Blood pressure levels of preterm infants in the first year of life

Systolic blood pressure was measured in 36 infants with median gestational age of 29 weeks (range 24–35 weeks) and birth weight of 1160 g (range 642–1500 g). Measurements were made at 1, 6 and 12 weeks, and subsequently at 12-weekly intervals during the first year of life. No infant developed chronic lung disease. Systolic blood pressure increased over the first 24 weeks ( p < 0.001) to a mean value of 95 mmHg, but did not change significantly over the next 24 weeks. These data provide a reference range of blood pressure levels during the first year of life for infants born at an early gestational age.     

巨噬细胞迁移抑制因子通过CC趋化因子配体2诱导巨噬细胞聚集

巨噬细胞迁移抑制因子最初是由于能抑制体外巨噬细胞随机迁移而被发现,现在它作为一种重要的调节因子参与一系列炎症性疾病过程.我们最近发现,巨噬细胞迁移抑制因子的缺失使一些由炎症介质诱发的白细胞-内皮细胞相互作用减弱,提示巨噬细胞迁移抑制因子在炎症反应中起作用的机制之一是促进白细胞聚集.……     

British HIV Association (BHIVA) national cohort outcomes audit of patients commencing antiretrovirals from naïve

Objectives

The aim of this work was to audit the extent to which routine HIV care in the UK conforms with British HIV Association (BHIVA) guidelines and specifically the proportion of patients starting highly active antiretroviral therapy (HAART) who achieve the outcome of virological suppression below 50 HIV‐1 RNA copies/mL within 6 months.

Methods

A prospective cohort review of adults with HIV infection who started antiretroviral therapy (ART) for the first time between April and September 2006 was carried out using structured questionnaire forms.

Results

A total of 1170 adults from 122 clinical sites participated in the review. Of these patients, 699 (59.7%) started ART at CD4 counts <200 cells/μL and 193 (16.5%) had not been tested for HIV drug resistance. Excluding patients with valid reasons for stopping short‐term ART, 795 (73.5%) of 1081 patients had an undetectable viral load (VL) at follow‐up. Detectable VL was strongly associated with pretreatment CD4 count below 50 cells/μL and pretreatment VL above 100 000 copies/mL, and was not associated with clinic location or case load. About a quarter of patients did not have a VL measurement during the first 6 weeks after starting ART.

Conclusions

The majority of patients who initiated ART at sites participating in this UK national audit were managed within the BHIVA guidelines and achieved virological suppression below 50 copies/mL around 6 months after commencing treatment. Poor VL outcomes were associated with very low CD4 cell count and/or high VL at baseline but not with clinic case load or location. There is an urgent need to diagnose patients at an earlier stage of their HIV disease.     

Frequent ongoing T-cell receptor rearrangements in childhood B- precursor acute lymphoblastic leukemia: implications for monitoring minimal residual disease

Crosslineage T-cell receptor delta (TCR delta) rearrangements are widely used as tumor markers for the follow up of minimal residual disease in childhood B-precursor acute lymphoblastic leukemia (ALL) by polymerase chain reaction (PCR). The major drawback of this approach is the risk of false-negative results due to clonal evolution. We investigated the stability of V delta 2D delta 3 rearrangements in a group of 56 childhood B-precursor ALL patients by PCR and Southern blot analysis. At the PCR level, V delta 2D delta 3-to-J alpha rearranged subclones (one pathway for secondary TCR delta recombination) were demonstrated in 85.2% of V delta 2D delta 3-positive patients tested, which showed that small subclones are present in the large majority of patients despite apparently monoclonal TCR delta Southern blot patterns. Sequence analysis of V delta 2D delta 3J alpha rearrangements showed a biased J alpha gene usage, with HAPO5 and J alpha F in 26 of 32 and 6 of 32 clones, respectively. Comparison of V delta 2D delta 3 rearrangement status between diagnosis and first relapse showed differences in seven of eight patients studied. In contrast, from first relapse onward, no clonal changes were observed in six patients studied. To investigate the occurrence of crosslineage TCR delta rearrangements in normal B and T cells, fluorescence-activated cell sorter-sorted peripheral blood CD19+/CD3- and CD19-/CD3+ cell populations from three healthy donors were analyzed. V delta 2D delta 3 rearrangements were detected at low frequencies in both B and T cells, which suggests that V delta 2-to-D delta 3 joining also occurs during normal B-cell differentiation. A model for crosslineage TCR delta rearrangements in B-precursor ALL is deduced that explains the observed clonal changes between diagnosis and relapse and is compatible with multistep leukemogenesis of B-precursor ALL.     

