Sleep-disordered breathing and epicardial adipose tissue in patients with heart failure.

BACKGROUND AND AIMS: Sleep-disordered breathing (SDB) is common in patients with heart failure (HF), contributes to the progression of cardiac disease, and is associated with adverse prognosis. Previous evidence indicates that epicardial adipose tissue (EAT) is independently associated with sleep apnea in obese individuals. We explored the relationship between SDB and EAT in HF patients. METHODS AND RESULTS: EAT thickness was assessed by echocardiography in 66 patients with systolic HF undergoing nocturnal cardiorespiratory monitoring. A significantly higher EAT thickness was found in patients with SDB than in those without SDB (10.7 ± 2.8 mm vs. 8.3 ± 1.8 mm; p = 0.001). Among SDB patients, higher EAT thickness was found in both those with prevalent obstructive sleep apnea (OSA) and those with prevalent central sleep apnea (CSA). Of interest, EAT thickness was significantly higher in CSA than in OSA patients (11.9 ± 2.9 vs. 10.1 ± 2.5 p = 0.022). Circulating plasma norepinephrine levels were higher in CSA than in OSA patients (2.19 ± 1.25 vs. 1.22 ± 0.92 ng/ml, p = 0.019). According to the apnea-hypopnea index (AHI), patients were then stratified in three groups of SDB severity: Group 1, mild SDB; Group 2, moderate SDB; Group 3, severe SDB. EAT thickness progressively and significantly increased from Group 1 to Group 3 (ANOVA p < 0.001). At univariate analysis, only left ventricular ejection fraction and AHI significantly correlated with EAT (p = 0.019 and p < 0.0001, respectively). At multivariate analysis, AHI was the only independent predictor of EAT (ß = 0.552, p < 0.001). CONCLUSIONS: Our results suggest an association between the presence and severity of sleep apneas and cardiac visceral adiposity in HF patients.

Acid-sensing ion channel 2 (ASIC2) is selectively localized in the cilia of the non-sensory olfactory epithelium of adult zebrafish.

Ionic channels play key roles in the sensory cells, such as transducing specific stimuli into electrical signals. The acid-sensing ion channel (ASIC) family is voltage-insensitive, amiloride-sensitive, proton-gated cation channels involved in several sensory functions. ASIC2, in particular, has a dual function as mechano- and chemo-sensor. In this study, we explored the possible role of zebrafish ASIC2 in olfaction. RT-PCR, Western blot, chromogenic in situ hybridization and immunohistochemistry, as well as ultrastructural analysis, were performed on the olfactory rosette of adult zebrafish. ASIC2 mRNA and protein were detected in homogenates of olfactory rosettes. Specific ASIC2 hybridization was observed in the luminal pole of the non-sensory epithelium, especially in the cilia basal bodies, and immunoreactivity for ASIC2 was restricted to the cilia of the non-sensory cells where it was co-localized with the cilia marker tubulin. ASIC2 expression was always absent in the olfactory cells. These findings demonstrate for the first time the expression of ASIC2 in the olfactory epithelium of adult zebrafish and suggest that it is not involved in olfaction. Since the cilium sense and transduce mechanical and chemical stimuli, ASIC2 expression in this location might be related to detection of aquatic environment pH variations or to detection of water movement through the nasal cavity.

Acid-sensing ion channels (ASICs) 2 and 4.2 are expressed in the retina of the adult zebrafish.

Acid-sensing ion channels (ASICs) are H(+)-gated, voltage-insensitive cation channels involved in synaptic transmission, mechanosensation and nociception. Different ASICs have been detected in the retina of mammals but it is not known whether they are expressed in adult zebrafish, a commonly used animal model to study the retina in both normal and pathological conditions. We study the expression and distribution of ASIC2 and ASIC4 in the retina of adult zebrafish and its regulation by light using PCR, in situ hybridization, western blot and immunohistochemistry. We detected mRNA encoding zASIC2 and zASIC4.2 but not zASIC4.1. ASIC2, at the mRNA or protein level, was detected in the outer nuclear layer, the outer plexiform layer, the inner plexiform layer, the retinal ganglion cell layer and the optic nerve. ASIC4 was expressed in the photoreceptors layer and to a lesser extent in the retinal ganglion cell layer. Furthermore, the expression of both ASIC2 and ASIC4.2 was down-regulated by light and darkness. These results are the first demonstration that ASIC2 and ASIC4 are expressed in the adult zebrafish retina and suggest that zebrafish could be used as a model organism for studying retinal pathologies involving ASICs.

