Laryngeal advancement surgery improves swallowing function in a reversible equine dysphagia model.

REASONS FOR PERFORMING STUDY: Pharyngeal dysphagia is a debilitating, sometimes fatal condition in horses, with multiple aetiologies. The pathophysiology is complex and not fully understood. Treatment is largely supportive. Laryngeal advancement surgery may diminish symptoms of dysphagia and improve swallowing in affected horses. OBJECTIVES: 1) to induce reversible moderate and marked pharyngeal dysphagia by regional anaesthesia of branches of the glossopharyngeal (IX), vagus (X) and hypoglossal (XII) nerves; 2) to characterise the dysphagia produced by each model; and 3) to determine whether laryngeal advancement surgery improves swallowing in these models. STUDY DESIGN: Experimental design using 6 adult horses. METHODS: Two dysphagia models were produced by blocking IX, the pharyngeal branch of X and XII unilaterally (moderate model) and only the pharyngeal branch of X bilaterally (marked model) within the guttural pouches. Both models were performed on each horse before and after surgery in order to assess the effectiveness of the surgical procedure as a potential treatment for pharyngeal dysphagia. Dysphagia was scored by partly blinded observers on a scale of 0-12 based on observations of eating (nonblinded), endoscopic examinations and fluoroscopic swallowing (blinded), where 0 = normal swallow and 12 = severe dysphagia with tracheal aspiration. Data were analysed by 3-factor ANOVA, with significance taken as P<0.05. RESULTS: Dysphagia models were reversible, and horses swallowed normally within 3 h of model induction. The marked dysphagia model impaired movement of feed from the base of the tongue to the oesophagus and caused severe airway contamination. The average dysphagia score (mean ± s.d.) for the marked dysphagia model was 10.6 ± 1.1 before surgery and 6.1 ± 4.3 after surgery (P = 0.007). Laryngeal advancement surgery did not significantly improve the dysphagia scores in the moderate model (P = 0.5). CONCLUSIONS: Laryngeal advancement surgery may improve swallowing and reduce aspiration in horses affected with diseases that cause pharyngeal dysphagia.

Incidence of support limb laminitis in horses treated with half limb, full limb or transfixation pin casts: a retrospective study of 113 horses (2000-2009).

REASONS FOR PERFORMING STUDY: To determine the incidence of support limb laminitis among horses treated with half limb, full limb or transfixation pin casts and determine potential risk factors. METHODS: Medical records of 113 horses treated with half limb, full limb or transfixation pin casts at an equine referral hospital from 2000 to 2009 were reviewed. Associations between potential risk factors and development of support limb laminitis were evaluated by bivariable and multivariable logistic regression analyses. RESULTS: Of the 113 horses that received casts, 14 (12%) developed confirmed support limb laminitis. The bodyweight of the horse and duration of casting in weeks were significantly associated with support limb laminitis. Horses requiring full limb casts or transfixation pin casts were more likely to develop this complication than horses requiring half limb casts. There were no significant associations between developing support limb laminitis and weightbearing capacity on presentation to the hospital, the limb affected (fore- or hind), whether there was a fracture present or breed of horse. CONCLUSIONS: Support limb laminitis is a relatively common complication among horses treated with half limb, full limb and transfixation pin casts. Greater durations of casting and higher bodyweights increase the likelihood of developing this complication. POTENTIAL RELEVANCE: Support limb laminitis may occur secondary to any painful unilateral lameness and is not necessarily more likely to develop in horses with severe orthopaedic conditions such as fractures. However, heavier horses, those requiring casts for longer periods of time and those that require a full limb or transfixation pin cast as opposed to a half limb cast should be considered to have an increased risk for developing support limb laminitis post operatively.

Short communication: methicillin-resistant Staphylococcus aureus detection in US bulk tank milk.

