unrelated donor transplants. Performance score and disease status were the major predictors of outcome. High-risk disease (ie, active AML or MDS with > 5% blasts) or even modest decreases in performance status were associated with poor outcomes. Patients with standard-risk leukemia (first or second complete remission) or MDS (< 5% blasts) had excellent outcomes despite unfavorable disease characteristics. CONCLUSION: Fludarabine and melphalan combined with in vivo alemtuzumab is a promising transplantation regimen for patients with AML or MDS and low tumor burden. For patients with active disease, this regimen provides at best modest palliation. Despite a low incidence of GVHD, transplantation is still associated with considerable nonrelapse mortality in patients with decreased performance status.

First Report of Chihuahua Pine Dwarf Mistletoe (Arceuthobium gillii) on Apache Pine (Pinus engelmannii).

Arceuthobium gillii Hawksw. & Wiens (Viscaceae) is primarily distributed in the Sierra Madre Occidental of Mexico from central Durango and northern Sinaloa into Chihuahua and Sonora (3). In Mexico, it commonly parasitizes Pinus leiophylla Schiede & Deppe var. leiophylla and var. chihuahuana (Engelm.) G. R. Shaw, P. lumholtzii Robinson & Fern., and P. herrerai Martinez and rarely infects P. arizonica Engelm. and P. cooperi Blanco (3). This dwarf mistletoe also occurs in the Chiricahua, Huachuca, Santa Rita, Rincon, and Santa Catalina Mountains of southern Arizona and the Animas Mountains of southwestern New Mexico (1,3). In the United States, A. gillii has only been reported to parasitize P. leiophylla var. chihuahuana (1,2,3). The host range of A. gillii has consistently not included P. engelmannii Carr. (2,3). However, we have located a small population of P. engelmannii naturally infected by A. gillii in the South Fork of Cave Creek, Chiricahua Mountains, Arizona. The infected P. engelmannii occurred approximately 2.6 km west of the South Fork Cave Creek Picnic Area, Coronado National Forest, along Forest Trail 243 (31°50'53″N, 109°12'14″W, elevation 1,670 m). Only four P. engelmannii were infected: one tree had four infections as indicated by small, dense witches' brooms and branches with large, spindle-shaped swellings, and three trees had one infection each based on the presence of brooms. One branch was collected from the tree with four infections because observations of the branch with binoculars indicated it had dwarf mistletoe shoots. Only four male shoots of A. gillii were on the branch and they were consistent morphologically with other male plants of this dwarf mistletoe collected from nearby P. leiophylla var. chihuahuana. The infected P. engelmannii were all growing in close association with P. leiophylla var. chihuahuana severely infected with A. gillii. The only other dwarf mistletoe reported to infect P. engelmannii in the Chiricahua Mountains is A. vaginatum (Willd.) Presl subsp. cryptopodum (Engelm.) Hawksw. & Wiens. This dwarf mistletoe was not present or anywhere near the Cave Creek population of P. engelmannii. In addition, plants of A. gillii can be easily distinguished morphologically from those of A. vaginatum subsp. cryptopodum by their color (2,3). Plants of A. gillii are consistently green to greenish brown, while plants of A. vaginatum subsp. cryptopodum are consistently orange to reddish brown. A specimen of A. gillii on P. engelmannii has been deposited at the Deaver Herbarium, Northern Arizona University, Flagstaff (Accession No. 75392). To our knowledge, this is the first report of A. gillii on P. engelmannii. This host-dwarf mistletoe combination is evidently very rare because other investigators have reported that P. engelmannii was probably immune to A. gillii (2,3). References: (1) F. G. Hawksworth and M. Weiss. Southwest. Nat. 20:418, 1975. (2) F. G. Hawksworth and D. Wiens. Brittonia 16:54, 1964. (3) F. G. Hawksworth and D. Wiens. Dwarf mistletoes: Biology, pathology, and systematics. USDA For. Serv. Agric. Handb. 709, 1996.

Toxic honey and the march up-country.

This article gives an account of the Greek warrior-historian Xenophon and his Anabasis (The March Up-Country), one of the most famous events in military history. It includes a description of how the Greek soldiers, after reaching apparent safety near the south-eastern Black Sea, were felled by a strange honey that rendered them as if dead for a day. Modern understanding of the toxicology of this honey is given, then an epilogue summarizing the rest of Xenophon's life.

Conditioning reduces metabolic rate and time to steady-state in the lizard Naultinus manukanus (Reptilia: Gekkonidae).

The rate of oxygen consumption (VO2) is commonly used as a measure of whole organism metabolic rate, but requires the animal to be motionless and at rest. Few studies have measured whether animals that appear motionless are truly at rest, or whether being in a novel environment elevates metabolic rate. We investigated whether conditioning of the gecko Naultinus manukanus to experimental procedures influenced the VO2 and probability of achieving a constant rate of oxygen consumption. Metabolic rate was measured at 24 degrees C in 22 individuals until a steady-state was achieved, or for 80 min if no steady-state was reached, once a day on 5 consecutive days (five trials). Geckos in the first trial, when compared with subsequent trials, had a significantly higher mass-adjusted VO2 (0.89+/-0.06 vs. 0.67+/-0.05 ml O2 h(-1), respectively), and time to reach a steady-state VO2 (66+/-8 vs. 47+/-3 min, respectively), as well as a significantly lower probability of reaching a steady-state VO2 (24% vs. 74%, respectively). We conclude that there may be hidden inaccuracies in studies that do not condition animals and that at least one conditioning trial should be used to obtain a metabolic rate at rest for small lizards.

