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Objective: Vitamin D deficiency, even subclinical, has been considered to worsen the skeletal damage in primary hyperparathyroidism (PHPT). Our study aimed to investigate the impact of vitamin D status on skeletal involvement in PHPT., Design and Measurements: A cross-sectional study was designed involving 62 female patients with PHPT. Serum total calcium (tCa), phosphate (P), creatinine (Cr) and total alkaline phosphatase activity (AP), together with 24-h (uCa 24 h) and spot fasting (uCa/Cr) urinary calcium, were measured by autoanalyser; ionized calcium (iCa) was assessed by an ion-specific electrode; intact parathyroid hormone (PTH) was measured by immunoradiometric assay (IRMA) and 25-hydroxyvitamin D (25-OHD) by radioimmunoassay (RIA). Bone mineral density (BMD) was measured by dual energy X-ray absorptiometry (DXA) at lumbar spine in 58 patients, and at femoral neck, Ward's triangle, greater trochanter, intertrochanteric line and total hip in 56 patients. The associations of all variables with age, 25-OHD, body mass index (BMI) and PTH were studied by linear multiple regression analysis, using progressively restricted models., Results: The model including age, 25-OHD, PTH and BMI showed significant regression with BMD values. PTH, age and BMI exerted a leading role in determining such a significance, while no significant regression was found between the parameters studied and 25-OHD; this was confirmed by Pearson's linear correlation analysis. The progressively restricted models showed significant regression of BMD at femoral neck, femoral intertrochanteric line and total hip with age, BMI and PTH. BMD measured at the Ward's triangle and greater trochanter showed significant regression with age and BMI, and that measured at lumbar spine with age., Conclusions: Our data indicate that in primary hyperparathyroidism patients the influence of 25-hydroxyvitamin D levels on bone mineral density, if any, was overwhelmed by the effects of parathyroid hormone excess, age and body mass index. The latter unequally affected bone mineral density of various measured sites with different composition.

Background and Aims: The aim of the study was to investigate the impact of fractures (i.e., hip, Colles, humeral and vertebral fractures), compared with that of other common diseases requiring hospitalization, on health care in the main hospital in Rome (Italy)., Methods: Hospital discharge forms, filled in according to the 9th International Classification of Diseases, were examined from 1996 to 1999. Data on fractures were compared with those related to other diseases which occupy a considerable proportion of hospital operating time in Italy: coronary heart disease (CHD), cerebrovascular disorders (CVD), diabetes mellitus (DM), chronic obstructive pulmonary disease (COPD) and breast cancer (BC)., Results: In all groups of patients, the mean age of females was significantly higher (p<0.0001) than that of males. Male patients with hip fractures had hospital stays significantly longer than females (p<0.0001), whereas women with Colles fractures had significantly (p<0.02) longer stays. When patients were divided according to age (i.e., over or under 60 years), mean hospital stays did not differ between younger and older patients in all groups except Colles fractures (p<0.001). Hip fractures in older patients showed striking in-hospital mortality. Throughout the study period, hip fractures accounted for the highest overall and per-patient costs. The number of female patients with fractures (and, obviously, breast cancer) was higher, while the opposite applied to the other disorders. Male patients with fractures, CHD and CVD were significantly younger than females (p<0.0001). When the percentage of deaths was added to that of patients discharged to other institutions, fractures showed the poorest outcome of any hospitalization event. Per-patient costs were remarkably higher for CHD, followed by fractures., Conclusions: Fractures represent a growing but often underestimated burden for hospital care in Italy; further studies are needed on this issue.

Little is known about how memories of new voluntary motor actions, also known as procedural memory, are formed at the molecular level. Our work examining acquisition of lever-pressing for food in rats has shown that activation of glutamate NMDA receptors, within broadly distributed but interconnected regions (e.g., nucleus accumbens core, prefrontal cortex, basolateral amygdala), is critical for such learning to occur. This receptor stimulation triggers intracellular cascades that involve protein phosphorylation and new protein synthesis. In support of this idea, we have found that posttrial inhibition of protein synthesis in the ventral striatum impairs learning, whereas posttrial NMDA receptor blockade does not. More recent data show extension of this network to the central amygdala, where infusions of NMDA antagonists also impair learning. We hypothesize that activity in this distributed network (including dopaminergic activity and perhaps muscarinic cholinergic activity) computes coincident events and thus enhances the probability that temporally related actions and events (e.g., lever pressing and delivery of reward) become associated. Such basic mechanisms of plasticity within this reinforcement learning network also appear to be profoundly affected in addiction.