Staphylococcus aureus is a major cause of mastitis in dairy cattle. This study estimated the herd prevalence of methicillin-resistant Staph. aureus (MRSA) among US dairy herds by testing bulk tank milk (BTM) samples using genotypic and phenotypic methods. A nationally representative sample of 542 operations had BTM cultured for Staph. aureus, and 218 BTM samples were positive upon initial culture. After 4 wk to 4 mo of frozen storage, 87% of 218 samples (n = 190) were still culture positive for Staph. aureus on blood agar, but none were positive for MRSA on the selective indicator medium CHROMagar MRSA. A duplex PCR was used to detect the Staph. aureus-specific nuc gene and the methicillin resistance gene, mecA, in mixed staphylococcal isolates from the 190 BTM samples that were positive for Staph. aureus after storage. Seven samples tested positive for nuc and mecA, and 2 samples tested positive for mecA only. MecA-positive Staphylococcus spp., but not MRSA, were subsequently isolated from 5 samples, whereas neither mecA-positive Staphylococcus spp. nor MRSA was isolated from the remaining 4 samples. Presence of methicillin-resistant, coagulase-negative Staphylococcus spp. may complicate the detection of MRSA by means of PCR on BTM. Bulk tank milk in the United States is not a common source of MRSA.

Autoimmunity to the M(r) 32,000 subunit of replication protein A in breast cancer.

PURPOSE: We sought to identify autoantigens recognized by antibodies in breast cancer patient sera with potential diagnostic or prognostic significance. EXPERIMENTAL DESIGN: Serum from a female breast cancer patient exhibiting a high titer antinuclear antibody was used to screen a HeLa cDNA expression library, leading to the cloning of a cDNA for the M(r) 32,000 subunit of replication protein A (RPA32). RPA32 expression and localization were assayed in autologous tumor by monoclonal antibody staining. A specific ELISA using recombinant protein was used to screen sera from 801 breast cancer patients and 65 controls. RESULTS: A relationship between anti-replication protein A (RPA) antibodies and the ductal breast carcinoma of the proband was suggested by overexpression and aberrant localization of RPA32 in tumor cells as compared with surrounding normal ductal tissue and by the presence of anti-RPA32 antibodies before the diagnosis. The prevalence of anti-RPA32 antibodies was significantly higher (P < 0.01) among breast cancer patients (87 of 801 patients) than among noncancer controls (0 of 65 controls). Similarly, anti-RPA32 antibodies were present in 4 of 39 patients with intraductal in situ carcinoma. No associations were found between anti-RPA antibodies and survival, occurrence of a second tumor, metastases, or antibodies to p53. Reactivity to RPA32 also was detected in sera from 3 of 47 patients with other cancers. CONCLUSIONS: In view of the central role of RPA in DNA replication, recombination, and repair, we suggest that autoimmunity to RPA32 may reflect molecular changes involved in the process of tumorigenesis. The finding of antibodies to RPA32 before diagnosis and their prevalence in in situ carcinoma suggest that they are potentially useful markers of early disease.

Identification of differentially expressed genes by serial analysis of gene expression in human prostate cancer.

Prostate cancer is the leading cause of cancer death in American males. To better understand the genetic bases of this disease, we have generated a comprehensive molecular profile of human prostate. The gene expression pattern in normal and prostate cancer tissues was analyzed by serial analysis of gene expression (SAGE). A total of 133,217 transcripts were analyzed, and 35,185 distinct SAGE tags were identified representing 19,287 genes. Comparison of the transcripts in normal and tumor tissue revealed 156 differentially expressed genes (P < 0.05), of which 88 genes were up-regulated and 68 genes were down-regulated in the tumor tissue. Based on SAGE data, we estimate that the transcriptome for human prostate is approximately 37,000. Several differentially expressed genes identified by SAGE were selected for confirmation using immunohistochemistry. Some genes (e.g., E2F4) were overexpressed in tumor epithelial cells and some (e.g., Daxx) were increased in tumor stroma. Further characterization of the role of E2F4 and Daxx as well as other differentially expressed genes may provide useful insights into the mechanism of prostate cancer development.

Crossing over is rarely associated with mitotic intragenic recombination in Schizosaccharomyces pombe.

