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Preliminary evidence supports the notion that COVID-19 patients may have an increased susceptibility to develop venous thromboembolism (VTE). However, the magnitude of this association still needs to be defined. Furthermore, clinical predictors of thrombogenesis, and the relationship with the inflammatory status are currently unknown. On this basis, we conducted a retrospective, observational study on 259 consecutive COVID-19 patients admitted to an academic tertiary referral hospital in Northern Italy between March 19th and April 6th, 2020. Records of COVID-19 patients with a definite VTE event were reviewed for demographic information, co-morbidities, risk factors for VTE, laboratory tests, and anticoagulation treatment. Twenty-five cases among 259 COVID-19 patients developed VTE (9.6%), all of them having a Padua score > 4, although being under standard anticoagulation prophylaxis since hospital admission. In the VTE subcohort, we found a significant positive correlation between platelet count (PLT) and either C reactive protein (CRP) (p p = 0.0013), while a significant inverse correlation was observed between PLT and mean platelet volume (p p p = 0.002 and p = 0.005, respectively). No significant difference was found in d-dimer levels between VTE and non VTE patients, while significantly higher levels of LDH (p = 0.04) and IL-6 (p = 0.04) were observed in VTE patients in comparison to non-VTE patients. In conclusion, our findings showed a quite high prevalence of VTE in COVID-19 patients. Raised inflammatory indexes and increased serum levels of pro-inflammatory cytokines should raise the clinical suspicion of VTE.

COVID-19 patients typically present with lower airway disease, although involvement of other organ systems is usually the rule. Hematological manifestations such as thrombocytopenia and reduced lymphocyte and eosinophil numbers are highly prevalent in COVID-19 and have prognostic significance. Few data, however, are available about the prevalence and significance of anemia in COVID-19. In an observational study, we investigated the prevalence, pathogenesis and clinical significance of anemia among 206 patients with COVID-19 at the time of their hospitalization in an Internal Medicine unit. The prevalence of anemia was 61% in COVID-19, compared with 45% in a control group of 71 patients with clinical and laboratory findings suggestive of COVID-19, but nasopharyngeal swab tests negative for SARS-CoV-2 RNA (p = 0.022). Mortality was higher in SARS-CoV-2 positive patients. In COVID-19, females had lower hemoglobin concentration than males and a higher prevalence of moderate/severe anemia (25% versus 13%, p = 0.032). In most cases, anemia was mild and due to inflammation, sometimes associated with iron and/or vitamin deficiencies. Determinants of hemoglobin concentration included: erythrocyte sedimentation rate, serum cholinesterase, ferritin and protein concentrations and number of chronic diseases affecting each patient. Hemoglobin concentration was not related to overall survival that was, on the contrary, influenced by red blood cell distribution width, age, lactate dehydrogenase and the ratio of arterial partial oxygen pressure to inspired oxygen fraction. In conclusion, our results highlight anemia as a common manifestation in COVID-19. Although anemia does not directly influence mortality, it usually affects elderly, frail patients and can negatively influence their quality of life.

Although many studies have been carried out in order to understand the implication of copper (Cu) in the pathogenesis of multiple sclerosis (MS), the exact role that this metal plays in the disease is not still clear. Because of the lack of information in this subject, the present study compared the serum and cerebrospinal (CSF) levels of copper in MS patients in respect to a control group, matched for age and sex, finding a significant increase of metal concentrations, in both biological fluids of MS subjects. To confirm the possible impairment of Cu metabolism, we analyzed ceruloplasmin (Cp) level and activity, seeing as this protein is an established peripheral marker in diseases associated with Cu imbalance. By comparing these two parameters between control and MS subjects, we found an increase of Cp levels, associated with a decrease in Cp activity, in the second group. By analysing these data, free copper levels were calculated, significantly increased in serum of MS subjects; the increase in free copper could be one of the predisposing factors responsible for the Cu altered levels in CSF of MS patients. At the same time, this alteration could be attributable to the inability to incorporate Cu by Cp, probably due to the high oxidative environment found in serum of MS patients. Overall, all these copper alterations may play a role in MS pathogenesis.