The lipid theory in the pathogenesis of calcific aortic stenosis.

AIMS: Biologically active phenomena, triggered by atherogenesis and inflammation, lead to aortic valve (AV) calcification. Lipids play an important role in activating the cell signaling leading to AV bone deposition. This review, based on evidence from animal and human studies, mainly focused on the involvement of lipids and atherogenic phenomena in the pathogenesis of calcific aortic stenosis (AS). DATA SYNTHESIS: The role of elevated low density lipoproteins for the risk of both vascular atherosclerosis and AS has been elucidated. Lipid disorders act synergistically with other risk factors to increase prevalence of calcific AS. Atherosclerosis is also involved in the pathogenesis of bone demineralization, a typical hallmark of aging, which is associated with ectopic calcification at vascular and valvular levels. Animal studies have recently contributed to demonstrate that lipids play an important role in AS pathogenesis through the activation of molecular cell signalings, such as Wnt/Lrp5 and RANK/RANKL/Osteprotegerin, which induce the transition of valvular myofibroblasts toward an osteogenic phenotype with consequent valvular bone deposition. Although all these evidence strongly support the lipid theory in AS pathogenesis, lipids lowering therapies failed to demonstrate in controlled trials a significant efficacy to slow AS progression. Encouraging results from animal studies indicate that physical activity may counteract the biological processes inducing AV degeneration. CONCLUSIONS: This review indicates a robust interplay between lipids, inflammation, and calcific AS. This new pathophysiological scenario of such an emerging valvular disease paves the way to the next challenge of cardiovascular research: "prevent and care aortic valve stenosis".

Electrocardiogram analysis in Anderson-Fabry disease: a valuable tool for progressive phenotypic expression tracking.

Background: Electrocardiogram (ECG) has proven to be useful for early detection of cardiac involvement in Anderson-Fabry disease (AFD); however, little evidence is available on the association between ECG alterations and the progression of the disease. Aim and Methods: To perform a cross sectional comparison of ECG abnormalities throughout different left ventricular hypertrophy (LVH) severity subgroups, providing ECG patterns specific of the progressive AFD stages. 189 AFD patients from a multicenter cohort underwent comprehensive ECG analysis, echocardiography, and clinical evaluation. Results: The study cohort (39% males, median age 47 years, 68% classical AFD) was divided into 4 groups according to different degree of left ventricular (LV) thickness: group A ≤ 9 mm (n = 52, 28%); group B 10-14 mm (n = 76, 40%); group C 15-19 mm (n = 46, 24%); group D ≥ 20 mm (n = 15, 8%). The most frequent conduction delay was right bundle branch block (RBBB), incomplete in groups B and C (20%,22%) and complete RBBB in group D (54%, p < 0.001); none of the patients had left bundle branch block (LBBB). Left anterior fascicular block, LVH criteria, negative T waves, ST depression were more common in the advanced stages of the disease (p < 0.001). Summarizing our results, we suggested ECG patterns representative of the different AFD stages as assessed by the increases in LV thickness over time (Central Figure). Patients from group A showed mostly a normal ECG (77%) or minor anomalies like LVH criteria (8%) and delta wave/slurred QR onset + borderline PR (8%). Differently, patients from groups B and C exhibited more heterogeneous ECG patterns: LVH (17%; 7% respectively); LVH + LV strain (9%; 17%); incomplete RBBB + repolarization abnormalities (8%; 9%), more frequently associated with LVH criteria in group C than B (8%; 15%). Finally, patients from group D showed very peculiar ECG patterns, represented by complete RBBB + LVH and repolarization abnormalities (40%), sometimes associated with QRS fragmentation (13%). Conclusions: ECG is a sensitive tool for early identification and long-term monitoring of cardiac involvement in patients with AFD, providing "instantaneous pictures" along the natural history of AFD. Whether ECG changes may be associated with clinical events remains to be determined.

Effects of circadian rhythm disruption on retinal physiopathology: Considerations from a consensus of experts.