Chromosomal rearrangements can result from crossing over during ectopic homologous recombination between dispersed repetitive DNA. We have previously shown that meiotic ectopic recombination between artificially dispersed ade6 heteroalleles in the fission yeast Schizosaccharomyces pombe frequently results in chromosomal rearrangements. The same recombination substrates have been studied in mitotic recombination. Ectopic recombination rates in haploids were approximately 1-4 x 10(-6) recombinants per cell generation, similar to allelic recombination rates in diploids. In contrast, ectopic recombination rates in heterozygous diploids were 2.5-70 times lower than allelic recombination or ectopic recombination in haploids. These results suggest that diploid-specific factors inhibit ectopic recombination. Very few crossovers occurred in ade6 mitotic recombination, either allelic or ectopic. Allelic intragenic recombination was associated with 2% crossing over, and ectopic recombination between multiple different pairing partners showed 1-7% crossing over. These results contrast sharply with the 35-65% crossovers associated with meiotic ade6 recombination and suggest either differential control of resolution of recombination intermediates or alternative pathways of recombination in mitosis and meiosis.

Isochromosome 8q formation is associated with 8p loss of heterozygosity in a prostate cancer cell line.

BACKGROUND: In advanced prostate cancer, loss of chromosomal regions on 8p is frequently associated with gain of 8q. We studied the gross chromosomal abnormalities associated with 8p loss of heterozygosity (LOH) in the prostate tumor cell line 1542 CP3Tx. The cell line was previously established from a primary prostatic adenocarcinoma by immortalization with a recombinant retrovirus carrying the E6 and E7 genes of human papilloma virus type 16. Allelotyping studies demonstrated LOH at multiple markers on 8p. METHODS: To investigate the relationship of 8p LOH to gross chromosomal rearrangements, and to screen for other genetic abnormalities in 1542 CP3Tx, we used comparative genomic hybridization (CGH), conventional karyotyping, fluorescence in situ hybridization (FISH), and allelotyping. RESULTS: CGH revealed loss of the entire 8p arm, associated with gain of the entire 8q arm. Other abnormalities included chromosome 4 loss and chromosome 11 gain. The karyotype showed an isochromosome (8q), monosomy 4, and trisomy 11. FISH and allelotyping confirmed and extended these results. CONCLUSIONS: These results demonstrate that i(8q) formation is a mechanism for associated 8p loss and 8q gain in prostate cancer. Furthermore, the small number of chromosomal abnormalities in this cell line indicates that immortalization of low-passage cultures with viral oncogenes provides a method for obtaining cell lines for studying genetic abnormalities in prostate cancer.

The M26 hotspot of Schizosaccharomyces pombe stimulates meiotic ectopic recombination and chromosomal rearrangements.

Homologous recombination is increased during meiosis between DNA sequences at the same chromosomal position (allelic recombination) and at different chromosomal positions (ectopic recombination). Recombination hotspots are important elements in controlling meiotic allelic recombination. We have used artificially dispersed copies of the ade6 gene in Schizosaccharomyces pombe to study hotspot activity in meiotic ectopic recombination. Ectopic recombination was reduced 10-1000-fold relative to allelic recombination, and was similar to the low frequency of ectopic recombination between naturally repeated sequences in S. pombe. The M26 hotspot was active in ectopic recombination in some, but not all, integration sites, with the same pattern of activity and inactivity in ectopic and allelic recombination. Crossing over in ectopic recombination, resulting in chromosomal rearrangements, was associated with 35-60% of recombination events and was stimulated 12-fold by M26. These results suggest overlap in the mechanisms of ectopic and allelic recombination and indicate that hotspots can stimulate chromosomal rearrangements.

Position- and orientation-independent activity of the Schizosaccharomyces pombe meiotic recombination hot spot M26.

The activity of the M26 meiotic recombination hot spot of Schizosaccharomyces pombe depends on the presence of the heptamer 5'-ATGACGT-3'. Transplacement of DNA fragments containing the ade6-M26 gene to other chromosomal loci has previously demonstrated that the heptamer functions in some, but not all, transplacements, suggesting that hot spot activity depends on chromosomal context. In this study, hot spot activity was tested in the absence of gross DNA changes by using site-directed mutagenesis to create the heptamer sequence at novel locations in the genome. When created by mutagenesis of 1-4 bp in the ade6 and ura4 genes, the heptamer was active as a recombination hot spot, in an orientation-independent manner, at all locations tested. Thus, the heptamer sequence can create an active hot spot in other chromosomal contexts, provided that the gross chromosomal structure is not altered; this result is consistent with the hypothesis that a specific higher-order chromatin structure is required for M26 hot spot activity.

