Participation of non-zinc finger residues in DNA binding by two nuclear orphan receptors.

Steroid-thyroid hormone receptors typically bind as dimers to DNA sequences that contain repeated elements termed half-sites. NGFI-B, an early response protein and orphan member of this receptor superfamily, binds to a DNA sequence that contains only one half-site (5'-AAAGGTCA-3'). A domain separate from the NGFI-B zinc fingers, termed the A box, was identified and is required for recognition of the two adenine-thymidine (A-T) base pairs at the 5' end of the NGFI-B DNA binding element. In addition, a domain downstream of the zinc fingers of the orphan receptor H-2 region II binding protein, termed the T box, determined binding to tandem repeats of the estrogen receptor half-site (5'-AGGTCA-3').

Effect of prescribed fire for tick control in California chaparral.

Prescribed fire was investigated as a method for controlling ixodid and argasid ticks in chaparral habitats in northern California. Two experimental and two adjacent control plots within a wildlife preserve were monitored for 1 yr postburn. Ticks were collected by flagging vegetation, by CO2-baited pitfall trap, and by live-trapping rodents. Twice as many rodents were caught at control sites compared with burn sites and no dusky-footed woodrats, Neotoma fuscipes Baird, were found in the treatment sites postburn. This species is known to be a reservoir of the agents of Lyme disease, Borrelia burgdorferi sensu stricto Johnson, Schmid, Hyde, Steigerwalt & Brenner, and human granulocytic anaplasmosis, Anaplasma phagocytophilum Dumler, Barbet, Bekker, Dasch, Palmer, Ray, Rikihisa, Rurangirwa. Six ixodid tick species were removed from rodents (Ixodes pacificus Cooley & Kohls, Ixodes jellisoni Cooley & Kohls, Ixodes spinipalpis Hadwen & Nuttall, Ixodes woodi Bishopp, Dermacentor occidentalis Marx, and Dermacentor parumapertus Neumann), two of which transmit bacterial zoonotic agents to people in the far-western United States. There was no decrease in number of ticks per animal trapped at either burn site compared with controls; in fact, the mean number of immature I. pacificus per rodent was significantly higher at one burn site than its control site. Soil refugia may protect ticks from fire-induced mortality; the argasid tick Ornithodoros coriaceus Koch, which lives in soil, was unaffected by the prescribed fire as were I. pacificus and D. occidentalis buried in packets 2.5 cm below ground. We conclude that although prescribed fires in chaparral habitats may diminish local rodent abundance, it does not decrease tick loads on rodents. Furthermore, burning chaparral does not result in a decreased abundance of adult ixodid ticks on vegetation and apparently does not affect argasid or ixodid ticks that are sheltered within soil refugia.

Gadolinium DTPA Enhancement Characteristics of the Rat Sciatic Nerve after Crush Injury at 4.7T.

BACKGROUND AND PURPOSE: Traumatic peripheral nerve injury is common and results in loss of function and/or neuropathic pain. MR neurography is a well-established technique for evaluating peripheral nerve anatomy and pathology. However, the Gd-DTPA enhancement characteristics of acutely injured peripheral nerves have not been fully examined. This study was performed to determine whether acutely crushed rat sciatic nerves demonstrate Gd-DTPA enhancement and, if so, to evaluate whether enhancement is affected by crush severity. MATERIALS AND METHODS: In 26 rats, the sciatic nerve was crushed with either surgical forceps (6- to 20-N compressive force) or a microvascular/microaneurysm clip (0.1-0.6 N). Animals were longitudinally imaged at 4.7T for up to 30 days after injury. T1WI, T2WI, and T1WI with Gd-DTPA were performed. RESULTS: Forceps crush injury caused robust enhancement between days 3 and 21, while clip crush injury resulted in minimal-to-no enhancement. Enhancement after forceps injury peaked at 7 days and was seen a few millimeters proximal to, in the region of, and several centimeters distal to the site of crush injury. Enhancement after forceps injury was statistically significant compared with clip injury between days 3 and 7 (P < .04). CONCLUSIONS: Gd-DTPA enhancement of peripheral nerves may only occur above a certain crush-severity threshold. This phenomenon may explain the intermittent observation of Gd-DTPA enhancement of peripheral nerves after traumatic injury. The observation of enhancement may be useful in judging the severity of injury after nerve trauma.

