Adequately Prepared? A Study Using an Innovative Computer Application to Measure Clinical Crown Convergence Angles Achieved by Students at a UK Dental School.

This study examined the total occlusal convergence angles created for full coverage crown preparations by students at a UK dental school. Working casts of 82 clinical crown preparations were scanned using a 3D scanner. Stereolithographic files were uploaded to Preppr, a crown preparation analysis application. Mean bucco-lingual convergence angle were 19.6° (+/-11.7) and mesial-distally 17.8° (+/-11.1). Smallest bucco-lingual convergence angles were achieved for canine teeth with the largest on molar teeth. The smallest mesio-distal values were on canine teeth with the largest on molar teeth. Ideal total convergence angles (4-14°) were achieved in 23% of bucco-lingual preparations and 33% of mesio-distal preparation. Results for clinically acceptable angles (10-20°) were 30% and 40% respectively. There were no statistically significant differences between tooth types for mean bucco-lingual values. (p=0.623), mesio-distal mean values were statistically different by tooth type (p=0.003). Mean values for mandibular molars were significantly higher than for maxillary incisors (p=0.001) and mandibular molars had significantly higher values than maxillary canines (p=0.045). Results in this study were comparable to those of other students and qualified clinicians, with a minority of preparation achieving ideal values.

First Report of Pepper chat fruit viroid in Traded Tomato Seed, an Interception by Australian Biosecurity.

Pepper chat fruit viroid (PCFVd), a species of Pospiviroid, was first discovered in a capsicum crop in the Netherlands in 2006 (4) and was then reported only in Thailand (2) and Canada. The mechanism of international spread was not known, but movement with traded seed was suspected. PCFVd is transmissible through capsicum seed (4) and very probably through tomato seed, like other pospiviroids. The viroid causes disease in capsicum and tomato and experiments by others indicate a capacity to cause disease in potato. It poses a biosecurity threat to crops internationally. PCFVd was intercepted by the Australian Government Department of Agriculture, Fisheries, and Forestry (DAFF) in five shipments of tomato seed (Solanum lycopersicum) exported from Israel and Thailand in September and October 2012. Batches of up to 20,000 seeds were sampled from each seed lot in a shipment and total nucleic acids were extracted from sub-samples, each of about 400 seeds, following a method similar to Hoshino et al. (1). PCFVd was initially detected when reverse transcription PCR using the generic pospiviroid primers Pospi1-FW and Pospi1-RE (3) produced amplicons of 189 bp, which were then sequenced. The PCFVd specific primers AP FW1 and AP RE2 (4) were used to amplify the remainder of the viroid genome, which was directly sequenced. Overlapping sequences were aligned to produce complete sequences of 349 bases, one from seed from Thailand and two from seed from Israel (GenBank: KC762952, KC762953, KC762954). Searches of the GenBank nucleotide non-redundant database indicated close matches with sequences from PCFVd isolates from tomato in Thailand (2); alignments generated by BLAST showed the sequences differed from those from Thailand at only 2 to 18 nucleotide positions, equating to 95 to 99% identity. PCFVd sequences from seed from Thailand were almost identical (>99%) to the sequences from seed from Israel. Many sub-samples were negative, indicating that the number of contaminated seeds was very small in some shipments. The positive sub-samples as a proportion of the total number of sub-samples tested from the five shipments was 1/1, 1/5, 1/1, 12/50, and 7/50. Tomato and capsicum seed are produced in many countries and often traded through second countries. The infected tomato seed shipments intercepted by DAFF were destroyed or re-exported following Australian regulations. Other countries were informed through the International Plant Protection Convention. This pest viroid has not been intercepted by Australian authorities before and has not been detected in recent Australian survey work (data not shown). References: (1) S. Hoshino et al. Res. Bull. Plant Prot. Japan 42:75, 2006. (2) K. Reanwarakorn et al. New Dis. Rep. 24:6, 2011 (3) J. Th. J. Verhoeven et al. EJPP 110:823, 2004. (4) J. Th. J. Verhoeven et al. Virus Res. 144:209, 2009.