Few biochemical and molecular details are available on microspore growth and development. In this work, a nuclease was partially purified from diffusates of barley (Hordeum vulgare L.) microspores by using concanavalin-A as ligand. The chromatographic preparation contained a 34-kDa protein with nucleolytic activity; the enzyme (called BMN: barley microspore nuclease) was very stable at pH8.0 and temperatures below 50 degrees C. Activity was highest at pH 5.6 and increased almost exponentially with temperature until a breakpoint between activity and stability was reached at 70 degrees C. Although BMN was able to cleave RNA, the enzyme showed a remarkable preference for DNA, especially in the single-stranded form. The best homopolymeric substrates were poly(dA) and poly(A), whereas poly(dC), poly(G) and poly(I) were almost completely uncleaved. When incubated with intact nuclei, BMN caused a nucleosomal DNA ladder of approximately 200 bp. On the basis of DNA laddering, substrate specificity, Mg2+ -dependence and best performance at apoplastic pH, BMN can be referred to as a putative apoptotic nuclease involved in pollen development.

It was previously demonstrated that some plant inhibitors are able to inactivate the bacterial proteinase subtilisin ; since information concerning the effect of plant inhibitors on other microbial proteinases remains limited, we decided to determine the activity of the soybean Kunitz and Bowman-Birk inhibitors (KI and BBI, respectively) on 14 proteinases of fungal and bacterial origin. The results show that microbial proteinases are frequently inhibited by KI and BBI and that proteinases with the same EC number (eg subtilisin Carlsberg and subtilisin BPN') may equally give different responses to the inhibitors. In particular, all serine proteinases examined were affected by both KI and BBI while metalloproteinases were not. Inhibition was also achieved on a range of microbial proteinases for which the mechanistic class is yet to be established ; the data suggest that they belong to the serine type. In one instance, activation instead of inhibition was noted.

Solutions of D-fructose and sucrose were heated, alone and in the presence of glycine. The reaction products were examined in terms of their effects on peroxidase (POD) activity. Compared with glucose mixtures, fructose products had a great inhibiting effect on POD activity, while in the case of Maillard reaction products (MRPs), the differences in antibrowning activity between fructose-glycine and glucose-glycine mixtures were slight. As regards the sucrose and sucrose-glycine solutions, a slight inhibiting effect on POD activity was observed only after sugar hydrolysis. Measurements of the reducing power of sugar and sugar-amino acid solutions showed a correspondence between antioxidant properties of caramelization and Maillard reaction products and their antibrowning effects.

A complex chemical transformation known as the Maillard reaction may occur in foods containing reducing sugars and compounds with free amino groups. This reaction is accelerated by heating but occurs also at room temperature. Some food flavors, colors and texture characteristics depend on this reaction, but reduced nutritive values of proteins are also reported. To evaluate the contribution of digestive enzymes inhibition to this effect, activities of some proteases (pepsin, trypsin and chymotrypsin) were tested in the presence of glucose-glycine solutions heated at 90C for different times. Pepsin was partially inhibited by glycine itself but Maillard reaction products (MRP) did not produce additional effect. MRP showed a limited inhibition on trypsin and chymotrypsin. Unfractionated MRP solution showed an inhibiting effect on trypsin of the same order as already reported by Oeste (1989) for the low molecular weight (LMW) fraction of glucose-lysine MRP. High molecular weight (HMW) MRP presented an additional inhibiting effect, beside that reported for LMW on chymotrypsin. This however only occurred in particular conditions.

A bacterial species (Leuconostc oenos), well suited to acidic pH values, is proposed as substrate for the spectrophotometric determination of lysozyme activity. The principal advantage of this organism as the substrate is the ease of investigating lysozyme behavior at acidic pH. The sensitivity is lower than with Micrococcus luteus, but data are highly reproducible.

The effect of glucose, fructose and sucrose aqueous solutions on polyphenol oxidase (PPO) and peroxidase (POD) activity was studied. Solutions with high concentrations of sugars showed a slight inhibiting effect on PPO while an activation was observed for POD. Glucose solutions, tested after sufficient heat treatments, showed an inhibiting action only on PPO activity. Maillard reaction products, obtained by heating a glucose/glycine solution, caused an unexpectedly strong inhibiting effect on both enzymes. The inhibition became more evident with increasing heating times of the glucose/glycine solution, reducing the activity of PPO and POD nearly to zero. Thus, the Maillard reaction products, already known for their antioxidant properties, were also shown to have a strong inhibiting effect on enzymatic browning.

Many assay procedures have been devised to measure lipolytic activity, but none is without problems. It is for this reason that new methods are still being proposed. In this work we have investigated the use of two esters of p-nitrophenol, the palmitic acid and lauric acid esters, as substrates for a highly sensitive high-performance liquid chromatographic method. Data on recovery, specific activity and reproducibility are reported only for the lauric ester, because the palmitic ester turned out to be a very poor substrate.

