Evaluation of the effectiveness of single nucleotide polymorphisms compared to microsatellite markers for parentage verification in Moroccan horses.

The International Society for Animal Genetics (ISAG) currently advocates for a transition towards single nucleotide polymorphism (SNP) markers as a potential alternative for equine parentage verification. To ascertain the efficacy of this transition, it is imperative to evaluate the performance of parentage testing using SNPs in juxtaposition with short tandem repeats (STRs). As per ISAG's recommendation, we used an equine genotyping-by-sequencing panel with 144 SNPs for this purpose. Equine parentage is currently realized using 16 microsatellites (STRs) excluding the LEX3 marker. In this study, 1074 horses were genotyped using the 144 SNPs panel, including 432 foals, 414 mares, and 228 stallions, from five different breeds: 293 Arabians, 167 Barbs, 189 Thoroughbreds, 73 Anglo-Arabians, and 352 Arabian-Barbs. As a result, two SNPs markers were eliminated from the panel system due to inconsistent amplification across all examined individuals leaving 142 SNPs markers for analysis. A comparative analysis between SNPs and STRs markers revealed that the mean expected heterozygosity was 0.457 for SNPs and 0.76 for STRs, while the mean observed heterozygosity stood at 0.472 for SNPs and 0.72 for STRs. Furthermore, the probability of identity was calculated to be 5.722 × 10-57 for SNPs and 1.25 × 10-15 for STRs markers. In alignment with the Hardy-Weinberg equilibrium in polyploids test, 110 out of the total SNPs were consistent with the Hardy-Weinberg equilibrium in polyploids test (p > 0.05). Employing both SNPs and STRs markers, the mean polymorphic information content was discerned to be 0.351 for SNPs and 0.72 for STRs. The cumulative exclusion probabilities for SNP markers exceeded 99.99%, indicating that the 142 SNPs panel might be adequate for parentage testing. In contrast, when utilizing STRs markers, the combined average exclusion probabilities for one and both parents were determined to be 99.8% and 99.9%, respectively. Our comprehensive study underscores the potential of SNPs in equine parentage verification, especially when compared to STRs in terms of exclusion probabilities. As a corollary, the application of SNPs for parentage verification and identification can significantly contribute to the conservation initiative for the five Moroccan horse breeds. Nonetheless, further research is required to address and replace the deficient SNPs within the panel.

Involvement of transcribed lncRNA uc.291 and SWI/SNF complex in cutaneous squamous cell carcinoma.

While non-melanoma skin cancers (NMSCs) are the most common tumours in humans, only the sub-type cutaneous squamous cell carcinoma (cSCC), might become metastatic with high lethality. We have recently identified a regulatory pathway involving the lncRNA transcript uc.291 in controlling the expression of epidermal differentiation complex genes via the interaction with ACTL6A, a component of the chromatin remodelling complex SWI/SNF. Since transcribed ultra-conserved regions (T-UCRs) are expressed in normal tissues and are deregulated in tumorigenesis, here we hypothesize a potential role for dysregulation of this axis in cSCC, accounting for the de-differentiation process observed in aggressive poorly differentiated cutaneous carcinomas. We therefore analysed their expression patterns in human tumour biopsies at mRNA and protein levels. The results suggest that by altering chromatin accessibility of the epidermal differentiation complex genes, down-regulation of uc.291 and BRG1 expression contribute to the de-differentiation process seen in keratinocyte malignancy. This provides future direction for the identification of clinical biomarkers in cutaneous SCC. Analysis of publicly available data sets indicates that the above may also be a general feature for SCCs of different origins.

Free-amino acid metabolic profiling of visceral adipose tissue from obese subjects.

