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Two mineral oils and 12 linear primary alcohols were studied, alone and in combination, to determine their contact toxicity to adult German cockroaches, Blattella germanica (L.) (Dictyoptera: Blattellidae). The more toxic oil, PD23 (LD50 = 1.45 mg per cockroach) was used for combination studies. Alcohols with carbon chain lengths of C3 and C8 through C12 were the most toxic, with LD50 values ranging from 0.3 to 0.6 mg. C1 (methanol) and C14 (1-tetradecanol) were least toxic, with LD50 values of 2.35 and 1.75 mg, respectively. Eight of the 12 combinations of a nonlethal dose of PD23 oil with an LD10 dose of alcohol produced significantly greater mortality than predicted under the assumption of additive effects. A sample of five synergistic oil + alcohol combinations, covering most of the alcohol carbon chain length range over which synergy occurred, was further studied by calculating LD50 values for three fixed mixture ratios (80:20, 50:50, and 20:80) of each combination. Results were analyzed using both graphical techniques (isobole analysis) and by nonlinear regression. At least one, but not necessarily all, of the three fixed ratio combinations of each oil + alcohol pairing indicated synergy. The conclusions drawn from the isobole and regression analyses were consistent.

Objective— To determine the effects of intraarticular administration of Samarium-153 (153Sm) bound to hydroxyapatite microspheres (153SmM) on an osteochondral chip–Ninduced synovitis. Study Design— Sixty days after implantation of autogenous osteochondral fragments in the middle carpal and metacarpophalangeal joints, 153SmM was administered into 1 joint of each type. The contralateral joints were used as untreated controls. Animals or Sample Population— Fifteen horses without preexisting joint disease were randomly divided into 2 groups (7 in the carpal group, 8 in the metacarpophalangeal group). Methods— Horses had osteochondral fragments that were harvested from the lateral ridge of the trochlea of the talus and implanted bilaterally into a middle carpal joint and a metacarpophalangeal joint; the opposite joint type served as a control. Sixty days later, 10 to 15 mCi of 153SmM (20 to 50 μm diam) was injected into the fragment-implanted joints. Three horses were treated with nonradioactive hydroxyapatite fragments. Horses were examined clinically until they were killed 14 or 30 days later. Control and treated joints were examined grossly and microscopically to determine the effects of 153SmM on synovial membrane and cartilage. Results— Intraarticular 153SmM caused a transient flare with lameness, effusion, and edema for 48 to 72 hours. Implanted osteochondral chips induced a synovitis characterized by variable degrees of joint damage and synovial infiltrate. Use of 153SmM resulted in synovectomy of variable depth and extent. Conclusions— Intraarticular 153SmM may be a useful method for synovectomy of inflamed synovial membrane. Clinical Relevance— With further testing, radioactive pharmaceuticals might become useful clinical treatments for persistent synovitis not responsive to conventional techniques.

Protein kinase C (PKC) is a major regulator of a broad range of cellular functions. Activation of PKC has been reported to stimulate Na+ transport across frog skin epithelium by increasing the apical Na+ permeability. This positive natriferic response has not been observed with other epithelial preparations, and could reflect the specific experimental conditions of different laboratories, or species or organ specificity of the response to PKC. In the present study, measurements were conducted with skins and urinary bladders from the same animals of two different species. The PKC activator TPA uniformly increased the transepithelial Na+ transport (measured as amiloride-sensitive short-circuit current, ISC, across skins from Rana temporaria and Bufo marinus, and inhibited ISC across bladders from the same animals. Inhibitors of PKC (staurosporine, H-7 and chelerythrine) partially blocked the TPA-induced stimulation of ISC across frog skin. The specificity of the PKC response by amphibian skin could have reflected an induction of moulting, similar to that observed with aldosterone. However, light micrographs of paired areas of frog skin revealed no evidence of the putative moulting. Separation of stratum corneum from the underlying stratum granulosum could be detected following application of aldosterone. We conclude that the effect of PKC on epithelial Na+ channels is organ, and not species specific. The stimulation of Na+ permeability in amphibian skin does not arise from sloughing of the stratum corneum. These observations are consistent with the hypothesis that the natriferic action arises from the calcium-independent isozyme of PKC previously detected in frog skin.

Amiloride-sensitive whole cell currents have been reported in M-1 mouse cortical collecting duct cells (Korbmacher et al., J. Gen. Physiol. 102: 761-793, 1993). We have confirmed that amiloride inhibits the whole cell currents but not necessarily the measured whole cell currents. Anomalous responses were eliminated by removing external Na+ and/or introducing paraepithelial shunts. The amiloride-sensitive whole cell currents displayed Goldman rectification. The ionic selectivity sequence of the amiloride-sensitive conductance was Li+ > Na+ >> K+. Growth of M-1 cells on permeable supports increased the amiloride-sensitive whole cell permeability, compared with cells grown on plastic. Single amiloride-sensitive channels were observed, which conformed to the highly selective low-conductance amiloride-sensitive class [Na(5)] of epithelial Na+ channels. Hypotonic pretreatment markedly slowed run-down of channel activity. The gating of the M-1 Na+ channel in excised patches was complex. Open- and closed-state dwell-time distributions from patches that display one operative channel were best described with two or more exponential terms each. We conclude that 1) study of M-1 whole cell Na+ currents is facilitated by reducing the transepithelial potential to zero, 2) these M-1 currents reflect the operation of Na(5) channels, and 3) the Na+ channels display complex kinetics, involving > or = 2 open and > or = 2 closed states.

Maternal cocaine injection causes fetal hypoxemia, hypertension, and increased cerebral blood flow (CBF) in sheep. To test the hypothesis that increased fetal CBF is not due solely to fetal hypoxemia, we injected cocaine directly into a fetal vein. A single dose of cocaine [1 (group 1; n = 7) or 2 (group 2; n = 8) mg/kg i.v.] was administered to chronically catheterized, unanesthetized, near-term fetal sheep. Fetal CBF (microspheres), arterial blood pressure (BP), O2 content, and cerebral O2 consumption (CMRo2) were measured at baseline, 30 s, and 2, 5, and 15 min after fetal cocaine injection. Fetal CBF increased 27 +/- 9% (SE) at 5 min in group 1 and returned to baseline by 15 min, whereas fetal CBF increased 57 +/- 8% at 5 min and remained elevated at 15 min in group 2. Fetal BP increased at 30 min in both groups and remained increased at 2 min in group 1 and at 5 min in group 2. Cerebrovascular resistance increased at 30 s in both groups and then decreased at 5 min only in group 2. Fetal hypoxemia was observed in group 2 5 min after cocaine injection (arterial PO2 decreased 24 +/- 5%), whereas no hypoxemia was noted in group 1. CMRO2 was unchanged in group 1 but increased in group 2 at 5 min (41 +/- 10%) and was associated with an increase in cerebral O2 extraction. Increases in myocardial and adrenal blood flows and reductions in both small and large intestinal blood flows were noted at 5 min in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumors have heterogeneous properties, which could be explained by the existence of hierarchically and biologically distinct tumor cells such as tumor-initiating cells (TICs). This model is clinically important, as TICs are promising targets for cancer therapies. However, TICs in spontaneous B-cell lymphoma have not been conclusively identified.Tumor cells with a progenitor phenotype exist in B-cell lymphoma, reflecting a hierarchical organization.Twenty-eight client-owned dogs with previously untreated B-cell lymphoma and 6 healthy dogs.This was a prospective study. Flow cytometry was used to identify lymphoid progenitor cells (LPCs) that coexpressed hematopoietic progenitor antigens CD34, CD117, and CD133, with lymphoid differentiation markers CD21 and/or CD22 in B-cell lymphoma. The polymerase chain reaction for antigen receptor rearrangements was used to analyze clonality and relatedness of tumor populations. A xenograft model with NOD/SCID/IL-2Rγ(-/-) mice was adapted to expand and serially transplant primary canine B-cell lymphoma.LPCs were expanded in lymph nodes from 28 dogs with B-cell lymphoma compared with 6 healthy dogs (P= .0022). LPCs contained a clonal antigen receptor gene rearrangement identical to that of the bulk of tumor cells. Canine B-cell lymphoma xenografts in recipient mice that maintained LPCs in the tumors were recurrently observed.These results suggest the presence of a hierarchy of tumor cells in B-cell lymphoma as has been demonstrated in other cancers. These findings have the potential to impact not only the understanding of lymphoma pathogenesis but also the development of lymphoma therapies by providing novel targets for therapy.