The circadian rhythms originate within the organism and synchronize with cyclic fluctuations in the external environment. It has been demonstrated that part of the human genome is under control of the circadian clock and that a synchronizer that helps to maintain daily rhythms is Melatonin, a neuro-hormone primarily synthesized by the pineal gland during the night. The chronic disruption of circadian rhythm has been linked to many conditions such as obesity, metabolic syndrome, type 2 diabetes, cancer, and neurodegenerative diseases. Studies in the mice showed that the disruption of the retinal circadian rhythm increases the decline during the aging of photoreceptors, accelerating age-related disruption of cone cell structure, function, and viability and that the melatonin receptor deletion seems to influence the health of retinal cells, speeding up their aging. In conclusion, preserving the circadian rhythms could be to add to the prevention and treatment of age-related degenerative retinal diseases, and although additional studies are needed, melatonin could be a valid support to favor this "chronoprotection action".

Potential role of IL-13 in neuroprotection and cortical excitability regulation in multiple sclerosis.

BACKGROUND: Inflammation triggers secondary neurodegeneration in multiple sclerosis (MS). OBJECTIVES: It is unclear whether classical anti-inflammatory cytokines have the potential to interfere with synaptic transmission and neuronal survival in MS. METHODS: Correlation analyses between cerebrospinal fluid (CSF) contents of anti-inflammatory cytokines and molecular, imaging, clinical, and neurophysiological measures of neuronal alterations were performed. RESULTS: Our data suggest that interleukin-13 (IL-13) plays a neuroprotective role in MS brains. We found, in fact, that the levels of IL-13 in the CSF of MS patients were correlated with the contents of amyloid-ß(1-42). Correlations were also found between IL-13 and imaging indexes of axonal and neuronal integrity, such as the retinal nerve fibre layer thickness and the macular volume evaluated by optical coherence tomography. Furthermore, the levels of IL-13 were related to better performance in the low-contrast acuity test and Multiple Sclerosis Functional Composite scoring. Finally, by means of transcranial magnetic stimulation, we have shown that GABAA-mediated cortical inhibition was more pronounced in patients with high IL-13 levels in the CSF, as expected for a neuroprotective, anti-excitotoxic effect. CONCLUSIONS: The present correlation study provides some evidence for the involvement of IL-13 in the modulation of neuronal integrity and synaptic function in patients with MS.

Outer retina dysfunction and choriocapillaris impairment in type 1 diabetes.

To study the outer retina morpho-functional characteristics and the choriocapillaris (CC) features in type 1 diabetic (T1D) patients, with and without signs of diabetic retinopathy (NPDR and NoDR). Twenty-five NPDR and 18 NoDR eyes were imaged by Optical Coherence Tomography Angiography. Ellipsoid zone (EZ) "normalized" reflectivity and CC perfusion density parameters, as flow deficits number (FDn), flow deficit average area (FDa) and flow deficit percentage (FD%), were analysed. Multifocal electroretinogram (mfERG) response amplitude densities (RADs) were measured. Mean EZ "normalized" reflectivity, CC FDn and FD% values, were similar (p > 0.05) in both groups, FDa was significant greater (p > 0.05) in NPDR compared with NoDR eyes. MfERG-RADs were similar in both groups. NPDR eyes showed a significant (p < 0.05) linear correlation between RADs and both, CC FDa and FD%. The EZ "normalized" reflectivity was negatively correlated with CC FD% in NoDR eyes. In NPDR T1D eyes a significant relationship between abnormal outer retina functional responses and CC impairment was observed, while in NoDR eyes the photoreceptor reflectivity was correlated to CC abnormalities. The outer retina dysfunction in NPDR correlated to CC drop-out let hypothesize that the outer retinal elements are functionally impaired in proportion to the CC vascular supply deficit.

Electrophysiological assessment of visual function in patients with non-arteritic ischaemic optic neuropathy.