Interest in adipose tissue pathophysiology and biochemistry have expanded considerably in the past two decades due to the ever increasing and alarming rates of global obesity and its critical outcome defined as metabolic syndrome (MS). This obesity-linked systemic dysfunction generates high risk factors of developing perilous diseases like type 2 diabetes, cardiovascular disease or cancer. Amino acids could play a crucial role in the pathophysiology of the MS onset. Focus of this study was to fully characterize amino acids metabolome modulations in visceral adipose tissues (VAT) from three adult cohorts: (i) obese patients (BMI 43-48) with metabolic syndrome (PO), (ii) obese subjects metabolically well (O), and (iii) non obese individuals (H). 128 metabolites identified as 20 protein amino acids, 85 related compounds and 13 dipeptides were measured by ultrahigh performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) and gas chromatography-/mass spectrometry GC/MS, in visceral fat samples from a total of 53 patients. Our analysis indicates a probable enhanced BCAA (leucine, isoleucine, valine) degradation in both VAT from O and PO subjects, while levels of their oxidation products are increased. Also PO and O VAT samples were characterized by: elevated levels of kynurenine, a catabolic product of tryptophan and precursor of diabetogenic substances, a significant increase of cysteine sulfinic acid levels, a decrease of 1-methylhistidine, and an up regulating trend of 3-methylhistidine levels. We hope this profiling can aid in novel clinical strategies development against the progression from obesity to metabolic syndrome.

Genetic variability of dromedary camel populations based on microsatellite markers.

Understanding existing levels of genetic variability of camel populations is capital for conservation activities. This study aims to provide information on the genetic diversity of four dromedary populations, including Guerzni, Harcha, Khouari and Marmouri. Blood samples from 227 individuals belonging to the aforementioned populations were obtained and genotyped by 16 microsatellite markers. A total of 215 alleles were observed, with the mean number of alleles per locus being 13.4 ± 6.26. All loci were polymorphic in the studied populations. The average expected heterozygosity varied from a maximum of 0.748 ± 0.122 in Guerzni population to a minimum of 0.702 ± 0.128 in Harcha population; Guerzni population showed the highest value of observed heterozygosity (0.699 ± 0.088), whereas Harcha population the lowest (0.646 ± 0.130). Mean estimates of F-statistics obtained over loci were FIS = 0.0726, FIT = 0.0876 and FST = 0.0162. The lowest genetic distance was obtained between Guerzni and Khouari (0.023), and the highest genetic distance between Harcha and Marmouri (0.251). The neighbour-joining phylogenetic tree showed two groups of populations indicating a cluster of Guerzni, Khouari and Marmouri, and a clear isolation of Harcha. The genetic distances, the factorial correspondence analysis, the analysis of genetic structure and the phylogenetic tree between populations revealed significant differences between Harcha and other populations, and a high similarity between Guerzni, Khouari and Marmouri. It is concluded from this study that the camel genetic resources studied are well diversified. However, the herd management, especially the random selection of breeding animals, can increase the level of genetic mixing between different populations, mainly among Guerzni, Khouari and Marmouri, that live in the same habitat and grazing area.

Clusterin silencing restores myoblasts viability and down modulates the inflammatory process in osteoporotic disease.

BACKGROUND: Targeting new molecular pathways leading to Osteoporosis (OP) and Osteoarthritis (OA) is a hot topic for drug discovery. Clusterin (CLU) is a glycoprotein involved in inflammation, proliferation, cell death, neoplastic disease, Alzheimer disease and aging. The present study focuses on the expression and the role of CLU in influencing the decrease of muscle mass and fiber senescence in OP-OA condition. METHODS: Vastus lateralis muscle biopsies were collected from 20 women with OP undergoing surgery for fragility hip fracture and 20 women undergoing arthroplasty for hip osteoarthritis. RESULTS: We found an overexpression of CLU in degenerated fibers in OP closely correlated with interleukin 6 (IL6) and histone H4 acetylation level. Conversely, in OA muscle tissues we observed a weak expression of CLU but no nuclear histone H4 acetylation. Ex vivo studies on isolated human myoblasts confirmed CLU overexpression in OP as compared to OA (p < 0.001). CLU treatment of isolated OP and OA myoblasts showed: modulation of proliferation, morphological changes, increase of histone H4 acetylation and induction of myogenin (MYOG) activation in OP myoblast only. In OP condition, functional knockdown of CLU by siRNA restores proliferative myoblasts capability and tissue damage repair, carried out by an evident upregulation of Transglutaminase 2 (TGM2). We also observed downmodulation of CX3CR1 expression with consequent impairing of the inflammatory infiltrate recruitment. CONCLUSIONS: Results obtained suggest a potential role of CLU in OP by influencing myoblasts terminal differentiation, epigenetic regulation of muscle cell differentiation and senescence. Moreover, CLU silencing points out its role in the modulation of tissue damage repair and inflammation, proposing it as a new diagnostic marker for muscle degeneration and a potential target for specific therapeutic intervention in OP related sarcopenia.

