Lymphocyte movements and interactions in microgravity

Cell-cell contacts and the formation of aggregates play an important role in the mitogen induced in-vitro activation of lymphocytes. The fact that the formation of cell aggregates is only slightly reduced in microgravity suggests that cells are moving and interacting also in space. Direct evidence was obtained for the first time in an experiment performed on a sounding rocket flight, where the movements and interactions of free-floating, non activated cells could be observed in real time in microgravity. In an experiment performed on the IML-2 mission in July 1994, the movements and interactions of human lymphocytes during activation with the mitogen Con A were studied in real time in microgravity.     

Early detection of cell metabolism with a silicon microsensor

A silicon microsensor (ISFET — Ion Sensitive Field Effect Transistor) has been used to detect the metabolism of a cell population cultured on a coverslip and positioned close to the sensor surface. The system output is analyzed as a function of cell density.     

Low molecular weight kininogen binds to platelets to modulate thrombin-induced platelet activation

The kininogens, high molecular weight kininogen (HK) and low molecular weight kininogen (LK), are multifunctional, single-gene products that contain bradykinin and identical amino-terminal heavy chains. Studies were performed to determine if LK would bind directly to platelets. 125I-LK specifically bound to gel-filtered platelets in the presence of 50 microM Zn2+. HK effectively competed with 125I-LK for the same binding site (Ki = 27 +/- 9 nM, n = 5). Similarly, the Ki for LK inhibition of 125I-LK binding was 12 +/- 1 nM (n = 3). Albumin, fibrinogen, factor XIII, and kallikrein did not inhibit 125I-LK binding to unstimulated platelets. 125I-LK (66 kDa) was not cleaved upon binding to platelets. The binding of 125I-LK to unstimulated platelets was found to be fully reversible by the addition of a 50 molar excess of unlabeled LK at both 10 and 20 min. LK binding to platelets was saturable with an apparent Kd of 27 +/- 2 nM (mean +/- S.E., n = 9) and 647 +/- 147 binding sites/platelet. Both LK and HK at plasma concentrations inhibited thrombin-induced platelet aggregation. LK and HK at about 5% of plasma concentration also inhibited thrombin-induced secretion of both stirred and unstirred platelets. Both kininogens were found to be noncompetitive inhibitors of proteolytically active thrombin binding to platelets. The kininogens did not inhibit D-phenylalanyl-prolyl-arginine chloromethyl ketone-treated thrombin from binding to platelets. These studies indicated that both kininogens have a region on their heavy chain which allows them to bind to platelets. Further, kininogen binding by its heavy chain modulates thrombin activation of platelets since it prevents proteolytically active thrombin from binding to its receptor.     

PcoB is a defense outer membrane protein that facilitates cellular uptake of copper

Copper (Cu) is one of the most abundant trace metals in all organisms, involved in a plethora of cellular processes. Yet elevated concentrations of the element are harmful, and interestingly prokaryotes are more sensitive for environmental Cu stress than humans. Various transport systems are present to maintain intracellular Cu homeostasis, including the prokaryotic plasmid‐encoded multiprotein pco operon, which is generally assigned as a defense mechanism against elevated Cu concentrations. Here we structurally and functionally characterize the outer membrane component of the Pco system, PcoB, recovering a 2.0 Å structure, revealing a classical β‐barrel architecture. Unexpectedly, we identify a large opening on the extracellular side, linked to a considerably electronegative funnel that becomes narrower towards the periplasm, defining an ion‐conducting pathway as also supported by metal binding quantification via inductively coupled plasma mass spectrometry and molecular dynamics (MD) simulations. However, the structure is partially obstructed towards the periplasmic side, and yet flux is permitted in the presence of a Cu gradient as shown by functional characterization in vitro. Complementary in vivo experiments demonstrate that isolated PcoB confers increased sensitivity towards Cu. Aggregated, our findings indicate that PcoB serves to permit Cu import. Thus, it is possible the Pco system physiologically accumulates Cu in the periplasm as a part of an unorthodox defense mechanism against metal stress. These results point to a previously unrecognized principle of maintaining Cu homeostasis and may as such also assist in the understanding and in efforts towards combatting bacterial infections of Pco‐harboring pathogens.     

Spliceosomal snRNAs in the unicellular eukaryote Trichomonas vaginalis are structurally conserved but lack a 5'-cap structure

Few genes in the divergent eukaryote Trichomonas vaginalis have introns, despite the unusually large gene repertoire of this human-infective parasite. These introns are characterized by extended conserved regulatory motifs at the 5' and 3' boundaries, a feature shared with another divergent eukaryote, Giardia lamblia, but not with metazoan introns. This unusual characteristic of T. vaginalis introns led us to examine spliceosomal small nuclear RNAs (snRNAs) predicted to mediate splicing reactions via interaction with intron motifs. Here we identify T. vaginalis U1, U2, U4, U5, and U6 snRNAs, present predictions of their secondary structures, and provide evidence for interaction between the U2/U6 snRNA complex and a T. vaginalis intron. Structural models predict that T. vaginalis snRNAs contain conserved sequences and motifs similar to those found in other examined eukaryotes. These data indicate that mechanisms of intron recognition as well as coordination of the two catalytic steps of splicing have been conserved throughout eukaryotic evolution. Unexpectedly, we found that T. vaginalis spliceosomal snRNAs lack the 5' trimethylguanosine cap typical of snRNAs and appear to possess unmodified 5' ends. Despite the lack of a cap structure, U1, U2, U4, and U5 genes are transcribed by RNA polymerase II, whereas the U6 gene is transcribed by RNA polymerase III.     