BACKGROUND AND PURPOSE: Our study aims to evaluate retinal function and neural conduction in post-retinal visual pathways of patients with non-arteritic ischaemic optic neuropathy (NION). METHODS: Twenty patients (mean age: 63.7 +/- 5.96 year) with NION and 20 age-similar control subjects were enrolled. Simultaneous recording of pattern electroretinograms (PERGs) and visual evoked potentials (VEPs), and Log of minimum angle resolution (MAR) visual acuity (VA) were assessed in NION patients and controls. RESULTS: Significantly (ANOVA, P < 0.01) abnormal PERG and VEP responses, delayed retinocortical time (RCT, difference between VEP P100 and PERG P50 implicit times), and reduced VA were found in NION patients with respect to control subjects. The delay in RCT was not significantly (Pearson's test, P > 0.01) correlated with the PERG impairment. The reduction in VA was significantly (Pearson's test, P < 0.01) correlated to the increase in VEP P100 implicit time and RCT, whereas no correlations (P > 0.01) were found with PERG abnormalities. CONCLUSIONS: Non-arteritic ischaemic optic neuropathy patients with a reduction in VA may present two different, unrelated impairments: a dysfunction of the inner retinal layer (abnormal PERG) and abnormal post-retinal neural conduction (abnormal VEP and RCT). The reduction in VA seems to be related to the post-retinal impairment and seems to be independent from the retinal dysfunction.

Cytidine-5'-diphosphocholine (Citicoline): a pilot study in patients with non-arteritic ischaemic optic neuropathy.

BACKGROUND AND PURPOSE: Our work evaluates visual function before and after treatment with cytidine-5-diphosphocholine (Citicoline) in patients with non-arteritic ischaemic optic neuropathy (NION). METHODS: Twenty-six patients in which at least 6 months elapsed from NION, were randomly divided into two age-similar groups: 14 patients had Citicoline (Cebrolux-Tubilux, Italy, 1600 mg/diem for 60 days, followed by a 120-day period of wash out, days 60-180) (T-NION); 12 patients had no treatment during the same period (NT-NION). At day 180, in T-NION a second period of treatment (days 181-240) followed by a wash-out (days 241-360) was performed. Fourteen age-matched healthy subjects provided normative data. In all patients, pattern-electroretinogram (PERG), visual evoked potentials (VEPs) and visual acuity (VA) measurements were performed at baseline and at days 60 and 180. In T-NION, further measurements were achieved at days 240 and 360. RESULTS: At baseline, NT-NION and T-NION patients showed abnormal PERGs and VEPs, and reduced VA, compared to controls. At the end of treatment (days 60 and 240), T-NION patients showed improvement (P < 0.01) of PERGs, VEPs parameters and VA, compared to pre-treatment values. After wash out, functional improvements persisted compared to baseline. No changes in NT-NION patients were observed. CONCLUSIONS: Our results suggest a beneficial effect of oral Citicoline in NION.

Reduction of optic nerve fiber layer thickness in CADASIL.

Our study aims to assess nerve fiber layer (NFL) thickness in patients affected by cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL). Six CADASIL patients (mean age 42 +/- 16 years, best corrected visual acuity >20/20 with refractive error between +/-3 diopters, intraocular pressure <18 mmHg) were enrolled. They were compared with 16 age-matched controls. In all subjects enrolled, NFL thickness was measured by optical coherence tomography (OCT). Three different measurements were taken in each quadrant (superior, inferior, nasal, and temporal) and averaged. The data from all quadrants (12 values averaged) were identified as NFL overall. In CADASIL eyes there was a reduction of NFL thickness in each quadrant and in the NFL overall evaluation compared with the values observed in control eyes. Our results suggest that in CADASIL patients there is a reduction of NFL thickness evaluated by OCT. This morphological abnormality could be ascribed to an impairment of the retinal vascular supply leading to a global neuroretinal involvement. These anatomical changes may precede the onset of the neurological clinical manifestations.

Asymmetric distribution of visual evoked potentials in patients with migraine with aura during the interictal phase.

PURPOSE: One of the most commonly described electrocortical phenomena in patients with migraine is an increased interhemispheric asymmetry, in response to different sensory stimuli. This study aims to evaluate the bioelectrical activity of both occipital cortices in patients with migraine with visual aura (MA) during the interictal period, and its possible relationship with visual symptoms. METHODS: The authors recorded visual evoked potentials (VEPs) simultaneously from the left (O1) and right (O2) occipital cortices (80% contrast 60', 30', 15', and 7.5' checkerboard stimuli reversed at 2 Hz) in 22 patients with MA and 20 control subjects. The main outcome measure was interhemispheric asymmetry (IA) for both implicit time and amplitude, defined as the difference between the left and right scalp derivation (in absolute values). RESULTS: IA was significantly different in patients with MA with respect to controls when employing 60' (p<0.001) and 15' (p<0.05) checkerboard stimuli for implicit times, and 60' (p<0.05) checkerboard stimuli for amplitudes. On the other hand, IA was not statistically different (p>0.05) in patients with MA with respect to controls when employing 30' and 7.5' checkerboards for both implicit times and amplitudes, and 15' checkerboards for amplitudes. No correlations were found between IA and age, onset of disease, attack frequency, or side of headache/aura. CONCLUSIONS: Patients with MA presented asymmetries in VEP responses not related to visual aura or to headache side during the pain-free phase. These abnormalities may be ascribed to abnormal visual information processing, resulting in a different cortical activation when both foveal and parafoveal stimuli are used.