Brucellosis associated with stillbirth in a bottlenose dolphin in Australia.

A captive adult female bottlenose dolphin presented with stillbirth. The placenta appeared oedematous. No other gross lesions were evident in the placenta or the stillborn calf. Histopathology revealed mild multifocal placentitis and foetal encephalitis. Brucella sp. was isolated from lung, liver, spleen and kidney. Sequence and phylogenetic analysis demonstrated this organism to be most similar to Brucella ceti sequence type (ST) 27. Brucella sp. DNA was detected in formalin-fixed paraffin-embedded placenta and brain by real-time PCR using primers targeting the IS711 gene. Immunohistochemical staining revealed Brucella sp. antigen in placental inflammation. This is the first report of isolation of Brucella sp. from a marine mammal in the Southern Hemisphere and the first report of marine Brucella-associated disease in Australia.

Spindle microtubule differentiation and deployment during micronuclear mitosis in Paramecium.

Spindles underwent a 12-fold elongation before anaphase B was completed during the closed mitoses of micronuclei in Paramecium tetraurelia. Two main classes of spindle microtubules have been identified. A peripheral sheath of microtubules with diameters of 27-32 nm was found to be associated with the nuclear envelope and confined to the midportion of each spindle. Most of the other microtubules had diameters of approximately 24 nm and were present along the entire lengths of spindles. Nearly all of the 24-nm microtubules were eliminated from spindle midportions (largely because of microtubule disassembly) at a relatively early stage of spindle elongation. Disassembly of some of these microtubules also occurred at the ends of spindles. About 60% of the total microtubule content of spindles was lost at this stage. Most, perhaps all, peripheral sheath microtubules remained intact. Many of them detached from the nuclear envelope and regrouped to form a compact microtubule bundle in the spindle midportion. There was little, if any, further polymerization of 24-nm microtubules after the disassembly phase. Polymerization of microtubules with diameters of 27-32 nm continued as spindle elongation progressed. Most microtubules in the midportions of well-elongated spindles were constructed from 14-16 protofilaments. A few 24-nm microtubules with 13 protofilaments were also present. The implications of these findings for spatial control of microtubule assembly, disassembly, positioning, and membrane association, that apparently discriminate between microtubules with different protofilament numbers have been explored. The possibility that microtubule sliding occurs during spindle elongation has also been considered.

Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line.

The doxorubicin-selected lung cancer cell line H69AR is resistant to many chemotherapeutic agents. However, like most tumor samples from individuals with this disease, it does not overexpress P-glycoprotein, a transmembrane transport protein that is dependent on adenosine triphosphate (ATP) and is associated with multidrug resistance. Complementary DNA (cDNA) clones corresponding to messenger RNAs (mRNAs) overexpressed in H69AR cells were isolated. One cDNA hybridized to an mRNA of 7.8 to 8.2 kilobases that was 100- to 200-fold more expressed in H69AR cells relative to drug-sensitive parental H69 cells. Overexpression was associated with amplification of the cognate gene located on chromosome 16 at band p13.1. Reversion to drug sensitivity was associated with loss of gene amplification and a marked decrease in mRNA expression. The mRNA encodes a member of the ATP-binding cassette transmembrane transporter superfamily.

A comparative study of the use of Exsikines and other advanced wound care products/modalities in the treatment of various wound types in efficacy.

