Osteoinductive squamous cell carcinoma associated with a putative novel papillomavirus on the digit of a cat.

CASE HISTORY AND CLINICAL FINDINGS: An approximately 10-year-old, castrated male domestic short-haired cat developed swelling and ulceration of the second digit of the right front paw. Radiographs revealed a spherical soft tissue swelling with irregular distal margins that contained multiple lacy mineral opacities. The digit was amputated and submitted for histology. No recurrence has been observed 7 months after amputation. PATHOLOGICAL AND MOLECULAR FINDINGS: Histology revealed a moderately well-circumscribed proliferation of well-differentiated squamous cells arranged in trabeculae and nests. Numerous thin spicules of osseous metaplasia were visible throughout the neoplasm. Around 70% of the neoplastic cells contained papillomavirus-induced cell changes including large amphophilic cytoplasmic bodies and cells with shrunken nuclei surrounded by a clear halo. Intense p16CDKN2A protein immunostaining was visible within the neoplastic cells, suggesting papillomavirus-induced changes in cell regulation. A DNA sequence from a putative novel Taupapillomavirus type was amplified from the neoplasm. DIAGNOSIS: Osteoinductive squamous cell carcinoma associated with a putative novel papillomavirus type. CLINICAL RELEVANCE: The findings in this case increase the number of papillomavirus types known to infect cats, and the squamous cell carcinoma had histological features that have not been previously reported. The neoplasm was not as invasive as is typical for a squamous cell carcinoma and excision appeared curative. This is the first report of an osteoinductive squamous cell carcinoma of the skin of cats and the neoplasm had a unique radiographic appearance.

Cardiac morphology of North Island brown kiwi (Apteryx mantelli).

AIMS: To investigate the cardiac anatomy of North Island brown kiwi (Apteryx mantelli) through heart morphometric parameters measured at post-mortem examination. METHODS: Morphometric cardiac parameters were established at post-mortem examination of 20 North Island brown kiwi. Birds were classified by gender and age (chicks vs. adults). Measurements included: body mass, heart mass, sternal length, midpoint thickness of left ventricular free wall, midpoint thickness of right ventricular free wall and ratios of heart mass to body mass, left ventricular length to sternal length, right ventricular length to sternal length, length of left ventricle to right ventricle, interventricular septal thickness relative to the sternal length and interventricular septal thickness relative to the left ventricular length. Unadjusted estimates of the median difference and their 95% CI were then reported at each age and sex for all the cardiac morphometric parameters and their ratios. RESULTS: The small sample size led to wide 95% CI for the median difference between gender and age for the cardiac morphometric measurements. Nevertheless, between adult female and male kiwi, the estimated population median differences for heart mass (2.2 (95% CI = -2.9-5.6) g), length (1.2 (95% CI = -2.2-5.6) mm), width (6.1 (95% CI = -1.0-8.2) mm), left ventricular free wall length (5.5 (95% CI = -0.5-8.8) mm) and right ventricular free wall length (2.6 (95% CI = -3.7-6.9) mm) were established. In adult North Island brown kiwi, the heart mass is 0.8 (95% CI = 0.7-0.8)% of the body mass. CONCLUSIONS: The precision of the differences noted in heart measurements recorded between male and female kiwi at each age was limited by the low sample size available for this study. This led to wide CI and an inability to adjust differences observed for gender by differences in other confounders such as body size. With this caveat, there is weak evidence that adult female kiwi have a larger heart size and mass than the adult males. CLINICAL RELEVANCE: These results can be used to improve the diagnosis of cardiac disease in kiwi at post-mortem examination and aid in interpretation of the results of echocardiography in live birds for the antemortem diagnosis of cardiac disorders.

Effects of novel 5-lipoxygenase inhibitors on the incidence of pulmonary adenomas in the A/J murine model when administered via nose-only inhalation.