Photodynamic therapy outcomes in a case of macular choroidal neovascularization secondary to Candida endophthalmitis.

PURPOSE: To evaluate the effects on visual function and choroidal neovascularization (CNV) progression in a case of subretinal CNV due to Candida endophthalmitis treated with a combination of photodynamic therapy (PDT) and drugs. METHODS: A 28-year-old one-eyed woman with CNV in the right eye came to our observation. The CNV developed as a consequence of Candida endophthalmitis. The CNV was treated with six PDT treatments with verteporfin in association with systemic steroid therapy with prednisone (100 mg/day to reduce) and fluconazole (800 mg/day to reduce). Visual acuity (VA) was assessed in pre-PDT conditions and after six PDT treatments (24 months of follow-up). RESULTS: Pre-PDT VA was 20/125; after 24 months of follow-up, six PDT re-treatments, and pharmacologic therapy, VA was stabilized at 20/100. CONCLUSIONS: In our case, associated PDT and drug therapies were safe and useful to maintain VA and to arrest CNV progression in the foveal region after 2 years of follow-up.

Management of breast cancer during pregnancy using a standardized protocol.

PURPOSE: No standardized therapeutic interventions have been reported for patients diagnosed with breast cancer during pregnancy. Of the potential interventions, none have been prospectively evaluated for treatment efficacy in the mother or safety for the fetus. We present our experience with the use of combination chemotherapy for breast cancer during pregnancy. PATIENTS AND METHODS: During the past 8 years, 24 pregnant patients with primary or recurrent cancer of the breast were managed by outpatient chemotherapy, surgery, or surgery plus radiation therapy, as clinically indicated. The chemotherapy included fluorouracil (1,000 mg/m2), doxorubicin (50 mg/m2), and cyclophosphamide (500 mg/m2), administered every 3 to 4 weeks after the first trimester of pregnancy. Care was provided by medical oncologists, breast surgeons, and perinatal obstetricians. RESULTS: Modified radical mastectomy was performed in 18 of the 22 patients, and two patients were treated with segmental mastectomy with postpartum radiation therapy. This group included patients in all trimesters of pregnancy. The patients received a median of four cycles of combination chemotherapy during pregnancy. No antepartum complications temporally attributable to systemic therapy were noted. The mean gestational age at delivery was 38 weeks. Apgar scores, birthweights, and immediate postpartum health were reported to be normal for all of the children. CONCLUSION: Breast cancer can be treated with chemotherapy during the second and third trimesters of pregnancy with minimal complications of labor and delivery.

Detection of circulating tumor cells in carcinoma patients by a novel epidermal growth factor receptor reverse transcription-PCR assay.

The epidermal growth factor receptor (EGFR) is overexpressed in 50-70% of human primary breast, lung, and colon carcinomas, whereas it is not usually expressed in hematopoietic cells. We developed a novel reverse transcription-PCR (RT-PCR)-Southern blot assay for the detection of circulating, EGFR mRNA-expressing tumor cells in carcinoma patients. The assay was set up by increasing the amount of cDNA step by step in the PCR reaction. The highest sensitivity and specificity were found when using 800 ng of cDNA in the PCR reaction. Peripheral blood samples from 91 patients with either colon (38), lung (30), or breast (23) carcinomas and from 38 healthy volunteers were analyzed. EGFR transcripts were found in 44 of 75 (59%) patients with metastatic carcinoma and in 4 of 38 (10.5%) healthy donors (P < 0.001; chi2 test). The expression of EGFR, cytokeratin 19, and carcinoembryonic antigen mRNA in blood samples from patients with metastatic colon carcinoma was compared. EGFR, cytokeratin 19, and carcinoembryonic antigen transcripts were found in 8 of 11 (73%), 3 of 11 (27%), and 5 of 11 (45%) patients, respectively. Furthermore, two of seven (29%) Dukes' B and five of nine (55%) Dukes' C colon carcinoma patients were found to express EGFR mRNA in the peripheral blood. All patients that expressed EGFR transcripts in the peripheral blood were found to express the EGFR protein in the corresponding primary carcinoma, as assessed by immunohistochemistry. These data suggest that the EGFR assay that we developed is a highly specific and sensitive technique to detect circulating tumor cells in patients affected by different carcinoma types.

