Role of palmitic acid on the isolation and properties of halorhodopsin

Purified halorhodopsin was isolated from Halobacterium halobium as previously described (Dusch!, A. et al. (1988) J. Bioi. Chern. 263, 17016-17022). Two purple bands were eluted from phenyl-Sepharose column, indicating the presence of differently retained halorhodopsin forms; the absorption spectra of the two halorhodopsin bands in the dark were not different. By gas chromatography /mass spectrometry we could identify palmitate (which is only a minor lipid component of archaeal cells) among lipids associated with purple fractions. Typically the palmitate content of the first eluted band was higher than that of the second, indicating a correlation between the palmitate content and the retention time; from one to two fatty acid molecules per halorhodopsin molecule were present depending on the fraction analysed. Very little or no palmitate was released from denaturated halorhodopsin. By adding palmitate to buffers used in the phenyl-Sepharose chromatography, only one sharp purple band was collected, corresponding to the less retained halorhodopsin fraction. Pentadecanoic fatty acid could also affect the halorhodopsin chromatography. Chromatography of halorhodopsin in the presence of beta-mercaptoethanol showed only one band, corresponding to the more retained halorhodopsin form. The two halorhodopsin fractions had different photoreactivity; the less retained halorhodopsin fraction (at higher palmitate content) showed an higher rate of decay of the absorbance at 570 nm upon illumination. By following the decay of the absorbance at 570 nm upon addition of alkali in the dark, we found that the two halorhodopsin fractions had different pKa values of deprotonation

Role of palmitic acid on the isolation and properties of halorhodopsin

AbstractPurified halorhodopsin was isolated from Halobacterium halobium as previously vescribed (Duschl, A. et al. (1988) J. Biol. Chem. 263, 17016–17022). Two purple bands were eluted from phenyl-Sepharose column, indicating the presence of differently retained halorhodopsin forms; the absorption spectra of the two halorhodopsin bands in the dark were not different. By gas chromatography/mass spectrometry we could iventify palmitate (which is only a minor lipid component of archaeal cells) among lipids associated with purple fractions. Typically the palmitate content of the first eluted band was higher than that of the second, indicating a correlation between the palmitate content and the retention time; from one to two fatty acid molecules per halorhodopsin molecule were present vepending on the fraction analysed. Very little or no palmitate was released from venaturated halorhodopsin. By adding palmitate to buffers used in the phenyl-Sepharose chromatography, only one sharp purple band was collected, corresponding to the less retained halorhodopsin fraction. Pentavecanoic fatty acid could also affect the halorhodopsin chromatography. Chromatography of halorhodopsin in the presence of β-mercaptoethanol showed only one band, corresponding to the more retained halorhodopsin form. The two halorhodopsin fractions had different photoreactivity; the less retained halorhodopsin fraction (at higher palmitate content) showed an higher rate of vecay of the absorbance at 570 nm upon illumination. By following the vecay of the absorbance at 570 nm upon addition of alkali in the dark, we found that the two halorhodopsin fractions had different pKa values of veprotonation

ROLE OF PALMITiC ACID ON ISOLATION AND PROPERTIES OF HALORHODOPSIN

Halorhodopsin, a photoactivated Cl- pump of halobacteria, shows several analogies with bovine rhodopsin. As it is known that bovine rhodopsin is acylated, we have examined the possibility that fatty acids ore covalently bound to halorhodopsin too. Purified halorhodopsin was isolated from Halobacterium halobium as previously described (Duschl Aet ol, 263:17016-17022, 1988). Two purple fractions were eluted from phenylsepharose column; the absorption spectra of the two halorhodopsin fractions in the dark were identical. The two fractions showed the same 20 kDa protein bond on SDS-PAGE (Blonck A, Oesterhelt D, 6: 265-273, 2987). Lipids released by alkaline hydrolysis of the two halorhodopsin fractions were esterified with HCI/MeOH, analysed by gas chromatography/mass spectrometry and quantitated using an internal standard. We have identified methyl palmitate among derivatized lipids released from both halorhodopsin fractions; the paImitate content of the f i r s t eluted fraction was higher than the second. By adding palmitate to buffers used in the phenylsephorose chromatography, only one sharp purple bond was collected. The halorhodopsin photoreaction, studied by an optical multichannel analyzer, was found to be dependent on the amount of palmitate associated to purified halorhodopsin

Novel therapeutic approaches in Rheumatoid Arthritis: Role of Janus Kinases Inhibitors

