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Mumps is a vaccine-preventable infectious disease diffuse worldwide. The implementation of mumps vaccination reduced largely the spread of infection. On 11,327 Medical School students the prevalence of mumps positive antibodies was evaluated according to dose/doses of vaccine, year of birth and sex. Compliance to mumps vaccine was low in students born before 1990 but increased consistently after this year, above all compliance to two doses, due to the implementation of the vaccine offer. Positivity of mumps antibodies is significantly (p &lt, 0.0001) lower in students vaccinated once (71.2%) compared to those vaccinated twice (85.4%). In addition, students born after 1995, largely vaccinated twice, showed a seropositivity near to 90%. Further, females had a significantly (p &lt, 0.0001) higher proportion of positive antibodies after vaccination than males, both one (74.6% vs. 64.7%) and two doses (86.8% vs. 82.9%). Finally, seropositivity after two vaccine doses remains high (86.1%) even 15 years after the second dose. In conclusion, the research highlighted that vaccination against mumps reaches a good level of coverage only after two doses of vaccine persisting at high levels over 15 years and induces a more significant response in females.

In 2017 in Italy, a number of vaccinations became mandatory or started to be recommended and offered free of charge. In this study, we aimed at assessing the coverage rates for those vaccinations in the pre-mandatory era among students at the School of Medicine of Padua University studying the degree course in medicine and surgery (future physicians) on the basis of the vaccination certificates presented during health surveillance. The vaccinations considered were those against pertussis, rubella, mumps, measles, varicella, Haemophilus influenzae type b (which became mandatory in 2017), pneumococcus, meningococcus C and meningococcus B (only suggested and offered for free since 2017). The study enrolled 4706 students of medicine and surgery. High vaccine uptake was observed, especially in younger students (born after 1990), with vaccines against pertussis, rubella, mumps and measles. Good completion for Haemophilus influenzae type b and meningococcus C was also observed. Very low coverage rates (all under 10%) for vaccination against varicella, pneumococcus and meningococcus B were observed. In conclusion, uptake for some non-mandatory vaccines was below the recommended threshold, although younger generations showed a higher uptake, possibly as a results of policy implemented at the national level. Our findings support the idea to consider health surveillance visits also as an additional opportunity to overcome confidence and convenience barriers and offer vaccine administration.

Before the introduction of universal vaccination, hepatitis B caused high morbidity and mortality, especially among healthcare workers. In the present study, the immune status against hepatitis B was assessed in a cohort of 11,188 students of the degree courses of the School of Medicine of the University of Padua (Italy) who had been subjected to mandatory vaccination in childhood or adolescence and who will be future healthcare workers. The variables that influence the antibody response to vaccination are mainly the age at which the vaccine was administered and sex. If vaccination was administered before one year of age, there is a high probability (around 50%) of having an antibody titer lower than 10 IU/L compared to those vaccinated after one year of age (12.8%). The time between vaccine and analysis is not decisive. Furthermore, female sex, but only if vaccination was administered after one year of age, shows a significant (p = 0.0008) lower percentage of anti-HBs below 10 IU/L and a greater antibody titer (p &lt, 0.0001). In conclusion, the differences related to the age of vaccination induce more doubts than answers. The only plausible hypothesis, in addition to the different immune responses (innate and adaptive), is the type of vaccine. This is not easy to verify because vaccination certificates rarely report it.