The nonlinear properties of an encapsulated microbubble of a contrast agent were studied theoretically and experimentally. A modified nonlinear differential equation (Herring equation) was used to describe the radial oscillation of the microbubble and solved numerically. It was found that the nonlinear resonance frequency, at which the peak radial oscillation amplitude occurs, was a decreasing function of the acoustic amplitude of a driving ultrasonic pulse. Optical images of the contrast agent microbubbles under various ultrasonic exposure conditions: 1. sham exposure; 2. 2-MHz spatial peak acoustic pressure = 200 kPa, I(SATA) = 260 mW/cm(2), duty cycle = 7.5%, repetition period = 0.0266 ms; 3. 0.5-MHz spatial peak acoustic pressure = 200 kPa, I(SATA) = 130 mW/cm(2), duty cycle = 7.5%, repetition period = 0.1067 ms; have also shown that the lower-frequency ultrasound (US) excitation (0.5 MHz) is more effective in disruption of the microbubbles due to acoustic inertial cavitation than the higher frequency US (2 MHz).

Purpose: To provide indications for currently approved treatments and future options for male osteoporosis, based on the pathogenetic mechanisms of bone loss in the male sex., Design: Review of the most significant data reported in the literature., Results: Unhealthy lifestyle habits should be modified. Testosterone replacement is indicated only in patients with diagnosis of hypogonadism. Based on the demonstrated pathogenetic role of estrogen lack in bone loss in men, either low doses of this hormone or selective androgen receptor modulators have been proposed for the treatment of male osteoporosis. Bisphosphonates are the only medications approved by Food and Drug Administration for idiopathic and glucocorticoid-induced osteoporosis in men. As far as anabolic treatments are concerned, there is not agreement on clinical utility of sodium fluoride. Short-term treatment with parathyroid hormone (PTH) seems to be safe and effective in patients with idiopathic osteoporosis, due to its anabolic action. Therapeutic use of growth hormone (GH) and insulin-like growth factor type I (IGF-I), both considered as potential anabolic agents, is still limited because of the high incidence of side effects and relatively transient efficacy, particularly of IGF-I., Conclusions: Treatments should be selected on the basis of anti-fracture efficacy of various medications, which has been demonstrated so far only for alendronate and risedronate. Although anabolic agents produce noticeable increase of bone mineral density, is still debated if they also reduce fracture incidence in males with osteoporosis.

The main clinical presentation of osteoporosis is fracture and its consequences. However a number of diseases and factors can induce bone loss and increase the risk of fracture. Therefore the clinical approach should be initially directed to exclude secondary osteoporosis. Vertebral fractures are the most common osteoporotic fractures; they are characterized by back pain, typical physical changes such as kyphosis and height loss, functional impairment and social decline. On the other hand, hip fracture is the most severe consequence of osteoporosis, because of its higher morbility and mortality. The main pathogenetic determinants of hip fracture are represented by both bone loss and several factors contributing to fall in the elderly. Moreover, a number of conditions are responsible for the high mortality rate following hip fracture. Colles' fracture is rarely hospitalized; however, most patients complain a complex algodystrophic syndrome which impairs the quality of life.

This investigation was undertaken to determine whether the preservation of bone mass in patients with mild primary hyperparathyroidism (PHPT) could be detected when measuring spine density in the lateral projection. We compared the bone mineral density (BMD) of L2-L4 utilizing the posterior-anterior (PA) and lateral projections in postmenopausal patients with PHPT and in a group of 27 postmenopausal normal women. Thirty-three consecutive postmenopausal patients with PHPT were studied; 25 were asymptomatic whereas the remaining 8 suffered complications related to the disease. Based upon the criteria established by the Consensus Conference on the Management of Asymptomatic PHPT, only 10 of the 25 asymptomatic patients could be considered affected by mild disease; the remaining patients were classified as having moderate disease. Patients with mild disease had mean lateral total BMD values (0.682 +/- 0.113 g/cm(2)) significantly higher than normal women (0.588 +/- 0.076, p<0.02) and patients with moderate disease (0.599 +/- 0.077, p<0.05). There were significant differences among the three groups in both PA L2-L4 and L1-L4 levels: patients with mild disease had significantly higher mean BMD values than patients with moderate disease and normal women, when either three or four vertebrae were considered. Interestingly, at this latter site, patients with moderate disease had significantly ( p<0.05) lower values than normal women. Our results indicate that patients with mild PHPT have a preservation of vertebral mass when compared with the other hyperparathyroid patients and normal women, when taking into account both the mainly trabecular portion and the whole vertebra. The finding that when the PA projection was assessed, BMD values of patients with moderate disease were significantly lower than those of normal women, might be attributed to the detrimental effect of raised parathyroid hormone levels on the cortical component of the vertebral body.