OBJECTIVE: To examine the efficacy of Exsikines (patent-pending) in the treatment of chronic wounds. DESIGN: Subjects were treated with Exsikines every 3 to 5 days, wound healing parameters were monitored and compared with other published studies. SETTING: A clinical practice. PATIENTS: A total of 21 patients (22 chronic wounds each identified etiologically) were randomly selected for this study, based on presentation of Stage II or III wounds using the Wagner wound classification system of at least 6 weeks' duration. The average wound age in this study was 12.48 months with a median wound age of 3 months. To reflect clinical practice scenarios, no other criteria for inclusion were required. The study was initiated with 24 patients with 24 chronic wounds. Three patients were removed from the study for nonadherence of the therapeutic protocol of Exsikines application. INTERVENTIONS: Baseline measurements were taken of all wounds, and time of persistence was noted. Exsikines, a patent-pending mixture of homologous-derived blood products containing human cytokines and other growth factors, was applied to the wounds at approximately 4-day intervals. MAIN OUTCOME MEASURES: Wound measurements were taken intermittently. Healing times, healing rates, recurrence rates, cost of treatments, and other parameters were determined. MAIN RESULTS: One hundred percent of the wounds were completely healed within an average of 53.5 days (median, 41.5 days). Recurrence monitoring after wound closure averaged 8.9 months. Recurrence in diabetic ulcers and pressure ulcers occurred at a rate of 7.14% and 16.67%, respectively. Wounds were documented by digital photography. CONCLUSION: In comparison to standard wound care, as well as other wound care products, Exsikines offers a very efficacious method of treating chronic wounds.

Primary tonsillar mast cell tumour in a dog.

CASE REPORT: A 6-year-old speyed female Bull Arab-cross dog was found to have a small tonsillar nodule. Histological examination revealed a well-differentiated mast cell tumour (MCT). At initial staging, no evidence of concurrent cutaneous or visceral MCTs was found on a complete blood count, a single lateral thoracic radiograph, abdominal ultrasound or cytology of the spleen and regional lymph nodes. A diagnosis of primary tonsillar MCT was made. At 40 months postoperatively, the dog is alive with no evidence of gross tumour progression, in contrast to some previous reports of rapid disease progression and metastasis in dogs with primary oral MCTs. CONCLUSION: To the authors' knowledge, no previous reports of a primary MCT of the tonsil in dogs exist in the veterinary literature.

Trypanosomiasis in an Australian little red flying fox (Pteropus scapulatus).

CASE REPORT: An adult female Australian little red flying fox (Pteropus scapulatus) presented with icterus and anaemia. Examination of a blood smear revealed numerous trypanosomes 20.4-30.8 µm long with tapered ends. Necropsy and histological findings were consistent with trypanosome infection of lymphoid tissue and intravascular haemolysis. Sequence and phylogenetic analysis demonstrated this trypanosome species to be genetically distinct and most similar to Trypanosoma minasense and Trypanosoma rangeli (with a genetic distance of 1% at the 18S rRNA locus for both). CONCLUSION: To the authors' knowledge this is the first report of a trypanosome infection associated with clinical disease in bats.

Characterization of the M(r) 190,000 multidrug resistance protein (MRP) in drug-selected and transfected human tumor cell.

Overexpression of multidrug resistance-associated protein (MRP) has been detected in resistant cell lines derived from a variety of tumor types. The deduced amino acid sequence of MRP suggests that it is a member of the ATP-binding cassette transmembrane transporter superfamily that may be glycosylated and/or phosphorylated [S. P. C. Cole et al., Science Washington, DC), 258: 1650-1654, 1992]. Recently, transfection of HeLa cells with MRP expression vectors has demonstrated that the protein is capable of increasing resistance to natural product drugs such as anthracyclines, Vinca alkaloids, and epipodophyllotoxins (C. E. Grant et al., Cancer Res., 54: 357-361, 1994). Although the resistance phenotype of the transfectants is similar to that of the human small cell lung cancer cell line, H69AR, from which MRP was originally cloned, the transfectants differ in their drug accumulation characteristics, relative resistance to certain drugs, and MRP mRNA:protein ratio. Such differences have also been observed among drug-selected cell lines that overexpress MRP, and the underlying causes of these variable phenotypes are presently not known. We have utilized polyclonal anti-MRP-peptide antibodies to compare MRP post-translational modification, stability, processing, and subcellular distribution in the HeLa transfectants and in the drug-selected H69AR cells. These studies establish that MRP in both the transfected and selected cells is an ATP-binding, integral membrane glycophosphoprotein with an apparent molecular weight of 190,000. No obvious differences were detected in the extent or type of glycosylation or the kinetics of processing and turnover of the protein that might contribute to the different characteristics of the transfected and drug-selected cells. Analyses of the subcellular distribution of MRP by isopyknic density gradient centrifugation revealed that approximately 80% of MRP in the HeLa transfectants was associated with a low density plasma membrane fraction while the comparable fraction in the drug-selected H69AR cells contained only approximately 50% of the protein. The remaining MRP and plasma membrane markers were codistributed in higher density fractions consistent with the presence of MRP in endocytotic vesicles. The relatively high proportion of MRP associated with these fractions in H69AR cells may contribute to the lack of an observable accumulation defect in these cells when compared with the transfectants.