Validation of locomotion scoring as a new and inexpensive technique to record circadian locomotor activity in large mammals.

BACKGROUND: The locomotor activity (LA) rhythm, widely studied in rodents, has not been fully investigated in large mammals. This is due to the high cost and the brittleness of the required devices. Alternatively, the locomotion scoring method (SM), consisting of attribution of a score to various levels of activity would be a consistent method to assess the circadian LA rhythm in such species. NEW METHOD: To test this, a SM with a score ranging from 0 to 5 has been developed and used in two domestic large mammals, the camel and the goat. One minute interval scoring was performed using visual screening and monitoring of infra-red camera recording videos and carried out by two evaluators. RESULTS: The SM provides a clear daily LA rhythm that has been validated using an automate device, the Actiwatch-Mini. The obtained curves and actograms were indeed highly similar to those acquired from the Actiwatch-Mini. Moreover, there were no statistical differences in the period and acrophase. The period was exactly of 24.0h and the acrophases occurred at 12h05 ± 00h03 and 12h14 ± 00h07 for the camel and at 13h13 ± 00h09 and 12h57 ± 00h09 for the goat using SM and Actiwatch-Mini respectively. COMPARISON WITH EXISTING METHODS: Compared to the automatic system, the SM is inexpensive and has the advantage of describing all types of performed movements. CONCLUSIONS: The new developed SM is highly reliable and sufficiently accurate to assess conveniently the LA rhythm and specific behaviors in large mammals. This opens new perspectives to study chronobiology in animal models of desert, tropical and equatorial zones.

Hacia la implementación de un protocolo nacional de alerta temprana basado en el monitoreo de los parámetros fisiológicos en pacientes internados / Towards the implementation of a national early warning protocol based on the monitoring of physiological parameters in hospitalized patients

INTRODUCCIÓN La escala NEWS (National Early Warning Score) clasifica a los pacientes según su probabilidad de deterioro. Fue desarrollada a partir de signos vitales (SV) obtenidos automáticamente. En Argentina, los SV son tomados manualmente; hay una percepción de que no son confiables y su capacidad predictiva es baja. OBJETIVOS Validar la capacidad de discriminación del NEWS a partir de SV, tal como se toman en el medio local. MÉTODOS Nueve hospitales ingresaron pacientes en forma prospectiva, relevando los SV, su hora y fecha, datos demográficos, co-morbilidades, eventos severos y muerte. Se calculó la escala NEWS con cada set de parámetros. Se realizó un análisis de regresión logística evaluando la capacidad del primer valor de NEWS de predecir un evento durante la internación independientemente de la edad, co-morbilidades y sexo. Mediante una curva ROC se analizó la capacidad de discriminación de la media del NEWS y, a través de árboles de decisión, se analizó el mejor valor de corte que predecía muerte y/o evento a las 12 y a las 24 horas. RESULTADOS Ingresaron 1705 pacientes, 869 mujeres, con edad de 18 a 100; 10% de los pacientes presentó algún evento y la mortalidad fue 3,5%. El 90% fue clasificado al ingreso como NEWS de bajo grado de deterioro (0-4), el 5% de riesgo moderado (5-6) y el 5% de riesgo alto (mayor a 7). El valor de NEWS al ingreso de la internación predijo el riesgo de presentar un evento severo durante ella (OR 3,40; IC 2,8-3,5), independientemente de la edad, sexo, y la presencia de co-morbilidades. La escala NEWS fue mejor predictora a las 12 horas previas de un evento que la edad sumada a las co-morbilidades; un valor mayor a 3 fue el predictor más "seguro" de muerte a las 12 horas y a las 24 horas. La media del NEWS durante la internación presentó un área bajo la curva de 0,90 para mortalidad y de 0,80 para evento severo. DISCUSIÓN La escala NEWS es una excelente predictora de la ocurrencia de eventos durante la internación, inclusive a partir de la toma tal como se realiza en este medio.

Clinical protocol with digital cad/cam chairside workflow for the rehabilitation of severely worn dentition patients.

