Structure and nucleotide sequence of Euphorbia characias copper/TPQ-containing amine oxidase gene

A cDNA encoding for a copper containing amine oxidase has been isolated and sequenced from young leaves of Euphorbia characias, a perennial mediterranean shrub. A single long open reading frame of 2068 pb encodes a protein composed of 653 amino acids with a molecular mass of about 74 kDa. A putative 24-aminoacid signal peptide precedes the sequence of the mature protein, with characteristics of a secretion signal peptide. Alignments of Euphorbia amine oxidase cDNA nucleotide sequence with that of amine oxidase from the seedlings of the pulses lentil, pea, and chickpea reveal several conserved regions, especially in the C-terminus, with a homology 90%–97%. The near 5 region shows several insertions, deletions, and different nucleotide sequence with ca. 60% homology. The enzyme contains 1%–2% carbohydrate deduced by deglycosylation experiments. Five cysteine residues are present in the deduced aminoacid sequence with a single disulfide bridge as judged by titration with cysteine reagents.     

Identification of the human salivary cystatin complex by the coupling of high-performance liquid chromatography and ion-trap mass spectrometry

Human salivary cystatins, five major (S, S1, S2, SA, SN) and two minor (C and D), are multifunctional proteins playing a different role in the oral environment. Salivary cystatin SN is able to effectively inhibit lysosomal cathepsins B, C, H and L and cystatin SA inhibits cathepsins C and L in vitro. These activities suggest, particularly for cystatin SN, an important role in the control of proteolytic events in vivo. Differently, cystatins S are involved, together with statherin, in the mineral balance of the tooth. Due to their distinct role, a reliable method for identification and quantification of the different cystatins, as well as of possible truncated and derived forms, could be helpful for the assessment of the status of the oral cavity. To this purpose high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI MS) was applied to the analysis of human saliva obtained from healthy subjects. All known salivary cystatins, with the exception of cystatin C, were detected. Strong evidence was also obtained for the presence in saliva of post-translational modified isoforms of cystatins, which may be related to donor habits. Cystatin SN and cystatins S, S1 and S2 were well separated by HPLC-ESI MS coupling from other components and thus this approach can be successfully applied to their quantification.     

Excited state charge-transfer dynamics study of poplar plastocyanin by ultrafast pump-probe spectroscopy and molecular dynamics simulation

We have applied ultrafast pump-probe spectroscopy to investigate the excited state dynamics of the blue copper protein poplar plastocyanin, by exciting in the blue side of its 600-nm absorption band. The decay of the charge-transfer excited state occurs exponentially with a time constant of approximately 280 fs and is modulated by well visible oscillations. The Fourier transform of the oscillatory component, besides providing most of the vibrational modes found by conventional resonance Raman, presents additional bands in the low frequency region modes, which are reminiscent of collective motions of biological relevance. Notably, a high frequency mode at approximately 508 cm(-1), whose dynamics are consistent with that of the excited state and already observed for other blue copper proteins, is shown to be present also in poplar plastocyanin. This vibrational mode is reproduced by a molecular dynamics simulation involving the excited state of the copper site.     

Plasma Free Hemoglobin and Microcirculatory Response to Fresh or Old Blood Transfusions in Sepsis

BackgroundFree hemoglobin (fHb) may induce vasoconstriction by scavenging nitric oxide. It may increase in older blood units due to storage lesions. This study evaluated whether old red blood cell transfusion increases plasma fHb in sepsis and how the microvascular response may be affected.MethodsThis is a secondary analysis of a randomized study. Twenty adult septic patients received either fresh or old (<10 or >15 days storage, respectively) RBC transfusions. fHb was measured in RBC units and in the plasma before and 1 hour after transfusion. Simultaneously, the sublingual microcirculation was assessed with sidestream-dark field imaging. The perfused boundary region was calculated as an index of glycocalyx damage. Tissue oxygen saturation (StO₂) and Hb index (THI) were measured with near-infrared spectroscopy and a vascular occlusion test was performed.ResultsSimilar fHb levels were found in the supernatant of fresh and old RBC units. Despite this, plasma fHb increased in the old RBC group after transfusion (from 0.125 [0.098–0.219] mg/mL to 0.238 [0.163–0.369] mg/mL, p = 0.006). The sublingual microcirculation was unaltered in both groups, while THI increased. The change in plasma fHb was inversely correlated with the changes in total vessel density (r = -0.57 [95% confidence interval -0.82, -0.16], p = 0.008), De Backer score (r = -0.63 [95% confidence interval -0.84, -0.25], p = 0.003) and THI (r = -0.72 [95% confidence interval -0.88, -0.39], p = 0.0003).ConclusionsOld RBC transfusion was associated with an increase in plasma fHb in septic patients. Increasing plasma fHb levels were associated with decreased microvascular density.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01584999","term_id":"NCT01584999"}}NCT01584999     

