Experimental Demonstration of Hadron Beam Cooling Using Radio-Frequency Accelerated Electron Bunches.

Cooling of beams of gold ions using electron bunches accelerated with radio-frequency systems was recently experimentally demonstrated in the Relativistic Heavy Ion Collider at Brookhaven National Laboratory. Such an approach is new and opens the possibility of using this technique at higher energies than possible with electrostatic acceleration of electron beams. The challenges of this approach include generation of electron beams suitable for cooling, delivery of electron bunches of the required quality to the cooling sections without degradation of beam angular divergence and energy spread, achieving the required small angles between electron and ion trajectories in the cooling sections, precise velocity matching between the two beams, high-current operation of the electron accelerator, as well as several physics effects related to bunched-beam cooling. Here we report on the first demonstration of cooling hadron beams using this new approach.

Re-emergent tremor in Parkinson's disease: the effect of dopaminergic treatment.

BACKGROUND AND PURPOSE: Patients with Parkinson's disease (PD) with resting tremor may be affected by a tremor that appears after a varying latency while a posture is maintained, a phenomenon referred to as re-emergent tremor (RET). The aim of the study was to evaluate the occurrence and clinical features of RET in patients with PD tested off and on treatment, and to compare the effect of dopaminergic treatment on RET with the effect on resting and action tremor. METHODS: We consecutively enrolled 100 patients with PD. Patients were clinically evaluated 24 h after withdrawal of therapy (off-treatment phase) and 60 min after therapy administration (on-treatment phase). We collected the demographic and clinical data of patients with PD. The severity of the disease was assessed by means of the Hoehn and Yahr scale and Movement Disorder Society-sponsored revision of the Unified Parkinson's Disease Rating Scale part III. We evaluated the latency, severity and body side affected both off and on treatment in patients with RET. RESULTS: Re-emergent tremor was present in 24% of the patients with PD off treatment and in 19% of the patients on treatment. Dopaminergic treatment reduced the clinical severity of RET. Dopaminergic treatment increased the number of patients with unilateral RET and reduced the number of those who had bilateral RET. RET and resting tremor responded similarly to dopaminergic treatment, whereas action tremor was less responsive. Patients with RET had milder motor symptoms than patients without RET both off and on treatment. CONCLUSIONS: Dopaminergic treatment modified RET occurrence, severity and body distribution. Dopaminergic depletion plays a role in the pathophysiology of RET.

Frequency distribution of six cytokine gene polymorphisms in North- and South-Italy.

Allelic and genotype frequencies of four cytokine genes were obtained from 738 subjects from North- and South-Italy. Populations were in Hardy-Weinberg equilibrium for all genes but significantly differed in the frequency of all SNPs and three haplotypes. In the MDS graph, they were plotted in separate positions close to Europeans and an Ivorian population, respectively.

Operational Head-on Beam-Beam Compensation with Electron Lenses in the Relativistic Heavy Ion Collider.

Head-on beam-beam compensation has been implemented in the Relativistic Heavy Ion Collider in order to increase the luminosity delivered to the experiments. We discuss the principle of combining a lattice for resonance driving term compensation and an electron lens for tune spread compensation. We describe the electron lens technology and its operational use. To date, the implemented compensation scheme approximately doubled the peak and average luminosities.

Embolization of left spermatic vein in non-obstructive azoospermic men with varicocele: role of FSH to predict the appearance of ejaculated spermatozoa after treatment.

PURPOSE: Varicocele repair in non-obstructive azoospermia (NOA) was occasionally associated to ejaculated spermatozoa independently from clinical and laboratory measures. We performed a prospective study in infertile men affected by NOA and left side varicocele to find whether or not the appearance of ejaculated spermatozoa after varicocele repair is predicted by baseline measures. METHODS: Patients with NOA and grade II, or grade III left side varicocele were submitted to hormone analysis and to scrotal color Doppler ultrasound (CDU). Azoospermia was confirmed in 23 patients aged 25-47 years who were than submitted to varicocele repair through a retrograde internal spermatic vein embolization. Patients were re-evaluated after 6 months. RESULTS: Six months after varicocele repair 12 patients (52.2 %) were still azoospermic (Group 1) while 11 patients (47.8 %) reported ejaculated spermatozoa (Group 2) [sperm count: 1.3 × 10(6)/mL; 0.5 × 10(6)/mL-1.6 × 10(6)/mL (median 25th-75th centiles)]. Serum baseline FSH was lower in Group 2 compared to Group 1 (p = 0.012), while no differences between groups were revealed for all other clinical and laboratory parameters. ROC analysis indicated that baseline FSH level predicted the appearance of ejaculated spermatozoa after treatment [AUC = 0.811; 95 % Confidence Interval (CI) 0.6-0.9; p = 0.0029]. A cut-off level of FSH <10.06 mIU/mL identified 82.0 % of cases with ejaculated spermatozoa with a specificity of 81.8 % and a sensitivity of 83.3 %. CONCLUSION: Selected patients with NOA may show ejaculated spermatozoa after a non-invasive repair of a left side varicocele, therefore avoiding testicular sperm extraction. Baseline serum FSH was a valuable predictor for ejaculated spermatozoa after treatment.