We describe a patient with multiple endocrine neoplasia type 1 characterized by the simultaneous occurrence of parathyroid cancer, parathyroid adenomas, and pancreatic gastrinoma, who presented with an episode of acute hypercalcemia. The rapid parathyroid hormone assay provided a basis for the diagnosis of parathyroid hyperfunction. Mediastinal metastasis of the parathyroid carcinoma was found at autopsy. However, the staining of pancreatic and gastric tissue for parathyroid hormone-related protein does not make it possible to exclude completely the contribution of this peptide in mediating the hypercalcemia. To our knowledge, this is the first reported case of parathyroid carcinoma as part of the multiple endocrine neoplasia type 1 syndrome.

Heme oxygenase (HO) catalyzes the rate-controlling step of physiologic heme catabolism, namely, the oxidation of the alpha-methene bridge of the macrocycle with formation of CO, Fe, and biliverdin. HO-1, the first isoform of HO to be identified, is highly inducible by a large number of physical and chemical factors. Many of these factors cause oxidative or other stresses to cells. In this work, we have studied the regulation of the chick HO-1 gene, using selected promoter--reporter constructs of the gene transiently or stably transfected into primary cultures of chick embryo liver cells or into the LMH line of chicken hepatoma cells. By use of deletional and mutational analyses, DNase protection, and electromobility shift DNA-binding assays, we identified a heretofore undefined regulatory region in the 5'-UTR of the chick HO-1 gene which confers up-regulation of reporter gene (luciferase) expression in the presence of heme and other selected metalloporphyrins. This new metalloporphyrin-responsive element (MPRE) was localized to a 200-bp region 3.8 to 3.6 kb upstream of the transcription starting point of the chick HO-1 gene. It responded particularly to heme and cobalt protoporphyrin with maximal inductions at 10-15 microM concentrations and 15-18 h of exposure. In contrast, sodium arsenite, a prototypical stress-type inducer of HO-1, led to down-regulation of the reporter gene down stream of MPRE. DNase analysis identified an 18-mer oligonucleotide that was required for the metalloporphyrin response (5'-(-3711)TATTGCAGCTGTGTGGGG-3'). Mutations at any of four sites within this oligonucleotide abrogated the metalloporphyrin-dependent up-regulation of reporter gene expression. Nuclear protein extracts of cells treated with heme or cobalt protoporphyrin showed specific enhanced binding to this 18-mer. We conclude that the chick HO-1 promoter region contains a unique sequence that subserves up-regulation of the gene by metalloporphyrins and propose the name "metalloporphyrin-responsive element" for this sequence.

To assess how two different serum markers of bone resorption may reflect changes in bone turnover, we compared age- and sex-related changes in serum C-terminal telopeptide of type I collagen (betaCTx) and tartrate-resistant acid phosphatase activity (TRAP) in 136 healthy men and 184 normal women. Serum levels of the two markers were also assessed in several groups of patients of both sexes presenting with the most common metabolic and endocrine bone diseases: established osteoporosis (n = 77), primary hyperparathyroidism (n = 44), glucocorticoid excess (n = 17), chronic renal failure (n = 39), active Paget's disease of bone (n = 5), humoral hypercalcemia of malignancy (n = 3), osteomalacia (n = 3), hyperthyroidism (n = 10), post-surgical hypoparathyroidism (n = 10), acromegaly (active disease, n = 8) and Cushing's syndrome (n = 10). In men the regression of betaCTx with age showed an initial decrease in bone resorption followed by an increase thereafter, starting from the sixth decade of life. No age-related change in serum TRAP activity was observed. In women, by contrast, a slight but significant linear correlation of both serum betaCTx and TRAP with age (r = 0.223, p<0.003 and r = 0.333, p<0.0001, respectively) was found, the two markers being positively correlated (r = 0.238, p<0.002). In each class of patients the mean Z-scores of betaCTx were significantly higher than those of TRAP activity. Moreover, compared with normal subjects, serum betaCTx seems to be characterized by a superior sensitivity relative to TRAP measurement, at least in the disorders studied.