Visual acuity and macular sensitivity in myopic eyes before and after laser in situ keratomileusis.

PURPOSE: To evaluate the changes in visual acuity (VA) and in macular sensitivity in myopic eyes subjected to laser in situ keratomileusis (LASIK) refractive surgery. METHODS: In 38 myopic eyes, VA by Snellen's table and macular sensitivity by scanning laser ophthalmoscope (SLO) microperimetry were assessed before and after 24 weeks after LASIK surgery. The myopic eyes were divided into three age-matched groups: Group A = from -5 diopters (D) to -7 D and normal SLO-macular sensitivity (15 eyes); Group B = from -8 D to -16 D and normal SLO-macular sensitivity (9 eyes); and Group C = from -8 D to -16 D and abnormal SLO-macular sensitivity. RESULTS: Group A and B eyes, at the first week after LASIK surgery, we observed a significant analysis of variance, p<0.01) reduction in VA and SLO-macular sensitivity with respect to baseline values, while after 12 and 24 weeks no differences (p>0.01) were found when compared to baseline. In Group C patients, at 1 and 4 weeks after surgical treatment, we observed a significant (p<0.01) reduction in VA and SLO-macular sensitivity with respect to baseline values. At 12 and 24 weeks the values of VA were reduced, but not significantly (p>0.01), while values of SLO-macular sensitivity were still significantly (p<0.01) reduced. CONCLUSIONS: LASIK could induce a reduction in VA and SLO-macular sensitivity in all myopic eyes during the 4 weeks following the surgery. This reduction is still present after 24 weeks only in eyes with the highest preoperative degree of myopia combined with the greatest reduction in macular sensitivity.

Three dimensional myocardial deformation and diagnosis of stable coronary artery disease.

To date, only one third of patients, with stable angina, undergoing coronary angiography demonstrated obstructive coronary artery disease (CAD). Thus, identifying high sensitivity and specificity, low-cost, non invasive tests is crucial. Here we present the case of a patient, at a high risk of CAD, undergoing coronary angiography because of positive exercise test and stress imaging results, with non obstructive coronary artery disease at angiography, confirmed by FFR. Interestingly, 3D speckle tracking, performed before angiography, assessed normal left ventricle deformation, predicting the absence of severe coronary artery lesions.

Conservation of local electric fields in the evolution of Cu,Zn superoxide dismutase.

The trend of the electric field and the value of the electric field flux, sensed by the superoxide substrate in the proximity of the active site, were found to be constant in three highly homologous Cu,Zn superoxide dismutases from ox, pig and sheep, which display large differences in net protein charge and distribution of electrically charged surface residues but very similar catalytic rate constants. The spatial relationship of charges on the protein surface apparently has been conserved during the evolution of this enzyme to create electrostatic facilitation of catalysis.

Is the activity-linked electrostatic gradient of bovine Cu, Zn superoxide dismutases conserved in homologous enzymes irrespective of the number and distribution of charges?

Electrostatic potential calculations have been performed on three different Cu, Zn superoxide dismutases (superoxide: superoxide oxidoreductase, EC 1.15. 1.1), in order to evaluate the degree of conservation of the pattern of electrostatic interactions between O2- and the active site recently pointed out in bovine Cu Zn SOD. The three Cu, Zn SODs that have been selected for this study, namely the bovine, ovine, and porcine enzymes, are highly homologous as to reasonably assume identical three-dimensional structure but display large differences in their net charge, as shown by their pI's, which span over a wide pH range: 8.0 (sheep), 6.5 (pig), 5.2 (ox). Despite such a large difference in the net protein charge and in the spatial arrangement of electrostatic charges, electrostatic potential calculations show that the electrostatic channel directing the negatively charged substrate toward the positive catalytic site is strictly preserved with the same features for the three proteins. This suggests that the electrostatic funnel for conducting small anions into the active site is a highly conservative property in the evolution of Cu, Zn SOD.