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial inflammation and hyperplasia, autoantibody production, cartilage and bone destruction and several systemic features. Cardiovascular, pulmonary, psychological, and muscle involvement are the main comorbidities of RA and are responsible for the severity of the disease and long-term prognosis. Pharmacological treatment of rheumatic diseases has evolved remarkably over the past years. In addition, the widespread adoption of treat to target and tight control strategies has led to a substantial improvement of outcomes, so that drug-free remission is nowadays a realistic goal in the treatment of RA. However, despite the availability of multiple therapeutic options, up to 40% of patients do not respond to current treatments, including biologics. Small-molecule therapies offer an alternative to biological therapies for the treatment of inflammatory diseases. In the past 5 years, a number of small-molecule compounds targeting Janus kinases (JAKs) have been developed. Since JAKs are essential for cell signaling in immune cells, in particular controlling the response to many cytokines, their inhibitors quickly became a promising class of oral therapeutics that proved effective in the treatment of RA. Tofacitinib is the first Janus kinase (JAK) inhibitor approved for the treatment of RA, followed more recently by baricitinib. Several other JAK inhibitors, are currently being tested in phase II and III trials for the treatment of a different autoimmune diseases. Most of these compounds exhibit an overall acceptable safety profile similar to that of biologic agents, with infections being the most frequent adverse event. Apart from tofacitinib, safety data on other JAK inhibitors are still limited. Long-term follow up and further research are needed to evaluate the general safety profile and the global risk of malignancy of these small molecules, although no clear association with malignancy has been reported to date. Here, we will review the main characteristics of JAK inhibitors, including details on their molecular targets and on the clinical evidences obtained so far in the treatment of RA

Ischaemic Stroke Occurring in a Patient Treated with Monoclonal Antibodies for COVID-19

Since the COVID-19 outbreak began, an association between COVID-19 and thrombotic diseases has been underlined. Although this association is more frequent with venous thromboembolism, ischaemic stroke has also been reported as a thrombotic complication in several cohorts of affected patients. Furthermore, the association between ischaemic stroke and COVID-19 has been considered a risk factor for early mortality. On the other hand, after the successful vaccination campaign, the incidence and the virulence of SARS-CoV-2 decreased, though it has been observed that COVID-19 may induce a severe infection in specific cohorts of frail subjects. For this reason, different drugs have been introduced of an antiviral action in order to improve the disease outcome of frail patients. In this field, with the arrival of a neutralizing monoclonal antibody against SARS-CoV-2, in particular, sotrovimab, a further chance to treat high-risk patients with mild-to-moderate COVID-19 arrived, achieving a concrete reduction in the risk of disease progression. We here report our clinical experience of an ischaemic stroke occurring a few minutes after the administration of sotrovimab for the treatment of moderate COVID-19 in a frail patient with chronic lymphocytic leukaemia. Other causes of ischaemic stroke were ruled out, and in order to evaluate the probability of a rare side effect, the Naranjo probability scale has also been utilized. In conclusion, among several side effects that have been described during the treatment of COVID-19 with sotrovimab, ischaemic stroke was not reported. Therefore, we here report a rare case of ischaemic stroke with early clinical manifestation after the administration of sotrovimab for the treatment of moderate COVID-19 in an immunocompromised patient for the first time

Severe Hypothyroidism due to the Loss of Therapeutic Efficacy of l-Thyroxine in a Patient with Esophageal Complication Associated with Systemic Sclerosis

BackgroundThyroid function abnormalities and thyroid autoantibodies have been frequently described in patients with systemic autoimmune diseases as systemic sclerosis (SSc). Serum TSH levels are higher in SSc patients with more severe skin diseases and a worse modified Rodnan skin score. Asymptomatic esophageal involvement due to SSc has never been described as a cause of severe hypothyroidism due to l-thyroxine (l-T4) malabsorption in patients with Hashimoto’s thyroiditis (HT) and SSc.Case reportHere, we report a case of a 56-year-old female affected by both SSc and HT who developed severe hypothyroidism due to the loss of therapeutic efficacy of l-T4. Therapeutic failure resulted from the altered l-T4 absorption because of SSc esophageal complications. Clinical findings improved after the administration of oral liquid l-T4. Thyroid function completely normalized with a full clinical recovery, the disappearance of the pericardial effusion and the improvement of the pulmonary pressure.ConclusionA recognition of a poor absorption is crucial in patients with hypothyroidism and SSc to reduce the risk of the subsequent adverse events. This case suggests the importance of clinical and laboratory surveillance in patients with SSc and HT because the systemic complications of these dysfunctions may worsen the prognosis of hypothyroid SSc/HT patients