Summary Complete thoracic radiographic examinations were performed on 7 horses ranging in age from 24 to 60 months, followed by in-situ lung fixation. Radiographs were examined by 3 radiologists for the presence, degree and distribution of generalised pulmonary patterns within a region of interest in the caudodorsal lung. Pulmonary tissue was obtained from 12 sites within a designated volume of interest in the caudodorsal lung, corresponding to the area of interest evaluated radiographically, and examined for the presence, character and severity of microscopic lesions. Radiographic findings within the volume of interest consisted of mild to moderate bronchial, bronchointerstitial, or interstitial pulmonary patterns. Interstitial and bronchointerstitial radiographic findings were related to severity of peribronchiolar mononuclear cell infiltrates, the degree of bronchiolar mucosal plication, and alveolar capillary and peribronchial blood vessel erythrocyte content. The severity of the interstitial radiographic pattern was inversely associated with the perceived diagnostic quality of the radiographic examinations. There was no evidence of spatial variation in the severity of the microscopic changes examined in this limited pulmonary region. Inter-rater reliability between radiologists was good in the assessment of diagnostic quality of the radiographic examinations but poor in assessing severity of the primary generalised pulmonary patterns within the radiographic region of interest.

The distal phalanx and metacarpal physis of both forelimbs of 32 Thoroughbred foals 3-32 weeks of age were radiographed to identify those limbs with osseous bodies at a palmar process (PP) of the distal phalanx. Osseous bodies (ossicles) were identified radiographically in 19% of the foals. Sixteen of the 32 foals were selected for microradiographic and histological evaluation: 6 foals with radiographic evidence of ossicles and 10 foals without. Fourteen ossicles of the PP were observed radiographically. Ossicles were either a triangular bone fragment at the palmar aspect of the distal angle of the PP, or an oblong bone fragment separated from P3 by a radiolucent line extending 1-3 cm from the incisure of the PP to the solar margin. One foal had radiographic evidence of bilateral distal metacarpal physitis. Seventeen of 35 PPs examined microradiographically were considered normal, in that the dorsal and solar cortices were thin with trabecular bone orientated parallel to the cortical surfaces, and there was a depression in the dorsal cortical surface (i.e. parietal sulcus). Abnormal microradiographic findings in the other 18 PPs included a fracture line extending from the dorsal cortical surfaces and trabecular bridging of the fracture gap. The fracture line was often continuous with the parietal sulcus. Microradiographic evidence of a fracture was found in 75% of foals evaluated. Normal histological findings in 16 PPs included thin dorsal and solar cortices with trabeculae orientated parallel to the cortical surfaces, parallel-fibred dense connective tissue attachments of the deep digital flexor tendon to the solar cortical surfaces, and a neurovascular bundle associated with the parietal sulcus.(ABSTRACT TRUNCATED AT 250 WORDS)

Maternal cocaine abuse has been associated with neonatal neurological and neurobehavioral problems of unknown pathogenesis. We administered a single intravenous dose of cocaine (2 mg/kg) to 12 unanesthetized pregnant sheep; their fetuses had been catheterized in utero 2 days before the study. We measured fetal cerebral blood flow (CBF), cerebral metabolic rate of O2 (CMRO2), mean arterial blood pressure (MAP), and blood gases before and 2, 5, 15, and 30 min after maternal cocaine injection. Fetal CBF increased by 37 +/- 33% (mean +/- SD) at 5 min and returned to baseline by 15 min. Regional brain blood flow changes paralleled CBF changes with the greatest increases occurring in cerebellum (54 +/- 43%) and brain stem (54 +/- 52%). Cerebral vascular resistance was decreased for cerebellum (22%) and brain stem (19%) but was unchanged for cerebral hemispheres and caudate. Increased CBF at 5 min was associated with a 20 +/- 9% increase in fetal MAP and a 38 +/- 13% decrease in fetal arterial O2 content. Fetal CMRO2 was unchanged. There was a decrease in fetal intestinal blood flow at 2 min, an increase in myocardial, adrenal, and renal blood flow at 5 min, and no change in placental blood flow. Maternal cocaine injection causes fetal hypoxemia, hypertension, and increased CBF. Possible mechanisms for cerebral vasodilation (in some areas) include hypoxemia, impaired autoregulatory response to increased blood pressure, and/or direct or indirect vascular effects of cocaine or its metabolites.

The aim of this study was to examine inhibin production in granulosa-theca cell tumours (GTCT). The experimental aims were: (i) to determine GTCT cell types that produce inhibin alpha- and betaA-subunits; (ii) to determine whether alpha- and betaA-subunit forms differ in GTCT fluid and normal equine follicular fluid (eFF); and (iii) to determine whether dimeric inhibin (alpha betaA) is present in GTCT plasma and tumour fluid. Plasma, tumour fluid and tumour tissue were collected from mares (n=6) with GTCT. Plasma and eFF were collected during the follicular phase from mares (n=4) undergoing normal cycles. Immunohistochemical examination of GTCT tumour sections showed strong inhibin alpha- and betaA-Subunit immunostaining in granulosa cells and polyhedral-shaped cells in the thecal-stromal layer. The presence of polyhedral-shaped cells was related to testosterone concentration in tumour fluid. Low molecular weight alpha-subunit forms were less abundant in tumour fluid than in eFF, whereas the amounts of betaA-subunit forms were similar in tumour fluid and eFF. Concentrations of betaA were increased in plasma from mares with GTCT and similar in tumour fluid and eFF. In summary, lower molecular weight alpha-subunit forms were less prominent in GTCT fluid than in eFF and concentrations of betaA were higher in GTCT plasma than in control plasma.

CA 125 is an antigenic determinant located on the surface of ovarian carcinoma cells and elevated in the serum of greater than 90% of patients with carcinoma. The antigen, derived from the ovarian epithelium, has been described as a mucinlike glycoprotein greater than 200 kd. To date little is known of the metabolic regulation or expression of this antigen in either normal or neoplastic tissues. New monoclonal antibodies that we describe here recognize both unique and similar epitopes to OC 125. These reagents may allow for a more complete definition of the structure and expression of the CA 125 complex. These antibodies recognize high-molecular weight (greater than 200 kd) subspecies and a lower-molecular-weight (68 kd) subspecies of the antigen and identify it in the cytoplasm and the extracellular matrix of CA 125-producing cells.

Activators of protein kinase C (PKC) stimulate Na+ transport (JNa) across frog skin. We have examined the effect of Ca2+ on PKC stimulation of JNa. Both the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and the diacyl-glycerol sn-1,2-dioctanoylglycerol (DiC8) were used as PKC activators. Blocking Ca2+ entry into the cytosol (either from external or internal stores) reduced the subsequent natriferic effect of the PKC activators. This negative interaction did not simply reflect saturation of activation of the apical Na+ channels, since the stimulations produced by blocking Ca2+ entry and adding cyclic AMP were simply additive. The Ca2+ dependence of the natriferic effect could have reflected either a direct action of cytosolic Ca2+ on PKC or an indirect action on the final receptor site (the Na+ channel). To distinguish between these possibilities, the TPA- and phospholipid-dependent kinase activity of broken-cell preparations was assayed. The kinase activity was not stimulated by physiological levels of Ca2+, and in fact was inhibited at millimolar concentrations of Ca2+. We conclude that the effects of Ca2+ on the natriferic response to PKC activators are indirect. Reducing cytosolic uptake of Ca2+ may have stimulated Na+ transport by a chemical modification of the apical channels observed in other tight epithelia. The usual stimulation of Na+ transport produced by PKC activators in frog skin may reflect the operation of a nonconventional form of PKC. This enzyme is Ca2+ independent and seems related to the nPKC or PKC epsilon observed in other systems.