Measles is a very contagious infectious disease, and vaccination is the only medical aid to counter the spread of the infection. The aim of this study was to evaluate the influence of vaccination schedule and type of vaccine, number of doses, and sex on the immune response. In a population of Italian medical students (8497 individuals born after 1980 with certificate of vaccination and quantitative measurement of antibodies against measles), the prevalence of positive antibodies to measles and antibody titer was measured. Vaccination schedule such as number of doses and vaccine type (measles alone or combined as measles, mumps and rubella (MMR)) and sex were the variables considered to influence the immune response. The vaccination schedule depends on the year of birth: students born before 1990 were prevalently vaccinated once and with measles vaccine alone (not as MMR). One dose of vaccine induces a significantly (p &lt, 0.0001) higher positive response and antibody titer than two doses, in particular when measles alone is used (p &lt, 0.0001). Females have a significantly higher percentage of positive response (p = 0.0001) than males but only when the MMR formulation was used. Multiple linear regression confirms that sex significantly influences antibody titer when only MMR is used, after one (p = 0.0002) or two (p = 0.0060) doses. In conclusion, vaccination schedule and, partially, sex influence immune response to measles vaccination. Most notably, the measles vaccine alone (one dose) is more effective than one and two doses of MMR.

Objective: Rubella is a very diffusive but relatively benign infectious disease unless contracted during pregnancy, when it causes congenital rubella syndrome. The aim of this research was to determine the prevalence and titer of antirubella antibodies in a population of future healthcare workers (students at the school of medicine).Methods: The cohort consisted of 11,022 students who underwent antibody analysis after the presentation of a vaccine certificate.Results: Vaccination compliance was very high, particularly in younger students (born after 1995), reaching almost 100% (at least one dose). Unvaccinated students born before 1990 had high seropositivity (>95%), but this percentage dropped to zero among the youngest students. Variables affecting antibody titer included year of birth and sex. Considering only vaccinated students, a greater antibody response was observed if the vaccine was administered between 8 and 10 years of age. Female sex was associated with more significant (p < 0.0001) positivity and higher antibody titer after one and two doses. However, this difference appeared less consistent in relation to year of birth.Conclusions: The studied population exhibited excellent vaccination compliance, high seropositivity, and high antibody titer. Vaccine and immune coverage were higher than what is deemed necessary to achieve herd immunity.

The present research evaluated the course of cobalt and chromium in the blood and urine after the revision of metal-on-metal with a ceramic-on-polyethylene total hip arthroplasty. Seven patients were enrolled for hip prosthesis revision owing to ascertained damage of the implant. Metals in the blood and urine were evaluated before and after the hip revision. The double measurement before the total hip revision revealed high levels of metal ions (on average, 88.1 µg/L of cobalt in the blood, 399.0 µg/g of creatinine cobalt in the urine, 46.8 µg/L of chromium in the blood, and 129.6 µg/g of creatinine chromium in the urine at the first measurements), with an increasing trend between the first and second dosage. Within a week after the hip revision, the levels of metal ions significantly decreased by approximately half. Four to six months after the operation, the cobalt levels were found near to the reference values, whereas the chromium levels reached 25% of the values measured before the revision. The revision of malfunctioning metal-on-metal implants produced a dramatic decrease of metal ions in biological fluids, although it did not completely rescue the chromium level.

The aim of this study was to evaluate the levels of As, Be, Bi, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Se, Tl, V, and Zn, by inductively coupled plasma-mass spectrometry (ICP-MS) in the urine of two groups of patients with two different types of metal-on-metal (MoM) total hip prostheses (ASR DePuy&reg, group A, 25 patients, total Met-Met System Lima&reg, group B, 28 patients). The determination of metals reflected a steady-state release (group A: 9 years after surgery and group B: 6 years after surgery). The results obtained confirmed the increase of Co and Cr urinary levels in both group when compared with the reference values for the general population adopted by the Italian Society of Reference Values (SIVR). In particular, Co and Cr levels exceeded the threshold values in urine, respectively, of 30 &mu, g and 21 &micro, g, adjusted to creatinine based on the threshold in whole blood of 7 &mu, g/L proposed by the Medicines and Healthcare Products Regulatory Agency (MHRA). Regarding the other investigated metals, significantly higher values were found in Group A than in Group B. These differences could be due to the type of hip prosthesis implanted, the longer period of time since the implantation, as well as many other factors such as diet, age, drug consumption, physical activity, or presence of dental fillings. The continuous monitoring over the years of metal concentrations in patients carrying a prosthesis could be useful to better identify the sources of these metals.