OBJECTIVES: The purpose of this paper is to investigate the effectiveness and to describe a clinical protocol with digital CAD CAM chairside workflow for the rehabilitation of severely compromised and worn dentitions. METHODS: This article reports 4 consecutive cases, where a clinical digital chairside workflow is used for the rehabilitation of severely compromised and worn dentitions. Advantages and limitations of this method compared with the traditional prosthetic protocol are also described and discussed. RESULTS: With all four patients treated with this protocol, we obtained a good aesthetic and functional result, improvement in chewing function, loss of cold sensitivity, better preservation of most of the left hard tissue and a good level of satisfaction. In a two-year follow-up, all patients also maintained the condition obtained with prosthetic chairside rehabilitation, resulting in almost 100% cumulative survival rate. CONCLUSION: Within the limitations of this study, we can assert that the aforementioned restorative treatment with digital CAD/CAM chairside workflow represents a valid alternative to rehabilitate this kind of patients, because it is a safe, predictable and personalized procedure but also it seems easier, faster and cheaper than traditional protocols.

Selección de un hongo filamentoso altamente productor de amilasas para ser usadas en detergentes biodegradables / Selection of filamentous fungus highly producer of amylases to be used in biodegradable detergents

Las amilasas (alfa-amilasa, EC 3.2.1.1 y glucoamilasa, EC 3.2.1.3) son enzimas extracelulares que hidrolizan el almidón en dextrinas hasta glucosa y tienen gran aplicación industrial, especialmente alimentaria; detergentes y en la producción de alcohol a partir de granos. El objetivo del trabajo es seleccionar un hongo filamentoso que presente alta producción de amilasas con características particulares para ser empleadas en biodetergentes. Se estudiaron los siguientes hongos: Penicillium expansum; P. digitatum; P. islandicum; Aspergillus clavatus; A. niger; A. ochraceus; A. fumigatus; A. flavus; A. oryzae; A. nidulans y Geotrichum candidum; Los ensayos se realizaron en un medio de hidrolizado de papa de descarte (variedad Spunta) suplementado con las siguientes sales: KH2 PO4 1,0; NaNO3 3,0; MgSO4 .7H2 O 0,5, a pH 4,0; se inoculó con 2 x106 conidios/mL y se incubaron a 25ºC en un agitador rotatorio a una velocidad de agitación de 250 rpm. Con los extractos enzimáticos parcialmente purificados con (NH4 )2 SO4 al 60 por ciento de saturación, se estudió el efecto del pH (2,5; 3,5; 4,0; 4,5; 5,0; 5,5; 6,0, 7,0 y 8,0) y la temperatura (20; 25; 30; 35 y 40ºC). Los resultados mostraron que la máxima producción de enzima (128 U/L) se obtuvo con Aspergillus niger, en las condiciones ensayadas, a las 48 h de incubación, con alto rendimiento de producto respecto a la biomasa (Yp/x= 18,3 U/g) y productividad volumétrica (Pdv=2,7 U/L). El análisis cualitativo de las enzimas del complejo amilolítico de A. niger mostró que las amilasas implicadas son alfa-amilasa y glucoamilasa y se caracterizaron por hidrolizar en un tiempo de 3 min. manchas mixtas de almidón y grasas de muestras textiles en un rango de pH 4,0 a 8,0 y de 20 a 40 ºC.