The objective of this study was to determine the effects of 5-lipoxygenase (5-LO) inhibitors on the incidence of benzo(a)pyrene-induced pulmonary adenomas in female A/J mice. Two novel compounds, S-29606 and S-30621, and the Food and Drug Administration-approved Zileuton were investigated. S-29606 and S-30621 were selected from a group of similar active structures on the basis of local versus systemic 5-LO inhibitory activity. Preliminary studies found them to lack oral bioavailability, in direct contrast to Zileuton. Treatment was initiated 1 week following exposure to the carcinogen benzo(a)pyrene. Both S-29606 and S-30621 were dosed via nose-only inhalation 5 days a week, for 16 weeks, whereas Zileuton was administered orally. Dose levels for S-29606 and S-30621 were determined to be 220 and 430 microg/kg for the low- and high-dose groups, respectively, whereas the dose of Zileuton was 245 mg/kg. Both test compounds exhibited a significant reduction of pulmonary adenomas, compared with a positive control for high and low doses, P < 0.05. Additionally, a dose response for both S-29606 and S-30621 was observed when compared with placebo. Despite a dose 575 times greater than that of the novel test compounds, orally administered Zileuton did not produce a reduction in adenoma occurrence. The findings of this study offer compelling preliminary data for the use of S-29606 and S-30621 in further investigations of the treatment of pulmonary adenomas and support the use of inhalation drug delivery as an alternate to oral delivery for these compounds.

MLYCD mutation analysis: evidence for protein mistargeting as a cause of MLYCD deficiency.

Malonyl-CoA decarboxylase (MLYCD) deficiency is an autosomal recessive disorder characterized by malonic aciduria, developmental delay, seizure disorder, hypoglycemia, and cardiomyopathy. Genomic sequencing of MLYCD in nine unrelated patients identified 16 of 18 pathogenic alleles, which are documented in the newly created Human MLYCD Allelic Variant Database (http://mlycd.hgu.mrc.ac.uk/). Fibroblast cell lines were available from eight of these patients and two previously reported patients with homozygous MLYCD mutations. Western blot analysis using antisera raised to a C-terminal peptide detected a 66-kDa band that was absent in six patients and substantially reduced in three patients. One patient showed an increase in protein levels with a prominent smeary 68-l83-kDa band. Immunocytochemical analysis of MLYCD-expressing patient cell lines showed apparent intracellular mislocalization. An extreme N-terminal mutation c.8G>A (p.G3D) mislocalized to the plasma membrane, suggesting that a novel targeting signal may reside in a four-amino acid conserved N-terminal motif. A 25-base deletion between the putative mitochondrial and peroxisomal initiating codons (M1 and M40) and a point mutation ablating the second of these (c.119T>C, p.M40T) both showed punctate perinuclear staining. As none of the three mislocalizing mutations are predicted to alter the catalytic function of the peptide, it seems likely that correct subcellular localization of MLYCD is critical for it to function normally.

cDNA cloning, genomic organization, and chromosomal localization of a novel human gene that encodes a kinesin-related protein highly similar to mouse Kif3C.

We report the cloning and characterization of a novel human kinesin-like gene with strong homology to the mouse kinesin Kif3c. The full-length cDNA contains an open reading frame of 2382 nucleotides encoding a predicted 793 amino acid peptide that includes a 389 amino acid motor domain conserved among other kinesins. PCR and DNA sequence analysis of PAC clones containing the human KIF3C sequence revealed that the gene contains 8 exons. All introns have the conserved GT and AG dinucleotides present at their donor and acceptor sites, respectively. We have localized KIF3C to chromosome band 2p23 by fluorescence in situ hybridization.

Isolation and characterization of a putative collagen gene from the potato cyst nematode Globodera pallida.

A cDNA clone encoding a full length putative collagen has been isolated in a screen of a mixed stage Globodera pallida expression library. Comparison of the deduced amino acid sequence of this molecule with other collagens suggests it is a cuticular collagen and a member of the col-8 subfamily of collagen genes. Northern blots show the gene is expressed specifically in gravid, adult females of the parasite as compared to second (invasive) stage juveniles and virgin females. Preliminary immunocytochemical studies indicate this collagen is present in areas other than the cuticle; these findings and the potential functional role of this collagen are discussed.

Analysis of production brewing strains of yeast by DNA fingerprinting.

Production brewing strains of the yeast Saccharomyces cerevisiae were analysed by DNA fingerprinting, using a Southern blotting and hybridization procedure and employing the Ty1-15 transposon as a probe. The ability to differentiate readily between strains was very dependent on the restriction enzyme used to digest the DNA prior to Southern blotting and hybridization; the enzymes EcoRI, PstI and SalI were found to be particularly useful in this respect. The method was applicable to the differentiation of both ale and lager yeasts, and was sufficiently sensitive to distinguish between very closely related strains. DNA fingerprinting by this approach confirmed, for example, that a flocculent strain isolated during a production-scale fermentation with a lager yeast was genotypically different from the parent.