Effect of nesiritide in patients with acute decompensated heart failure.

BACKGROUND: Nesiritide is approved in the United States for early relief of dyspnea in patients with acute heart failure. Previous meta-analyses have raised questions regarding renal toxicity and the mortality associated with this agent. METHODS: We randomly assigned 7141 patients who were hospitalized with acute heart failure to receive either nesiritide or placebo for 24 to 168 hours in addition to standard care. Coprimary end points were the change in dyspnea at 6 and 24 hours, as measured on a 7-point Likert scale, and the composite end point of rehospitalization for heart failure or death within 30 days. RESULTS: Patients randomly assigned to nesiritide, as compared with those assigned to placebo, more frequently reported markedly or moderately improved dyspnea at 6 hours (44.5% vs. 42.1%, P=0.03) and 24 hours (68.2% vs. 66.1%, P=0.007), but the prespecified level for significance (P≤0.005 for both assessments or P≤0.0025 for either) was not met. The rate of rehospitalization for heart failure or death from any cause within 30 days was 9.4% in the nesiritide group versus 10.1% in the placebo group (absolute difference, -0.7 percentage points; 95% confidence interval [CI], -2.1 to 0.7; P=0.31). There were no significant differences in rates of death from any cause at 30 days (3.6% with nesiritide vs. 4.0% with placebo; absolute difference, -0.4 percentage points; 95% CI, -1.3 to 0.5) or rates of worsening renal function, defined by more than a 25% decrease in the estimated glomerular filtration rate (31.4% vs. 29.5%; odds ratio, 1.09; 95% CI, 0.98 to 1.21; P=0.11). CONCLUSIONS: Nesiritide was not associated with an increase or a decrease in the rate of death and rehospitalization and had a small, nonsignificant effect on dyspnea when used in combination with other therapies. It was not associated with a worsening of renal function, but it was associated with an increase in rates of hypotension. On the basis of these results, nesiritide cannot be recommended for routine use in the broad population of patients with acute heart failure. (Funded by Scios; ClinicalTrials.gov number, NCT00475852.).

Chitosan nanoparticles are efficient carriers for delivering biodegradable drugs to neuronal cells.

Chitosan nanoparticles (NPs) are biocompatible drug carriers able to cross the blood-brain barrier and represent a promising drug delivery system to the central nervous system. We used chitosan NPs to deliver the D-Ala2-D-Leu5-enkephalin (DADLE) to neuronal cells in vitro. DADLE is a hypometabolising synthetic opioid potentially useful for biomedical applications, but its short plasmatic half-life makes its in vivo administration ineffective. Here, we demonstrate by immunoelectron microscopy that (1) chitosan NPs are capable to deliver the opioid to neuronal cells; (2) DADLE is released from the internalised, opioid-loaded NPs up to 48 h; (3) in the nucleus, DADLE binds the transcription/splicing sites; (4) cells treated with DADLE-loaded NPs undergo a decrease in transcription factor amounts and proliferation rate without damage to cell organelles. In this model, chitosan NPs protected the loaded opioid from degradation, thereby prolonging its intracellular effects. These findings suggest that these NPs are efficient for the systemic and tissue administration of opioids in vivo.

Diagnosis of hepatic steatosis and steatohepatitis in people with new-onset type 2 diabetes: a multidisciplinary approach.