Heme oxygenase-1, the major inducible isoform of heme oxygenase (HO), can be induced by heme and numerous other physical and chemical factors, many of which cause cellular 'stress'. This has led to the realization that HO-1 is a major highly conserved stress or heat shock protein. Recent work has implicated activation of mitogen-activated protein kinases and other kinases in the mechanism of induction of HO-1, and suggested that signal transduction pathways through tyrosine kinases are involved in induction of HO-1 gene expression by stress inducers. We hypothesized that phenylarsine oxide (PAO), an inhibitor of protein tyrosine phosphatases (PTPs), might up-regulate the HO-1 gene. Here, we show that a remarkably brief (1-15 min) exposure of normal hepatocytes to low concentrations (0.5-3 microM) of PAO produces a marked increase in mRNA and protein of HO-1. This increase is comparable to the level obtained by addition of heme (20 microM), and occurs without producing changes in cellular glutathione levels or stabilization of HO-1 message. Preincubation of cells with inhibitors of protein synthesis decreased the ability of PAO to increase levels of HO-1 mRNA, suggesting that the inductive effect requires de novo protein synthesis. Addition of thiol donors abrogated the PAO-mediated induction of HO-1 in a dose dependent fashion. Addition of genistein, a tyrosine kinase inhibitor, blunted the induction produced by both PAO and heme. After brief incubations with PAO or heme, cell extracts showed comparable increases in levels of protein tyrosine phosphorylation in general, and specifically in ZAP70 kinase. Our results are consistent with the proposition that induction of HO-1 by PAO involves inhibition of specific PTP(s), and that the mechanisms of induction of HO-1 by PAO and by heme may share some common pathways.

Induction of expression of heme oxygenase-1 (HO-1) has been studied in primary cultures of chick embryo liver cells and in the LMH line of avian hepatoma cells. Cells were transiently transfected with selected constructs containing portions of the 5'-untranslated (promoter) region of the HO-1 gene linked to luciferase as reporter gene. LMH cells that had been stably transfected with selected wild type or mutant constructs were also studied. Metalloporphyrins, especially Fe protoporphyrin (heme) and Co protoporphyrin strongly induced luciferase expression in both types of transfected cells. Low concentrations of Zn mesoporphyrin, an inhibitor of HO activity, exerted a synergistic effect on heme-, but not Co protoporphyrin-dependent induction. The antioxidant and &bond;SH donor N-acetyl cysteine had little effect on the metalloporphyrin-dependent inductions of HO-1, in contrast to its marked inhibitory effect on the sodium arsenite-dependent induction of the HO-1 gene. Deletional analysis showed that the key element(s) required for the metalloporphyrin-dependent induction of HO-1 is located between -3.6 and -5.6 kb upstream of the transcription starting point. Data from electrophoretic mobility shift and site-directed mutagenesis experiments excluded a role for consensus AP-1 binding elements at -1576, -3647, or -4578 in the inductions produced by heme or Co protoporphyrin., (Copyright 2000 Academic Press.)

Previously, chick heme oxygenase-1 (cHO-1) gene was cloned by us and two regions important for induction by sodium arsenite were identified. These two regions were found to contain consensus sequences of an AP-1 (-1580 to -1573) and a MRE/cMyc complex (-52 to -41). In the current study, the roles of these two elements in mediating the sodium arsenite or cobalt chloride dependent induction of cHO-1 were investigated further. DNA binding studies and site-directed mutagenesis studies indicated that both the AP-1 and MRE/cMyc elements are important for the sodium arsenite induction, while cobalt chloride induction involves only the AP-1 element. Electrophoretic mobility shift assays showed that nuclear protein binding to the AP-1 element was increased by both sodium arsenite or cobalt chloride treatment, whereas the binding of proteins to the MRE/cMyc element showed a high basal expression in untreated cells and the binding activity was only slightly increased by sodium arsenite treatment. Site-directed mutagenesis studies showed that, to completely abolish sodium arsenite induction, both the AP-1 and MRE/cMyc elements must be mutated; mutation of either element alone resulted in only a partial effect. In contrast, a single mutation at AP-1 element was sufficient to reduce the cobalt chloride induction almost completely. The MRE/cMyc complex plays a major role in the basal level expression, and shares some similarities to the upstream stimulatory factor element (USF) identified in the promoter regions of mammalian HO-1 genes and other stress regulated genes. Because sodium arsenite is known to cause oxidative stress and because activation of AP-1 proteins has been shown to be a key step in the oxidative stress response pathway, we also explored the possibility that the induction of the cHO-1 gene by sodium arsenite is mediated through oxidative stress pathway(s) by activation of AP-1 proteins. We found that pretreatment with antioxidants (N-acetyl cysteine or quercetin) reduced the induction of the endogenous cHO-1 message or cHO-1 reporter construct activities induced by sodium arsenite or cobalt chloride. These antioxidants also reduced the protein binding activities to the AP-1 element in the electrophoretic mobility shift assays. In summary, induction of the cHO-1 gene by sodium arsenite or cobalt chloride is mediated by activation of the AP-1 element located at -1,573 to -1,580 of the 5'UTR.