Two serine-proteases (P I and P II ) were isolated from the larval midgut of Spodoptera littoralis by affinity chromatography. These hydrolysed casein and BApNA and were strongly inhibited by TLCK. On SDS-PAGE, an apparent molecular weight of 27 kDa was determined for both proteases. When mass spectrometric analysis was performed directly on the eluted fractions, one main component per peak was observed, having a molecular mass of 24,359±2 Da (P I ) and 24,448±2 Da (P II ). The enzymes had their maximum activity and stability at pH 10 and 4, respectively. P I was significantly more active than P II at 20°C or less; it was also more resistant to denaturation by heat (55 versus 50°C). A breakpoint between activity and stability was recorded at 30°C for both proteases. P II was strongly inhibited by aprotinin, leupeptin and KI, and partly inactivated by LBTI and chymostatin; P I was unaffected by LBTI and chymostatin and was also less susceptible to aprotinin and KI. On the basis of substrate degradation and inactivation rates, it was concluded that P I and P II are very different from the two proteases found in another strain of S. littoralis . Though P I and P II were found to belong to the same mechanistic class of bovine trypsin, they were not activated by Ca 2+ over the concentration range 0–20 mM. It is suggested that transgenic plants harbouring genes coding for effective P I inhibitors should have a high level of resistance towards this insect pest.

Autoimmune endocrinopathies are characterised by an increased number of peripheral blood lymphocytes (PBL) expressing activation/ memory markers on their surface. The aim of this study was to determine whether a similar finding could be detected in a group of 11 paediatric and young adult patients suffering from autoimmune polyglandular syndrome type 1 (APS1), also called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), as very few data have previously been reported in this field. The control group was made up of 11 sex- and age-matched healthy subjects. Fifteen lymphocyte subsets were compared, in terms of percentage and absolute number, and statistical analysis was performed by the Mann-Whitney test. Measurement of T (CD3+), B (CD19+), natural killer (NK, CD3-CD16/56+), CD4+ and CD8+ T lymphocytes showed that patients with APS1 had a higher percentage and absolute count of T lymphocytes: this was entirely due to the statistically larger CD3+CD4+ fraction. Patients with APS1 also had slightly fewer B and NK lymphocytes, but the difference was negligible. Comparison of CD4+ subpopulations bearing activation and naive/memory antigens (marked by CD69, CD25, anti-HLA-DR, CD45RA and CD45RO) showed that patients with APS1 had generally larger percentages and absolute counts of these subsets: however, only the percentage and absolute size of the CD4+CD25+ subset (p = 0.0354 and p = 0.0151, respectively), and the absolute number of the CD4+ anti-HLA-DR+ and CD4+ CD45RO+ subsets (p = 0.0193 and p = 0.0209, respectively) were significantly higher. Interestingly, patients with APS1 also had significantly fewer CD8+CD11b+ and CD3-CD8+ cells. In conclusion, PBL distribution in APS1 resembles that of other autoimmune diseases. Further studies are needed to confirm and possibly extend these data.

Many biochemical and molecular details are available for soybean seed proteinase inhibitors, but little is known about the quantitative regulation of structural genes. In order to fill this gap, a complete set of diallel crosses was made between inbred lines characterised by the same inhibitor array coupled with a different inhibitor content in the raw flour. The Hayman analysis revealed the presence of different regulatory elements in the parental lines, giving rise to both additive and dominance variation. Dominant alleles were found to decrease the trypsin inhibitory activity (TI activity) shown by the raw flours and appeared to have higher frequencies than recessive alleles. Significant maternal effects were also detected, particularly in crosses of low-TI activity female x high-TI activity male; maternal effects strengthened the role of the regulatory genes transferred by the female parent to the hybrid. Data fitted the simple additive-dominance model with genes independent in both action and distribution. Narrow and broad heritability values were 54% and 82% respectively, thus indicating the feasibility of lowering TI activity through selection.

A total of 168 strains of lactic acid bacteria were isolated from Italian raw ham and screened for antagonistic activity against Listeria monocytogenes by using an agar spot assay. Only one strain of Lactococcus lactis subsp. lactis produced antagonistic effects other than inhibition by low pH and hydrogen peroxide. The proteinaceous nature of the compound produced by L. lactis B10 was demonstrated by its inactivation by proteolytic enzymes. The cell-free culture super-natants (filtered and heat-treated) showed a bactericidal mode of action. Bacteriocin produced by L. lactis B10 was inhibitory to other lactic acid bacteria and one strain of Staphylococcus aureus, but not to the gram-negative bacteria tested.