Pneumonia due to Chlamydia pecorum in a Koala (Phascolarctos cinereus).

Chlamydiosis is a common infectious disease of koalas (Phascolarctos cinereus), but Chlamydia spp. have not yet been demonstrated to cause pneumonia in these animals. A juvenile male koala died following an episode of respiratory disease. At necropsy examination, the lung tissue was consolidated. Microscopical lesions in the lung included pyogranulomatous bronchopneumonia, proliferation of bronchiolar and alveolar epithelium and interstitial fibrosis. Hyperplastic bronchiolar epithelial cells contained aggregates of small basophilic punctate organisms, which were confirmed as chlamydiae by transmission electron microscopy and immunohistochemistry. Real-time polymerase chain reaction identified these as Chlamydia pecorum. This report provides the best evidence to date of chlamydial infection causing pneumonia in a koala, and the first evidence that C. pecorum is capable of infecting the bronchiolar epithelium of the koala.

Type C Niemann-Pick disease: cellular uncoupling of cholesterol homeostasis is linked to the severity of disruption in the intracellular transport of exogenously derived cholesterol.

A uniquely attenuated disruption of cholesterol homeostasis has been characterized in certain Niemann-Pick, type C (NP-C) fibroblasts. Uptake of LDL-cholesterol by cultured fibroblasts derived from two clinically affected brothers with this variant biochemical phenotype led to less intracellular accumulation of unesterified cholesterol than found in other typical cell lines. This limited cholesterol lipidosis in the variant NP-C cells reflected cholesterol processing errors that differed from the cellular lesions in classical NP-C cells in the following ways: (1) a more limited intracellular distribution of the excessive unesterified cholesterol; (2) shorter and more transient delays in the induction of cholesterol-mediated homeostatic responses; and (3) more efficient intracellular transport of exogenously derived cholesterol to the plasma membrane and the endoplasmic reticulum. Activation of acyl-CoA cholesterol acyltransferase (ACAT) was greater than 100-fold in both control and NP-C fibroblasts when cell cultures were preconditioned with 25-hydroxycholesterol, but the subsequent esterification of exogenous non-lipoprotein [3H]cholesterol remained deficient in all NP-C cells. In the variant NP-C cells conditioned with the oxysterol, this esterification of exogenous [3H]cholesterol was less affected than in classical NP-C cultures. The NP-C mutation affects a broad spectrum of metabolic responses related to the processing of exogenously derived cholesterol. Among this pleiotropic array of deficient responses, retarded intracellular cholesterol transport appears most closely linked to the primary mutation. This conclusion is supported by two current observations: (1) the degree to which sterol transport is affected in mutant cells in turn reflects the extent to which cholesterol-homeostatic responses are compromised; and (2) sterol transport remains deficient despite concurrent normal activation of other cellular responses, such as cholesterol esterification.

Clinical effects of the use of a bipolar vessel sealing device for soft palate resection and tonsillectomy in dogs, with histological assessment of resected tonsillar tissue.