Active and inactive transplacement of the M26 recombination hotspot in Schizosaccharomyces pombe.

The ade6-M26 mutation of the fission yeast Schizosaccharomyces pombe creates a meiotic recombination hotspot that elevates ade6 intragenic recombination approximately 10-15-fold. A heptanucleotide sequence including the M26 point mutation is required but not sufficient for hotspot activity. We studied the effects of plasmid and chromosomal context on M26 hotspot activity. The M26 hotspot was inactive on a multicopy plasmid containing M26 embedded within 3.0 or 5.9 kb of ade6 DNA. Random S. pombe genomic fragments totaling approximately 7 Mb did not activate the M26 hotspot on a plasmid. M26 hotspot activity was maintained when 3.0-, 4.4-, and 5.9-kb ade6-M26 DNA fragments, with various amounts of non-S. pombe plasmid DNA, were integrated at the ura4 chromosomal locus, but only in certain configurations relative to the ura4 gene and the cointegrated plasmid DNA. Several integrations created new M26-independent recombination hotspots. In all cases the non-ade6 DNA was located > 1 kb from the M26 site, and in some cases > 2 kb. Because the chromosomal context effect was transmitted over large distances, and did not appear to be mediated by a single discrete DNA sequence element, we infer that the local chromatin structure has a pronounced effect on M26 hotspot activity.

Complete and partial glycophospholipid anchors are found on a fusion protein consisting of luteinizing hormone beta subunit followed by a carboxyl-terminal domain of Thy-1.

The Thy-1 antigen is anchored to the cell surface by a carboxyl-terminal glycophospholipid moiety. To investigate the extent of anchor addition which occurs when such proteins cannot move efficiently to the cell surface, we have expressed a recombinant fusion protein composed of 107 amino-terminal amino acids of bovine luteinizing hormone beta subunit and 46 COOH-terminal amino acids of murine Thy-1 (Thy-1.2 allele). Although the limited amount of fusion protein transported to the cell surface is glycophospholipid-anchored, most of the protein accumulates in an intracellular, endoglycosidase H-sensitive form. The intracellular protein has an unusual structure that contains ethanolamine but does not bind detergent, suggesting either that anchor addition proceeds via a hydrophilic partial intermediate, or that anchor-degradative enzymes exist along the secretory path.

Disruption of N-linked glycosylation of bovine luteinizing hormone beta-subunit by site-directed mutagenesis dramatically increases its intracellular stability but does not affect biological activity of the secreted heterodimer.

The single site for N-linked glycosylation of the beta-subunit of bovine LH (LH beta) was disrupted by oligonucleotide-directed mutagenesis to assess its potential roles in the biosynthesis, transport, and hormonal activity of the LH alpha/beta heterodimer. Pulsechase studies performed with stably transfected Chinese hamster ovary cells that expressed both alpha-subunit (fully glycosylated) and nonglycosylated LH beta revealed that turnover, transport, and secretion of newly synthesized, nonglycosylated LH beta were effectively blocked over a 22-h span. Free nonglycosylated LH beta, like free wild-type LH beta, was sequestered inside the cell; therefore, the intracellular retention of uncombined LH beta-subunit is not due to a signal located within the N-glycan moiety. Nevertheless, an older pool of unlabeled, nonglycosylated LH beta-subunit was available for combination with newly synthesized alpha-subunit, as verified by immunoprecipitation of radiolabeled alpha-subunit from cell lysates and culture medium with anti-LH beta-antiserum. This heterodimer displayed normal kinetics of secretion (t 1/2 = 2.4 h) as compared to fully glycosylated LH (t 1/2 = 2.1 h). The wild-type and mutant forms of LH were also purified from culture supernatants of the two cell lines, and were compared for their relative abilities to stimulate progesterone secretion in cultured rat Leydig cells. Both proteins displayed similar potency (ED50 = 32 vs. 41 ng/ml, respectively) and maximal stimulation of progesterone release Pmax = 2.7 vs 2.5 micrograms/ml), indicating that N-linked glycosylation of the LH beta-subunit does not play a significant role in LH signal transduction. Collectively, these results indicate that N-linked glycosylation is important for intracellular degradation of free LH beta, but is not essential for either its assembly with alpha-subunit or the transport and secretion of biologically active heterodimer.