AIMS: Non-Alcoholic-Fatty-Liver-Disease (NAFLD) is the most common cause of chronic liver disease in Western countries; closely linked to obesity and type 2 diabetes (T2DM), it is an additional cardiovascular risk factor. The aim of this study is to investigate the prevalence of NAFLD at T2DM onset. METHODS: 122 newly diagnosed T2DM patients were enroled; NAFLD was diagnosed using ultrasound and fibrosis risk calculated with an FIB4-score. Intermediate and high-risk patients were referred to a hepatologist and underwent transient elastography (TE). RESULTS: At T2DM diagnosis, 25% of patients were overweight, 47% were obese; ultrasound steatosis was present in 79% of patients; the average FIB-4 score was 1.4 (0.7). The NAFLD population was characterised by higher presence of obesity (60%, p 0.06); hypertension (56%, p 0.00); AST (26.3 (23.6) UI/L; p 0.00); ALT (49.3(41.0) UI/L p 0.00); FIB-4 score (1.6 (0.8); p 0.00). Among patients referred to a hepatologist, at TE, 65% had severe steatosis, 22% significant fibrosis and 25% advanced fibrosis. CONCLUSION: This is the first proposal of a NAFLD screening model at T2DM diagnosis. The high prevalence of fibrosis found at the early stage T2DM confirms the compelling need for early management of NAFLD through cost-effective screening and long-term monitoring algorithms.

Understanding the role of cerebellum in early Parkinson's disease: a structural and functional MRI study.

Increasing evidence suggests that the cerebellum may have a role in the pathophysiology of Parkinson's disease (PD). Hence, the scope of this study was to investigate whether there are structural and functional alterations of the cerebellum and whether they correlate with motor and non-motor symptoms in early PD patients. Seventy-six patients with early PD and thirty-one age and sex-matched healthy subjects (HS) were enrolled and underwent a 3 T magnetic resonance imaging (MRI) protocol. The following MRI analyses were performed: (1) volumes of 5 cerebellar regions of interest (sensorimotor and cognitive cerebellum, dentate, interposed, and fastigial nuclei); (2) microstructural integrity of the cerebellar white matter connections (inferior, middle, and superior cerebellar peduncles); (3) functional connectivity at rest of the 5 regions of interest already described in point 1 with the rest of brain. Compared to controls, early PD patients showed a significant decrease in gray matter volume of the dentate, interposed and fastigial nuclei, bilaterally. They also showed abnormal, bilateral white matter microstructural integrity in all 3 cerebellar peduncles. Functional connectivity of the 5 cerebellar regions of interest with several areas in the midbrain, basal ganglia and cerebral cortex was altered. Finally, there was a positive correlation between abnormal functional connectivity of the fastigial nucleus with the volume of the nucleus itself and a negative correlation with axial symptoms severity. Our results showed that structural and functional alterations of the cerebellum are present in PD patients and these changes contribute to the pathophysiology of PD in the early phase.

Ultrastructural detection of photosensitizing molecules by fluorescence photoconversion of diaminobenzidine.

Photodynamic therapy is a moderately invasive therapeutic procedure based on the action of photosensitizers (PSs). These compounds are able to absorb light, and dissipate energy through photochemical processes leading to the production of oxidizing chemical species (singlet oxygen, free radicals or reactive oxygen species) which can damage the cell molecular structures eventually inducing cell death. To increase the entering through the plasma membrane, a PS with suitable chemical structure can be modified by addition of chemical groups (e.g., acetate or phosphate): this affects both the fluorescence emission and of the photosensitizing properties of the native PS. The modified compounds behave as fluorogenic substrates (FSs), since inside the cell the bound groups can be enzymatically removed and the fluorescence and photosensitizing properties of the native molecules are restored. With the aim to detect the subcellular localization of photoactive molecules at transmission electron microscopy, we loaded cultured HeLa cells with two different FSs, Rose Bengal acetate (RB-Ac) or Hypocrellin B acetate (HypB-Ac), and took advantage of the photophysical properties of the intracellularly restored PS molecules to obtain the photoconversion of diaminobenzidine (DAB) into an electrondense product. We demonstrated that RB-Ac and HypB-Ac are mostly internalized by endocytosis, and are converted into the native PSs already at the cell surface. Endocytosed PS molecules apparently follow the endosomes-lysosome route, being found in endosomes, lysosomes and multivescicular bodies; PS molecules were also detected in the cytosol. This ultrastructural localization of the photoactive molecules is fully consistent with the multiorganelle photodamage observed after irradiation in culture of RB-Ac- or HypB-Ac-loaded cells. Due to the very short half-life of the oxidizing chemical species and their limited mobility, DAB deposits do localize in close proximity of the very place where photoactive molecules elicited the production of reactive oxygen species upon light irradiation. Therefore, DAB photoconversion promises to be a suitable tool for directly visualizing in single cells the PS molecules at high resolution, helping to elucidate their mode of penetration into the cell as well as their dynamic intracellular redistribution and organelle targeting.