OBJECTIVE: To investigate whether soft palate resection and tonsillectomy with a bipolar vessel sealing device (BVSD) improves clinical respiratory score. To document histopathological changes to tonsillar tissue following removal with a BVSD. METHODS & RESULTS: Case series of 22 dogs with clinical signs of upper respiratory obstruction related to brachycephalic airway syndrome. Soft palate and tonsils were removed using a BVSD. Alarplasty and saccullectomy were also performed if indicated. A clinical respiratory score was assigned preoperatively, 24-h postoperatively and 5 weeks postoperatively. Excised tonsillar samples were measured and then assessed histologically for depth of tissue damage deemed to be caused by the device. Depth of tissue damage was compared between two power settings of the device. Soft palate resection and tonsillectomy with a BVSD lead to a significant improvement in respiratory scores following surgery. Depth of tissue damage was significantly less for power setting 1 compared with power setting 2. Using power setting 1, median calculated depth of tonsillar tissue damage was 3.4 mm (range 1.2-8.0). One dog experienced major complications. CONCLUSION: Soft palate resection and tonsillectomy with a BVSD led to significant improvement in clinical respiratory score.

The incidence of postoperative pain in children.

The incidence of pain in 170 children recovering from surgery was surveyed in two major teaching hospitals along with an analysis of analgesic medication prescribed and administered. Analgesic medication was not ordered for 16% of the patients and narcotic analgesic medication ordered was not given for 39% of the patients. In 29% of the patients, where an order for "narcotic or non-narcotic analgesic medication' was written, the non-narcotic drug was given exclusively. Irrespective of the treatments received, only 25% of the patients were pain free on the day of surgery and 13% reported severe pain. By the first postoperative day, 53% reported no pain but 17% still reported severe pain. There appeared to be no relationship between ages of patients and severity of pain reported. Analysis of orders written for postoperative analgesics revealed extremely variable prescribing habits of the medical staff and that doses frequently were too small and/or too infrequent. The majority of orders were written "PRN' which often was interpreted by nursing staff as "as little as possible.' Nursing staff also preferred not to give narcotic medications but substituted non-narcotic analgesics, even soon after surgery. Many of the children surveyed became withdrawn and this was interpreted as coping with pain. Others expressed a dread of "the needle' as a way of administering analgesics and preferred to suffer pain to an injection. We have concluded that there is considerable scope to improve pain management in children after surgery. This improvement must be based on improved education of medical and nursing staff in contemporary clinical pharmacology and on improved communication between staff, parents and patients.

Interleukin-2-diphtheria toxin fusion protein prolongs murine islet cell engraftment.

Allograft rejection is dependent upon complex cell-mediated processes, the primary effectors of which are activated T cells. As the expression of the cell surface protein interleukin 2 receptor is primarily limited to the subset of stimulated T cells, therapeutic agents that target this molecule may provide highly selective immunosuppression. A newly constructed chimeric IL-2 diphtheria toxin fusion protein specifically binds to and poisons activated T cells bearing the high-affinity IL-2R. We describe the in vivo effects of IL-2 toxin in preventing rejection of a crude pancreatic islet preparation transplanted across major and minor histoincompatibility barriers. IL-2 toxin administered once daily as the sole immunosuppressive agent prolongs islet graft survival and decreases the severity of the early mononuclear cell infiltrate into the graft site. Long-term survival of transplanted islets (greater than 100 days) was achieved following a short course (10 days) of more-intensive IL-2 toxin treatment. Thus IL-2 toxin, a highly selective immunosuppressive agent, leads to prolonged islet cell engraftment while sparing the resting or memory subset of the entire T cell repertoire.

Dose-related mechanisms of immunosuppression mediated by murine anti-CD3 monoclonal antibody in pancreatic islet cell transplantation and delayed-type hypersensitivity.