The transcriptional response of the human chorionic gonadotropin beta-subunit gene to cAMP is cycloheximide sensitive and is mediated by cis-acting sequences different from that found in the alpha-subunit gene.

Cyclic AMP stimulates transcription of the genes encoding the alpha- and beta-subunits of human CG (hCG). Although the cis-acting cAMP response element (CRE) of the alpha-subunit gene has been extensively characterized, relatively little is known about the putative response element of the hCG beta gene. Here, we demonstrate that cAMP stimulation of hCG beta gene transcription requires ongoing protein synthesis, whereas cAMP stimulation of alpha-subunit gene transcription does not. These results suggest that cAMP-stimulated transcription of the alpha and hCG beta genes may involve different cis-acting elements and trans-acting factors. Accordingly, we have constructed a series of deletion mutants consisting of fragments of the hCG beta promoter-regulatory region linked to the bacterial chloramphenicol acetyl transferase gene. To potentiate detection of a CRE, we constructed vectors containing the Rous sarcoma virus enhancer to augment transcriptional activity of the hCG beta-promoter. We also used vectors designed to determine whether regions containing a putative CG beta CRE could confer cAMP responsiveness to a heterologous promoter. These constructs were evaluated for activity by transfection into choriocarcinoma (BeWo) cells. Transient expression of these vectors identifies a broad region which renders transcription of the chloramphenicol acetyl transferase gene responsive to cAMP. Surprisingly, division of the hCG beta 5'-flanking and untranslated regions into smaller fragments led to loss of cAMP responsiveness, suggesting that the hCG beta CRE may be comprised of multiple domains dispersed across the proximal 650 base pairs of CG beta 5'-flanking and untranslated sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Cerebral infarctions studied by [123I]iodoamphetamine. Clinical aspects of the findings on single photon emission computed tomography and transmission computed tomography.

We have used single photon emission computed tomography (SPECT) and the radioisotope [123I]-iodoamphetamine to study 16 patients with cortical and central cerebral infarctions. In 12 cases SPECT, in addition to changes at the site of infarction, showed areas of deficient uptake in other regions of the brain. Diaschisis as well as infarctions, not detected on the transmission computed tomography scanning, seem to explain these findings.

Cyclic AMP regulation of the human glycoprotein hormone alpha-subunit gene is mediated by an 18-base-pair element.

cAMP regulates transcription of the gene encoding the alpha-subunit of human chorionic gonadotropin (hCG) in choriocarcinoma cells (BeWo). To define the sequences required for regulation by cAMP, we inserted fragments from the 5' flanking region of the alpha-subunit gene into a test vector containing the simian virus 40 early promoter (devoid of its enhancer) linked to the bacterial chloramphenicol acetyltransferase (CAT) gene. Results from transient expression assays in BeWo cells indicated that a 1500-base-pair (bp) fragment conferred cAMP responsiveness on the CAT gene regardless of position or orientation of the insert relative to the viral promoter. A subfragment extending from position -169 to position -100 had the same effect on cAMP-induced expression. Furthermore, the entire stimulatory effect could be achieved with an 18-bp synthetic oligodeoxynucleotide corresponding to a direct repeat between positions -146 and -111. In the absence of cAMP, the alpha-subunit 5' flanking sequence also enhanced transcription from the simian virus 40 early promoter. We localized this enhancer activity to the same -169/-100 fragment containing the cAMP response element. The 18-bp element alone, however, had no effect on basal expression. Thus, this short DNA sequence serves as a cAMP response element and also functions independently of other promoter-regulatory elements located in the 5' flanking sequence of the alpha-subunit gene.

The gene for the beta subunit of bovine luteinizing hormone encodes a gonadotropin mRNA with an unusually short 5'-untranslated region.