Inflammatory, Serological and Vascular Determinants of Cardiovascular Disease in Systemic Lupus Erythematosus Patients

Background and aim: Systemic lupus erythematosus (SLE) is associated with increased risk of cardiovascular disease (CVD). Among many mechanisms, accelerated atherosclerosis, endothelial dysfunction, and hypercoagulability play a main role. Here, we investigate whether inflammatory, serological and clinical markers of SLE determine and correlate with arterial stiffness in SLE patients. Materials and methods: Routine blood samples, inflammatory mediators, specific antibodies, and 24 h proteinuria were measured in 43 SLE patients and 43 age and sex-matched controls using routine laboratory assays. We also assessed arterial stiffness by measuring radial artery applanation tonometry-derived augmentation index (AI), normalized AI (AIx@75), aortic pulse pressure, central systolic, diastolic and peripheral blood pressure. Results: SLE patients showed a significantly greater arterial stiffness vs. controls, as demonstrated by the significantly higher AIx@75 and aortic pulse pressure. Interestingly, regression analysis showed that age, systolic pulse pressure, inflammatory markers (erythrocyte sedimentation rate and C-reactive protein), daily dose of glucocorticoids, and cumulative organ damage positively correlated with arterial stiffness. Conclusions: SLE patients show increased arterial stiffness which correlates with markers of inflammation, that is involved in early alterations in arterial walls. Applanation tonometry can be used to screen SLE patients for subclinical vascular damage to implement prevention strategies for CVD

Soccer helps in controlling the development of psoriasis in Italian second league players

We evaluated the impact of regular agonistic physical activity on psoriasis in soccer players of the Italian second league. Particularly, an observational cross‐sectional investigation was conducted by analysing a group of 323 Italian second league soccer players (consecutively enrolled during medical check‐up at training sessions) and 357 sex, age and body mass index matched controls (chaperones of patients attending the outpatient clinic of Department of Dermatology and Department of Allergy and Clinical Immunology, University of Naples Federico II) from July 2015 to May 2016. Our data may suggest that regular and intense sport activity might be an environmental factor which can positively influence psoriasis pathogenesis, thus limiting its outbreak

Prefrontal dysfunction in post-COVID-19 hyposmia: an EEG/fNIRS study

Introduction: Subtle cognitive dysfunction and mental fatigue are frequent after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, characterizing the so-called long COVID-19 syndrome. This study aimed to correlate cognitive, neurophysiological, and olfactory function in a group of subjects who experienced acute SARS-CoV-2 infection with persistent hyposmia at least 12 weeks before the observation.Methods: For each participant (32 post-COVID-19 patients and 16 controls), electroencephalography (EEG) and functional near-infrared spectroscopy (fNIRS) data were acquired using an integrated EEG-fNIRS system during the execution of a P300 odd-ball task and a Stroop test. The Sniffin' Sticks test was conducted to assess subjects' olfactory performance. The Montreal Cognitive Assessment (MoCA) and the Frontal Assessment Battery (FAB) were also administered.Results: The post-COVID-19 group consisted of 32 individuals (20 women and 12 men) with an average education level of 12.9 +/- 3.12 years, while the control group consisted of 16 individuals (10 women and 6 men) with an average education level of 14.9 +/- 3.2 years. There were no significant differences in gender (X-2 = 0, p = 1) or age between the two groups (age 44.81 +/- 13.9 vs. 36.62 +/- 11.4, p = 0.058). We identified a lower concentration of oxyhemoglobin (p < 0.05) at the prefrontal cortical level in post-COVID-19 subjects during the execution of the Stroop task, as well as a reduction in the amplitude of the P3a response. Moreover, we found that post-COVID-19 subjects performed worst at the MoCA screening test (p = 0.001), Sniffin's Sticks test (p < 0.001), and Stroop task response latency test (p < 0.001).Conclusions: This study showed that post-COVID-19 patients with persistent hyposmia present mild deficits in prefrontal function, even 4 months after the end of the infection. These deficits, although subtle, could have long-term implications for quality of life and cognitive wellbeing. It is essential to continue monitoring and evaluating these patients to better understand the extent and duration of cognitive impairments associated with long COVID-19