The bovine plasma retinol-binding protein. Amino acid sequence, interaction with transthyretin, crystallization and preliminary X-ray data

1. The primary structure of bovine plasma retinol-binding protein (RBP) has been determined and found to be more than 90% identical to human and rabbit RBPs, and more than 80% identical to rat RBP. Main changes in amino acid sequence are observed in two regions on the surface of the protein molecule (residues 138-148 and 169-183). 2. The interactions of bovine RBP with bovine and human transthyretins were investigated using the technique of fluorescence polarization. Bovine RBP was able to form high affinity complexes (K'd = 0.34 +/- 0.02 microM) with both bovine and human transthyretins, displaying a stoichiometry of approximately 2 molecules RBP/molecule transthyretin in both cases. The sites that participate in protein-protein interactions are thus very similar, and this tends to exclude the involvement of the superficial regions more significantly substituted in mammalian RBPs (residues 138-151 and 167-183) in the protein-protein recognition. 3. Bovine RBP has been crystallized (space group P2(1)2(1)2(1), with a = 4.61 nm, b = 4.91 nm, c = 7.61 nm) and the crystals are suitable for high-resolution X-ray diffraction studies.     

Prognostic impact of coronary microcirculation abnormalities in systemic sclerosis: a prospective study to evaluate the role of non-invasive tests

Introduction

Microcirculation dysfunction is a typical feature of systemic sclerosis (SSc) and represents the earliest abnormality of primary myocardial involvement. We assessed coronary microcirculation status by combining two functional tests in SSc patients and estimating its impact on disease outcome.

Methods

Forty-one SSc patients, asymptomatic for coronary artery disease, were tested for coronary flow velocity reserve (CFR) by transthoracic-echo-Doppler with adenosine infusion (A-TTE) and for left ventricular wall motion abnormalities (WMA) by dobutamine stress echocardiography (DSE). Myocardial multi-detector computed tomography (MDCT) enabled the presence of epicardial stenosis, which could interfere with the accuracy of the tests, to be excluded. Patient survival rate was assessed over a 6.7- ± 3.5-year follow-up.

Results

Nineteen out of 41 (46%) SSc patients had a reduced CFR (≤2.5) and in 16/41 (39%) a WMA was observed during DSE. Furthermore, 13/41 (32%) patients showed pathological CFR and WMA. An inverse correlation between wall motion score index (WMSI) during DSE and CFR value (r = -0.57, P <0.0001) was observed; in addition, CFR was significantly reduced (2.21 ± 0.38) in patients with WMA as compared to those without (2.94 ± 0.60) (P <0.0001). In 12 patients with abnormal DSE, MDCT was used to exclude macrovasculopathy. During a 6.7- ± 3.5-year follow-up seven patients with abnormal coronary functional tests died of disease-related causes, compared to only one patient with normal tests.

Conclusions

A-TTE and DSE tests are useful tools to detect non-invasively pre-clinical microcirculation abnormalities in SSc patients; moreover, abnormal CFR and WMA might be related to a worse disease outcome suggesting a prognostic value of these tests, similar to other myocardial diseases.     

Isolation and characterization of microsatellites in Primula glaucescens Moretti and their cross-species amplification in P. spectabilis Tratt.

The hexaploid herbaceous species Primula glaucescens Moretti and Primula spectabilis Tratt. (2n = 6x = 66) are two endemics of the Italian Pre-Alps protected by national and international laws. In order to plan conservation strategies for natural populations and to study the influence of the latest glaciation on them we developed a set of microsatellites markers for the endangered Primula species: ten primer pairs allowed analysis of polymorphic disomic loci in P. glaucescens samples and seven of them also amplified polymorphic disomic markers in P. spectabilis. Kirsten Wolff and Giorgio Binelli contributed equally to the paper     

Primary cortical neuronal cultures reduce cellular energy utilization during anoxic energy deprivation

It has been widely hypothesized that neurons reduce cellular energy use in response to periods of energy deprivation. To test this hypothesis, we measured rates of energy use under normoxia and anoxia in immature (6 days in vitro) and mature (13 days in vitro) neuronal cultures. During anoxic incubation immature and mature cultures reduced cellular energy use by 80% and 45%, respectively. Reduced cellular energy use dramatically affected ATP depletion in neuronal cultures under anoxia. Intracellular ATP stores were expected to deplete within 3 min of anoxia. However, ATP was maintained at decreased but stabilized concentrations for at least 3 h. The capacity of neuronal cultures to reduce cellular energy use during anoxia correlated with their sensitivity towards simulated ischemia. Immature cultures, with the largest capacity to reduce cellular energy use, survived simulated ischemia 2.5 times longer than mature cultures. The addition of glutamate receptor antagonists to mature cultures further decreased cellular energy use during anoxia and significantly extended their survival time under simulated ischemia. This study verifies that primary cortical neuronal cultures reduce cellular energy use during energy deprivation. Additionally, we show that maturation of glutamate receptor activity increases non-depressible energy demand in neuronal cultures.