Heterogeneity of alpha 1-acid glycoprotein in rheumatoid arthritis.

alpha 1-Acid glycoprotein (AGP) or orosomucoid is a major serum glycoprotein, of unknown physiological function, which is classified as one of the positive acute phase reactants since its plasma concentration becomes elevated two- to five-fold in certain disease states. Additionally, the proportions and identities of the five asparaginyl-linked complex oligosaccharide chains are altered during several physiological and pathological conditions, which may be functionally significant. The key to studying the structural heterogeneity of AGP is to develop a procedure that will isolate AGP without structural degradation. We have developed a method for the purification of AGP, using procedures unlikely to damage the glycoprotein structure, which was utilised to isolate AGP from samples of normal and rheumatoid plasma. The effectiveness of the purification procedure was examined by enzymatically deglycosylating each sample of AGP and separating the released oligosaccharides by chromatography on a pellicular high-performance anion-exchange (HPAE) resin at pH 13. The analytical profile for normal AGP was consistent with that previously reported thus indicating that the purification procedure did not denature the oligosaccharide chains of AGP. Additionally, there was a noticeable difference between the profiles for AGP from normal and rheumatoid plasma.

Regulation of phosphatidylinositol breakdown and leukotriene synthesis by endogenous prostaglandins in resident mouse peritoneal macrophages.

Mouse peritoneal macrophages synthesize large amounts of prostaglandins and leukotrienes in response to certain inflammatory stimuli. Lipopolysaccharide and phorbol esters stimulate prostaglandin formation but not leukotriene synthesis. Zymosan and the calcium ionophore, A23187, stimulate the formation of both prostaglandins and leukotrienes, as well as the phospholipase C-catalyzed breakdown of phosphoinositides. We have examined the interrelationships among phosphoinositide breakdown and prostaglandin and leukotriene synthesis in resident mouse peritoneal macrophages. We demonstrate that macrophages synthesize basally prostaglandin (PG)E2 and PGI2 and that these products begin to accumulate from the time of initial plating of the macrophages. The presence of these prostaglandins imparts a downward modulation of zymosan-stimulated phosphoinositide breakdown and, as a result, a downward modulation on leukotriene formation. Inhibition of the basal release of prostaglandin by indomethacin resulted in enhanced zymosan-stimulated phosphoinositide breakdown and an exactly corresponding enhancement of leukotriene release. This enhancement, resulting from the inclusion of indomethacin at the time of plating, was reversed by also including PGE2, PGI2, or dibutyryl cAMP. Dibutyryl cAMP, when added in the presence of zymosan and in the absence of indomethacin treatment, inhibited phosphoinositide breakdown and leukotriene synthesis in a parallel fashion, with no effect on prostaglandin release. These data demonstrate that phospholipase C activation is regulated in part by prostaglandin tone and that leukotriene synthesis, unlike prostaglandin synthesis, is dependent on phosphoinositide breakdown.

Crystallization and characterization of two crystal forms of the B800-850 light-harvesting complex from Rhodopseudomonas acidophila strain 10050.

Two different crystal forms of the B800-850-antenna complex from Rhodopseudomonas acidophila strain 10050 have been grown. This complex is an integral membrane protein and is isolated as an oligomeric assembly with a molecular weight of approximately 84 kDa. This assembly contains six alpha/beta apoprotein pairs, 18 molecules of bacteriochlorophyll a and nine molecules of carotenoid. The first crystal form has dimensions unit cell a = b = 75.8 A, c = 97.5 A with the space group P4 and diffracts to a resolution of 12.0 A. The second crystal form is rhombohedral with dimensions unit cell a = 121.1 A, alpha = 60 degrees, space group R32 and diffracts to a resolution of 3.5 A. Native data have been processes in both cases, to an Rmerge value of 9.0 to 11.0%. The X-ray data suggest that the asymmetric unit, in both crystal forms, contains one 84 kDa antenna complex.

Accumulation of lysophosphatidylinositol in RAW 264.7 macrophage tumor cells stimulated by lipid A precursors.