Non-occupational lead poisoning is not rare, mainly occurring in domestic situations in children, but also in adults. Lead poisoning was observed in a 65 years-old woman non-exposed to risk that caught our attention with a diagnostic suspicion of acute intermittent porphyria according to recurrent episodes of abdominal pain and neuropathy of upper limbs. Acute intermittent porphyria was excluded by a laboratory investigation that showed instead severe lead poisoning. After several thorough examinations of the domestic environment, the source of intoxication has been detected in some cooking pots that released high concentrations of lead. Ethylenediamine tetracetic acid disodium calcium therapy (three cycles) reduced consistently blood lead concentration and, after one year, neuropathy was almost entirely recovered.

Total hip arthroplasty (THA) with metal-on-metal (MoM) bearings have shown problems of biocompatibility linked to metal ion release at the local level causing an adverse reaction to metal debris (ARMD) and at a systemic level. The aim of this study was to evaluate clinical and radiological outcomes, and metal ion concentrations in the blood and urine of patients who underwent THA with the LIMA Met-Met hip system. Patients with ceramic-on-ceramic (CoC) bearings were included as a control group. In this study, 68 patients were enrolled: 34 with MoM THAs and 34 with CoC THAs. Patients were evaluated clinically (Harris Hip Score, SF-36) and radiologically at a median of 7.4 years after surgery. Whole blood and urinary cobalt and chromium levels were also assessed. Both types of implants were comparable in terms of clinical and functional results. Ion levels were significantly higher in the MoM group compared with CoC group 7 years after surgery. No correlations were found between metal ion levels and patient demographics, functional and radiological outcomes, and prosthesis features. Patient monitoring is thus advised to establish if prosthesis revision is necessary, especially in the case of MoM THA.

Healthcare workers are a population exposed to several infectious diseases, and an immunization programme is essential for the maintenance of good vaccination coverage to protect workers and patients. A population of 10,653 students attending degree courses at Padua Medical School (medicine and surgery, dentistry and health professions) was screened for vaccination coverage and antibody titres against rubella, mumps, and measles. The students were subdivided into five age classes according to their date of birth: those born before 1980, between 1980 and 1985, between 1986 and 1990, between 1991 and 1995, and after 1995. Vaccination coverage was very low in students born before 1980, but the rate of positive antibody titre was high due to infection in infancy. Increasing date of birth showed increased vaccination coverage. In contrast, immune coverage was high for rubella (more than 90%) but not for mumps and measles (approximately 80%). An &ldquo, anomaly&rdquo, was observed for mumps and measles in the cohort born between 1991 and 1995, probably due to the trivalent vaccine formulation. Students born after 1990 showed vaccination coverage that exceeded 90%. It is therefore very likely that we will have a future generation of healthcare workers with optimal vaccination coverage.

Aims: The goal of the present study was to establish the efficacy of the measles vaccine and the validity of the vaccination schedule adopted in Italy. Materials & methods: The following procedures are adopted: analyze the compliance to the vaccination schedule; assess the seroprevalence of measles antibodies according to the year of birth and the number of doses; and investigate the persistence of positive antibodies post-vaccination. We gathered and elaborated data of both vaccination history and seroprevalence against measles in a large population of students (4195) belonging to the Medical School of Padua University (Italy). Results: Our results reveal a requirement for a two-dose vaccination schedule to ensure protection from the disease. Nevertheless, these results clearly indicate that the percentage of seropositivity reached using the two-dose strategy is below the percentage (95%) that ensures optimal population immunity. Conclusion: It is uncertain whether immune coverage persists when circulating antibodies vanish, but two vaccine doses should prevent disease outbreaks.