Glycogen synthase kinase 3 beta (GSK-3beta) was one of the first kinases identified and studied, initially for its role in the regulation of glycogen synthesis. Over the past decade, interest in GSK-3beta has grown far beyond glycogen metabolism, and this is due in large measure to the critical role that GSK-3beta plays in the regulation of many other cellular processes, particularly cell proliferation and apoptosis. GSK-3beta has been shown to regulate the proteolysis and sub-cellular compartmentalization of a number of proteins directly involved in the regulation of cell cycling, proliferation, differentiation and apoptosis. GSK-3beta also regulates the degradation of proteins that regulate gene expression and thus affects a variety of important cell functions. Specifically, GSK-3beta controls the degradation of beta-catenin, the main effector of Wnt that regulates haematopoiesis and stem cell function. In this case GSK-3beta is a negative regulator of Wnt. In contrast, GSK-3beta positively regulates NF-kappaB, another important biochemical pathway also involved in the regulation of multiple aspects of normal and aberrant haematopoiesis. GSK-3beta regulates degradation of IkappaB, a central inhibitor of NF-kappaB. In this way, GSK-3beta acts to control the resistance of leukaemic cells to chemotherapy through the modulation of NF-kappaB, a critical factor in maintaining leukaemic cell growth. In addition, GSK-3beta regulates the pro-inflammatory activity of NF-kappaB. As GSK-3beta is a pleiotropic regulator, inhibitors may increase the range of novel anti-leukaemic and anti-inflammatory drugs that control immune response.

Vulvovaginitis in the prepubertal child may present with itch, irritation, pain, dysuria or discharge as a result of poor hygiene, contact irritants, sexual abuse or skin disease involving the genital area. Common infectious causes include threadworms, group A beta-haemolytic streptococci and Haemophilus influenzae. A full history and examination are essential. Investigations may include swabs, midstream urine, biopsy, ultrasound and examination under anaesthesia.

It has been known for10 years that there are two alleles of the human ornithine decarboxylase (ODC) gene, defined by a polymorphic PstI RFLP in intron 1. We have sequenced a large portion of each of the two alleles, including some of the 5' promoter region, exon 1, intron 1, and exon 2, and determined that a single nucleotide polymorphism at base +317 (relative to transcription start site) is responsible for the presence or absence of the PstI restriction site. We have developed two genotyping assays, a PCR-RFLP assay and a high-throughput TaqMan-based method, and determined the ODC genotype distribution in900 North American DNA samples. On the basis of its location between two closely spaced Myc/Max binding sites (E-boxes), we speculated that the single nucleotide polymorphism at base +317 could have functional significance. Results of transfection assays with allele-specific reporter constructs support this hypothesis. The promoter/regulatory region derived from the minor ODC allele (A allele) was more effective in driving luciferase expression in these assays than the identical region from the major allele (G allele). Our results suggest that individuals homozygous for the A allele may be capable of greater ODC expression after environmental exposures, especially those that up-regulate c-MYC expression.

Objective—To assess the suitability of lithium dilution as a method for measuring cardiac output in anesthetized horses, compared with thermodilution and transesophageal Doppler echocardiography. Animals—6 horses (3 Thoroughbreds, 3 crossbreeds). Procedure—Cardiac output was measured in 6 anesthetized horses as lithium dilution cardiac output (LiDCO), thermodilution cardiac output (TDCO), and transesophageal Doppler echocardiographic cardiac output (DopplerCO). For the LiDCO measurements, lithium chloride was administered IV, and cardiac output was derived from the arterial lithium dilution curve. Sodium nitroprusside, phenylephrine hydrochloride, and dobutamine hydrochloride were used to alter cardiac output. Experiments were divided into 4 periods. During each period, 3 LiDCO measurements, 3 DopplerCO measurements, and 3 sets of 3 TDCO measurements were obtained. Results—70 comparisons were made between LiDCO, DopplerCO, and triplicate TDCO measurements over a range of 10 to 43 L/min. The mean (± SD) of the differences of LiDCO – TDCO was –0.86 ± 2.80 L/min; LiDCO = –1.90 + 1.05 TDCO (r = 0.94). The mean of the differences of DopplerCO – TDCO was 1.82 ± 2.67 L/min; DopplerCO = 2.36 + 0.98 TDCO (r = 0.94). The mean of the differences of LiDCO – DopplerCO was –2.68 ± 3.01 L/min; LiDCO = –2.53 + 0.99 DopplerCO (r = 0.93). Conclusions and Clinical Relevance—These results indicate that lithium dilution is a suitable method for measuring cardiac output in horses. As well as being accurate, it avoids the need for pulmonary artery catheterization and is quick and safe to use. Monitoring cardiac output during anesthesia in horses may help reduce the high anesthetic mortality in this species. (Am J Vet Res 2000;61:731–737)

The hair follicle is characterized by cyclic transformations from active growth and hair fiber production through regression into a resting phase. The growth phase, known as anagen, is associated with rapid rates of cell turnover, and variations in the rate of DNA synthesis in mouse skin throughout the hair cycle are accompanied by changes in the activity of ornithine decarboxylase (ODC), a key enzyme in the synthesis of polyamines, which are actively involved in regulation of normal cell division, differentiation, and growth. Previously, a transgenic mouse was created that overexpressed ODC in the skin using a K6 promoter. The first hair cycle in neonatal transgenic mice appeared to be normal, but by the third week of postnatal life transgenic pups begin to progressively lose hair. The lower portion of the hair follicle was progressively replaced with enlarging cystic structures located in the deep dermis, and the transgenic mice exhibited excessive growth of skin mass resulting in pronounced wrinkling and folding. Interestingly, these findings bore striking resemblance to the rhino mouse phenotype and to human patients with papular atrichia, a rare congenital ectodermal disorder characterized by progressive and irreversible hair loss in early childhood. The similarities in phenotype between transgenic mice and human atrichia with papular lesions suggest that ODC transgenics may represent a useful model for studying this disorder. It appears that ODC plays a functionally important, yet still obscure role in a complex metabolic pathway that is critical in hair follicle function not only in mice, but in humans as well.

Mitochondrial ribosomal proteins (MRPs) are required for the translation of all 13 mitochondrial encoded genes in humans. It has been speculated that mutations and polymorphisms in the human MRPs may be a primary cause of some oxidative phosphorylation disorders or modulate the severity and tissue specificity of pathogenic mitochondrial DNA mutations. Although the sequences of most of the yeast MRPs are known, only very few mammalian and nearly no human MRPs have been completely characterized. MRPs differ greatly in sequence, and sometimes biochemical properties, between different species, not allowing easy recognition by sequence homology. Therefore, the Mammalian Mitochondrial Ribosomal Consortium is using a direct approach of purifying individual mammalian (bovine) MRPs, determining their N-terminal and/or internal peptide sequences using different protein sequencing techniques, and using the resulting sequence information for screening expressed sequence tags and genomic data bases to determine human, mouse, and rat homologues of the bovine proteins. Two proteins of the large and three proteins of the small ribosomal subunit have been analyzed in this manner. Three of them represent "new," i.e. formerly unknown mammalian mitochondrial ribosomal protein classes. Only one of these three different MRPs shows significant sequence similarities to known ribosomal proteins. In one case, the corresponding human genomic DNA sequences were found in the data bases, and the exon/intron structure was determined.