N2,O3-Diacylglucosamine 1-phosphate (lipid X), a monosaccharide precursor of Escherichia coli lipid A, was used to stimulate RAW 264.7 macrophage tumor cells, and the effects on macrophage phospholipid metabolism were examined. The addition of E. coli lipid X to the medium of cells that had been uniformly labeled with 32Pi resulted in a 4-8-fold increase in the level of lysophosphatidylinositol. This effect was maximal at 5 microM lipid X. Lysophosphatidylinositol levels reached a maximum 45 min after stimulation, followed by a gradual decline to near normal levels within 2 h. The formation of lysophosphatidylinositol was dependent upon extracellular calcium and was almost completely inhibited when cycloheximide was added at the time of stimulation. The addition of the disaccharide lipid A precursor IVA, commercial lipopolysaccharide (1 microgram/ml), phorbol 12-myristate 13-acetate (10(-7) M), or calcium ionophore A23187 (10(-6) M) to these cells resulted in a similar increase in lysophosphatidylinositol levels, but phosphatidic acid was inactive. The stimulation by IVA and phorbol myristate acetate was blocked by cycloheximide, but the stimulation by lipopolysaccharide was only partially blocked. The stimulation by A23187 was unaffected by cycloheximide. The increase in lysophosphatidylinositol levels might be related to the stimulation of arachidonate release and prostaglandin synthesis that is also observed in cells treated with lipid A precursors. The disaccharide precursor, IVA, was at least 100 times more effective than lipid X at stimulating lysophosphatidylinositol formation and prostaglandin release. The relative ability of lipid X and IVA to stimulate these cells correlated well with their effects on other lipopolysaccharide-responsive systems. Macrophage tumor cells also had the ability to inactivate lipid X by dephosphorylating it.

Uteroglobin inhibits phospholipase A2 activity.

Although progesterone is known to produce quiescence in the mammalian uterus, the mechanism of this effect is not clearly understood. Here, we report that uteroglobin, a progesterone-induced small molecular weight (16K) protein, inhibits phospholipase A2(PLA2) derived from porcine pancreas as well as from the RAW 264.7 macrophage cell line. We speculate that progesterone may exert its antimotility effects on the uterus via uteroglobin which, by inhibiting PLA2, decreases arachidonic acid release and subsequently reduces prostaglandin levels in this organ. This may explain why progesterone is so vital for the maintenance of pregnancy in almost all mammals.

The activation of protein kinase C by biologically active lipid moieties of lipopolysaccharide.

The monosaccharide lipid A precursor, N2,O3-diacylglucosamine 1-phosphate (Escherichia coli lipid X), has been shown previously to be a potent B-lymphocyte mitogen. We now report that lipid X interacts with macrophages, stimulating turnover of phosphatidylinositol, deacylation of phospholipids, and release of arachidonic acid. In addition, the monosaccharide lipid X, the incomplete lipid A disaccharides found in KDO-deficient mutants, and crude free lipid A by itself activate protein kinase C isolated from RAW 264.7 macrophages. This activation is augmented by diglyceride, a product of phosphatidylinositol turnover. Like the lipid X-induced mitogenesis of B-lymphocytes, lipid X activation of macrophages and the cell-free activation of protein kinase by lipid X require the presence of the O-linked hydroxymyristoyl residue at position 3. We suggest, therefore, that some of the biological effects of lipid A may be mediated by its interaction with protein kinase C.

Inhibition of the release of prostaglandins, leukotrienes and lysosomal acid hydrolases from macrophages by selective inhibitors of lecithin biosynthesis.

The release of the inflammatory mediators, prostaglandins (PGs), leukotrienes (LT) and lysosomal acid hydrolases (LAH), by macrophages is stimulated by endocytic stimuli such as zymosan. This process can be interfered with by specific inhibitors of phosphatidylcholine (PC) biosynthesis. The diphenylsulfone dapsone and three analogs selectively inhibited [14C]choline incorporation into PC but had varied effects on inhibition of mediator release by macrophages. Dapsone inhibited the release of PGs, LT and LAH, whereas the three closely related structural analogs inhibited LAH release only, with little or no effect on PG production.

Protein kinase activation of phospholipase A2 in sonicates of mouse peritoneal macrophages.

Mouse peritoneal macrophages have a phospholipase A2 activity which is optimally active at pH 8.5 (PLA8.5), requires 2 mM Ca2+ and is capable of hydrolyzing arachidonic acid from phosphatidylcholine and phosphatidylethanolamine. The specific activity of PLA8.5 can be greatly increased in macrophage sonicates by their incubation at 37 degrees C. This augmentation of PLA8.5 activity occurs maximally at pH 7.5, requires Ca2+, and is inhibited by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid and EDTA. The sulfhydryl-specific reagents N-ethylmaleimide and p-hydroxymercuribenzoate inhibit PLA8.5 activation but have no effect on the fully activated PLA8.5 enzyme itself. PLA8.5 activation is also augmented by ATP and is inhibited by pretreatment of the sonicates with ATPase and by beta-gamma-methylene ATP. The addition of the catalytic subunit of bovine heart cAMP-dependent protein kinase to macrophage sonicates in the presence of 1 mM reduced glutathione augments PLA8.5 activation. These data suggest that a protein kinase may be involved in the activation of PLA8.5 in mouse macrophage sonicates.