Multiparametric evaluation of preoperative MRI in early stage breast cancer: prognostic impact of peri-tumoral fat.

PURPOSE: Obesity is associated with adverse outcomes in breast cancer patients. Fat-specific cytokines (adipokines) have been proposed as key drivers of breast cancer progression, invasion, and metastasis. We aimed at assessing correlations between peri-tumoral fat, quantified on magnetic resonance imaging (MRI) and pathologic factors potentially impacting therapy recommendations. METHODS: We retrospectively reviewed records of 63 patients with early stage breast cancer who underwent preoperative MRI imaging using appropriately weighted series for breast and tumor contouring. Fat volumes were generated through voxel intensity filtering. The peri-tumoral region was defined as the intersection of a 1-cm spherical extension around the tumor and the breast contour. Peri-tumoral fat was defined as the fraction of a fat content in this volume. Surgical pathology records were used to extract clinical data. Statistical analyses were conducted using Pearson and Spearman correlation coefficients. RESULTS: Among reviewed patients, 45 had T1 tumors (1.22 ± 0.85 cm diameter) and 18 had T2 tumors (2.08 ± 1.06 cm). Axillary lymph nodes were dissected in 31 and positive in 17 patients analyzed. Peri-tumoral fat ratio ranged between 25 and 99 %. Peri-tumoral fat ratio significantly correlated with the nodal-positive ratio of positive axillary lymph nodes (r = 0.532). Peri-tumoral fat ratio demonstrated optimally prominent correlation among obese patients upon body mass index categorical stratification. CONCLUSIONS: In women with early stage breast cancer, peri-tumoral fat correlates positively with the ratio of pathologically involved axillary nodes. This work highlights a novel method for quantitating peri-tumoral fat content. Preoperative breast MRI may be utilized to predict extent of axillary disease.

Creating seamless junctions independent of restriction sites in PCR cloning.

A method is described for the efficient cloning of any given DNA sequence into any desired location without the limitation of naturally occurring restriction sites. The technique employs the polymerase chain reaction (PCR) combined with the capacity of the type-IIS restriction endonuclease (ENase) Eam1104I to cut outside its recognition sequence. Primers that contain the Eam1104I recognition site (5'-CTCTTC) are used to amplify the DNA fragments being manipulated. Because the ENase is inhibited by site-specific methylation in the recognition sequence, all internal Eam1104I sites present in the DNA can be protected by performing the PCR amplification in the presence of 5-methyldeoxycytosine (m5dCTP). The primer-encoded Eam1104I sites are not affected by the modified nucleotides (nt) since the newly synthesized strand does not contain any cytosine residues in the recognition sequence. In addition, the ENase's ability to cleave several bases downstream from its recognition site allows the removal of superfluous, terminal sequences from the amplified DNA fragments, resulting in 5' overhangs that are defined by the nt present within the cleavage site. Thus, the elimination of extraneous nt and the generation of unique, non-palindromic sticky ends permits the formation of seamless junctions in a directional fashion during the subsequent ligation event.

Life cycle of Ixodes pacificus (Acari: Ixodidae): timing of developmental processes under field and laboratory conditions.

The developmental timing of Ixodes pacficus Cooley & Kohls, the primary vector of the Lyme disease spirochete and the agent of human granulocytic ehrlichiosis in the far-western United States, was determined under field and laboratory conditions. During their seasonal peaks of abundance, each of the three parasitic stages of L. pacificus, both fed and unfed, was placed inside silk-screen packets. These packets were apportioned between four topographic exposures of two hilltop sites in northwestern California. The sites differed in vegetational composition and elevation: the first (elevation 390 m) was dominated by woodland-grass, the second (elevation 914 m) by chaparral. The timing of oviposition, larval eclosion, molting, and mortality were recorded in the field every 2-3 wk for 2.5 yr. Microenvironmental temperatures were measured on all four exposures at both sites. Accelerated developmental rates of all three stages were correlated with warmer soil temperatures and the time of placement in the field. In the laboratory, replete female I. pacificus maintained under uniform environmental conditions sustained constant preovipositional and prehatch periods independent of date-of-feeding. In the field, all unfed stages survived through one active feeding season with most larvae and nymphs remaining in behavioral diapause between late summer and early spring. No life stage survived through two active feeding periods which suggests that cohorts do not overlap. We concluded that I. pacificus takes a minimum of 3 yr to complete its life cycle in northwestern California.