Atopy patch tests are useful to predict oral tolerance in children with gastrointestinal symptoms related to non-IgE-mediated cow's milk allergy.

Atopy patch tests (APTs) have been proposed for the diagnostic approach in children with non-IgE-mediated cow's milk allergy and gastrointestinal symptoms. We aimed to investigate the benefit of APTs in predicting oral tolerance in these patients. We prospectively evaluated 172 subjects with a sure diagnosis of non-IgE-mediated CMA and gastrointestinal symptoms (97 boys, 56.4%; age, 6.37 m; range, 2-12 m). At diagnosis, 113/172 (65.7%) children had positive APTs to cow's milk proteins (CMP). After 12 months of exclusion, diet APTs were repeated immediately before OFC. APTs significantly correlated (P < 0.001) with the OFC outcome (r 0.579). Diagnostic accuracy was sensitivity of 67.95%, specificity of 88.3%, PPV of 82.81%, NPV of 76.85%, and a +LR of 5.80. APTs are a valuable tool in the follow-up of children with non-IgE-mediated CMA-related gastrointestinal symptoms by contributing in determining whether an OFC can safely be undertaken.

Absence of an independent association between serum uric acid and left ventricular mass in Caucasian hypertensive women and men.

BACKGROUND AND AIM: Experimentally uric acid may induce cardiomyocyte growth and interstitial fibrosis of the heart. However, clinical studies exploring the relationship between serum uric acid (SUA) and left ventricular (LV) mass yielded conflicting results. The aim of our study was to evaluate the relationships between SUA and LV mass in a large group of Caucasian essential hypertensive subjects. METHODS AND RESULTS: We enrolled 534 hypertensive patients free of cardiovascular complications and without severe renal insufficiency. In all subjects routine blood chemistry, including SUA determination, echocardiographic examination and 24 h ambulatory blood pressure (BP) monitoring were obtained. In the overall population we observed no significant correlation of SUA with LV mass indexed for height(2.7) (LVMH(2.7)) (r = 0.074). When the same relationship was analysed separately in men and women, we found a statistically significant correlation in female gender (r = 0.27; p < 0.001), but not in males (r = -0.042; p = NS). When we grouped the study population in sex-specific tertiles of SUA, an increase in LVMH(2.7) was observed in the highest tertiles in women (44.5 ± 15.6 vs 47.5 ± 16 vs 55.9 ± 22.2 g/m(2.7); p < 0.001), but not in men. The association between SUA and LVMH(2.7) in women lost statistical significance in multiple regression analyses, after adjustment for age, 24 h systolic BP, body mass index, serum creatinine and other potential confounders. CONCLUSIONS: Our findings do not support an independent association between SUA and LV mass in Caucasian men and women with arterial hypertension.

Characterization of chitinolytic bacteria and histological aspects of Shell Disease Syndrome in European spiny lobsters (Palinurus elephas) (Fabricius 1787).

The present research reports the first description of Shell Disease Syndrome in European spiny lobsters Palinurus elephas (Fabricius 1787), which occurred in an experimental aquaculture facility in Sicily (Italy). Both bacterial characterization and histopathological examination of the exoskeleton at site of lesions was carried out. Infected specimens showed tail fan erosions, and in one case uropod ulceration and complete loss of periods. Identified species included: Listonella anguillarum 50.5%, Vibrio parahaemolyticus 27.5% and Vibrio alginolyticus 22%. Microscopic evaluation of lesions indicate the presence of inflammatory responses, which include melanization and pseudomembrane formation, similar to those described for other crustaceans affected by SDS.

Rice protein-concentrate meal as a potential dietary ingredient in practical diets for blackspot seabream Pagellus bogaraveo: a histological and enzymatic investigation.

Field and laboratory studies were conducted to evaluate the intestinal responses to partial replacement of fish meal with rice protein concentrate (RPC) in practical diets for blackspot seabream Pagellus bogaraveo. Two experimental diets were formulated to be isoproteic and isoenergetic with an increasing level of RPC (20 and 35%, respectively) and were tested against a fish meal-based control diet (RPC0). The diets showed similar features for growth performances and both intestinal histology and digestive enzymes. This study confirmed that RPC does not induce intestinal mucosa alterations in this fish. The dietary RPC supplement caused a significant increase in trypsin activity, whereas lipase activity was reduced.