Objective— To determine the effects of Samarium-153 bound to hydroxyapatite microspheres (153SmM) when injected into the metacarpophalangeal and metatarsophalangeal joints of horses. Study Design— Horses were injected with 153SmM in metacarpophalangeal and metatarsophalangeal joints with the diagonal contralateral joints used as untreated controls. Animals or Sample Population Twelve adult horses without pre-existing disease involving the metacarpo/metatarsophalangeal joints. Methods— Horses were divided into three groups: high-dose Samarium-153 (12.5 to 17.0 millicurie [mCi]), intermediate dose (6.5 to 12.0 mCi), and low dose (3.5 to 6.0 mCi). Horses were examined daily for 7 days postinjection for clinical abnormalities, lameness, and surface and systemic radiation levels. One horse from each group was euthanatized at 14, 30, and 60 days postinjection and the effects of the 153SmM examined microscopically in the cartilage and synovial membrane. Results— Intraarticular153SmM caused inflammation characterized by lameness, effusion, and regional edema for 48 to 72 hours. Minimal levels of active 153SmM were identified in the blood or urine and were well below the maximal tolerance of 1 mCi. Microscopically the radiation caused no effects on the articular cartilage. The synovectomy created was good but not ideal in that some areas did have necrosis into the subintimal regions and a few islands of intact intimal cells persisted. Conclusions— The use of 153SmM is an effective means of targeting the synovial intimal cells with minimal extrasynovial leakage of radiation. Clinical Relevance— The metacarpophalangeal and metatarsophalangeal joints of the horse can be safely treated with 153SmM without damage to the cartilage or significant extracapsular leakage.

Four different classes of mammalian mitochondrial ribosomal proteins were identified and characterized. Mature proteins were purified from bovine liver and subjected to N-terminal or matrix-assisted laser-desorption mass spectroscopic amino acid sequencing after tryptic in-gel digestion and high pressure liquid chromatography separation of the resulting peptides. Peptide sequences obtained were used to virtually screen expressed sequence tag data bases from human, mouse, and rat. Consensus cDNAs were assembled in silico from various expressed sequence tag sequences identified. Deduced mammalian protein sequences were characterized and compared with ribosomal protein sequences of Escherichia coli and yeast mitochondria. Significant sequence similarities to ribosomal proteins of other sources were detected for three out of four different mammalian protein classes determined. However, the sequence conservation between mitochondrial ribosomal proteins of mammalian and yeast origin is much less than the sequence conservation between cytoplasmic ribosomal proteins of the same species. In particular, this is shown for the mammalian counterparts of the E. coli EcoL2 ribosomal protein (MRP-L14), that do not conserve the specific and functional highly important His(229) residue of E. coli and the corresponding yeast mitochondrial Rml2p.

This study sought to determine if extensive trimming of the front hooves of foals results in a higher incidence of palmar process fractures compared to untrimmed foals, and to characterise the clinical course of foals with palmar process fractures with physical findings, hoof measurements and radiography. Twenty foals age 4-8 weeks of multiple breeds were examined every 2 weeks over a 12 week period. Ten foals had both front hooves extensively trimmed every 4 weeks, while 10 foals remained untrimmed. Palmar process fractures occurred in 4 trimmed and 3 untrimmed foals. Four foals with fractures had brief lameness (Grades I-II/V) and hoof tester sensitivity was found inconsistently with fractures. All foals with fractures were sound at the end of the study. Fractures healed based on radiographic evaluations in an average of 8.4 weeks. Two of 3 foals with fractures developed club-footed conformation. None of the measured hoof parameters significantly differed between foals with and without fractures. We conclude that extensive trimming of the heels did not appear to affect the occurrence of palmar process fractures in this group of foals.

Prophylactic hemodialysis has been employed in the treatment of 15 patients with acute renal failure due to acute tubular necrosis (12), bilateral renal cortical necrosis (two), and poststreptococcal glomerulonephritis (one). Dialyses, usually lasting six hours each, were begun before clinical evidence of uremia developed in each patient and/or before the nonprotein nitrogen reached 200 mg.%, and were repeated daily or often enough to maintain the nonprotein nitrogen below 150 mg.%. The hypothesis underlying this technic postulates (1) that wasting, sepsis and impaired wound healing in these patients may reflect tissue injury by the same dialyzable toxic agents which produce the uremic symptoms that are readily reversible by dialysis, and (2) that repeated dialyses should therefore prevent both clinical uremia and the later, often lethal sequelae. The results contrast dramatically with our own past experience in treating patients with acute renal failure with a carefully executed medical regimen together with hemodialysis on conventional indications. Except in one instance of crush injury with progressive intracerebral damage, and one brief occasion in another individual, these patients experienced a stable, convalescent clinical course, remained free of uremic symptoms or chemical imbalances, ate at least three meals daily which were unrestricted in amount and composition, and were ambulatory between dialyses unless confined to bed by associated disease. Wounds healed well. Infection either did not occur, or subsided after appropriate therapy. Fluid restriction was liberalized by means of ultrafiltration with dialysis. Regional heparinization of only the extracorporeal circuit eliminated actual or impending bleeding as a contraindication to dialysis. Chronic vessel cannulation made the frequent dialyses possible, but may have provided the route for repeated, transient bacterial contamination of the blood stream in the first hour of many dialyses. Marked anemia, despite reticulocytosis, moderate to mild weight loss and some mental deficit persisted in spite of the general clinical improvement and well-being. Three patients with tubular necrosis died after seven, 11 and 26 days of oliguria; both patients with bilateral renal cortical necrosis also succumbed, on the seventy-third and ninety-second days of renal failure, and after 29 and 40 dialyses, respectively. At autopsy, evidence of sepsis was conspicuously absent. The remaining 10 patients survived. Thus some, but not all, clinical manifestations of acute renal failure appear to be favorably influenced by prophylactic dialysis treatment. Our initial experience in this group of 15 patients does not of course prove that freedom from complications and a significantly better outlook for survival can be assured to patients with acute renal failure by these methods. However, it seems to offer a reasonable hope of this possibility which we cannot attach to management by medical measures alone, or by dialysis on conventional indications. If this hope is realized in greatly extended, subsequent series, then it seems inevitable that some form of prophylactic dialysis, or some equally effective alternative, should be adopted in treating the majority of patients with acute renal failure.

In standard mouse strains, a high proportion (more than 90%) of epidermal tumors produced by initiation with 7,12-dimethylbenz[a]anthracene and promotion with a variety of chemical agents contain an activating mutation in codon 61 (A182--T) of the c-Ha-ras gene. We analyzed the ras mutational spectra in 69 tumors induced by DMBA in a unique transgenic model, the K6/ODC mouse. In this model, low-dose DMBA treatment is sufficient per se for tumor induction, so tumor promotion with chemical agents is not required. In contrast to previous studies in standard mouse strains, our study showed that less than 50% of epidermal tumors from K6/ODC mice contained an activating codon 61 c-Ha-ras mutation (A182--T). This result was obtained in mice initiated either as newborns (when the transgene is not expressed) or as adults (when the transgene is fully expressed). Analysis of other codon hot-spots and other ras genes revealed the presence of three codon 12 and 20 codon 61 (A182--T) mutations in the c-Ki-ras gene in the 36 tumors that did not have c-Ha-ras mutations. We concluded that promotion in this model, by means of constitutive ornithine decarboxylase expression, causes the clonal expansion of a population of initiated cells not promoted by chemical agents.

Elevated polyamine levels are characteristic of many types of neoplastic cells and tissues. We demonstrate that in transgenic mice overexpressing ornithine decarboxylase in skin, changes in tissue polyamine levels, particularly putrescine, control the development and maintenance of the neoplastic phenotype. A specific inhibitor of the transgene, alpha-difluoromethylornithine (DFMO), reversibly blocked the appearance of squamous papillomas after carcinogen treatment. Furthermore, treatment of papilloma-bearing mice with DFMO caused rapid tumor regression, also in a reversible manner. Although the rate of apoptosis in papillomas was unaffected by acute DFMO treatment, tumor cell proliferation was rapidly decreased after drug treatment. Conversely, proliferation of normal epidermal keratinocytes was unaffected by DFMO treatment. The regulatory polyamine in this model appears to be putrescine, the immediate product of ornithine decarboxylase. These results demonstrate that elevated polyamine levels are required for both the development and maintenance of the neoplastic phenotype in skin.