Life history of Ixodes (Ixodes) jellisoni (Acari: Ixodidae) and its vector competence for Borrelia burgdorferi sensu lato.

Ixodes (Ixodes) jellisoni Cooley & Kohls, a nonhuman biting and little known tick, is one of 4 members of the I. ricinus complex in the United States. A localized population of I. jellisoni inhabiting a grassland biotope in Mendocino County, CA, was studied from 1993 to 1997. Rodent trapping in all seasons revealed that the only host of both immature and adult I. jellisoni was the heteromyid rodent Dipodomys californicus Merriam. Field investigations suggested that I. jellisoni is nidicolous in habit, and laboratory findings demonstrated that it reproduces parthenogenetically. Known parthenogenetic females (n = 4) produced an average of 530 eggs of which 74% hatched, which was comparable to the fecundity and fertility of wild-caught females (n = 8). After the transstadial molt, 57 F1 or F2 nymphs derived from 2 wild-caught or 4 laboratory-reared, unmated females produced only females. Ixodes jellisoni males were not found on 112 wild-caught D. californicus individuals that were captured an average of 2 times. Collectively, these findings suggest that I. jellisoni may be obligatorily parthenogenetic. Borrelial isolates were obtained from 85% of 58 D. californicus and 33% of 21 I. jellisoni females removed from this rodent. None of the 7 infected female ticks passed borreliae ovarially to its F1 larval progeny. Eight D. californicus and 5 I. jellisoni-derived isolates that were genetically characterized belonged to 2 restriction pattern groups of Borrelia burgdorferi s.l. Neither restriction pattern group has been assigned to a particular genospecies yet. After placement on naturally infected D. californicus, noninfected larval ticks acquired and transstadially passed spirochetes as efficiently as (group 1 borreliae) or 6 times more efficiently (group 2 borreliae) than Ixodes pacificus Cooley & Kohls. As few as 1-4 infected I. jellisoni nymphs were capable of transmitting group 1 or group 2 borreliae to naive D. californicus. We conclude that I. jellisoni is a competent vector of both restriction fragment groups when D. californicus is used as the animal model.

Scintigraphic comparison of intra-arterial injection and distal intravenous regional limb perfusion for administration of mesenchymal stem cells to the equine foot.

REASONS FOR PERFORMING STUDY: Intra-arterial (i.a.) and intravenous (i.v.) regional limb perfusions (RLP) through the median artery and cephalic vein, respectively, have been previously investigated for administration of mesenchymal stem cells (MSCs) to the equine distal limb. Limitations due to thrombosis of the arteries after i.a. RLP and poor distribution of MSCs to the foot with i.v. RLP were observed. These techniques need to be modified for clinical use. OBJECTIVES: Evaluate the distribution, uptake and persistence of radiolabelled MSCs after i.a. injection through the median artery without a tourniquet and after i.v. RLP through the lateral palmar digital vein. STUDY DESIGN: In vivo experimental study. METHODS: (99m) Tc-HMPAO-labelled MSCs were injected through the median artery of one limb and the lateral palmar digital vein of the other limb of 6 horses under general anaesthesia. No tourniquet was used for the i.a. injection. A pneumatic tourniquet was placed on the metacarpus for i.v. injection. Scintigraphic images were obtained up to 24 h after injection. RESULTS: Intra-arterial injection resulted in MSCs retention within the limb despite the absence of a tourniquet and no thrombosis was observed. Both i.a. injection and i.v. RLP led to distribution of MSCs to the foot. The i.a. injection resulted in a more homogeneous distribution. The MSC uptake was higher with i.v. RLP at the initial timepoints, but no significant difference was present at 24 h. CONCLUSIONS: Both i.a. injection through the median artery without a tourniquet and i.v. RLP performed through the lateral palmar digital vein under general anaesthesia are safe and reliable methods for administration of MSCs to the equine foot. The i.a. technique is preferred owing to the better distribution, but is technically more challenging. The feasibility of performing these techniques on standing horses remains to be investigated.