Relationships between metabolic syndrome and left ventricular mass in hypertensive patients: does sex matter?

Several studies documented an association between metabolic syndrome (MetS) and left ventricular (LV) hypertrophy. However, only in a few of these studies the impact of MetS on left ventricular mass (LVM) was separately analysed by gender, with conflicting results. The aim of our study was to verify, in a wide sample of essential hypertensive patients, the influence of gender, if any, on the relationship between MetS and LVM. We enrolled 475 non-diabetic subjects (mean age: 46 +/- 11 years), with mild-to-moderate essential hypertension, of whom 40% had MetS, defined on the basis of Adult Treatment Panel III (ATPIII) criteria. All the patients underwent a 24-h ambulatory blood pressure monitoring and an echocardiogram. LVM indexed for height (2.7) (LVMH (2.7)) was significantly (P < 0.001) higher in women with MetS (n=83) than in those without it (n=97; 54+/-17 vs 42+/-11 g m(-2.7)). An equally significant difference in LVMH (2.7) was documented also in male gender between the two groups with (n=105) and without MetS (n=190; 51+/-14 vs 43+/-11 g m(-2.7); P < 0.001). The relationship between MetS and LVMH (2.7) remained statistically significant (P < 0.001) in both sexes, in multiple regression analyses, even after adjustment for potential confounding factors. Our results seem to suggest that the relationship between MetS and LVM is not significantly affected by gender, being LVM increased in both hypertensive women and men with MetS.

Bone regeneration: in vitro evaluation of the behaviour of osteoblast-like MG63 cells placed in contact with polylactic-co-glycolic acid, deproteinized bovine bone and demineralized freeze-dried bone allograft.

Insufficient bone density of the alveolar crests, caused by loss of the dental elements, sometimes impedes the primary stability of an integrated bone implant. The techniques of bone regeneration allow to obtain a sufficient quantity of alveolar bone to permit the implant rehabilitation of the edentulous crests. Today several grafting materials are available and they have different characteristics, according to their structure, which influence the different behaviour of the grafting materials to the bone and the implant surface. The aim of this study is to evaluate the interaction between a human osteosarcoma MG63 cell line and three different biomaterials: polylactic-co-glycolic acid (PLAGA), deproteinized bovine bone and demineralised freeze-dried bone allograft (DFDBA). From this study a different behaviour emerges of the osteoblast-like MG63 cells in relation to the sublayer on which these cells were placed in culture. The results of the study, in fact, demonstrate that the most osteoconductive material of the three analysed is the DFDBA, followed by DPBB. On the contrary, the PLGA, because of its roughness, does not seem to represent a valid support for cell growth, and does not encourage any morphologic modification in tumor cells. Furthermore, deproteinized bovine bone shows a differentiating effect which could lead to hypothesise an osteoconductive capacity of this biomaterial. Further studies should be carried out with the aim of explaining the results obtained.

Krill oil, vitamin D and Lactobacillus reuteri cooperate to reduce gut inflammation.

Current research into original therapies to treat intestinal inflammation is focusing on no-drug therapies. KLD is a mixture of krill oil (KO), probiotic Lactobacillus reuteri (LR), and vitamin D (VitD3). The aim of this study was to assess in vitro and in vivo the potential cooperative effects of KLD in reducing gut inflammation. Colorectal adenocarcinoma cell lines, CACO2 and HT29, and C57BL/6 mice were used for in vitro and in vivo analyses, respectively. Cells were exposed to cytomix (interferon gamma + tumour necrosis factor alpha (TNF-α)) to induce inflammation or co-exposed to cytomix and KO, LR and VitD3 alone or to cytomix and KLD. Animals were treated for 7 days with dextran sodium sulphate (DSS) to induce colitis or with DSS and KLD. In vitro assays: F-actin expression was analysed by immunofluorescence; scratch test and trans-epithelial electric resistance test were performed to measure wound healing; adhesion/invasion assays of adhesive and invasive Escherichia coli (AIEC) bacteria were made; mRNA expression of TNF-α, interleukin (IL)-8 and vitamin D receptor (VDR) was detected by quantitative PCR. In vivo assays: body weight, clinical score, histological score and large intestine weight and length were estimated; mRNA expression of TNF-α, IL-1ß, IL-6, IL-10 by quantitative PCR; VDR expression was detected by quantitative PCR and immunohistochemistry. In vitro: KLD restores epithelial cell-cell adhesion and mucosal healing during inflammation, while decreases the adhesiveness and invasiveness of AIEC bacteria and TNF-α and IL-8 mRNA expression and increases VDR expression. In vivo: KLD significantly improves body weight, clinical score, histological score and large intestine length of mice with DSS-induced colitis and reduces TNF-α, IL-1ß and IL-6 mRNA levels, while increases IL-10 mRNA and VDR levels. KLD has significant effects on the intestinal mucosa, strongly decreasing inflammation, increasing epithelial restitution and reducing pathogenicity of harmful commensal bacteria.

