A new species of cellular slime mold from southern Portugal based on morphology, ITS and SSU sequences.

Sampling soils to look for dictyostelids in southern Portugal we found an isolate that has a morphology that differed from any previously described species of the group. We sequenced the internally transcribed spacer (ITS) and small subunit (SSU) genes of the nuclear ribosomal RNA and found that both sequences are distinct from all previously described sequences. Phylogenetic analyses place the new species in dictyostelid Group 3 (Rhizostelids) together with D. potamoides, with which it shares 65.8% identity for ITS and 96.6% for SSU. In this paper we describe a new species of cellular slime mold, Dictyostelium ibericum, based on morphological and molecular characters. It is a small species with polar granules in its spores.

Characterization of a novel pestivirus originating from a pronghorn antelope.

A unique pestivirus, isolated from a pronghorn antelope (Antilocopra americana), was characterized. Serum neutralization studies suggested that this virus was antigenically related to pestiviruses. Genomic characteristics, unique to pestiviruses, indicated that this virus belongs to the Pestivirus genus. These characteristics included the organization of the 5' untranslated region (5'-UTR), the presence and length of a viral Npro coding region, conservation of cysteine residues in Npro, conservation of predicted amino acid sequences flanking the cleavage sites between viral polypeptides Npro and C and between C and Erns and conservation of predicted hydrophobicity plots of Npro protein. While this data indicated the virus belongs to the Pestivirus genus, phylogenetic analysis in 5'-UTR, Npro and E2 regions suggested that it is the most divergent of the pestiviruses identified to date. This conclusion was also supported by the amino acid identity in coding regions. The corresponding values were much lower for the comparison of pronghorn pestivirus to other pestivirus genotypes than only between previous recognized genotypes. These results suggest the virus isolated from pronghorn antelope represents a new pestivirus genotype. It also represents the only pestivirus genotype first isolated from New World wildlife.

Manipulation of frontal EEG asymmetry through biofeedback alters self-reported emotional responses and facial EMG.

Individual differences in resting asymmetrical frontal brain activity have been found to predict subsequent emotional responses. The question of whether frontal brain asymmetry can cause emotional responses has yet to be addressed. Biofeedback training designed to alter the asymmetry of frontal brain activity was therefore examined. Eighteen right-handed female participants were randomly assigned to receive biofeedback training designed to increase right frontal alpha relative to left frontal alpha (n = 9) or to receive training in the opposite direction (n = 9). Five consecutive days of biofeedback training provided signals of reward or nonreward depending on whether the difference between right (F4) and left (F3) frontal alpha exceeded a criterion value in the specified direction. Systematic alterations of frontal EEG asymmetry were observed as a function of biofeedback training. Moreover, subsequent self-reported affect and facial muscle activity in response to emotionally evocative film clips were influenced by the direction of biofeedback training.

Fundus albipunctatus and retinitis punctata albescens in a pedigree with an R150Q mutation in RLBP1.

Fundus albipunctatus (FA; OMIM 136880) is a rare form of apparently stationary night blindness characterized by the presence of myriad symmetrical round white dots in the fundus with a greater concentration in the midperiphery. A distantly similar but distinct clinical entity, retinitis punctata albescens (RPA), is also characterized by aggregation of irregular white flecks but is progressive and evolves to generalized atrophy of the retina. We studied 4 consanguineous kindreds diagnosed with FA from Saudi Arabia. Given the substantial phenotypic variation and overlap between different flecked retinal dystrophies, we evaluated all known genes associated with such conditions by both genetic analysis and direct sequencing. In one kindred, KKESH-099, we identified a homozygous R150Q alteration in RLBP1, the gene encoding the cellular retinaldehyde binding protein, associated previously with both recessive retinitis pigmentosa (arRP) and RPA. Examination of several patients aged 3-20 years over a 9-year period presented no evidence for either RP or RPA. In contrast, clinical examination of individuals with the same mutation in their fourth and fifth decade revealed signs consistent with RPA. The data suggest that the R150Q mutation in RLBP1 may result in RPA with slow progression. More importantly, younger individuals diagnosed with the milder disorder FA thought to be stationary may evolve to a more devastating and progressive phenotype.

Isolation of bovine viral diarrhea virus from a free-ranging mule deer in Wyoming.