Distribution and persistence of technetium-99 hexamethyl propylene amine oxime-labelled bone marrow-derived mesenchymal stem cells in experimentally induced tendon lesions after intratendinous injection and regional perfusion of the equine distal limb.

REASONS FOR PERFORMING STUDY: Intralesional (i.l.) injection is currently the most commonly used technique for stem cell therapy in equine tendon injury. A comparison of different techniques of injection of mesenchymal stem cells for the treatment of tendon lesions is required. OBJECTIVES: We hypothesised that vascular perfusion of the equine distal limb with mesenchymal stem cells (MSCs) would result in preferential distribution of MSCs to acute tendon injuries. STUDY DESIGN: In vivo experimental study. METHODS: Lesions were surgically induced in forelimb superficial digital flexor tendons of 8 horses. Three or 10 days after lesion induction, technetium-99 hexamethyl propylene amine oxime-labelled MSCs were injected via i.v. or intra-arterial (i.a.) regional limb perfusion (RLP) at the level of the distal antebrachium and compared to i.l. injection. Mesenchymal stem cell persistence and distribution within the forelimb and tendon lesions was assessed with scintigraphy for 24 h. RESULTS: Lesion uptake was higher with i.l. injection than with RLP, but MSC persistence decreased similarly over time in all 3 techniques. Intra-arterial RLP resulted in a better distribution of MSCs and a higher uptake at the lesion site than i.v. RLP. Limbs perfused i.a. on Day 10 showed greater accumulation of MSCs in the lesion than limbs perfused on Day 3. Arterial thrombosis occurred in 50% of the i.v. RLP limbs and in 100% of the i.a. RLP limbs, which led to clinical complications in one horse. CONCLUSIONS AND POTENTIAL RELEVANCE: Compared with i.l. injection, RLP results in lower uptake but similar persistence of MSCs at the site of tendon lesions. A time dependent accumulation of MSCs was identified with i.a. RLP. The i.a. RLP appears more advantageous than the i.v. RLP in terms of distribution and uptake. However, the described i.a. technique produced arterial thrombosis and thus cannot currently be recommended for clinical use.

Tick infestation and spotted-fever group Rickettsia in shelter dogs, California, 2009.

In response to an outbreak of Rocky Mountain spotted fever (RMSF) in Baja California in early 2009, dogs at two shelters in neighbouring Imperial County, California, were evaluated for ectoparasites. Brown dog ticks (Rhipicephalus sanguineus), a recognized vector for RMSF, were found on 35 (30%) of 116 dogs but all ticks tested negative for Rickettsia rickettsii by PCR.

Scintigraphic evaluation of intra-arterial and intravenous regional limb perfusion of allogeneic bone marrow-derived mesenchymal stem cells in the normal equine distal limb using (99m) Tc-HMPAO.

REASONS FOR PERFORMING STUDY: Mesenchymal stem cells (MSCs) are commonly injected intralesionally for treatment of soft tissue injuries in the horse. Alternative routes of administration would be beneficial for treatment of lesions that cannot be accessed directly or to limit needle-induced iatrogenic damage to the surrounding tissue. OBJECTIVES: The purpose of our study was to evaluate MSC distribution after intra-arterial (IA) and intravenous (IV) regional limb perfusions (RLP) using scintigraphy. We hypothesised that MSCs would persist in the distal limb after tourniquet removal and that both techniques would lead to diffuse MSC distribution. METHODS: Six horses were used in the study. MSCs were labelled with hexamethyl propylene amine oxime (HMPAO) and technetium-99m. RLP was performed through the median artery of one forelimb and the cephalic vein of the opposite limb under general anaesthesia. The tourniquet was left in place for 45 min. Scintigraphic images were obtained at 0, 45, 75 min, 6 h and 24 h post injection. RESULTS: Distribution of labelled MSCs through the entire distal limb was achieved with all 6 IA RLP, but 3 out of 6 IV RLP showed poor or absent uptake distal to the metacarpus. Mesenchymal stem cell persistence was 39% (30-60%) and 28% (14-50%) (median [minimum-maximum]) at 6 h for IA and IV RLP, respectively. Severe arterial thrombosis occurred in one horse after IA RLP. CONCLUSIONS: Both IA and IV RLP of the distal limb result in MSC persistence in perfused tissues. The IA perfusion resulted in more reliable cell distribution to the pastern and foot area. POTENTIAL RELEVANCE: Regional limb perfusion of MSCs might be used in cases where intralesional injection is not possible or in order to avoid iatrogenic needle damage. Further work is needed to assess the safety of IA RLP before its clinical use.