Suppression of a defect in the 5' untranslated leader of mitochondrial COX3 mRNA by a mutation affecting an mRNA-specific translational activator protein.

Translation of the Saccharomyces cerevisiae mitochondrial COX3 mRNA, encoding subunit III of cytochrome c oxidase, specifically requires the action of the nuclear gene products PET54, PET122, and PET494 at a site encoded in the 612-base 5' untranslated leader. To identify more precisely the site of action of the translational activators, we constructed two large deletions of the COX3 mRNA 5' untranslated leader. Both deletions blocked translation without affecting mRNA stability. However, one of the large deletions was able to revert to partial function by a small secondary deletion within the remaining 5' leader sequences. Translation of the resulting mutant (cox3-15) mRNA was still dependent on the nuclear-encoded specific activators but was cold sensitive. We selected revertants of this mitochondrial mutant at low temperature to identify genes encoding proteins that might interact with the COX3 mRNA 5' leader. One such revertant carried a missense mutation in the PET122 gene that was a strong and dominant suppressor of the cold-sensitive defect in the mRNA, indicating that the PET122 protein interacts functionally (possibly directly) with the COX3 mRNA 5' leader. The cox3-15 mutation was not suppressed by overproduction of the wild-type PET122 protein but was very weakly suppressed by overproduction of PET494 and slightly better suppressed by co-overproduction of PET494 and PET122.

Product of Saccharomyces cerevisiae nuclear gene PET494 activates translation of a specific mitochondrial mRNA.

The product of Saccharomyces cerevisiae nuclear gene PET494 is known to be required for a posttranscriptional step in the accumulation of one mitochondrial gene product, subunit III of cytochrome c oxidase (coxIII). Here we show that the PET494 protein probably acts in mitochondria by demonstrating that both a PET494-beta-galactosidase fusion protein and unmodified PET494 are specifically associated with mitochondria. To define the PET494 site of action, we isolated mutations that suppress a pet494 deletion. These mutations were rearrangements of the mitochondrial gene oxi2 that encodes coxIII. The suppressor oxi2 genes had acquired the 5'-flanking sequences of other mitochondrial genes and gave rise to oxi2 transcripts carrying the 5'-untranslated leaders of their mRNAs. These results demonstrate that in wild-type cells PET494 specifically promotes coxIII translation, probably by interacting with the 5'-untranslated leader of the oxi2 mRNA.

Interactions among three proteins that specifically activate translation of the mitochondrial COX3 mRNA in Saccharomyces cerevisiae.

The PET54, PET122, and PET494 proteins, which are associated with the yeast inner mitochondrial membrane, specifically activate translation of the mitochondrially encoded COX3 mRNA. We used the two-hybrid system to test whether pairs of these proteins, when fused to either the GAL4 DNA-binding or transcriptional activating domain, can physically associate as measured by the expression of the GAL4-dependent reporter, lacZ. PET54 and PET122 interacted in this system, and an amino-terminally truncated PET494 fragment showed an interaction with PET54. We also detected functional interactions between PET54 and PET122 genetically: a pet54 missense substitution (Phe to Gly at position 244) that caused a severe respiratory defect was suppressed both by a missense substitution affecting PET122 (Gly to Val at position 211) and by overproduction of wild-type PET122. Both Gly and Ala, substituted at PET54 position 244, disrupted the two-hybrid interactions with PET122 and PET494. While Ala at PET54 position 244 caused only a modest respiratory phenotype alone, it caused a severe respiratory defect when combined with a cold-sensitive mitochondrial mutation affecting the COX3 mRNA 5' leader. This synthetic defect was suppressed by a missense substitution in PET122 and by overproduction of wild-type PET122, indicating functional interactions among PET54, PET122, and the mRNA. Taken together with previous work, these data suggest that a complex containing PET54, PET122, and PET494 mediates the interaction of the COX3 mRNA with mitochondrial ribosomes at the surface of the inner membrane.