A noncytopathic type 1a bovine viral diarrhea virus (BVDV) was isolated from a free-ranging yearling female mule deer (Odocoileus hemionus) from northwestern Wyoming (USA). The mule deer was emaciated, weak, and salivating, and Arcanobacterium pyogenes was cultured from lung abscesses. Bovine viral diarrhea virus was isolated from lung, however, BVDV antigen was not detected by immunohistochemistry. The BVDV genotype was determined by reverse transcriptase polymerase chain reaction and the RNA sequences from the 5'UTR and E2 genes compared with sequences of a type 1a BVDV isolated from cattle from the same area as the deer. The sequences from the deer BVDV were distinct from those of the bovine type 1a BVDV, but similar to other bovine type 1a BVDVs. Seventy-four (60%) of 124 sera collected from mule deer in this area had serum neutralizing antibody titers to type 1a BVDV of > or = 1:32. The high prevalence of seropositive mule deer and isolation of BVDV suggests that this virus circulates in the mule deer population. The isolate described in this report is the second reported BVDV isolate from free-ranging deer in North America and the first from a mule deer.

Outpatient penile aspiration and epinephrine irrigation for young patients with sickle cell anemia and prolonged priapism.

The optimal management of prolonged priapism for patients with sickle cell anemia (SCA) has not been established. We prospectively studied in an outpatient setting the efficacy and safety of a procedure that employs aspiration of blood from the corpora cavernosa and irrigation with a dilute epinephrine solution under local anesthesia to relieve priapism in young patients with SCA. If hydration and analgesics failed to produce detumescence or if priapism had lasted >4 hours, the protocol was activated in the emergency room or clinic. Fifteen patients with homozygous SCA (Hb SS) were treated on 39 occasions; 10 patients were treated once, 1 patient twice, 2 patients 3 times, 1 patient 6 times, and 1 patient 15 times. Median age of patients at first treatment was 14.3 years (range, 3.9-18.3 years). The procedure was successful in producing immediate detumescence on 37 of 39 occasions (95% efficacy, 95% confidence intervals (CI): 81%-99%). No serious immediate or long-term side effects were observed. None of the patients who demonstrated detumescence required hospitalization. The 2 patients whose priapism persisted after aspiration and irrigation presented with episodes lasting >24 hours. All evaluable patients whose priapism resolved after aspiration and irrigation self-reported normal erectile function at a median of 40 months (range, 3-58 months) after the last procedure. Thus, aspiration of the corpora cavernosa followed by irrigation with dilute epinephrine is effective in producing immediate and sustained detumescence and should be the initial therapy employed for patients with SCA and prolonged priapism. (Blood, 2000; 95:78-82)

Prevalence of priapism in children and adolescents with sickle cell anemia.

A questionnaire survey was conducted of patients with homozygous sickle cell anemia (Hb SS) and sickle cell beta(0)-thalassemia (Hb S-beta(0)) between 5 and 20 years of age to determine the prevalence and characteristics (number of episodes, timing, duration, cause, or precipitating event) of priapism. Ninety-eight male patients or their parents were surveyed by the same male investigator using a structured verbal interview, which was modified according to the age of the patient. Ninety-four patients had Hb SS and four Hb S-beta(0) thalassemia. Eleven (11%) patients were known to have experienced priapism previously. In response to the questionnaire, 16 of the remaining 87 (18%) patients reported having had priapism on one or more occasions. The actuarial probability of experiencing priapism by 20 years of age was 89% (+/- 9%). The mean age at the initial episode was 12 years, the mean number of episodes per patient was 15.7 (median, 1; range, 1-100), and the mean duration of an episode was 125 minutes. Episodes typically occurred around 4:00 am, and 75% of the patients surveyed had at least one episode starting during sleep or upon awakening from sleep. The prevalence of priapism in children and adolescents with SCA is much higher than previously described. Since early intervention and treatment may prevent irreversible penile fibrosis and impotence, patients and parents should be educated about this complication in advance of its occurrence.

Detection of Alexandrium tamarensis by rapid PCR analysis.

Alexandrium tamarensis is a toxigenic dinoflagellate found in coastal waters worldwide. A critical factor in alleviating the health and economic threats posed by this species is the development of a rapid and reliable method for detection. This study stream-lined a labor- and resource-intensive protocol for the isolation of A. tamarensis ribosomal DNA (rDNA). Subcultures of A. tamarensis were established in water samples from several coastal Atlantic sites. A commercial DNA isolation kit protocol for cultured cells was used for isolation of the dinoflagellate DNA. Samples were amplified by PCR using primers specific for a 700-bp sequence of A. tamarensis rDNA. It was determined that this method facilitated the detection of 10(-4) ng/microL of A. tamarensis DNA. Furthermore, the kit enabled A. tamarensis to be isolated from the water sources with little signal degradation. This is a valuable technique for the rapid detection of A. tamarensis, even before cell numbers are large enough for morphological identification.