The amylases (alpha-amylase, EC 3.2.1.1 and glucoamylase, EC 3.2.1.3) are extracellular enzymes that hydrolyze starch into dextrins to glucose and have great application industrial, especially food, detergents and in the production of alcohol from grains. The objective of the study is to select a filamentous fungus that present high production of amylases showing attractive features to be used in biodetergentes. Were studied following fungus: Penicillium expansum; P. digitatum; P. islandicum; Aspergillus clavatus; A niger; A. ochraceus; A. fumigatus; A. flavus; A. oryzae; A. nidulans and Geotrichum candidum. The tests were conducted in the medium of hydrolyzed potato discard (variety Spunta) supplemented with the following sales: KH2 PO4 , 1.0; NaNO3 , 3.0 and MgSO4 .7H2 O, 0.5, to pH 4.0. Were inoculated with 2 x 106 conidia/ mL and incubated at 25 °C on a rotary Shaker at a speed of 250 rpm. With partially purified enzyme extracts with (NH4 )2 SO4 at 60 percent of saturation, we studied the effect of pH (2.5; 3.5; 4.0; 4.5; 5.0; 5.5; 6.0, 7.0 and 8.0) and temperature (20; 25; 30; 35, and 40 ° C). The results showed that the maximum production of enzyme (128 U/L) was obtained with Aspergillus niger, under the conditions tested, at 48 h of incubation, with high product formation rate with respect to biomass (Yp/x = 18.3 U/g) and volumetric productivity (Pdv = 2,7 U/ hL). The qualitative analysis of the enzymes of the complex amylolític of A. niger showed that involved amylases are α-amylase and glucoamylase and characterized by hydrolyze in 3 min spots mixed starch and fats of textile samples over a range of pH 4.0 to 8.0 and 20 to 40 ° C.

ΔNp63 targets cytoglobin to inhibit oxidative stress-induced apoptosis in keratinocytes and lung cancer.

During physiological aerobic metabolism, the epidermis undergoes significant oxidative stress as a result of the production of reactive oxygen species (ROS). To maintain a balanced oxidative state, cells have developed protective antioxidant systems, and preliminary studies suggest that the transcriptional factor p63 is involved in cellular oxidative defence. Supporting this hypothesis, the ΔNp63α isoform of p63 is expressed at high levels in the proliferative basal layer of the epidermis. Here we identify the CYGB gene as a novel transcriptional target of ΔNp63 that is involved in maintaining epidermal oxidative defence. The CYGB gene encodes cytoglobin, a member of the globin protein family, which facilitates the diffusion of oxygen through tissues and acts as a scavenger for nitric oxide or other ROS. By performing promoter activity assays and chromatin immunoprecipitation, reverse transcriptase quantitative PCR and western blotting analyses, we confirm the direct regulation of CYGB by ΔNp63α. We also demonstrate that CYGB has a protective role in proliferating keratinocytes grown under normal conditions, as well as in cells treated with exogenous hydrogen peroxide. These results indicate that ΔNp63, through its target CYGB has an important role in the cellular antioxidant system and protects keratinocytes from oxidative stress-induced apoptosis. The ΔNp63-CYGB axis is also present in lung and breast cancer cell lines, indicating that CYGB-mediated ROS-scavenging activity may also have a role in epithelial tumours. In human lung cancer data sets, the p63-CYGB interaction significantly predicts reduction of patient survival.

PIR2/Rnf144B regulates epithelial homeostasis by mediating degradation of p21WAF1 and p63.

ΔNp63 is a transcription factor that is critical for the development of stratified epithelia and is overexpressed or amplified in >80% of squamous cell carcinomas (SCCs). We identified the RING finger E3 ubiquitin ligase PIR2/Rnf144b as a direct transcriptional target of ΔNp63α and showed that its expression parallels that of ΔNp63α in keratinocytes, SCC cell lines and SCCs. We used primary keratinocytes as a model system to investigate the function of PIR2/Rnf144b in stratified epithelia. Depletion of PIR2/Rnf144b severely impaired keratinocyte proliferation and differentiation, associated with accumulation of p21(WAF1/CIP1); a known target of PIR2/Rnf144b. More importantly, we found that PIR2/Rnf144b binds and mediates proteasomal degradation of ΔNp63α, generating a hitherto unknown auto-regulatory feedback loop. These findings substantiate PIR2/Rnf144b as a potentially critical component of epithelial homeostasis, acting downstream of ΔNp63α to regulate cellular levels of p21(WAF1/CIP1) and ΔNp63α.

Frequency of the severe combined immunodeficiency disease gene among horses in Morocco.

Severe combined immunodeficiency disease (SCID) of horses is an autosomal, recessive hereditary disease occurring among Arabian or crossbred Arabian horses. The genetic defect responsible was previously identified as a 5-base pair deletion in the gene encoding the catalytic subunit of the DNA dependant protein kinase (DNA-PKcs). This study was carried out to determine the frequency of SCID and identify horses carrying the gene for SCID among Arabian and Arabian crossbred stallions and mares in Morocco using a DNA-based test. Twenty-one horses were SCID carriers: 14 (7%) Arabians, 6 (4%) Arab-Barbs and one (33%) Anglo-Arab. After analysing their genealogy, 3 imported stallions were identified that disseminated the mutant gene of DNA-PKcs in Morocco.