Population surveys of Sulawesi crested black macaques (Macaca nigra) were conducted on the Indonesian islands of Sulawesi and Bacan in 1992-1994 to assess the status of natural populations and determine habitat and anthropogenic factors affecting their population densities. We surveyed five sites for primates, including undisturbed and disturbed habitats. Data were collected on habitat structure and composition at two undisturbed and one disturbed forest site in which the primates were surveyed. The highest density of macaques was found in primary forest at Gunung Sibela Nature Reserve on Bacan (170.3 individuals/km2). Population density in logged forest on Bacan was high but significantly less than primary forest (133.4 individuals/km2). The high density of crested black macaques in primary forest on Bacan is best explained by the high carrying capacity found in primary forest. The lower food quantity and quality of food resources found in logged forest correlated with lower primate densities compared to primary forest. However, the large population of macaques in logged forest demonstrates the conservation value of such forest. Densities on Sulawesi at Tangkoko-Batuangas-DuaSudara Nature Reserve (TBDS) showed a continuing decline since earlier surveys. Primate densities were highest near the protected center of Tangkoko Reserve (66.7 individuals/km2). The peripheral areas of Batuangas and DuaSudara, even though adjacent and continuous, showed lower population densities of 46.4 and 23.5 individuals/km2, respectively. The best explanation for the continued decline of Macaca nigra populations at TBDS is hunting. Unless conservation measures are implemented immediately, M. nigra on Sulawesi risks extinction in the near future.

In multistage tumorigenesis models, ornithine decarboxylase (ODC) is usually dysregulated at some point during tumor promotion, an early stage of carcinogenesis. To address the question whether constitutive overexpression of ODC would be a sufficient condition for tumor promotion, mice with high levels of ODC expression targeted to epidermal keratinocytes were used in skin tumorigenesis experiments. Transgenic mice with ODC targeted to hair follicle keratinocytes were much more sensitive than littermate controls to initiation with a single low dose of carcinogen; in fact, such mice no longer required treatment with tumor promoters for tumors to develop. Targeting ODC overexpression to both interfollicular and follicular keratinocytes did not further enhance tumor yield. Our results suggest that most, if not all, target cells for chemical carcinogens in the skin reside in hair follicles, and ODC overexpression is sufficient to activate such cells to expand clonally to form epidermal tumors.

To determine the potential clinical usefulness of duplex Doppler estimation of arcuate artery resistive index (a measure of intrarenal blood flow impedance) for diagnosis of aminoglycoside-induced nephrotoxicosis.30 adult, female, mixed-breed dogs, allotted to 3 groups of 10 dogs each as: toxic dosage of gentamicin, therapeutic dosage of gentamicin, and saline solution sham equivalent in volume to that of the toxic dosage of gentamicin.After baseline screening to establish normalcy (serum biochemical analysis, endogenous creatinine clearance determination, urinalysis, urine protein-to-creatinine ratio, urine culture, gray-scale sonography, and percutaneous ultrasound-guided renal biopsy), results of arcuate artery resistive index determination were compared with serum creatinine and urine specific gravity values on a Monday-Wednesday-Friday data collection schedule for 10 days. Endogenous creatinine clearance determination, ultrasound-guided renal biopsy, and urine culture were repeated at the end of data collection in all 3 groups.Significant differences in resistive index measurements were not observed, despite clinicopathologic and renal biopsy results compatible with severe acute tubular necrosis in dogs of the toxic dosage group.Duplex Doppler sonography of arcuate artery blood flow impedance, expressed as the resistive index, appears to have poor clinical usefulness as a diagnostic tool in this disorder.Normal arcuate artery resistive index values obtained in dogs for which aminoglycoside-induced nephrotoxicosis is suspected do not exclude the disorder. If abnormal arcuate artery resistive index values are obtained for such dogs, further evaluation for nephropathies other than aminoglycoside-induced nephrotoxicosis may be considered.

The M-1 cell line is derived from the mouse cortical collecting duct and displays the low-conductance, highly Na(+)-selective channel activity of the alpha,beta, gamma-heterotrimeric epithelial Na+ channel (ENaC). The short-circuit current (Isc) across M-1 monolayers was 89 +/- 4 microA/cm2, and the transepithelial conductance was 2.1 +/- 0.2 mS/cm2. Isc was abolished by blocking the Na+ pump with ouabain. Both Isc and transepithelial conductance (gT) were inhibited by benzamil > amiloride >> dimethylamiloride. Under our experimental conditions, vasopressin, vasopressin, forskolin, and dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) had no detectable effects on Isc or gT. Increasing apical Na+ entry with nystatin increased Isc. The possible regulation of the M-1 Na+ channel by cAMP-activated protein kinase A (PKA) was further examined with excised inside-out patches. The open-time probability (Po) was not fixed, displaying substantial variance. Perfusion with ATP itself, with the catalytic subunit of PKA with ATP, or with alkaline phosphatase had no consistent effect on Po, the unitary current, or the kinetics of the M-1 Na+ channel. The data are consistent with the concept that PKA stimulates ENaCs by phosphorylating a site with access to but not within the apical membrane patch during cell-attached and excised-patch studies.

Six 15- to 20-year-old obese cynomolgus monkeys were determined to have diabetes mellitus. These monkeys were hyperglycemic and hypertriglyceridemic, yet remained nonketotic for several years before requiring clinical intervention. A significant decrease in glucose disappearance and an increased area under the glucose disappearance curve were found in response to intravenous glucose tolerance testing. Basal hyperinsulinemia was found in three animals that, in response to glucose stimulation, had a blunted insulin response, resulting in an overall decrease in the area under the insulin curve. Two animals died during the study and had extensive amyloid infiltration of pancreatic islets. The combined findings of old age, obesity, hyperinsulinemia, improvement in response to caloric restriction, and islet amyloidosis are consistent with type 2 diabetes mellitus.

Our studies confirm the common occurrence of a unique form of apolipoprotein AI (apoAI)-derived vascular amyloidosis in dogs that appears to be unrelated to other disease conditions, but is associated with aging. Vascular amyloid deposits were most frequently located within the intima and media of medium-sized pulmonary arteries, and were not confirmed in any other tissues. Pulmonary vascular amyloid immunoreactive with antiserum to purified N-terminal (1-71) canine apoAI amyloid protein was demonstrated retrospectively in 12.8% of necropsied dogs (N = 243) 10 years of age or older. In a subsequent expanded 1-year prospective study of necropsied dogs (N = 231) of all ages, apoAI-derived pulmonary vascular amyloid deposits were demonstrated in 0.7% of dogs < 10 years of age and in 22% of dogs 10 years of age or older. The incidence of this form of amyloid in dogs 10 years of age or older was significantly associated with advancing age (P < 0.00001). However, significant differences regarding gender, breed, or the frequency of selected common disease conditions were not observed when the dogs with apoAI-derived amyloid were compared with control dogs. The possibility that this new form of senile apoAI-derived amyloidosis is a manifestation of an age-associated aberration in apoAI metabolism or is related to a mutant form of apoAI is the subject of ongoing investigations.

Ornithine decarboxylase, a critical regulatory enzyme for polyamine biosynthesis, is highly inducible by growth-promoting stimuli in mouse epidermis but the enzyme level is only transiently elevated due to rapid turnover of the protein. Here we report that constitutive overexpression of the enzyme in the skin of transgenic mice causes several phenotypic abnormalities. Effects observed include development of dermal follicular cysts, excessive skin wrinkling, enhanced nail growth, alopecia, and spontaneous tumor development. These results indicate that up-regulation of polyamine biosynthesis can profoundly disturb skin homeostasis and alter susceptibility to neoplastic development.