ABSTRACT Since 1992, the inclusion of HBV vaccination has been recommended by the WHO for all immunization programs implemented by nations. In Europe, the introduction of HBV vaccination has markedly reduced the incidence of acute HBV, and before the introduction of HBV vaccine, healthcare workers (HCW) were at considerable risk of infection. The present review discusses the main problems regarding three fundamental issues in hospital settings: prevention of HBV in HCW, protection induced by vaccination (problems regarding nonresponders) and risk for HCW exposed to blood-borne pathogens (occupational risk). The screening of HBV markers plays a decisive role in evaluating the degree of immune coverage in subjects exposed to biological risk and permits an increase in immune coverage through vaccine implementation.

The urinary excretion rate is calculated based on short-term, defined time sample collections with a known sample mass, and this measurement can be used to remove the variability in urine concentrations due to urine dilution. Adjustment to the urinary excretion rate of hippuric acid was evaluated in 31 healthy volunteers (14 males and 17 females). Urine was collected as short-term or spot samples and tested for specific gravity, creatinine and hippuric acid. Hippuric acid values were unadjusted or adjusted to measurements of specific gravity, creatinine or urinary excretion rate. Hippuric acid levels were partially independent of urinary volume and urinary flow rate, in contrast to specific gravity and creatinine, which were both highly dependent on the hippuric acid level. Accordingly, hippuric acid was independent on urinary specific gravity and creatinine excretion. Unadjusted and adjusted values for specific gravity or creatinine were generally closely correlated, especially in spot samples. Values adjusted to the urinary excretion rate appeared well correlated to those unadjusted and adjusted to specific gravity or creatinine values. Thus, adjustment of crude hippuric acid values to the urinary excretion rate is a valid procedure but is difficult to apply in the field of occupational medicine and does not improve the information derived from values determined in spot urine samples, either unadjusted or adjusted to specific gravity and creatinine.

Despite hepatitis B virus (HBV) immunization, a percentage of healthy individuals display an antibody titre below the threshold for clinical protection (10 IU/L). In order to predict the existence of this inducible immunological response, the precise anti-HBs titre required to achieve protection in immunized patients with waned HBs antibodies must first be determined. A total of 4486 vaccinated students attending the University of Padova Medical, Science, and Veterinary School were recruited for study between 2004 and early 2012. The baseline concentration of anti-HBs was measured at enrolment. Participants displaying anti-HBs titre < 10 IU/L at the follow-up examination (mean 10.8 years) were given a booster vaccination and retested 20-30 days later. At enrolment, 87.6% of the 4486 vaccinated subjects showed persistence of anti-HBs higher than 10 IU/L. Of the 279 booster-vaccinated subjects, 94.6% achieved the cut-off titre. Booster-induced immunological response was correlated to the pre-booster titre level, with ≥ 2 IU/L ensuring a robust positive response and less than 2 IU/L being associated with the probability of developing insufficient levels of antibodies. Pre-booster antibody titre higher than 2 IU/L in adults might be predictive of an anamnestic response to booster vaccination, whereas titres below this value may indicate likelihood of non-response.

For many years, toxicological investigations have shown that the sensitivity of kidney to xenobiotics evolves depending on the stage of life. The increasing requirement for information on the potential nephrotoxic effect of drugs during human embryonic development, childhood, adulthood and senescence has potentiated toxicological studies in vivo. Rodents, specifically rats, are the primary animal models used in toxicology testing. Despite the popularity of this approach, there are a number of doubts about the appropriateness of rats for the examination of changes in toxicological responses during different stages of life. This perspective tackles the issue of evaluating whether rats fail to adequately mimic the human kidney response to xenobiotic agents through a critical analysis of the literature. We conclude that rats constitute a good model for toxicological investigations during embryonic development, youth and adulthood. However, senescent rats frequently undergo spontaneous kidney degeneration caused by chronic progressive nephropathy, making them a poor model for the study of kidney responses to xenobiotics.