Insights into the role of the histidines in the structure and stability of cytochrome c.

In this paper we investigate the role played by each histidine in the amino acid sequence of yeast iso-1-cytochrome c (with the exception of H18, the residue axially coordinated to the heme iron) in determining the protein structure and stability. To this end, we have generated and characterized the double mutants H26Y/H33Y, H26Y/H39K and H33Y/H39K obtained from the C102T variant of the protein, which retain only one histidine side chain in the amino acid sequence. In particular, the H39K mutation inserts a lysine at position 39 as in the sequence of equine cytochrome c. The H26Y/H33Y/H39K triple mutant, which lacks all three histidines, was also produced and its spectroscopic properties are compared with those of the double mutants. The data highlight the critical role played by H26 in determining protein stability. Recombinant horse cytochrome c and the corresponding H26Y mutant were also generated and characterized. Since equine cytochrome c exhibits higher stability than the yeast protein, this provides a valuable opportunity to understand the role played by the invariant H26 residue in determining structure and stability.

Endothelium and inflammation.

During the past decade, our understanding of the pathophysiology of coronary heart disease (CAD) has undergone a remarkable evolution. To date atherosclerosis is considered an inflammatory disease, whose the endothelial dysfunction represents an early key event. When the arterial endothelium encounters certain bacterial products or risk factors, such as dyslipidemia, vasoconstrictor hormones involved in hypertension, the products of glycoxidation associated with hyperglycemia, or proinflammatory cytokines derived from excess adipose tissue, these cells increase the expression of adhesion molecules that promote the sticking of blood leukocytes to the inner surface of the arterial wall. Once in the arterial intima these cells communicate with endothelium and smooth muscle cells, under the influence of mediators of inflammation and immunity, such as the cytokines and complements components, prostanoids and leukotrienes. Thus, the activated endothelium promotes the development of the atherosclerotic disease process, i.e., vascular inflammation and thrombosis by producing vasoconstrictor substances, by inducing the expression of adhesive receptors for leukocytes and platelets, the production of tissue factor and endothelin, and by increasing the production of the plasminogen activator inhibitor-1. Emerging data support the concept that assessment of endothelial vasomotion may be a useful biomarker for atherosclerotic vascular disease.

CRP is or is not a reliable marker of ischaemic heart disease?

In recent years a growing body of evidence has emphasized the role of C-reactive protein (CRP) as a marker of future cardiovascular events. CRP is a pentameric molecule widely utilized as a marker of infections and inflammation. The evidence that inflammation plays an important role in the pathogenesis of coronary artery disease and in plaque destabilization has lead to use of CRP as a marker of cardiovascular disease as well. First described as a component of the inflammatory pathway in acute coronary syndromes, CRP has been consistently found to be associated with the risk of future events in no-ST elevation acute coronary syndromes, independently of other risk factors, including troponine. Subsequently CRP has been described as a powerful marker of risk of future events in large populations of apparently healthy subjects. So far there is very little doubt that CRP represents a reliable marker of cardiovascular events, but some issues remain unanswered such as why CRP is a good marker of cardiovascular events and whether or not a better inflammatory marker exists. It must be stressed that CRP, because of its analytical and biological properties and the large amount of available data, is the only inflammatory marker accepted for clinical use.

ATP specifically drives refolding of non-native conformations of cytochrome c.