Nuclear mitochondrial pseudogenes as molecular outgroups for phylogenetically isolated taxa: a case study in Sphenodon.

'Living fossil' taxa, by definition, have no close relatives, and therefore no outgroup to provide a root to phylogenetic trees. We identify and use a molecular outgroup in the sole extant lineage of sphenodontid reptiles, which separated from other reptiles 230 million years ago. We isolated and sequenced a partial nuclear copy of the mitochondrial cytochrome b gene. We confirm the copy is indeed not mitochondrial, is older than all extant mitochondrial copies in Sphenodon (tuatara), and is therefore useful as a molecular outgroup. Under phylogenetic analysis, the nuclear copy places the root of the tuatara mitochondrial gene tree between the northern and the southern (Cook Strait) groups of islands of New Zealand that are the last refugia for Sphenodon. This analysis supports a previous mid-point rooted mitochondrial gene tree. The mitochondrial DNA tree conflicts with allozyme analyses which place a Cook Strait population equidistant to all northern and other Cook Strait populations. This population on North Brother Island is the only natural population of extant S. guntheri; thus, we suggest that the current species designations of tuatara require further investigation.

Chromosomes of tuatara, Sphenodon, a chromosome heteromorphism and an archaic reptilian karyotype.

We examined karyotypes of the endemic New Zealand reptile genus Sphenodon (tuatara) from five populations, finding a karyotype unchanged for at least one million years. Animals karyotyped were from five geographically distinct populations, representing three groups, namely S. guntheri, S. punctatus (Cook Strait group), and S. punctatus (northeastern North Island group). All five populations have a diploid chromosome number of 2n = 36, consisting of 14 pairs of macrochromosomes and four pairs of microchromosomes. Chromosomal differences were not found between the five populations nor between female and male animals, except for one animal with a structural heteromorphism. Similarity between Sphenodon and Testudine karyotypes suggests an ancestral karyotype with a macrochromosome complement of 14 pairs and the ability to accumulate variable numbers of microchromosome pairs. Our research supports molecular phylogenies of the Reptilia.

First Report of Arceuthobium hondurense in Oaxaca, Mexico.

Honduran dwarf mistletoe (Arceuthobium hondurense Hawksw. & Wiens) has only been found in four locations in Honduras: west of Zamorano, east of Lepaterique, Cusuco National Park, and Celaque National Park (1,2). At one time it was believed that this mistletoe could be in danger of extinction (1). However, it has also been reported in two locations in Chiapas, Mexico (3). In December 2000, ≈1 km north of Suchixtepec, Oaxaca, Mexico, near Route 175 (elevation 2,770 m), we collected a dwarf mistletoe parasitizing Pinus tecunumanii (Schw.) Eguiluz et Perry that was morphologically similar to A. hondurense (1). This population initially had been classified as A. nigrum Hawksw. & Wiens (1), but is now classified as A. hondurense based on morphology (male flower color and stigma length) (1) and analysis of nuclear rDNA internal transcribed spacer (ITS) sequences from Honduras (GenBank Accession No. AF325969) and Oaxaca (GenBank Accession No. AY055215). A comparison of these two A. hondurense ITS sequences indicated they are very closely related to each other, whereas both are distinct from A. nigrum (GenBank Accession No. L25693). Specimens of A. hondurense from Oaxaca were deposited at the Deaver Herbarium, Northern Arizona University, Flagstaff. To our knowledge, this is the first report of A. hondurense in Oaxaca, Mexico, and extends its known distribution west across the Isthmus of Tehuantepec from central Chiapas by ≈400 km. References: (1) F. G. Hawksworth and D. Wiens. Dwarf Mistletoes: Biology, Pathology, and Systematics. USDA Agric. Handb. 709, 1996. (2) R. Mathiasen et al. Phytologia 85:268, 1998. (3) R. Mathiasen et al. Plant Dis. 85:444, 2001.

Psychosocial adjustment of patients and caregivers prior to allogeneic bone marrow transplantation.

There are many studies that examine the psychosocial adjustment of survivors of bone marrow transplantation (BMT). On the other hand, there are relatively few studies that examine the psychosocial adjustment of patients prior to BMT, and even fewer that focus on the psychosocial adjustment of the patient's caregiver. The purpose of the present study was to assess performance status and psychosocial adjustment to illness, mood and stress response of patients and caregivers prior to admission for allogeneic BMT. Forty patients and their 39 caregivers were assessed using standardized measures. One-fourth of the patients reported clinical levels of psychosocial maladjustment on the Psychosocial Adjustment to Illness Scale and had greater adjustment problems than BMT survivors. Approximately one-third (35%) and one-quarter (23%) of the patients reported significant symptoms of intrusive and avoidance stress responses, respectively on the Impact of Events Scale. Caregivers reported more impairments in family relationships than patients, but overall reported similar distress to that of patients. Information about the pre-BMT process appears to be critical to understanding the psychosocial impact that BMT can have on patients and their caregivers.