Several esterase inhibitors, not capable of causing peripheral neuropathy by themselves, exacerbate organophosphate-induced delayed polyneuropathy (OPIDP) and other axonopathies. This effect was called promotion of axonopathies and it was found not to be associated with inhibition of neuropathy target esterase (NTE), the molecular target of OPIDP. The search for an esterase as the target of promotion has started long ago, when an eterogeneous group of esterases-hydrolysing phenyl valerate (PV) was identified in hen's sciatic nerve by means of selective inhibitors. Correlation studies in vivo indicated that the target of promotion may have been among the proteins present in the soluble fraction. When this soluble PV-esterase activity was separated on a Sephacryl-S-300 column, correlation was found between promotion and its inhibition in vivo. The electrophoretic analysis of this fraction indicated the presence of several proteins. Subsequent ion-exchange chromatography identified a protein of about 80 kDa molecular weight that was associated with PV-esterase activity. The inhibition of this activity did also correlate with promotion. The sequence of this protein identified it as ovotransferrin, but commercial preparations of ovotransferrin were found to lack PV-esterase activity. Binding experiments on this purified PV-activity and on commercial ovotransferrin using radiolabelled promoters were inconclusive. Titration of this PV-activity showed that about 20-30% of it is resistant to high concentrations of several inhibitors, suggesting heterogeneity of the fraction. In fact, bi-dimensional electrophoresis indicated the presence of several proteins. Finally, in vivo correlation experiments with p-toluensulfonyl fluoride showed that whereas this chemical does not promote OPIDP induced by dibutyl dichlorovinyl phosphate, it does inhibit about 80% of this PV-activity. In conclusion, available data indicate that the target of promotion is unlikely to be ovotransferrin. However, all promoters identified so far are esterase inhibitors suggesting that the target of promotion might be, indeed, a protein with esteratic activity.

Blood metal ion levels in 72 patients with large head metal-on-metal hip arthroplasty were studied to determine the correlation between the values measured in whole blood and urine. Urinary cobalt and chromium levels of 30μg and 21μg, respectively, adjusted to creatinine were found to correspond to the 7μg/l cut-off value that has been accepted in whole blood. Cobalt and chromium levels in whole blood and urine both significantly correlated with increased acetabular component inclination angle over 50 degrees and pain scores. There was no correlation with socket anteversion angle or femoral head diameter. The data support the use of urinary measurement of metal ions adjusted to creatinine to monitor patients with large head metal-on-metal total hip arthroplasty.

The impact of sexual hormones on kidney metabolism may be relevant to modulate the effect of nephrotoxic substances. In the present research the effects of castration, testosterone, and estradiol on renal oxidative metabolism [cytochrome P-450 (CYP)], glutathione pathway [glutathione content (GSH), glutathione S-transferases (GST) activities, and cysteine-conjugate s-lyase [as glutamine transaminase K (GTK) activity] have been studied. Naive male rats have a significantly lower GSH content but show a significantly higher GST activities and CYP content than females, whereas no sex difference was for GTK activity. Castration significantly reduces GSH content and GTK activity in females and CYP content in males, partially but significantly restored by testosterone. Testosterone significantly increases GSH content and GTK activity in males and GTK activity and CYP content in females. Estradiol increases GSH content in males, whereas decreases GST activities in females and CYP content in both sexes. Castration followed by testosterone treatment increases GTK activity in both sexes whereas increases CYP content and reduces GSH content in females. Castration followed by estradiol treatment increases GSH content and reduces GST activities in males and CYP content in both sexes. In conclusion, the results suggest that sex hormones influence metabolic pathways of the kidney and that they are probably responsible of the sex- related differences of chemical-induced nephrotoxicity. An univocal model to define sex-related toxicity of xenobiotic substances is far to be identified.