Alpha-II-spectrin after controlled cortical impact in the immature rat brain.

Proteolytic processing plays an important role in regulating a wide range of important cellular functions, including processing of cytoskeletal proteins. Loss of cytoskeletal proteins such as spectrin is an important characteristic in a variety of acute central nervous system injuries including ischemia, spinal cord injury and traumatic brain injury (TBI). The literature contains extensive information on the proteolytic degradation of alpha-II-spectrin after TBI in the adult brain. By contrast, there is limited knowledge on the characteristics and relevance of these important processes in the immature brain. The present experiments examine TBI-induced proteolytic processing of alpha-II-spectrin after TBI in the immature rat brain. Distinct proteolytic products resulting from the degradation of the cytoskeletal protein alpha-II-spectrin by calpain and caspase 3 were readily detectable in cortical brain parenchyma and cerebrospinal fluid after TBI in immature rats.

Ants as first intermediate hosts of Mesocestoides on San Miguel Island, USA.

This study tested the hypotheses that ants (Formicidae) function as a first intermediate host of Mesocestoides (Cestoda: Mesocestoididae) and that deer mice (Peromyscus maniculatus) develop metacestode infections after ingesting cysticercoid or procercoid-infected ants. Field studies were conducted at an island fox (Urocyon littoralis littoralis) breeding facility located on San Miguel Island, California Channel Islands National Park, USA, where > 40% of captive foxes were infected with adult Mesocestoides. Eight percent (8%) of deer mice at the fox pen site were infected with Mesocestoides metacestodes while none were infected at a distant site where foxes were absent (campground), thereby indicating the potential localized presence of a first intermediate host. To test whether ants from San Miguel Island contained Mesocestoides DNA, a polymerase chain reaction (PCR)-based diagnostic assay was developed using nested primers that could detect a single hexacanth larva within pooled samples of ten ants. Ants (Lasius niger and Tapinoma sessile) collected near the fox breeding facility were tested using the nested-PCR assay. Seven of 223 pooled samples of L. niger (3.1%) and 2 of 84 pooled samples of T. sessile (2.4%) tested positive for Mesocestoides DNA, while none of the ants were positive at the campground site. Positive samples were sequenced and found to match DNA sequences from Mesocestoides obtained from island fox and deer mice. Finally, to determine whether ants function as a first intermediate host for Mesocestoides, colony-raised deer mice (n = 47) were fed L. niger (n = 3860) or T. sessile (n = 339) collected from the San Miguel Island fox breeding facility. No mouse became infected with Mesocestoides metacestodes after ingesting ants. While both L. niger and T. sessile from SMI were positive for Mesocestoides DNA, they were not infective to deer mice in the laboratory.

Molecular mapping of murine I region recombinants. III. Crossing over at two discrete sites within the beta 1-beta 2 intron of the E beta gene.

The murine MHC provides a unique genetic system for studying meiotic recombination. A large number of murine H-2 recombinants cross over within a stretch of the E beta gene referred to as the E beta hot spot. The crossing over of eight such recombinants, derived from the s and k haplotypes, was studied at the nucleotide level. A 3-kb stretch of DNA, 3' to the beta 1 exon of the E beta gene, was sequenced after amplification of the genomic DNA from B10.S (one of the parental strains) by polymerase chain reaction. A number of sequence variations were identified with respect to B10.A (the other parental strain). Examination of these sequence variations by RFLP, simple sequence length polymorphism, as well as direct sequencing after polymerase chain reaction-amplification of genomic DNA from the recombinants led to unambiguous identification of the cross-over sites. Although all eight recombinants crossed over within the beta 1-beta 2 intron, two discrete nonoverlapping sites were involved. Five of the recombinants B10.BASR1, B10.ASR1, B10.ASR12, B10.HTT, and B10.S(9R) crossed over within a maximum of 395 bp of DNA 3' to the beta 1 exon. The remaining three recombinants B10.ASR7, B10.ASR11, and B10.S(8R) crossed over within 950 bp of DNA, adjacent to the cross-over site noted above. Each of these stretches of DNA was completely identical in the two parental haplotypes precluding further dissection of the cross-over sites. These cross-over sites are within those reported for the b and k recombination.