A human biovar of Chlamydia trachomatis was used to develop a murine model of ocular chlamydial infection. The inbred mouse model will allow detailed immunologic studies during ocular infection, and use of a human biovar for infection may aid in identification of appropriate vaccine strategies against chlamydial infections.BALB/c, C3H/HeN, and C57B1/6J mice (n = 5 to 10 mice/group) were topically infected in the conjunctiva with C serovar of C. trachomatis. The effects were tested of single and repeated infection with 5000 inclusion-forming units (IFU) in 5 microliters and different inoculum doses. Conjunctival surfaces of both eyes were swabbed for microbiologic signs (isolation culture or direct fluorescent antibody staining) of infection over 4 to 6 weeks. Conjunctivae were removed for histopathologic study, and lymphocytes from draining cervical lymph nodes and spleens were tested for chlamydia-specific proliferative responses. Serum was obtained from all mice and tested for anti-chlamydial antibodies.BALB/c and C3H/HeN mice developed dose-dependent microbiologic, histopathologic, and immunologic evidence of ocular infection. Eyes of mice were culture-positive from day 7 through at least day 21, with the peak of infection at days 10 to 14 after infection. Histopathologically, the development of conjunctival subepithelial mononuclear infiltration, exudate, and loss of goblet cells occurred within 1 week. Dose-dependent lymphoproliferative responses to whole chlamydial elementary bodies were observed; anti-chlamydial antibody was detected by immunoblotting only in infected mice.Several strains of inbred mice are susceptible to human chlamydial biovars and may provide a useful alternative disease model in which to study the immunopathogenesis of ocular chlamydial infection and test of vaccine candidates derived from clinically relevant human biovars.

As shown in infected humans and in animal models of chlamydial infection, the major outer membrane protein (MOMP) of Chlamydia trachomatis is immunogenically potent. The purpose of this investigation was to test in the cynomolgus monkey model of trachoma a new extract of MOMP as a candidate vaccine against ocular chlamydial infection.The nonionic detergent octyl-beta-D glucopyranoside (OGP) was used to extract MOMP from purified C. trachomatis (serovar C) elementary bodies. Protective immunization with OGP-MOMP by mucosal and systemic routes was compared in the cynomolgus monkey model of trachoma. All control and immunized monkeys were challenged by topical application of infectious C. trachomatis to the conjunctivae 35 days after the initiation of immunization.Immunization with OGP-extracted MOMP successfully induced chlamydia-specific local and systemic immunity to MOMP and to whole organism before challenge and early clearance of infection by systemically immunized monkeys. Although ocular disease was not significantly reduced in either immunized group compared to control animals, the lowest clinical and microbiologic disease scores developed in two animals in the mucosal group with the highest immunoglobulin A tear antibody titers at day 0 to 14, whereas higher tear and serum immunoglobulin G correlated with reduced disease in the systemically immunized group.These data demonstrate that despite evidence of vigorous MOMP-specific and other chlamydia-specific serologic and cell-mediated immunity, as well as anamnestic serologic responses to chlamydia, vaccination with OGP-MOMP was only partially protective against chlamydial ocular disease. The partial protection correlated best with tear immunoglobulin A responses after mucosal immunization and with local and systemic immunoglobulin G responses after peripheral immunization, suggesting that alternative chlamydial antigens may have to be considered in future vaccine development to induce more effective protective immunity and that evaluation of efficacy must be appropriate to route of immunization.

Interstitial and bronchointerstitial pulmonary patterns are commonly observed in thoracic radiographs of Thoroughbreds. Prominent interstitial and bronchointerstitial pulmonary patterns are observed in clinically normal horses, and in horses with respiratory tract disease. Until recently, the relevance of these pulmonary patterns was not known. Previous studies indicated that bronchiolitis, bronchiolar epithelial hyperplasia, epithelial metaplasia, and bronchial arteriolar recruitment correlated strongly with the prominence of the interstitial and bronchointerstitial pulmonary patterns observed radiographically. We examined the content and distribution of collagen in the lungs of 7 clinically normal Thoroughbreds in race training. After standardized fixation, lung tissue was treated with a compound that selectively stains collagen. Standard morphometric techniques were used to determine the volume density of parenchymal tissue and parenchymal airspace, mean linear intercept (estimate of alveolar size), alveolar surface area-to-volume ratio, percentage of parenchyma composed of collagen, percentage of airway wall composed of collagen, and airway wall thickness. These values were compared with radiographic and histopathologic scores obtained from the same horses. The volume density of parenchymal tissue and small airway wall thickness correlated strongly with the prominence of the bronchial and bronchointerstitial pulmonary patterns observed radiographically. Small airway thickness was also highly correlated with the perceived prominence of the interstitial pulmonary patterns observed radiographically, and morphometric estimates of parenchymal tissue and parenchymal collagen. There were also strong correlations between the volume density of parenchymal tissue, the percentage of parenchymal collagen, peribronchiolar mononuclear cell infiltrates, and bronchiolar mucosal plication estimates.(ABSTRACT TRUNCATED AT 250 WORDS)

To study the molecular mechanisms that control patterning of the heart tube during early cardiogenesis, we have used the ventricular myosin regulatory light chain (MLC-2v), which is expressed in the ventricular segment of the primitive heart tube, as a genetic marker for ventricular specification in rodents. To assess whether the atrial isoform, MLC-2a, could also serve as a chamber-specific marker, we cloned an atrial MLC-2 cDNA (554 base pairs) which displayed homology to the human MLC-2a cDNA at both the nucleotide (87%) and amino acid (95%) levels. Northern blot, reverse transcriptase-linked polymerase chain reaction, RNase protection, and Western blot analysis revealed atrial restricted expression in the adult mouse heart, very low levels in aorta, and no detectable expression in ventricle, skeletal muscle, uterus, or liver. In situ hybridization studies during mouse embryogenesis revealed cardiac specific expression throughout days 8-16 postcoitum, with atrial restricted expression from day 12 and qualitatively greater atrial expression than ventricular from day 9. Thus, preferential pattern of expression in the atria occurs prior to septation. The MLC-2a gene was differentially regulated when compared with MLC-2v expression during embryonic stem cell cardiogenesis in vitro with MLC-2a transcript levels detectable from day 6 in suspension cultures as compared with day 9 for MLC-2v. The region-specific expression of the MLC-2a and MLC-2v genes in their respective chambers during early cardiogenesis provides genetic markers for chamber specification (atrial and ventricular) in both the in vitro and in vivo context.

Infectious crystalline keratopathy is a distinctive clinical entity characterized by bacterial replication within the corena without inflammation. The authors report on a patient with infectious crystalline keratopathy due to Streptococcus pneumoniae serotype 11F. They used this isolate to study the contribution of the pneumococcal polysaccharide capsule to the pathogenesis of the infectious crystalline keratopathy.Aliquots containing 10(6) colony-forming units of pneumococci serotype 11F, serogroup 9 or 15, were inoculated into New Zealand white rabbit corneas. The corneas were examined at 24, 48, and 72 hours. Representative corneas were excised at 24 hours and processed for histopathologic analysis.Pauci-inflammatory crystalline lesions developed in all corneas inoculated with the serotype 11F ocular isolate by 24 hours. Suppurative keratitis developed in control corneas inoculated with serogroup 9 or 15 pneumococci. The nonocular 11F isolates produced lesions with some features compatible with infectious crystalline keratopathy.Different pneumococcal serotypes vary in their ability to produce infectious crystalline keratopathy. Because serotype differences reflect differences in the antigenic polysaccharides of the bacterial capsule, this study suggests that properties of the pneumococcal capsule may influence the initial development of infectious crystalline keratopathy.