Transactivation of a ribosomal gene by simian virus 40 large-T antigen requires at least three activities of the protein.

Simian virus 40 large-T antigen transactivates the ribosomal genes which are transcribed by RNA polymerase (pol I), as well as genes that are dependent on either pol II or pol III. This report identifies regions and activities of T antigen that are required to transactivate a pol I-dependent rat ribosomal gene promoter. By using the rat ribosomal gene (rDNA) promoter linked to a chloramphenicol acetyltransferase gene, we show that at least three separable T-antigen regions are necessary to achieve wild-type levels of transactivation of rDNA in transiently transfected monkey cells. One activity depends on the region of T antigen shared with small-t antigen (T/t common region). A second activity maps to amino acids 109 to 626 and is highly sensitive to mutational inactivation. Complementation analyses suggest that at least one activity in this region is independent of and must be in cis with the activity within the T/t common region. In addition, a functional nuclear localization signal is required for maximal T-antigen-mediated transactivation of rat rDNA. The three activities work in concert to override cellular species-specific controls and transactivate the rat ribosomal gene promoter. Finally, we provide evidence that although the tumor suppressor protein Rb has been shown to repress a pol I-dependent promoter, transactivation of the rat rDNA promoter does not depend on T antigen's ability to bind the tumor suppressor product Rb.

Ultrasound biomicroscopy of globes from young adult pigs.

OBJECTIVE: To determine anterior chamber ocular measurements of adult porcine globes without histologic fixation by use of ultrasound biomicroscopy scanning. SAMPLE POPULATION: 25 porcine globes obtained at an abattoir. PROCEDURE: Globes were packed on ice for transport. In the laboratory, globes were trimmed, rinsed with antibiotic solution, secured on a single gauze-fold in a latex holder, then were ultrasonogrammed unreformed. Ultrasound biomicroscopy scanning was done, using a 50-MHz transducer, 17-mm cup, and 2% methyl cellulose. RESULTS: Average young adult pig external ocular measurements were: nasal-temporal corneal diameter, 16.61 mm; superior-inferior corneal diameter, 14.00 mm; nasal-temporal globe diameter, 25.48 mm; superior-inferior globe diameter, 24.48 mm; and axial length 21.64 mm. Ultrasound biomicroscopy anterior chamber measurements were: iris sulcus, 30.45 degrees; ciliary sulcus, 18.89 degrees; central corneal thickness, 0.98 mm; corneal thickness at limbus, 1.19 mm; central iris thickness, 0.58 mm; iris tip to ciliary apex, 1.73 mm; iris tip to iris sulcus origin, 3.83 mm; iris tip to ciliary sulcus origin, 2.98 mm; anterior chamber depth from iris tip to cornea, 2.21 mm; central anterior chamber depth, 2.47 mm; ciliary process mid-thickness, 0.65 mm; ciliary process apex to origin of iris sulcus, 2.32 mm; ciliary process apex to origin of ciliary sulcus, 1.34 mm; zonular bundle diameter, 0.10 mm; and interzonular bundle space, 0.11 mm. CONCLUSIONS: Anatomic anterior chamber measurements and relations in porcine globes can be used to describe trauma, confirm existence of lesions, and help explain theory. CLINICAL RELEVANCE: Ultrasound biomicroscopy is a clinical decision aid facilitating noninvasive anatomic or pathologic description without histologic fixation.

Vaccination of black-footed ferret (Mustela nigripes) x Siberian polecat (M. eversmanni) hybrids and domestic ferrets (M. putorius furo)against canine distemper.

An inactivated canine distemper vaccine with adjuvant and a modified-live virus (MLV) vaccine were evaluated using black-footed ferret (Mustegla nigripes) x Siberian polecat (Mustela eversmanni) hybrids us surrogates for endangered black-footed ferrets. For comparative purposes, we also vaccinated domestic ferrets (Mustela putorius furo) with the MLV vaccine. Response to vaccination was measured by clinical observation, hematology, dynamics of serum virus neutralizing antibodies, and challenge with virulent canine distemper virus. No clinical signs attributable to the vaccines were observed. Transient leukopenia occurred in hybrid ferrets that received MLV vaccine and there was marked lymphopenia for approximately 52 days post-vaccination. Lymphopenia was present for approximately 21 days in domestic ferrets vaccinated with MLV vaccine. Neutralizing antibodies against canine distemper virus were detected 14 days post-vaccination in hybrids receiving MLV vaccine and most titers were > 1:1024 for the 791 days of the study. Antibody titers in hybrids vaccinated with the inactivated vaccine were significantly lower. All eight hybrid ferrets that received MLV vaccine survived challenge with virulent canine distemper virus without clinical disease. However, one of seven hybrids vaccinated with the inactivated vaccine developed canine distemper and was euthanized; two other hybrids became clinically ill but survived. The MLV vaccine may be useful in prevention of canine distemper in black-footed ferrets, but until additional studies of efficacy and safety are completed, use of the inactivated vaccine is appropriate.