Patients with any of the acute porphyrias may suffer from acute attacks. If these patients are treated with certain drugs, such as barbiturates, the likelihood of developing an attack is increased. Patients treated with antidepressants or benzodiazepine-type anxiolytics also could be placed at increased risk of developing porphyric attacks because little is known about the potential for some of these drugs to induce attacks. Primary cultures of chick embryo liver cells were used to study the effects of selected antidepressants and anxiolytics on porphyrin accumulation. Cells were treated with desferrioxamine (to partially block heme synthesis, simulating conditions encountered in porphyric patients) and increasing concentrations (3.16-1000 microM) of the evaluated drugs. Twenty hours later, porphyrin accumulation was measured. The drugs included four antidepressants and five benzodiazepine-type anxiolytics. The antidepressants bupropion and nefazodone significantly increased porphyrin accumulation when given with desferrioxamine, whereas neither fluoxetine nor paroxetine increased porphyrin accumulation. The benzodiazepine-type anxiolytic agents oxazepam, lorazepam, diazepam, triazolam, and midazolam all significantly increased porphyrin accumulation when given with desferrioxamine. Dose-response studies showed that diazepam, midazolam, and triazolam produced significant increases even at the lowest concentration tested (3.16 microM), whereas lorazepam and oxazepam required higher concentrations (>/=10 microM). These studies suggest that patients with acute porphyrias may be at greater risk for developing porphyric attacks when treated with bupropion or nefazodone compared with fluoxetine or paroxetine, and that the evaluated benzodiazepine derivatives should be administered with caution. Among the latter, low doses of lorazepam and oxazepam may be safer than those of diazepam, midazolam, and triazolam.

An overview of the metabolic response to acute traumatic brain injury is presented. The consequences of hypermetabolism, hypercatabolism, and an altered immune function are discussed. Once a person with acute traumatic brain injury develops this hyperdynamic state, the resultant excessive protein breakdown ensues. This can lead to malnutrition. The feeding methods used to prevent malnutrition are discussed, along with the proper alimentation to provide to diminish the hyperdynamic state and improve immune function.

When patients with acute porphyrias are treated with antihypertensives and analgesics, they could be placed at increased risk of developing porphyric attacks, since little is known about the potential for many of these drugs to induce these attacks. We used primary chick embryo liver cells, which maintain intact heme synthesis and regulation, to study the effects of antihypertensives and analgesics on porphyrin accumulation. Cells were treated with desferrioxamine to block heme synthesis partially, simulating conditions encountered in porphyric patients. Typically, cells were treated for 20 hr with the test drugs (3.16 to 1000 microM), along with desferrioxamine. Porphyrins were measured spectrofluorometrically, as uro-, copro,- and protoporphyrin. The evaluated drugs included six antihypertensives (two calcium channel blockers, an angiotensin receptor antagonist, and three inhibitors of angiotensin converting enzyme) and eight analgesics. Of the calcium channel blockers tested, nifedipine greatly increased porphyrin accumulation, whereas diltiazem caused only a slight increase. Losartan (an angiotensin receptor antagonist), captopril, or lisinopril (two angiotensin converting enzyme inhibitors) produced only small increases in porphyrin accumulation. In contrast, enalapril (another angiotensin converting enzyme inhibitor) substantially increased porphyrin accumulation when given in high concentrations. Among the analgesics tested, fentanyl and tramadol produced the highest porphyrin accumulations. Nalbuphine, hydrocodone, oxycodone, and dezocine were moderately or weakly porphyrogenic, whereas buprenorphine and morphine did not increase porphyrin accumulation. These studies suggest that patients with acute porphyrias may be at greater risk for developing porphyric attacks when treated with nifedipine (compared with diltiazem), enalapril (compared with captopril or lisinopril), and tramadol (compared with the other analgesics).