Certain esterase inhibitors, such as carbamates, phosphinates and sulfonyl halides, do not cause neuropathy as some organophosphates, but they may exacerbate chemical or traumatic insults to axons. This phenomenon is called promotion of axonopathies. Given the biochemical and toxicological characteristics of these compounds, the hypothesis was made that the target of promotion is a phenyl valerate (PV) esterase similar to neuropathy target esterase (NTE), the target of organophosphate induced delayed polyneuropathy. However, attempts to identify a PV esterase in hen peripheral nerve have been, so far, unsuccessful. We tested several esters, other than PV, as substrates of esterases from crude homogenate of the hen peripheral nerve. The ideal substrate should be poorly hydrolysed by NTE but extensively by enzyme(s) that are insensitive to non-promoters, such as mipafox, and sensitive to promoters, such as phenyl methane sulfonyl fluoride (PMSF). When phenyl benzoate (PB) was used as substrate, about 65% of total activity was resistant to the non-promoter mipafox (up to 0.5 mM, 20 min, pH 8.0), that inhibits NTE and other esterases. More than 90% of this resistant activity was sensitive to the classical promoter PMSF (1 mM, 20 min, pH 8.0) with an IC{sub 50} of about 0.08 mM (20more » min, pH 8.0). On the contrary, the non-promoter p-toluene sulfonyl fluoride caused only about 10% inhibition at 0.5 mM. Several esterase inhibitors including, paraoxon, phenyl benzyl carbamate, di-n-butyl dichlorovinyl phosphate and di-isopropyl fluorophosphate, were tested both in vitro and in vivo for inhibition of this PB activity. Mipafox-resistant PMSF-sensitive PB esterase activity(ies) was inhibited by promoters but not by non promoters and neuropathic compounds.« less

Mitochondria possess an inner membrane channel, the permeability transition pore, which is inhibited by cyclosporin A (CsA) and by matrix protons. As suggested recently by our laboratory, pore closure by these inhibitors may be due to dissociation of mitochondrial cyclophilin (CyP-M), a matrix peptidyl-prolyl-cis-trans isomerase, from its putative binding site on the pore. Unbinding of CyP-M would follow a CsA-dependent or proton-dependent change in conformation of the CyP-M molecule. It is interesting that upon binding of CsA the enzymatic activity of CyP-M is inhibited, but it is not clear whether this event plays a role in pore inhibition. Here we report experiments designed to further test the role of CyP-M in pore function. Our results indicate that CyP-M-dependent and independent mechanisms of pore activation may exist, and that the peptidylprolyl-ds-rrafts-isomerase activity of CyP-M is not necessarily involved in pore modulation by CyP-M. (Mol Cell Biochem 174: 181–184,1997)

Survival of tumor cells is favored by mitochondrial changes that make death induction more difficult in a variety of stress conditions, such as exposure to chemotherapeutics. These changes are not fully characterized in tumor mitochondria, and include unbalance of the redox equilibrium, inhibition of permeability transition pore (PTP) opening through kinase signaling pathways and modulation of members of the Bcl-2 protein family. Here we show that a novel chemotherapeutic, the Gold(III)-dithiocarbamato complex AUL12, induces oxidative stress and tumor cell death both favoring PTP opening and activating the pro-apoptotic protein Bax of the Bcl-2 family. AUL12 inhibits the respiratory complex I and causes a rapid burst of mitochondrial superoxide levels, leading to activation of the mitochondrial fraction of GSK-3α/β and to the ensuing phosphorylation of the mitochondrial chaperone cyclophilin D, which in turn facilitates PTP opening. In addition, following AUL12 treatment, Bax interacts with active GSK-3α/β and translocates onto mitochondria, where it contributes to PTP induction and tumor cell death. These findings provide evidence that targeting the redox equilibrium maintained by mitochondria in tumor cells allows to hit crucial mechanisms that shield neoplasms from the toxicity of many anti-tumor strategies, and identify AUL12 as a promising chemotherapeutic compound.