Infectious keratoconjunctivitis in free-ranging mule deer (Odocoileus hemionus) from Zion National Park, Utah.

An epizootic of infectious keratoconjunctivitis (IK) was studied opportunistically in free-ranging mule deer (Odocoileus hemionus) from Zion National Park, Utah (USA), from November 1992 to March 1994. Moraxella sp. and Chlamydia sp. were isolated from the conjunctiva of two of seven deer. In addition, Thelazia californiensis occurred on the conjunctivas of six of seven deer. Based on field observations, adults appeared to be affected clinically at a higher incidence during both years as opposed to juveniles. Corneal opacity was the most apparent clinical sign from 1992 to 1993. However, in the following year, blepharospasm and epiphora were noted more often. We were also able to document the clinical recovery of three affected deer. In addition, Moraxella sp. was recovered from the eyes of a clinically unaffected deer 1 year after the epizootic occurred.

Simian virus 40 large T antigen contains two independent activities that cooperate with a ras oncogene to transform rat embryo fibroblasts.

The simian virus 40 large T antigen immortalizes growing primary cells in culture. In addition, this viral oncoprotein cooperates with an activated ras protein to produce dense foci on monolayers of rat embryo fibroblasts (REF). The relationship between independent immortalization and cooperative transformation with ras has not been defined. Previously, two regions of T antigen were shown to contain immortalization activities. An N-terminal fragment consisting of amino acids 1 to 147 immortalizes rodent cells (L. Sompayrac and K. J. Danna, Virology 181:412-415, 1991). Loss-of-function analysis indicated that immortalization depended on integrity of the T-antigen segments containing amino acids 351 to 450 and 533 to 626 (T. D. Kierstead and M. J. Tevethia, J. Virol. 67:1817-1829, 1993). The experiments described here were directed toward determining whether these same T-antigen regions were sufficient for cooperation with ras. Initially, constructs that produce T antigens containing amino acids 176 to 708 (T176-708) or 1 to 147 were tested in a ras cooperation assay. Both polypeptides cooperated with ras to produce dense foci on monolayers of primary REF. These results showed that T antigen contains two separate ras cooperation activities. In order to determine the N-terminal limit of the ras cooperation activity contained within the T176-708 polypeptide, a series of constructs designed to produce fusion proteins containing T-antigen segments beginning at residues 251, 301, 337, 351, 371, 401, 451, 501, 551, 601, and 651 was generated. Each of these constructs was tested for the capacity to cooperate with ras to produce dense foci on REF monolayers. The results indicated that a polypeptide containing T-antigen amino acids 251 to 708 (T251-708) was sufficient to cooperate with ras, whereas the more extensively truncated products were not. The abilities of the N-terminally truncated T antigens to bind p53 were examined in p53-deficient cells infected with a recombinant vaccinia virus expressing a phenotypically wild-type mouse p53. The results showed that polypeptides containing T-antigen amino acids 251 to 708, 301 to 708, 337 to 708, or 351 to 708 retained p53-binding capacity. The introduction into the T251-708 polypeptide of deletions that either prevented p53 binding (dl434-444) or did not prevent p53 binding (dl400) abrogated ras cooperation. These results indicated that although p53 binding may be necessary for ras cooperation, an additional, as-yet-undefined activity contained within the T251-708 polypeptide is needed.

Chronic generalized obliterative arteriopathy in cattle: a sequel to sheep-associated malignant catarrhal fever.