Herpes simplex virus type 1 (HSV-1) ICP0 is required for efficient viral gene expression during lytic infection, especially at low multiplicities. A series of cellular activities that can substitute for ICP0 has been identified, suggesting that when the activity of ICP0 is limiting, these activities can substitute for ICP0 to activate viral gene expression. The cellular activities may be especially important during reactivation of HSV from neuronal latency when viral gene expression is initiated in the absence of prior viral protein synthesis. Consistent with this hypothesis, we have identified an inducible activity in cells of neural lineage (PC12) that can complement the low-multiplicity growth phenotype of an ICP0 null mutant, n212. Pretreatment of PC12 cells with nerve growth factor (NGF) or fibroblast growth factor (FGF) prior to infection produced a 10- to 20-fold increase in the 24-h yield of n212 but only a 2- to 4-fold increase in the yield of wild-type virus relative to mock treatment. Slot blot analysis of nuclear DNA isolated from infected cells treated or mock treated with NGF indicated that NGF treatment does not significantly affect viral entry. The NGF-induced activity in PC12 cells was expressed transiently, with peak complementing activity observed when cells were treated with NGF 12 h prior to infection. Addition of NGF 3 h after infection had little effect on virus yield. The NGF-induced cellular activity was inhibited by pretreatment of PC12 cells with kinase inhibitors that have high specificity for kinases involved in NGF/FGF-dependent signal transduction. RNase protection assays demonstrated that the NGF-inducible PC12 cell activity, like that of ICP0, functions to increase the level of viral mRNA during low-multiplicity infection. These results suggest that activation of viral transcription by ICP0 and transcriptional activation of cellular genes by NGF and FGF utilize common signal transduction pathways in PC12 cells.

Induction of heme oxygenase (HO) has been proposed as a protective cellular mechanism against oxidative damage. In previous work (Tyrrell et al., Carcinogenesis [1993] 14, 761-765), portions of the 5' promoter region of the human HO-1 gene linked to the reporter gene chloramphenicol acetyl transferase (CAT), had been transiently expressed in HeLa cells. To extend the study of human HO gene expression into primary liver cells, these reporter gene fusion constructs, containing 121 or 1416 base pairs of the untranscribed 5'-upstream sequences of the human HO-1 gene, were used along with pSV beta-Gal plasmid to dually transfect primary cultures of chick embryo liver cells (CELC). The transfected cells were treated with selected metals, heme, phorbol ester, and chemical agents that produce oxidative stress (H2O2 or sodium arsenite). Reporter gene activities were measured 18-20 h later. Our major findings are: (1) these HO-CAT constructs were expressed in CELC; (2) unlike HeLa cells, the expression of CAT was detected in CELC without the need for the SV40 enhancer; (3) sodium arsenite and cobalt chloride induced the expression of the HO-CAT constructs whereas heme had no effect on or decreased CAT expression for all of the transfected constructs; (4) study of endogenous chick HO-1 gene expression in CELC showed that HO-1 responded to sodium arsenite treatment in a dose-dependent fashion, and the response was rapid and transient. We conclude that, in chick liver cell cultures, induction of the HO-1 gene by heme is fundamentally different from that produced by transition metals or sodium arsenite. Furthermore, the results suggest that expression of the HO-1 gene is highly conserved across species.

Heme oxygenase catalyzes the first and rate-controlling step in heme catabolism. One of the two forms of heme oxygenase (heme oxygenase-1) has been shown to be increased by heme, metals, and in some systems, by certain environmental stresses. However, it remains uncertain whether heme induces hepatic heme oxygenase-1 by a general stress response, or a specific heme-dependent cellular response. The work communicated here explores this issue by examining possible mechanisms whereby heme and other metalloporphyrins induce heme oxygenase-1 in normal liver cells. Primary cultures of chick embryo liver cells were tested for their ability to increase heme oxygenase mRNA after exposure to selected metalloporphyrins (heme, chromium mesoporphyrin, cobalt protoporphyrin and manganese protoporphyrin). The ability of antioxidants to decrease metalloporphyrin-mediated induction of heme oxygenase-1 mRNA was also tested. Our results indicate that: 1) the increase in heme oxygenase-1 mRNA mediated by heme or other metalloporphyrins may involve a short-lived protein(s) since the increase was prevented by several inhibitors of protein synthesis; and 2) in normal liver cells, heme-dependent oxidative stress does not play a key role in the heme-mediated induction of heme oxygenase-1. We conclude that heme and other non-heme metalloporphyrins induce heme oxygenase-1 through a mechanism requiring protein synthesis, not because metalloporphyrins increase cellular oxidative or other stress.