The permeability transition, a sudden permeability increase of the inner mitochondrial membrane that is greatly favored by Ca2+ accumulation, has puzzled mitochondrial scientists for more than 40 years. It is now recognized that this phenomenon is mediated by opening a high conductance channel (the mitochondrial permeability transition pore) whose open-closed transitions are highly regulated. Through the pore mitochondria may participate in intracellular signalling, and release proteins involved in amplification of the cell death cascade triggered by a variety of physiological and pathological stimuli. Yet, the basic questions of the molecular nature of the permeability transition pore, its physiological role and its very occurrence in vivo remain a matter of intense debate. This short review is meant to summarize our current views on the mitochondrial permeability transition, its perspectives, and our strategies to resolve at least some of the outstanding issues about its nature and function.

Mitochondria of higher cells contain a channel, the permeability transition pore (PTP), that, when open, increases the permeability of the inner membrane to ions and small molecules. The PTP may be of importance in cellular calcium homeostasis and in the execution of programmed cell death, and the definition of the molecular structure of the PTP is, therefore, an urgent problem in cell biology. Because there has been so little structural information to rely on, theories on the nature of the PTP have been unconstrained and ideas have ranged from degradation of membrane phospholipids to cross-linking of known carrier proteins. The discovery of the high-affinity inhibitor cyclosporin A (CsA) allowed a clear pharmacologic definition of the PTP, and paved the way to purification of cyclophilin D (CyP-D), a matrix peptidyl prolyl cis-trans isomerase that seems to mediate PTP inhibition by CsA. CyP-D is a soluble protein, however, and the nature of the ion-conducting channel remains undefined. Here, we review the information provided by the strategy of site-selective modification of the PTP with specific chemical reagents.

This review summarizes recent work on the regulation of the permeability transition pore, a cyclosporin A-sensitive mitochondrial channel that may play a role in intracellular calcium homeostasis and in a variety of forms of cell death. The basic bioenergetics aspects of pore modulation are discussed, with some emphasis on the links between oxidative stress and pore dysregulation as a potential cause of mitochondrial dysfunction that may be relevant to cell injury.

Energized mitochondria in sucrose medium take up a Ca2+ pulse but do not show opening of the permeability transition pore (MTP) upon membrane depolarization by uncoupler. This is due to locking of the pore in the closed conformation by matrix acidification and fast Ca2+ efflux following membrane depolarization (Petronilli, V., Cola, C., & Bernardi P. (1993) J. Biol. Chem. 268, 1011-1016). Here we show that addition of diethyl pyrocarbonate (DPC) prior to membrane depolarization restores the ability of uncoupler to induce MTP opening. Since DPC does not modify the rate and extent of matrix acidification and the rate and extent of Ca2+ release following addition of uncoupler, its effects on pore opening appear to be due to modification of histidyl residues regulating the pore open-closed probability. This hypothesis was confirmed in studies with deenergized mitochondria incubated in potassium thiocyanate medium. While at acidic pH values pore opening is otherwise prevented, DPC allows Ca2(+)-dependent pore opening at pH 6.5 in a process that maintains full sensitivity to cyclosporin A. Pore induction by DPC can be completely prevented and partially reversed by hydroxylamine, indicating that the effect of DPC can be specifically traced to carbethoxylation of histidyl residue(s) rather than to reaction with tyrosyl or sulfhydryl groups, while the possible involvement of lysyl residues cannot be excluded. Since DPC increases the pore open probability even at matrix pH values between 7.0 and 7.7, we propose that reversible protonation of one or more histidyl residues on the matrix side of the MTP plays a role in the physiological modulation of pore opening.