Although still controversial, several studies in humans have suggested That dietary protein restriction may slow the rate of progression of chronic renal failure. Thus, the influence of dietary protein on renal function and progression of renal failure has been the subject of numerous studies in several animal models of chronic renal failure, including rodents, dogs, and baboons. Because of the high incidence of chronic renal failure in aged cats, and the high dietary protein requirements of cats, we studied the effects of dietary protein intake on renal function, proteinuria, and renal morphology in cats with reduced renal mass.Partial (5/6) nephrectomy was performed in 14 young adult female cats. Sham surgical procedures were performed in eight control cats. Control cats and cats with 5/6 nephrectomy were randomly assigned to diets containing either 27.6% (low) or 51.7% (high) protein and studied for 1 year.Renal mass reduction by 5/6 decreased glomerular filtration rate by 2/3 and significantly increased proteinuria. Cats with remnant kidneys had significantly higher systolic and mean blood pressures than control cats. Increased dietary protein/calorie intake significantly increased glomerular filtration rate and proteinuria in all cats. Glomerular filtration rates remained stable in all cats over the year of study. However, high protein/calorie intake resulted in significant renal morphologic injury in remnant kidney cats that was prevented by dietary protein/calorie restriction. Light and electron microscopic glomerular changes in remnant kidney cats fed the high protein diet were similar to changes previously reported in rats and dogs with remnant kidneys.Dietary protein/calorie restriction limits proteinuria and glomerular injury in cats with remnant kidneys in a fashion similar to that reported in rats. However, the remnant kidney model in the cat appeared to be associated with a slower rate of progression compared with kidney model in the cat appeared to be associated with a slower rate of progression compared with rats.

To determine the effects of long-term dietary protein restriction in cats with chronic renal failure (CRF), 4 healthy adult cats and 7 cats with surgically induced CRF were fed a high-protein (HP, 51.7% protein) diet and 4 healthy adult cats and 7 cats with surgically induced CRF were fed a low-protein (LP, 27.6% protein) diet for 1 year. Cats with induced CRF that were fed the LP diet had reduced serum urea nitrogen concentrations, despite lower glomerular filtration rates, compared with cats with CRF fed the HP diet. Despite five-sixths reduction in renal mass, reduced glomerular filtration rate, and azotemia, 13 of the 14 cats with induced CRF retained the ability to concentrate urine and produced urine with a specific gravity1.035. Cats fed the HP diet consumed significantly more calories than did cats fed the LP diet, presumably because the HP diet was more palatable. As a result of the lower caloric intake in cats fed the LP diet, these cats were protein and calorie restricted, compared with cats fed the HP diet. Cats fed the HP diet weighed significantly more than did cats fed the LP diet. Mean hematocrit and mean serum albumin concentration were significantly lower in control cats and in cats with CRF fed the LP diet, compared with control cats and cats with CRF fed the HP diet. Hypokalemia developed in 4 of 7 cats with CRF fed the HP diet (containing 0.3% potassium); hypokalemia did not develop in control cats fed the same diet or in cats with CRF fed the LP diet containing 0.4% potassium. Excessive kaliuresis, hypomagnesemia, and metabolic acidosis did not appear to contribute to the hypokalemia. Subsequent supplementation of the HP diet with potassium gluconate prevented hypokalemia in cats with CRF.

Lameness examinations and radiography of the distal phalanx and associated soft-tissue structures of the front feet of 103 Thoroughbred racehorses, 4 to 9 years old, were performed to determine normal radiographic appearance and morphometry. Of 103 horses examined, 41 were used in the study that were without clinical signs of foot problems or lameness, had raced at least twice prior to radiography, and had raced at least twice more in the 6 months after radiography. Lateromedial and dorsoproximal-palmarodistal radiographic views of each front distal phalanx were used to measure 28 bone and soft-tissue structures, and to evaluate 14 radiographic findings. Significant differences were not observed between left and right digits for any radiographic determination. Mean thickness of the soft tissues dorsal to the distal phalanx, which provides an evaluation of the epidermal laminae, was 14.6 +/- 1.0 mm when measured adjacent to the distal aspect of the distal phalanx. Most horses had straight, smooth hoof walls that were parallel to the dorsal cortex of the distal phalanx. The mean degree of palmar rotation of the distal phalanx was -0.5 +/- 1.3, and none was rotated more than 4 degrees. The dorsal cortex was smooth and straight, without bone deposition or reaction in either digit for only 5 of the 41 horses. Active bone formation was seen unilaterally along the middle portion of the dorsal cortex in 7 horses, and along the distal portion of the dorsal cortex in 4 of the phalanges from 3 horses. New bone formation along the distal dorsal cortex was often accompanied by resorption of the palmar cortex. For 26 of the 31 horses without active bone deposition, smooth inactive bone formation along the midportion of the dorsal cortex was identified in 1 or both distal phalanges. Bone at the solar margin of the distal phalanx was uniformly dense and finely trabeculated, without evidence of resorption or fractures. Severe irregularity of the solar margin was not found in any digit, and the margin of both phalanges was smooth in 8 horses. Various degrees of solar margin irregularity were observed in the other 33 horses. The mean number of vascular canals within the distal phalanx was 8.4 +/- 1.7, and the diameter of the largest canal was 3.4 +/- 0.6 mm.(ABSTRACT TRUNCATED AT 400 WORDS)

The N-terminus of a mutant form of apolipoprotein AI [apoAI] has previously been shown to be the subunit protein of amyloid fibrils in a human kindred with a form of familial amyloid polyneuropathy (FAP, type III) and in a recently reported kindred with a form of non-neuropathic hereditary amyloidosis. In this study, we demonstrate by amino-acid sequence analysis, that a form of vascular amyloidosis occurring in the lungs of aged dogs is derived from a N-terminal fragment of apoAI and that no amino acid substitution is present in this confirmed sequence. This represents the first documentation of apoAI as a precursor for a form of amyloidosis in animals, and provides the first documentation of apoAI as a precursor for amyloid fibrils in a form of age-associated ("senile") amyloidosis. Secondary structure prediction analysis of the N-terminal regions of normal human and dog apoAI indicated a propensity for beta-pleated sheet conformation, and thus amyloidogenesis, in 40 and 45% of the respective sequences. These results suggest that apoAI (like transthyretin) may serve as an amyloid precursor protein for both familial and senile forms of amyloidosis. ApoAI should, therefore, be considered as a potential amyloid precursor when forms of human senile amyloidosis of unknown origin are evaluated.

The source of a previously described radiolucent crescent in the flexor cortex of the distal sesamoid (navicular) bone on the palmaro45 degrees proximal-palmarodistal oblique (Pa45 degrees Pr-PaDio) clinical radiographic projection was investigated in 48 forelimb navicular bones from 24 Thoroughbreds by use of high-detail radiography and x-ray computed tomography (CT). Twenty-five of these bones also were evaluated, using microradiography and histologic examinations. Of these 25 bones, 5 had been labeled in vivo with fluorochrome markers. Tetrachrome-stained 100-microns-thick nondecalcified sections of these 5 bones were examined, using epifluorescence microscopy. A reinforcement line of compacted cancellous bone, parallel and several millimeters deep to the flexor cortex in the region of the flexor central eminence, was visualized by CT in 42 of 48 navicular bones and by microradiography in 23 of 25 navicular bones investigated. Variable degrees of compaction were observed in the cancellous bone between the flexor cortex and the reinforcement line. High-detail skyline radiographic projections and reconstructed CT images indicated a crescent-shaped lucency within the flexor central eminence of the flexor cortex in the bones in which the reinforcement line was identified, but the cancellous bone between the reinforcement line and the flexor cortex had not been compacted. The radiolucent crescent seen in the flexor central eminence of the navicular bone on the Pa45 degrees Pr-PaDiO projection was not caused by the concave defect or synovial fossa of the flexor central eminence overlying the flexor cortex, as was described.(ABSTRACT TRUNCATED AT 250 WORDS)

cyr61 is a growth factor-inducible immediate early gene initially identified in serum-stimulated mouse fibroblasts. It encodes a member of an emerging family of cysteine-rich secreted proteins that includes a connective tissue growth factor. We show here that cyr61 is expressed in the developing mouse embryo and extraembryonic tissues. In the placenta, cyr61 is expressed in regions of trophoblastic origin, including the ectoplacental cone and the trophoblastic giant cells. In the midgestation embryo, cyr61 is expressed in the smooth muscle vessel walls of the arterial circulatory system. Most notably, expression is found in developing cartilaginous elements, including the limbs, ribs, and prevertebrae. In addition, regions of the chondrocranium and craniofacial elements, such as Meckel's cartilage, also express cyr61. Thus, cyr61 transcript is found in mesenchymal cells of both mesodermal and ectodermal origin during their differentiation into chondrocytes. The temporal and spatial regulation of cyr61 expression and the biochemical features of its encoded protein suggest that cyr61 may be important for the normal growth, differentiation, or morphogenesis of the cartilaginous skeleton of the embryo.