Although anti-CD3 mAb therapy is used extensively in clinical transplantation, the dose-related effects and mechanisms of action are not clearly defined. We have examined the dose-related effects of an antimurine CD3 mAb, 145-2C11, in pancreatic islet cell allograft and the delayed type hypersensitivity reaction models of T-cell-dependent immunity. Low-dose anti-CD3 therapy (0.5 micrograms/day) administered over several days mediated superficially equal, effective clinical immunosuppression as a single high-dose intravenous injection (400 micrograms). T cells harvested from animals treated with high-dose anti-CD3 were unresponsive to in vitro restimulation. In contrast, T cells isolated from low-dose treated animals retained in vitro proliferative capacity when restimulated with polyvalent anti-CD3 mAb. The terminal complement components were not required to support in vivo immunosuppression mediated by anti-CD3 mAb as C5 deficient mice were immunosuppressed by the administration of this mAb. In some pancreatic islet cell allograft recipients, permanent engraftment, but not tolerance, was achieved. Replacement of donor leukocytes produced acute rejection in hosts bearing long-term, well-accepted grafts. Prolonged anti-CD3 mAb treatment may provide sufficient time for replacement or inactivation of donor leukocytes.

A performance evaluation of commercial fibrinogen reference preparations and assays for Clauss and PT-derived fibrinogen.

The wide availability of fibrinogen estimations based on the prothrombin time (PT-Fg) has caused concern about the variability and clinical utility of fibrinogen assays. In a multi-centre study, we investigated fibrinogen assays using various reagents and analysers. Clauss assays generally gave good agreement, although one reagent gave 15-30% higher values in DIC and thrombolysis. Two commercial reference preparations had much lower potencies than the manufacturers declared, and plasma turbidity influenced parallelism in some Clauss assays. PT-Fg assays gave higher values than Clauss and showed calibrant dependent effects, the degree of disparity correlating with calibrant and test sample turbidity. Analyser and thromboplastin dependent differences were noted. The relationship between Clauss and PT-Fg assays was sigmoid, and the plateau of maximal PT-Fg differed by about 2 g/l between reagents. ELISA and immunonephelometric assays correlated well, but with a high degree of scatter. Antigen levels were higher than Clauss, but slightly lower than PT-Fg assays, which appeared to be influenced by degraded fibrinogen. Clauss assays are generally reproducible between centres, analysers and reagents, but PT-Fg assays are not reliable in clinical settings.

Synergistic induction of delta-aminolevulinic acid synthase activity by N-ethylprotoporphyrin IX and 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-isobutylpyridine .

The levels of the cellular free heme pool in chick embryo hepatocyte culture were lowered using N-ethylprotoporphyrin IX (N-ethylPP) and analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC), and the effect on delta-aminolevulinic acid synthase (ALAS) was examined. N-EthylPP, which lowers cellular heme levels by inhibiting ferrochelatase activity, produced an induction of ALAS activity to 444% of control at 3 hr after its administration. 4-Ethyl DDC, which lowers heme levels by destroying the heme moiety of cytochrome P-450 and lowering ferrochelatase activity, caused an induction of ALAS to 565% of control at 12 hr after administration. 4-Isobutyl DDC, which lowers heme levels by destroying the heme moiety of cytochrome P-450, induced the activity of ALAS to 289% of control at 3 hr after administration. This indicates that ferrochelatase inhibition is a more important mechanism of heme lowering than alkylation of cytochrome P-450 heme when both heme-depleting mechanisms are acting in chick embryo liver cells. It was anticipated that administration of a combination of 4-isobutyl DDC plus N-ethylPP would mimic the effect of 4-ethyl DDC. However, this combination induced ALAS activity to levels that were much greater than those observed after 4-ethyl DDC (1257% of control at 12 hr). This synergistic induction may be attributable to lowering of free heme levels to the point where transcription, translation, and translocation of ALAS are all derepressed.

Elevation of delta-aminolevulinic acid synthase and cytochrome PB1 P450 messenger RNA levels by dihydropyridines, dihydroquinolines, sydnones, and N-ethylprotoporphyrin IX.