Malignant catarrhal fever (MCF) in cattle is generally associated with a short clinical course and a high case fatality rate (90-95%). The lesions in cattle that survive acute MCF for a prolonged period or appear to recover have not been documented. In a naturally occurring outbreak of MCF in a herd of beef cattle in Wyoming, 7 of 84 yearling heifers (8.3% of replacement herd) and 2 of 230 cows (0.9% of cow herd) developed clinical signs of pyrexia, mucopurulent discharge, bilateral keratitis, and weight loss following contact with ewes that had lambed 34-62 days earlier. Six of 9 affected cattle were examined postmortem following clinical signs (CS) that developed 2-150 days earlier. Three cattle with CS for < or = 39 days had lesions of regional lymphadenopathy and widespread severe segmental lymphoid arteritis-phlebitis that were typical of acute MCF, and proliferative intimal lesions were present in a small proportion of arteries at days 20 and 39 of CS. By contrast, 3 cattle that survived to 90, 105, and 150 days after clinical onset had distinctive arterial lesions in multiple organs, characterized by proliferative concentric fibrointimal plaques, disrupted inner elastic lamina, focally atrophic tunica media, and vasculitis of variable severity. Immunohistochemical and ultrastructural examination of intimal plaques identified the predominant cellular component to be smooth muscle cells with a contractile phenotype. No viral structures were seen. Serologic studies, using a competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) that detects antibody to an epitope broadly conserved among isolates of the MCF virus, found that 2 chronically affected cattle were serologically positive between days 42 and 100 of CS, with seroconversion in 1 animal between days 52 and 73 of CS. Seroprevalence was 7.9% in the 76 remaining healthy animals of the replacement heifer herd and 40% (75% in adult sheep and 4% in lambs) in the in-contact sheep flock 77 days after onset of CS in the index case. This episode suggests that, in addition to the common and well recognized acute form of MCF in cattle, this viral infection encompasses a disease spectrum that includes chronic disease and partial to "complete" clinical recovery, and in recovered animals chronic obliterative arteriopathy is the preeminent lesion.

Atypical presentation of Coats disease.

PURPOSE: To highlight the possibility of intraocular calcification in Coats disease and evaluate the ultrasound and computed tomographic findings. METHOD: A 7.5-year-old boy had a 2-week history of unilateral glaucoma and leukocoria with retinal detachment, suggestive of Coats disease. The possibility of retinoblastoma, however, could not be excluded by ultrasound or computed tomographic examination, which revealed a retinal detachment overlying a subretinal mass with calcification. The blind, painful eye was subsequently enucleated. RESULTS: Histopathologically, there were telangiectatic retinal vessels in a fold of the detached retina peripherally and proteinaceous exudate in the subretinal space containing cholesterol clefts and foamy histiocytes, characteristic of Coats disease. There was also a fibro-osseous nodule in the macular area that correlated with intraocular calcification clinically. CONCLUSION: This case provides the first documentation of ancillary corroboration of intraocular bone formation in Coats disease, which, although rare, is an important consideration in the differential diagnosis of retinoblastoma.

Continuous cardiac output monitoring by the Fick method.

Current methods for longitudinal assessment of cardiac output in severely ill patients are intermittent only and in many respects appear unsatisfactory. We have developed a computerized on-line system for continuous Fick cardiac output monitoring, utilizing fiberoptic arterial and pulmonary arterial probes with a metabolic analyzer for VO2. In 15 patients, cardiac output ranged 1.9-6.8 L/min and 12 were within 5% of thermodilution values. Continuous output monitoring during interventions in two patients (saline infusion and coronary angioplasty) illustrate the utility of the technique. Two additional patients had unsatisfactory VO2 data due to low airflow velocity. The results of this pilot study suggest that cardiac output monitoring by the Fick method may have clinical and investigational uses in intensive care units and during cardiac catheterization or surgical procedures.

Bovine respiratory syncytial virus-specific immune responses in cattle following immunization with modified-live and inactivated vaccines. Analysis of proliferation and secretion of lymphokines by leukocytes in vitro.

Cattle were immunized with vaccines containing modified-live or inactivated bovine respiratory syncytial virus (BRSV) and lymphocyte proliferative responses and cytokine secretion were monitored sequentially. Compared to pre-inoculated values, significant increases in proliferative responses to modified-live BRSV were detectable by Day 7 after the primary immunization with the vaccine containing inactivated BRSV, and by 7 days after the second immunization with modified-live virus. After a third immunization with the respective vaccines, proliferative responses to live BRSV were significantly higher in the group that received modified-live vaccine compared to the group that received inactivated vaccine. Proliferative responses to live BRSV corresponded with the presence of interleukin-2 (IL-2) in the supernatants from BRSV-stimulated leukocyte cultures and there were significantly higher levels of IL-2 in cultures from the group that received modified-live BRSV. An interferon species with the characteristics of interferon-alpha was also present in the supernatants from leukocyte cultures and there were no significant differences between the groups of vaccines. The predominant phenotype of proliferating cells in BRSV-stimulated leukocyte cultures derived from both groups of bovine vaccines was a BoCD4+ T-lymphocyte. These in vitro data suggest that both types of vaccines are capable of stimulating cell-mediated immune responses to BRSV in cattle.