Hepatic 5-aminolevulinic acid synthase, the first and normally rate-controlling enzyme of heme biosynthesis, is regulated by heme. One of the known mechanisms whereby increased cellular heme regulates 5-aminolevulinic acid synthase is by decreasing the stability of its mRNA. In primary cultures of chick embryo liver cells, we tested whether a decrease in cellular heme might increase 5-aminolevulinic acid synthase mRNA stability and whether heme or other metalloporphyrins could reverse this stabilization. We found that: (a) The stability of 5-aminolevulinic acid synthase mRNA was markedly increased by inhibitors of heme biosynthesis, namely, 4,6-dioxoheptanoic acid or deferoxamine; (b) This increased stability of 5-aminolevulinic acid synthase mRNA was reversed by the addition of heme (10 microM) or by the combination of zinc mesoporphyrin (50 nM), an inhibitor of heme oxygenase, and heme (200 nM); (c) Repression of 5-aminolevulinic acid synthase mRNA levels by zinc mesoporphyrin (10 microM) was due to inhibition of heme oxygenase, rather than a direct, heme-like, effect of zinc mesoporphyrin on 5-aminolevulinic acid synthase mRNA; (d) Among the several non-heme metalloporphyrins tested, only zinc mesoporphyrin and chromium mesoporphyrin significantly decreased 5-aminolevulinic acid synthase mRNA without increasing heme oxygenase mRNA.

Heme oxygenase catalyzes the degradation of heme into biliverdin, carbon monoxide, and iron. Two forms of this enzyme, heme oxygenase-1 and -2, have been identified; only heme oxygenase-1 is subject to induction by heme, metal ions, and other chemical and physical perturbations (e.g. drugs, oxidants, and heat shock). Primary chick embryo liver cells are widely used for the study of heme metabolism because of their ease of preparation, low cost, and high degree of similarity to human heme metabolism. Nonetheless, this system has some limitations: new cultures must be prepared every week; the resulting cell populations are non-homogeneous; and cells are short-lived, limiting the feasible duration of time course and transfection studies. LMH cells are the first chicken hepatoma cell line to be established. The aim of this study was to characterize the regulation of heme oxygenase-1 in LMH cells, and to compare this regulation to that previously described in primary chick embryo liver cells. The induction of heme oxygenase-1 was assessed by measuring changes in mRNA levels or enzyme activities in response to several treatments, including heme, heavy metals, sodium arsenite, and heat shock, which have been shown to increase the expression of heme oxygenase. Similarities were observed with respect to regulation of heme oxygenase-1 expression in primary hepatocytes and LMH cells. We report the first measurable heat shock response of heme oxygenase-1 in CELC or LMH cells; and show that LMH cells are a useful model for the study of heme oxygenase-1 regulation.

Temperature is a key environmental cue for Yersinia enterocolitica as well as for the two other closely related pathogens, Yersinia pestis and Yersinia pseudotuberculosis. Between the range of 30 degrees C and 37 degrees C, Y. enterocolitica phase-varies between motility and plasmid-encoded virulence gene expression. To determine how temperature regulates Y. enterocolitica motility, we have been dissecting the flagellar regulatory hierarchy to determine at which level motility is blocked by elevated temperature (37 degrees C). Here we report the cloning, DNA sequences, and regulation of the two main regulators of Class III flagellar genes, fliA (sigma F) and flgM (anti-sigma F), and a third gene, flgN, which we show is required for filament assembly. Identification of the Y. enterocolitica fliA and flgM genes was accomplished by functional complementation of both S. typhimurium and Y. enterocolitica mutations and by DNA sequence analysis. The Y. enterocolitica fliA gene, encoding the flagellar-specific sigma-factor, sigma F, maps immediately downstream of the three flagellin structural genes. The flgM and flgN genes, encoding anti-sigma F and a gene product required for filament assembly, respectively, map downstream of the invasin (inv) gene but are transcribed in the opposite (convergent) direction. By using Northern blot analyses we show that transcription of both fliA and flgM is immediately arrested when cells are exposed to 37 degrees C, coincident with the timing of virulence gene induction. Unlike S. typhimurium flgM mutants, Y. enterocolitica flgM mutants are fully virulent.