A series of compounds that increase the activity of delta-aminolevulinic acid synthase (ALAS) in chick embryo hepatocyte cultures were studied for their effects on steady-state levels of mRNA for ALAS and phenobarbital-inducible cytochrome PB1 P450. N-Ethylprotoporphyrin IX (N-EtPP), which is believed to lower heme levels by inhibition of ferrochelatase (FC), had little effect on steady-state ALAS mRNA levels. 3,5-Diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4- isobutylpyridine (4-isobutyl DDC), which is believed to lower heme levels by repetitive destruction of the heme moiety of cytochrome P450, increased steady-state levels of ALAS mRNA levels approximately 2-fold. 3,5-Diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine (4-ethyl DCC) which inhibits FC activity and destroys the heme moiety of cytochrome P450, increased ALAS mRNA levels approximately 4-fold. A combination of N-EtPP and 4-isobutyl DDC produced a synergistic increase in ALAS mRNA levels to approximately 6-fold over control levels. The synergistic increase in ALAS activity observed previously with this combination can be explained, at least in part, by a synergistic increase in ALAS mRNA levels. Other porphyrinogenic agents, which function as mechanism-based inactivators of cytochrome P450 and elevate ALAS activity, were found to elevate ALAS mRNA. These compounds included 3-[2-(2,4,6-trimethylphenyl)thioethyl]-4-methylsydnone (TTMS), 2,4-diethyl-2-methyl-1,2-dihydroquinoline (DMDQ), and 2,2,4-trimethyl-1,2,dihydroquinoline (TMDQ). The elevation of ALAS mRNA by these porphyrinogenic agents is probably due to their lowering of cellular heme levels by a combination of ferrochelatase inhibition and repetitive destruction of the heme moiety of cytochrome P450. The lowering of heme levels should result in an enhancement of ALAS mRNA half-life as it has been demonstrated by others that heme shortens the half-life of ALAS mRNA. It was of interest that some of these drug treatments also caused an elevation in steady-state levels of cytochrome PB1 P450 mRNA; the exception was TTMS, which along with its analogue 3-(2-phenylethyl)-4-methylsydnone (PEMS), did not alter cytochrome PB1 P450 mRNA levels. Increases in steady-state levels of cytochrome PB1 P450 mRNA subsequent to increases in steady-state levels of ALAS mRNA were observed with 4-ethyl DDC, 4-isobutyl DDC, DMDQ, and TMDQ. The data obtained with N-EtPP and a combination of N-EtPP and 4-isobutyl DDC on cytochrome PB1 P450 mRNA levels do not support the contention that heme functions as a positive regulator of cytochrome P450 gene expression.

Effects of 3-(2-phenylethyl)-4-methylsydnone and related sydnones on heme biosynthesis.

3-[2-(2,4,6-Trimethylphenyl)thioethyl]-4-methylsydnone (TTMS) and 3-(2-phenylethyl)-4-methylsydnone (PEMS) cause mechanism-based inactivation of rat hepatic microsomal cytochrome P-450 and the formation of N-alkylprotoporphyrins in rat liver. In the present study, we have shown that both TTMS and PEMS cause mechanism-based inactivation of chick embryo hepatic microsomal cytochrome P-450. TTMS also caused the inhibition of ferrochelatase activity, the accumulation of protoporphyrin IX, and an increase in the activity of delta-aminolevulinic acid synthase in chick embryo liver cell culture. PEMS was devoid of effect on ferrochelatase activity, porphyrin accumulation, and delta-aminolevulinic acid synthase activity. There are two possible explanations for the lack of effect of PEMS on heme biosynthesis: (1) the ring-A- and/or ring-B-substituted regiosomers of the N-phenylethyl- and N-phenylethenylprotoporphyrins which are produced during the mechanism-based inactivation of cytochrome P-450 by PEMS are too bulky to fit into the active site of ferrochelatase to inhibit its activity, in contrast to the N-vinylprotoporphyrin formed from TTMS; and (2) the N-alkylprotoporphyrins produced consist of the ring-C- and/or ring-D-substituted regioisomers, which are not inhibitors of ferrochelatase activity.