An increasing body of evidence ascribes to misfolded forms of cytochrome c (cyt c) a role in pathophysiological events such as apoptosis and disease. Here, we examine the conformational changes induced by lipid binding to horse heart cyt c at pH 7 and study the ability of ATP (and other nucleotides) to refold several forms of unfolded cyt c such as oleic acid-bound cyt c, nicked cyt c, and acid denatured cyt c. The CD and fluorescence spectra demonstrate that cyt c unfolded by oleic acid has an intact secondary structure, and a disrupted tertiary structure and heme environment. Furthermore, evidence from the Soret CD, electronic absorption, and resonance Raman spectra indicates the presence of an equilibrium of at least two low-spin species having distinct heme-iron(III) coordination. As a whole, the data indicate that binding of cyt c to oleic acid leads to a partially unfolded conformation of the protein, resembling that typical of the molten globule state. Interestingly, the native conformation is almost fully recovered in the presence of ATP or dATP, while other nucleotides, such as GTP, are ineffective. Molecular modeling of ATP binding to cyt c and mutagenesis experiments show the interactions of phosphate groups with Lys88 and Arg91, with adenosine ring interaction with Glu62 explaining the unfavorable binding of GTP. The finding that ATP and dATP are unique among the nucleotides in being able to turn non-native states of cyt c back to native conformation is discussed in the light of cyt c involvement in cell apoptosis.

Rupture of the hydrogen bond linking two Omega-loops induces the molten globule state at neutral pH in cytochrome c.

His26Tyr and His33Tyr mutants were obtained from the Cys102Thr variant of yeast iso-1-cytochrome c. Spectroscopic studies show that a mutation at position 26 at pH 7.0 enhances flexibility of the peptide, alters the heme pocket region and the axial coordination to heme-iron, and reduces protein stability. The His26Tyr mutant shows properties typical of the molten globule. Further, formation of an axially misligated minor low spin species occurs with partial displacement of Met80, the axial ligand of the heme-iron in the native protein. The pK(a) determined for the alkaline transition of this mutant is 7.48 (+/- 0.05), approximately 0.5 lower than that of the wild-type protein. Hence, the alkaline conformer is populated at pH 7.0, and the sixth ligand of the misligated species is proposed to be a lysine. Furthermore, a reduction in catalytic activity indicates that the functional properties are altered. The results suggest that the structural and functional changes observed in the His26Tyr mutant are because the mutation frees the two Omega-loops that, in the native protein, are linked by the hydrogen bond between His26 and Glu44. Hence, one may infer that the His26-Glu44 hydrogen bond is essential for the rigidity and stability of the native protein. In its absence, the heightened flexibility of the peptide fold results in conversion of the macromolecule to a molten globule state, even at neutral pH. Ligand exchange at the sixth coordination position of the heme-iron(III) observed as the minor species (i.e., the alkaline conformer) is therefore induced by a long-range effect. This result is of interest since mutations reported to date, which stabilize the alkaline conformer, all occur in the loop including Met80. By contrast, only very minor spectroscopic (and, thus, structural) changes are observed for the His33Tyr mutant. This suggests that His33 does not form intramolecular bonds considered important for the protein structure and stability, and is consistent with the high variability of residues at position 33 in cytochromes c.

Heme pocket disorder in myoglobin: reversal by acid-induced soft refolding.

The protein folding process of heme proteins entails generation of not only a correct global polypeptide structure, but also a correct, functionally competent heme environment. We employed a variety of spectroscopic approaches to probe the structure and dynamics of the heme pocket of a recombinant sperm whale myoglobin. The conformational characteristics were examined by circular dichroism, time-resolved fluorescence spectroscopy, FTIR spectroscopy, and optical absorption spectroscopy in the temperature range 300-20 K. Each of these spectroscopic probes detected modifications confined exclusively to the heme pocket of the expressed myoglobin relative to the native protein. The functional properties were examined by measuring the kinetics of CO binding after flash-photolysis. The kinetics of the expressed myoglobin were more heterogeneous than those of the native protein. Mild acid exposure of the ferric derivative of the recombinant protein resulted in a protein with "nativelike" spectroscopic properties and homogeneous CO binding kinetics. The heme pocket modifications observed in this recombinant myoglobin do not derive from inverted heme. In contrast, when native apomyoglobin is reconstituted with the heme in vitro, the heme pocket disorder could be attributed exclusively to 180 degrees rotation of the bound heme [La Mar, G. N., Toi, H., and Krishnamoorthi, R. (1984) J. Am. Chem. Soc. 106, 6395-6401; Light, W. R., Rohlfs, R. J., Palmer, G., and Olson, J. S. (1987) J. Biol. Chem. 262, 46-52]. We conclude that exposure to low pH decreases the affinity of globin for the heme and allows an extended conformational sampling or "soft refolding" to a nativelike conformation.