Hypopituitarism secondary to penetrating head trauma is extremely rare, and its diagnosis may be delayed for several years. We present a patient who developed hypopituitarism secondary to a transfacial gunshot wound and who experienced damage to the hypophysis secondary to transmission of energy and bullet fragments. The importance of a computerized tomographic scan of the head in facial gunshot wound is discussed.

Mifepristone (RU-486), a potent progesterone receptor antagonist and inducer of cytochromes P-450, is currently in use in Europe, particularly as a post-coital oral contraceptive. Soon it will be available in the United States, as well. Since progesterone has been implicated in the pathogenesis of acute attacks of porphyria, the use of RU-486 or related compounds might be considered in porphyric patients. However, as with other cytochrome P-450 inducers, RU-486 may have the ability to precipitate or exacerbate attacks of acute porphyria. The acute porphyrias in relapse are associated with an increase in activity of delta-aminolevulinic acid synthase, the first and normally rate-controlling enzyme in heme biosynthesis. We have used primary cultures of chick embryo liver cells to test the ability of RU-486 to induce delta-aminolevulinic acid synthase activity and mRNA, cytochromes P-450, porphyrin accumulation, and heme oxygenase. We found that RU-486, at concentrations observed in human plasma after a single oral dose, induced the mRNA and activity of delta-aminolevulinic acid synthase, both by itself and in the presence of deferoxamine, a potent iron chelator that inhibits ferrochelatase. RU-486 and deferoxamine together also produced significant accumulations of protoporphyrin. These results indicate that RU-486 may pose a risk in patients with known acute porphyria and should be used with caution. RU-486 increased the concentration of total cytochrome P-450, and the activity of erythromycin demethylase, an activity specifically catalyzed by cytochrome P-450 3A. Unlike several other porphyrogens (e.g. hydantoins, barbiturates), RU-486 does not produce accumulation of uroporphyrin or induction of heme oxygenase in chick embryo liver cells.

The inv gene encodes the protein invasin, which is the primary invasion factor for Yersinia enterocolitica in vitro and in vivo. Previous studies of Yersinia species have shown that inv expression and entry into mammalian cells are temperature regulated. Invasin production is reduced at the host temperature of 37 degrees C as compared to production at ambient temperature; consequently, this study was initiated to determine whether other host environmental signals might induce inv expression at 37 degrees C. An inv::phoA translational fusion was recombined on to the Y. enterocolitica chromosome by allelic exchange to monitor inv expression. Molecular characterization of expression of the wild-type inv gene and the inv::phoA fusion showed that invasin is not produced until early stationary phase in bacteria grown at 23 degrees C. Y. enterocolitica grown at 37 degrees C and pH 5.5 showed levels of inv expression comparable to those observed in bacteria grown at 23 degrees C. An increase in Na+ ions caused a slight increase in expression at 37 degrees C. However, expression at 37 degrees C was unaffected by anaerobiosis, growth medium, calcium levels, or iron levels. Additionally, Y. enterocolitica expressed invasin in Peyer's patches two days after being introduced intragastrically into BALB/c mice. These results suggest that invasin expression in Y. enterocolitica may remain elevated early during interaction with the intestinal epithelium, a site at which invasin was shown to be necessary.

Epidemiological data suggest that the ability to invade the intestinal epithelium of mammals is an essential virulence determinant of Yersinia enterocolitica. The chromosomally encoded Y. enterocolitica 8081v invasion gene, inv, was disrupted to assess its role in pathogenesis. The inv mutant was unable to invade cultured epithelial cells as efficiently as wild type. Furthermore, when mice were infected intragastrically, the inv mutant was extremely deficient at penetrating the murine intestinal epithelium. Analysis of the course of infection showed that the inv mutant had distinct differences relative to wild type in the distribution of visible infectious foci and in tissue colonization; however, the mutant and wild-type strains had similar median lethal doses for both orally and intraperitoneally infected mice. The invasion defect of the inv mutant was fully complemented in vitro and in vivo by introduction of the wild-type inv gene in trans. The inv gene product, invasin, appears to play a vital role in promoting entry during the initial stage of infection. During the subsequent establishment of a systemic infection, invasin may be of secondary importance, since the Y. enterocolitica inv mutant was as proficient as wild-type at causing a fatal infection in mice. The possible role of invasin in a naturally occurring Y. enterocolitica infection is discussed.