Quantitation of myeloperoxidase (MPO) activity by guaiacol peroxidation (GP) assay is profoundly affected by the peroxidase present in eosinophils (EPO) that contaminate the granulocyte suspensions. Inclusion of 3-amino-1,2,4-triazole (AMT) in the GP assay permits quantitation of MPO activity in mixed neutrophil-eosinophil suspension because of the differential inhibition of EPO and MPO by AMT. Results show that: (1) the peroxidase activity of eosinophil-free granulocyte suspensions is not appreciably affected by AMT; (2) in the presence of AMT the peroxidase activities of granulocyte preparations containing different numbers of eosinophils are similar on a neutrophil basis, regardless of the number of eosinophils and correspond with the activity of eosinophil-free granulocyte suspensions; (3) AMT almost completely inhibits the activity of partially purified EPO, only slightly affecting the catalytic activity of partially purified MPO; (4) AMT completely inhibits the residual peroxidase activity of granulocyte suspensions from MPO-deficient subjects contributed by contaminating eosinophils. The GP assay in the presence of AMT was used to study the pattern of hereditary transmission of MPO deficiency. The genealogy derived on the basis of this assay was compatible with an autosomal recessive inheritance, in agreement with previously reported results, while no definite pattern of inheritance could be established by use of the GP assay without AMT. We suggest that the GP assay supplemented with AMT is the method of choice for detection of MPO deficiency, particularly partial deficiency.

A rapid and specific high-performance liquid chromatographic assay for the quantitative determination of angiotensin-converting enzyme activity is described. Hippuryl- l -histidyl- l -leucine (Hip—His—Leu) is used as substrate and the released hippuric acid is measured. The procedure is accurate and precise and no extraction is required.

(1) The effect of phospholipids on a preparation containing the ATPase complex and the adenine nucleotide carrier is studied in the presence of ligands known to affect the conformation of these components of the mitochondrial inner membrane. (2) When ATPase activity is abolished by phospholipid depletion, the reactivation induced by phosphatidylcholine is prevented by the simultaneous addition of ATP. ADP partially reproduces the ATP effect. AMP, GTP, UTP, and Pi are ineffective. (3) The influence of ATP is associated with reduced phospholipid binding to the membrane fragments and is reversible. The ATP effect on reconstitution is not manifest when phosphatidylcholine is added together with negatively charged phospholipids. (4) Carboxyatractyloside does not modify the phospholipid-ATPase complex interaction but bongkrekic acid is as effective as ATP. In the presence of ADP, the influence of bongkrekic acid is considerably increased. (5) It is concluded that the binding of ATP to the adenine nucleotide carrier enables the complex to select between the charged and uncharged phospholipids. As a result of the carrier conformational change, the ATPase complex is induced to prefer a negatively charged phospholipid environment.

1. 1. The activation by phospholipids of particulate mitochondrial ATPase from rat liver was studied on a preparation extracted from submitochondrial particles with cholate. 2. 2. Lysophosphatidylcholine and the highly acidic phospholipids diphosphatidylglycerol, phosphatidylinositol and phosphatidylserine stimulated the ATPase at low concentration (50–60 μg/mg protein); phosphatidylethanolamine was less effective whereas phosphatidylcholine exhibited the lowest activity. 3. 3. The saturation curves of phospholipids acting at low concentration showed in most cases a deviation from normal Michaelis-Menten kinetics. This devation was absent or less manifest with phosphatidylethanolamine and phosphatidylcholine. Competition between phospholipids and oligomycin was seen with acidic phospholipids or mixtures of neutral and acidic phospholipids. 4. 4. Similarly to oligomycin, dicyclohexylcarbodiimide, tributyltin and chlorpromazine competitively inhibited the activation produced by phosphatidylserine, phosphatidylinositol and diphosphatidylglycerol. 5. 5. It is concluded that both the hydrophobic-hydrophilic balance and the negative charge of phospholipids are important in the interaction between phospholipids and protein at the level of particulate mitochondrial ATPase. The competition between phospholipids and inhibitors of mitochondrial ATPase suggests that the activation induced by phospholipids is involved in the inhibitory effect.

A simple method for measuring angiotensin-converting enzyme activity in human serum was developed. Samples were incubated with hippurylhistidylleucine and the liberated hippuric acid was determined directly by reversed phase ion-pair high-performance liquid chromatography with UV spectrometric detection.