Lysosomotropic agents. 4. Carbobenzoxyglycylphenylalanyl, a new protease-sensitive masking group for introduction into cells.

Bioactive primary and secondary amines, when acylated with the Z-Gly-Phe group, are transported into pinocytic cells, such as macrophages, P-815 mastocytoma, SV-40 3T3, and leukemia 1210, much faster than the parent compounds. Amines such as lysosomotropic detergents [R. A. Firestone, J. M. Pisano, and R. J. Bonney, J. Med. Chem., 22, 1130 (1979) and nitrogen mustard, which are deactivated by acylation, are unmasked by enzymic action intracellularly, probably in lysosomes because an acidic pH maximum in activity exists which acts only on the L isomer. The added polarity and molecular weight brought about by acylation prevents the amines' normally facile entry into cells by simple diffusion, restricting it to an active-transport mechanism.

The selective release of phospholipase A2 by resident mouse peritoneal macrophages.

Resident mouse peritoneal macrophages have three phospholipase activities: a phospholipase A2 active at pH 4.5, a Ca2+-dependent phospholipase A2 active at pH 8.5 and a phosphatidylinositol-specific phospholipase C activity. When macrophages are exposed to zymosan in culture, the cellular activity of pH-4.5 phospholipase A2 is diminished in a manner dependent on zymosan concentration and time of exposure, whereas the cellular activities of pH-8.5 phospholipase A2 and phospholipase C remain unchanged. The depletion of pH-4.5 phospholipase A2 activity from the cell is paralleled by a quantitative recovery of this activity in the culture medium in a manner similar to the cellular depletion and extracellular recovery of two lysosomal enzymes. This release is specifically elicited by an inflammatory substance such as zymosan, since macrophages incubated with 6 micrometer latex spheres retain pH-4.5 phospholipase A2 activity and lysosomal enzyme activities intracellularly.

Identification and characterization of a phospholipase C activity in resident mouse peritoneal macrophages. Inhibition of the enzyme by phenothiazines.

Resident mouse peritoneal macrophages contain a phospholipase C of high activity that is specific for phosphatidylinositol. The activity has a neutral pH optimum, is Ca(2+)-dependent and has a maximum reaction velocity of 525nmol/h per mg of protein. Certain phenothiazines are potent inhibitors of this activity.

[Identification and characterization of two phospholipase A2 activities in resident mouse peritoneal macrophages].

Resident mouse peritoneal macrophages synthesize and release large amounts of prostaglandins in response to inflammatory stimuli. Release of prostaglandin E2 and 6-oxoprostaglandin F1 alpha occurs at a rate of 1 nmol/h per mg of cell protein. The mechanisms by which substrate arachidonic acid is released have yet to be established. We have therefore initiated studies to characterize those enzymes that can catalyse its release from phospholipid and may be of significance at the cellular level. We report initially the characterization of two phospholipase A2 activities in homogenates of mouse peritoneal macrophages. The first is active at pH 4.5 and is not dependent on Ca2+. The second is Ca2+-dependent and is optimally active at pH 8.5. Either phospholipase A2 activity is capable of hydrolysing [14C] arachidonic acid from [14C] arachidonic acid-labelled phospholipids in quantities sufficient to account for the amounts of prostaglandins by macrophages in culture. Phospholipid substrates are prepared from mouse LM fibroblasts in serum-free Higuchi medium containing radiolabelled phospholipid precursors. Single-labelled phospholipids bear the 14C label in the arachidonic acid moiety. Dual-labelled phospholipids bear a 14C label in the polar head group and a 3H label in the arachidonic acid moiety. Experiments with dual-labelled substrates establish that both phospholipase activities are of the A2 type as indicated by the equimolar recovery of [3H] arachidonic acid and [14C] lysophospholipid. Studies with aqueous sonicated dispersions of purified [14C] arachidonic acid-labelled phospholipid or mixed liposomal substrates formed from mixtures of cellular polar lipids reveal that the pH 4.5 activity hydrolyses phosphatidylethanolamine and phosphatidylcholine more efficiently when they are present in a mixture of other polar lipids. The pH 8.5 activity, however, hydrolyses the purified phospholipids more efficiently.