Amyloid deposits characteristically associated with pancreatic islets of those species (e.g., humans, cats, and monkeys) that develop age-associated forms of diabetes have been shown to represent a concentrated and polymerized form of a previously unknown islet-derived protein identified either as IAPP or amylin. IAPP, a highly conserved and carboxy-terminally amidated 37 amino acid polypeptide with approximately 45% amino acid sequence identity to CGRP, is produced by islet beta cells and is cosecreted with insulin in response to glucose and other secretagogues. Prepro-IAPP is synthesized in beta cells as an 89 to 93 amino acid molecule, and mature IAPP appears to be formed by enzymatic processing similar to that involved in the formation of insulin. Glucose-stimulated IAPP secretion generally parallels that of insulin and, on a molar basis, IAPP represents about 1% of the amount of insulin secreted. A significant dissociation of IAPP and insulin secretion (associated with relatively greater upregulation of IAPP secretion) is observed in response to marked hyperglycemia, suggesting that IAPP and insulin expression are differentially regulated. The amyloidogenicity of IAPP in only a very limited number of species is importantly related to the amino acid residues inherently found in the 20-29 region of IAPP from those species. The 25-28 region of human and cat IAPP is identical in structure and appears to be the most important amyloidogenic sequence common to the human and cat. In vitro fibrillogenesis studies have shown that amino acid substitutions in this region especially affect the amyloidogenicity of IAPP. Studies in dogs and cats suggest that aberrations in beta cell synthesis (or processing) of IAPP may lead to an increased concentration of IAPP in the local milieu, thus providing a second prerequisite for the self aggregation of IAPP to form islet amyloid. IAPP has been implicated to have physiological roles in glucose regulation, hemodynamics, calcium homeostasis, and as an anorectic agent. The major current interest in IAPP concerns its potential relationships to glucose metabolism and the development of type 2 diabetes. Evidence has been provided which indicates that IAPP can inhibit glucose-stimulated insulin secretion by beta cells, and that IAPP can also potentially contribute to the pathogenesis of type 2 diabetes by increasing hepatic glucose output and by inducing peripheral insulin resistance.(ABSTRACT TRUNCATED AT 400 WORDS)

Destructive lesions of the axial region of the proximal sesamoid bones were identified by radiography in eight fetlocks and seven lame adult horses. Lameness ranged from 2 to 5 (mean 4; scale 1 to 5) at the time of examination, with a duration of 10 days to two years (mean 5.6 months). Destructive lesions involved both proximal sesamoid bones when examined radiographically and were situated primarily at the level of the mid-body and apical region of the axial borders. Some lesions were cystic, whereas others eroded the axial border more diffusely. Scintigraphy revealed markedly increased activity within the proximal sesamoid bones of the clinically lame limb of four of the five horses examined. In four horses, post mortem computed tomography revealed axial border bone destruction and cavitary lesions within cancellous bone of affected proximal sesamoid bones. Lesions seen by computed tomography were larger than those identified on radiographic examination. Cavitary lesions not seen radiographically were identified in the proximal sesamoid bones of two clinically unaffected fetlocks examined for comparison in two of the seven horses. Evidence of acute, subacute or chronic/reparative osteomyelitis of the axial region of the proximal sesamoid bones was seen in the 10 fetlocks identified as abnormal from radiography or computed tomography. Also, three horses had septic synovitis of the flexor sheath of the clinically affected limb; of these, two had septic arthritis of the fetlock joint.

Clinical records and thoracic radiographs of 19 horses with a confirmed pathologic diagnosis of silicosis were reviewed. These horses had histories of varying degrees of chronic weight loss, exercise intolerance, and respiratory distress. At the time of presentation, two horses were asymptomatic. Ten horses were geldings and nine were female. The mean age of the 19 horses was 10.7 +/- 5.5 years. Fourteen horses were identified as being from the Monterey-Carmel Peninsula of midcoastal California. An abnormal, structured interstitial pulmonary pattern was identified on thoracic radiographs in each horse. The interstitial pulmonary changes were classified as miliary (13 horses), reticulonodular (4), or linear interstitial (2), and were best visualized dorsally and caudodorsally. In addition to the abnormal interstitial pulmonary pattern, areas of pulmonary consolidation were evident caudodorsally in seven horses. Other thoracic radiographic features included: hilar lymphadenopathy (4 horses), pleural effusion/thickening (4), cranial mediastinal lymphadenopathy (2), hyperinflation (1), and a discrete pulmonary mass (1). Necropsy findings in eight horses and results of lung biopsies in an additional five horses showed a diffuse, multifocal, granulomatous pneumonia with areas of pulmonary fibrosis. Cellular infiltrates included predominantly macrophages with intracellular and/or extracellular crystalline material, occasional lymphocytes, and giant cells. Similar cellular changes were also identified, during necropsy, in the hilar and tracheobronchial lymph nodes in each of the eight horses, although gross enlargement of the lymph nodes was present in only six horses. The radiographic and pathologic findings of these 19 horses are consistent with chronic or the accelerated forms of silicosis that are recognized in humans.

Squamous cell carcinoma of the hoof wall, with resultant invasion of the right hind distal phalanx, was identified in a 15-year-old Thoroughbred stallion. The clinical features included a chronic grade 2/5 right hind limb lameness and a sessile dorsal hoof wall mass that was not sensitive to palpation. Radiography revealed a well-circumscribed circular lucency within the distal phalanx, beneath the clinically noticed hoof wall mass. These features were considered to be characteristic of a hoof wall keratoma. Surgical intervention was done 10 months later. The histologic diagnosis at the time of surgery was squamous cell carcinoma. Eight months after surgery, progressive tumor invasion of the distal phalanx resulted in a pathologic articular fracture. This case highlights the need for accurate histologic diagnosis of equine hoof wall masses to differentiate between benign and malignant conditions.

Mammalian mitochondrial ribosomes possess a binding site for guanine nucleotides. GTP binds in unit stoichiometry and with high affinity (Kd = 15.3 +/- 2.8 nM) to the small subunit of bovine mitochondrial ribosomes. This binding activity survives high salt washes, indicating that the nucleotide binds to an integral site within this subunit. GDP also binds to the small subunit with high affinity (Kd = 17 +/- 5.8 nm) and in unit stoichiometry. The GTP binding activity can be competed with GDP but not appreciably by other nucleotides, indicating that both GTP and GDP bind specifically and to the same site. The non-hydrolyzable analogs of GTP, guanylyl-5'-imidophosphate, and guanylyl-(beta,gamma-methylene)- diphosphonate also bind to the small subunit, but with reduced affinity. These results indicate that mammalian mitochondrial ribosomes, unlike other ribosomes, are able to interact directly with guanosine triphosphate, suggesting that the bound GTP may be involved in a novel regulatory mechanism in mitochondrial protein synthesis.