Grafting on a Non-Transgenic Tolerant Tomato Variety Confers Resistance to the Infection of a Sw5-Breaking Strain of Tomato spotted wilt virus via RNA Silencing

RNA silencing controls endogenous gene expression and drives defensive reactions against invasive nucleic acids like viruses. In plants, it has been demonstrated that RNA silencing can be transmitted through grafting between scions and silenced rootstocks to attenuate virus and viroid accumulation in the scions. This has been obtained mostly using transgenic plants, which may be a drawback in current agriculture. In the present study, we examined the dynamics of infection of a resistance-breaking strain of Tomato spotted wilt virus (RB-TSWV) through the graft between an old Apulian (southern Italy) tomato variety, denoted Sl-Ma, used as a rootstock and commercial tomato varieties used as scions. In tests with non-grafted plants, Sl-Ma showed resistance to the RB-TSWV infection as viral RNA accumulated at low levels and plants recovered from disease symptoms by 21 days post inoculation. The resistance trait was transmitted to the otherwise highly susceptible tomato genotypes grafted onto Sl-Ma. The results from the analysis of small RNAs hallmark genes involved in RNA silencing and virus-induced gene silencing suggest that RNA silencing is involved in the resistance showed by Sl-Ma against RB-TSWV and in scions grafted on this rootstock. The results from self-grafted susceptible tomato varieties suggest also that RNA silencing is enhanced by the graft itself. We can foresee interesting practical implications of the approach described in this paper.     

Reliable and Accurate CD4+ T Cell Count and Percent by the Portable Flow Cytometer CyFlow MiniPOC and “CD4 Easy Count Kit-Dry”, as Revealed by the Comparison with the Gold Standard Dual Platform Technology

Background

An accurate and affordable CD4+ T cells count is an essential tool in the fight against HIV/AIDS. Flow cytometry (FCM) is the “gold standard” for counting such cells, but this technique is expensive and requires sophisticated equipment, temperature-sensitive monoclonal antibodies (mAbs) and trained personnel. The lack of access to technical support and quality assurance programs thus limits the use of FCM in resource-constrained countries. We have tested the accuracy, the precision and the carry-over contamination of Partec CyFlow MiniPOC, a portable and economically affordable flow cytometer designed for CD4+ count and percentage, used along with the “CD4% Count Kit-Dry”.

Materials and Methods

Venous blood from 59 adult HIV+ patients (age: 25–58 years; 43 males and 16 females) was collected and stained with the “MiniPOC CD4% Count Kit-Dry”. CD4+ count and percentage were then determined in triplicate by the CyFlow MiniPOC. In parallel, CD4 count was performed using mAbs and a CyFlow Counter, or by a dual platform system (from Beckman Coulter) based upon Cytomic FC500 (“Cytostat tetrachrome kit” for mAbs) and Coulter HmX Hematology Analyzer (for absolute cell count).

Results

The accuracy of CyFlow MiniPOC against Cytomic FC500 showed a correlation coefficient (CC) of 0.98 and 0.97 for CD4+ count and percentage, respectively. The accuracy of CyFlow MiniPOC against CyFlow Counter showed a CC of 0.99 and 0.99 for CD4 T cell count and percentage, respectively. CyFlow MiniPOC showed an excellent repeatability: CD4+ cell count and percentage were analyzed on two instruments, with an intra-assay precision below ±5% deviation. Finally, there was no carry-over contamination for samples at all CD4 values, regardless of their position in the sequence of analysis.

Conclusion

The cost-effective CyFlow MiniPOC produces rapid, reliable and accurate results that are fully comparable with those from highly expensive dual platform systems.