Ion chromatographic determination of sulfites in foods.

Ion chromatography (IC) is shown to be a promising technique for the determination of sulfites (SO2, SO2/3-) in foods. Results of a 10 min flash distillation and 10 min IC determination compare favorably with the results from the conventional Monier-Williams method for total sulfite in a variety of food matrices. The IC technique also provides a wealth of additional information, such as (1) sulfite and sulfate (oxidized sulfite) content of the spiking or treatment solution, (2) residual sulfite applied to the food after oxidation losses in the treatment process, (3) free sulfite in foods, and (4) total sulfite in foods. As a further check on the Monier-Williams method, the sulfate content of the trapping solution can be determined by IC. Because the IC technique traps the liberated SO2 in a non-oxidizing rather than an oxidizing medium, it is considered free from interfering sulfides and organic sulfur-containing groups which can give false positives in the Monier-Williams method. IC thus offers a high speed, more sensitive, and cost-effective alternative to conventional techniques for the determination of sulfite in foods.

A genetic method for defining DNA-binding domains: application to the nuclear receptor NGFI-B.

A method is described that allows for rapid and efficient generation of functional mutations in DNA-binding domains of proteins. The target DNA-binding domain is attached to the Gal4p transcriptional-activating domain and expressed in yeast. The binding site recognized by the target domain is placed upstream of a gene that produces a protein toxic to yeast cells, so that the chimeric protein activates its expression, providing a selection against DNA-binding domain function. The chimeric protein also activates expression of a gene necessary for histidine prototrophy, using a second DNA-binding domain included in the chimera (lexA), providing a selection against general activator mutations. Therefore, requiring growth in the absence of histidine focuses mutations to the target DNA-binding domain. This method was applied to the DNA-binding domain of the nuclear receptor NGFI-B. Nearly all mutations obtained concurred with previous studies of NGFI-B and other nuclear receptors, verifying the functional validity of the mutational profile obtained. In addition, by coupling this selection scheme with the two-hybrid system [Chien, C.-t., Bartel, P. L., Sternglanz, R. & Fields, S. (1991) Proc. Natl. Acad. Sci. USA 88, 9578-9582], mutations that alter protein interaction domains could also be obtained.

In vivo mutational analysis of the NGFI-A zinc fingers.

NGFI-A is a mammalian transcription factor that contains zinc fingers similar to those observed in several other proteins, including NGFI-C and Krox-20. To define precisely the DNA binding domain of NGFI-A, we selected mutants using a chimeric transcriptional activator that contains the NGFI-A zinc finger domain sandwiched between the lexA DNA binding domain and the GAL4 transcriptional activating domain. Expression of this lexA-NGFI-A-GAL4 (LAG) trimeric protein in yeast significantly retarded their growth, unlike an activator containing only the lexA and GAL4 components. This suggested that LAG inappropriately regulates genes in yeast that contain NGFI-A binding sites. Yeast that contained LAG reverted to wild-type growth at high frequency by inactivation of LAG. The mutations recovered from these revertants were specifically limited to the 83-residue NGFI-A zinc finger domain by requiring that the lexA and GAL4 portions of the LAG chimera remain functional. Nearly all of the 93 mutants obtained contained single missense mutations that mapped within the zinc fingers to residues thought to be important for zinc finger function. Deletion analysis of native NGFI-A verified that residues distant from the zinc fingers do not influence DNA binding, thus establishing the minimal functional DNA binding domain. Interestingly, many zinc finger residues ascribed specific functions by x-ray crystallography were never mutated in yeast, implying that the identity of these residues is not critical. Surprisingly, not all of the mutations tested significantly impaired NGFI-A-specific DNA binding, suggesting that the function of these zinc fingers is more diverse than previously recognized.