Both cDNA and genomic clones encoding the beta subunit of bovine luteinizing hormone (LH) have been isolated and characterized. The nucleotide sequence was determined for the entire gene and 776 base pairs of 5'-flanking sequence. The mRNA cap site and polyadenylation site were mapped by primer extension and S1 nuclease protection, respectively. The bovine LH beta spans less than 1.1 kilobase pairs and has three exons encoding a 550 nucleotide mRNA (excluding the poly(A) tail). Bovine LH beta is a single-copy gene, in contrast to human LH beta, which is a member of the LH/chorionic gonadotropin beta subunit multigene family. Comparison of the bovine LH beta gene with the human LH beta/chorionic gonadotropin gene family reveals a high degree of nucleotide sequence homology, both within the genes and in the 5'-flanking sequences. Despite this extensive sequence conservation, there is a major difference between the two species in the selection of a promoter site. As a result, the bovine LH beta gene produces an mRNA with an usually short 5'-untranslated region of only 6-11 nucleotides.

Turnover of acetylcholinesterase in innervated and denervated rat diaphragm.

The acetylcholinesterase (AChE) in rat diaphragms was labelled by intravenous injection of echothiophate in order to evaluate the turnover of AChE in innervated and denervated muscle in vivo. Echothiophate diethylphosphorylates AChE thereby inactivating it. Labelled (diethylphosphorylated) enzyme is rapidly and quantitatively reactivated with 1-methyl-2-hydroxyiminomethylpyridinium (2-PAM), so labelled (diethylphosphorylated) AChE was conveniently measured as 2-PAM-reactivatable AChE activity. In homogenates in vitro, label is lost spontaneously (diethylphosphorylated AChE spontaneously reactivates) with a half-time of 27 h. In innervated diaphragm, labelled non-end-plate AChE is lost with a half-time of 13 h. When correction is made for the spontaneous loss of label on the basis of in vitro measurements, this data indicates that non-end-plate AChE turns over with a half-time of about 26 h. In innervated diaphragm, labelled end-plate-specific AChE is lost more slowly than non-end-plate AChE and at a rate essentially identical to the rate of spontaneous loss of label in vitro. The rate of loss of labelled non-end-plate AChE is essentially identical in 18 h denervated and in paired innervated diaphragms. The rate of loss of labelled end-plate-specific AChE is significantly faster in 18 h denervated diaphragms than in paired innervated diaphragms. On the basis of these observations, hypotheses concerning the mechanisms of the denervation-induced decreases in non-end-plate and end-plate-specific AChE are formulated and discussed.

Gamma camera detection of 99mTC-plasmin in the diagnosis of deep-vein thrombosis.

99mTc-Plasmin scintigraphy of suspected vein thrombosis registered by a gamma camera was compared with phlebography. The results indicate that the plasmin test can be an alternative method to phlebography. The plasmin test registered by a gamma camera has a high sensitivity but relatively low specificity; this agrees with the results of previous investigations regarding registration with a single detector.

Expression of alpha subunit and luteinizing hormone beta genes in the ovine anterior pituitary. Estradiol suppresses accumulation of mRNAS for both alpha subunit and luteinizing hormone beta.

Bovine cDNA clones containing coding sequences for growth hormone, prolactin, alpha subunit, and luteinizing hormone beta (LH beta) have been used to quantitate their respective mRNA concentrations in anterior pituitary glands isolated from ovariectomized ewes, or from ovariectomized ewes treated for three weeks with estradiol. Concentrations of mRNAs for prolactin or growth hormone remained unchanged in either physiological state. In contrast, treatment with estradiol resulted in a 98% decrease of mRNA for LH beta, relative to untreated animals. This change in mRNA was associated with a similar decrease in the concentrations of pituitary and serum LH. Administration of estradiol also led to a reduction (86%) of alpha subunit mRNA. These results suggest that estrogen regulates the expression of the genes encoding both the alpha and LH beta subunit prior to translation. Furthermore, the pronounced effect of estradiol on the concentrations of mRNAs for alpha subunit and LH beta suggest that the assembly of mature glycoprotein hormones may not be limited solely by the rate of accumulation of the beta subunit.