1. 1. Phosphatidylcholines of different acyl-chain composition and a preparation of ATPase complex depleted of phospholipids have been employed in order to evaluate the contribution of lipid bilayer to the assembly of this multisubunit component of mitochondrial membrane. 2. 2. At the minimal requirement for bilayer assembly (dinonanoylphosphatidylcholine, mixtures of lysophosphatidylcholine and phosphatidylcholine), fragments with oligomycin-insensitive ATPase activity are reconstituted. Conformational changes with dislocation of ATPase complex subunits may explain these results. 3. 3. At increased strength of acyl-chain interaction (dilauroylphosphatidylcholine and higher homologues), the damage to the ATPase complex is prevented but this is not sufficient to achieve functional restoration. Bilayers with a tendency to coalesce and fuse aggregate in large amounts with the complex and yield low ATPase reactivation. Bilayers of high stability yield complexes with physiological content of phospholipids and efficient ATPase activity. Transition between these two possibilities is found at sixteen carbon acyl-chains. Only at this chain length does the cholate dialysis procedure of reconstitution become feasible. 4. 4. It is concluded that a minimum of 16 carbon atoms in each chain are required to organize a bilayer structurable to maintain the ATPase complex conformation and to sustain the transmembrane position of the whole assembly.

1. 1. Kidney (Na+ + K+)-stimulated ATPase was depleted of phospholipids by extraction with lubrol and inserted in lipid structures of known composition. Both ouabain-sensitive ATPase and phosphatase reactions could be partially restored by lipid replacement. 2. 2. Lipid vesicles of natural and synthetic negative phospholipids proved to be effective. The low activity of uncharged liposomes was increased when negative charges were included into the bilayer structure. 3. 3. Reactivation by negative phospholipids was accompanied by spontaneous re-assembly of a stable lipid · protein complex. By contrast, the interaction of lipid deficient ATPase complex with uncharged lamellae was possible only after sonication of lipid-protein suspension. Reactivation did not ensue. 4. 4. The ouabain-sensitive ATPase reactivated by synthetic dioleoylphosphatidylglycerol yielded curvilinear Arrhenius plots. The same pattern was seen with the original undepleted microsomal preparation. A discontinuity close to the temperature of fluid-order transition was found with dimyristoyl phosphatidylglycerol. 5. 5. It is concluded that reassembly of lipid-deficient ( Na + + K + )-stimulated ATPase requires the addition of diacylphospholipids with fluid acyl-chains and negatively charged polar heads able to assemble in an expanded lamellar configuration.

The interaction of bovine heart mitochondrial oligomycin-sensitive ATPase (Serrano, R., Kranner, B. L., and Racker, E. (1976) J. Biol. Chem. 251, 2453-2461) with phospholipids has been examined by labeling the subunits exposed to lipids with photoreactive radioactive phospholipids. A subunit of Mr = 29,000 and some polypeptides in the range of 6,000 to 13,000 daltons were labeled. F1-ATPase subunits did not interact with the photoactive probes. This result is compared with the different pattern of labeling obtained with another mitochondrial ATPase preparation (Galante, Y.M., Wong, S. Y., and Hatefi, Y. (1979) J. Biol. Chem. 254, 12372-12378), which is devoid of the 29,000 component.

Mixed phospholipids, purified phosphatidylcholine and phosphatidylserine restored the ATPase activity of submitochondrial particles pretreated with dicyclohexylcarbodiimide (DCCD). Pretreatment of mixed phospholipids with DCCD resulted in preparations which were able to transfer the inhibitor to submitochondrial particles. Phosphatidylcholine alone was fully effective. Phosphatidylserine, which apparently forms a stable bond with DCCD, was inactive. These findings suggest different functions of individual phospholipids in the DCCD-induced inhibition. The observation that phosphatidylserine displays unusual high activity in the restoration of ATPase activity in phospholipid-depleted submitochondrial particles indicates that it plays an important role in the activation of mitochondrial ATPase and deserves attention as costituent of the final receptor site for DCCD.

1. 1. The stimulation of mitochondrial particulate ATPase by natural phosphatidylcholines was studied in a phospholipid-free preparation from bovine heart obtained by extraction with cholate. The low stimulatory activity of these phospholipids was increased by: (a) introduction of negatively charged amphiphatic substances into the zwitterionic liposomes; (b) addition of Cl − to the incubation medium. 2. 2. Liposomes of acidic phospholipids or phosphatidylcholine containing anionic amphipaths prevented the ATP plus Mg 2+ -induced decrease of ATPase activity in Mg-ATP particles which have a high content of ATPase inhibitor. 3. 3. Acidic phospholipids, in contrast to phosphatidylcholine, prevented the inhibition of soluble and particulate ATPase by purified ATPase inhibitor. 4. 4. It is concluded that the structure of the lipid-water interface is of primary importance in the stimulation produced by liposomes of diacylphospholipids on particulate mitochondrial ATPase. This effect is associated with the removal of inhibition induced by endogenous ATPase inhibitor on phospholipid-depleted preparations.