Mammalian mitochondria possess an inner membrane channel, the permeability transition pore (MTP), which can be inhibited by nanomolar concentrations of cyclosporin (CS) A. The molecular basis for MTP inhibition by CSA remains unclear. Mitochondria also possess a matrix cyclophilin (CyP) with a unique N-terminal sequence (CyP-M). To test the hypothesis that it interacts with the MTP, we have studied the interactions of CyP-M with rat liver mitochondria by Western blotting with a specific antibody against its unique N terminus. Although sonication in isotonic sucrose at pH 7.4 releases a large proportion of CyP-M, a sizeable CyP-M fraction sediments with submitochondrial particles at 150,000 × g. We show that the interactions of this CyP-M pool with submitochondrial particles are disrupted (i) by the addition of CSA, which inhibits the pore, but not of CSH, which does not, and (ii) by acidic pH condition, which also leads to selective inhibition of the MTP; furthermore, we show that the effect of acidic pH on CyP-M binding is prevented by diethylpyrocarbonate, which fully prevents the inhibitory effect of H+ on the MTP (Nicolli, A., Petronilli, V., and Bernardi, P.(1993) Biochemistry 32, 4461-4465). These data suggest that CyP-M binding is involved in opening of the MTP and that pore inhibition by CSA and protons may be due to unbinding of CyP-M from its putative binding site on the MTP. A role for CyP-M in MTP regulation is also supported by a study with a series of CSA derivatives with graded affinity for CyP. We show that with each derivative the potency at inhibition of the peptidylprolyl cis-trans-isomerase activity of CyP-M purified to homogeneity is similar to that displayed at inhibition of MTP opening, relative to that displayed by CSA. Decreased binding to CyP-M (but not CyP-A) and decreased efficiency at MTP inhibition is obtained by substitutions in position 8 while a 4-substituted, nonimmunosuppressive derivative is as effective as the native CSA molecule, indicating that calcineurin is not involved in MTP inhibition by CSA.

Mitochondria from a variety of sources possess a regulated inner membrane channel, the permeability transition pore (MTP), which is responsible for the 'permeability transition', a sudden permeability increase to solutes with molecular masses < or = 1500 Da, most easily observed after Ca2+ accumulation. The MTP is a voltage-dependent channel blocked by cyclosporin A with Ki in the nanomolar range. The MTP open probability is regulated by both the membrane potential and matrix pH. The probability of pore opening increases as the membrane is depolarized, while it decreases as matrix pH is decreased below 7.3 through reversible protonation of histidine residues. Many physiological and pathological effectors, including Ca2+ and ADP, modulate MTP operation directly through changes of the gating potential rather than indirectly through changes of the membrane potential (Petronilli, V., Cola, C., Massari, S., Colonna, R. and Bernardi, P. (1993) J. Biol. Chem. 268, 21939-21945). Here we present recent work from our laboratory indicating that (i) the voltage sensor comprises at least two vicinal thiols whose oxidation-reduction state affects the MTP gating potential; as the couple becomes more oxidized the gating potential increases; conversely, as it becomes more reduced the gating potential decreases; (ii) that MTP opening is fully reversible, as mitochondria maintain volume homeostasis through several cycles of pore opening/closure; and (iii) that the mechanism of MTP inhibition by cyclosporin A presumably involves a mitochondrial cyclophilin but does not utilize a calcineurin-dependent pathway.

Addition of A23187 plus EDTA to rat liver mitochondria induces a common uniport pathway for monovalent cations. In this study, we have carried out a detailed characterization of the flow/force relationship for K+ transport along this pathway under steady state conditions. In the presence of EDTA, the K+ conductance is a linear function of external K+ in the range 0-20 mM K+, with a slope of 0.15 nmol of K+ x mg of protein-1 x min-1 x mV-1. The K+ conductance is inhibited by Mg2+ in the range 10(-9)-10(-6) M, while K+ flux is stimulated by the sulfhydryl group reagent mersalyl. Uniport activity can be detected in native mitochondria. These findings are compatible with the notion that electrophoretic K+ flux across the inner membrane takes place via a regulated K+ uniport with the potential of transporting K+ at rates in excess of 600 nmol x mg of protein-1 x min-1.