Increased surface expression of the membrane glycoprotein IIb/IIIa complex induced by platelet activation. Relationship to the binding of fibrinogen and platelet aggregation

Platelet activation altered the binding of three monoclonal antibodies (monovalent Fab' fragment) directed against the glycoprotein (GP) IIb/IIIa complex. An increased binding of two- to threefold occurred after stimulation with thrombin or phorbol myristate acetate (PMA), with slight but significant increase in the dissociation constants (Kd) of two antibodies (LJ-CP8 and LJ-P9). In contrast, no statistically significant changes were observed with ADP-stimulated platelets. The increased binding of LJ-CP3, but not of the other two antibodies, to activated platelets decreased by 30% to 40% in the presence of EDTA at 22 to 25 degrees C. Platelets stimulated by thrombin or PMA bound more fibrinogen than did those stimulated by ADP, and significant differences in the extent but not in the affinity of fibrinogen binding were observed with various platelet agonists. When the pool of GP IIb/IIIa molecules exposed on the surface of unstimulated platelets was reacted with the monoclonal antibody LJ-CP3 to block ADP-induced fibrinogen binding and platelet aggregation, stimulation with thrombin or PMA still induced substantial binding of antibody and fibrinogen, and aggregation ensued. Therefore, platelets exposed to "strong" agonists exhibit an increased number of surface-oriented epitopes associated with GP IIb/IIIa. The GP IIb/IIIa molecules bearing these newly exposed epitopes are functional in that they can bind fibrinogen and mediate platelet aggregation.     

Mechanistic basis for ubiquitin modulation of a protein energy landscape

Ubiquitin is a common posttranslational modification canonically associated with targeting proteins to the 26S proteasome for degradation and also plays a role in numerous other nondegradative cellular processes. Ubiquitination at certain sites destabilizes the substrate protein, with consequences for proteasomal processing, while ubiquitination at other sites has little energetic effect. How this site specificity—and, by extension, the myriad effects of ubiquitination on substrate proteins—arises remains unknown. Here, we systematically characterize the atomic-level effects of ubiquitination at various sites on a model protein, barstar, using a combination of NMR, hydrogen–deuterium exchange mass spectrometry, and molecular dynamics simulation. We find that, regardless of the site of modification, ubiquitination does not induce large structural rearrangements in the substrate. Destabilizing modifications, however, increase fluctuations from the native state resulting in exposure of the substrate’s C terminus. Both of the sites occur in regions of barstar with relatively high conformational flexibility. Nevertheless, destabilization appears to occur through different thermodynamic mechanisms, involving a reduction in entropy in one case and a loss in enthalpy in another. By contrast, ubiquitination at a nondestabilizing site protects the substrate C terminus through intermittent formation of a structural motif with the last three residues of ubiquitin. Thus, the biophysical effects of ubiquitination at a given site depend greatly on local context. Taken together, our results reveal how a single posttranslational modification can generate a broad array of distinct effects, providing a framework to guide the design of proteins and therapeutics with desired degradation and quality control properties.

Ubiquitin is an 8.5-kDa protein appended to target proteins as a posttranslational modification (PTM). Typically, ubiquitin is conjugated to the primary amine of substrate lysine residues, though noncanonical linkages to serine and cysteine also exist in vivo. Ubiquitin itself contains seven lysine residues, which allows building of ubiquitin chains with various linkages and topologies. Ubiquitination is most typically associated with targeting condemned proteins to the 26S proteasome for degradation; however, it is also involved in a large and ever-growing list of crucial regulatory, nondegradative cellular processes (1). A complex and highly regulated enzymatic cascade attaches ubiquitin to substrates and therefore plays a key role in determining the specific downstream effects of an individual ubiquitination event. There are several hundred E3 ligases, the terminal enzymes in this cascade (2), which give rise to broad proteome coverage and allow for some level of site specificity (3, 4).Multiple different ubiquitin chain linkages and topologies bind with high affinity to proteasomal ubiquitin receptors and promote degradation (5–8). However, the presence of a ubiquitin tag alone is not sufficient to ensure proteasomal degradation. In fact, a substantial proportion of ubiquitin-modified proteins that interact with the 26S proteasome are ultimately released (9, 10) and not degraded. The proteasome also relies on substrate conformational properties, initiating degradation at an unstructured region on the condemned protein (11, 12). Much work has been done to understand the requirements of this unstructured region with regard to length, sequence composition, and topological position (13–15), yet at least 30% of known proteasome clients lack such a region (16). While evidence suggests that well-folded proteins are processed by diverse cellular unfoldases, such as Cdc48/p97/VCP (17, 18), an intriguing possibility is that the ubiquitin modification itself can modulate the conformational landscape and thus regulate proteasome substrate selection. Simulations have suggested that ubiquitination can destabilize the folded state of the substrate protein, thereby allowing it to more readily adopt unfolded or partially unfolded conformations (19, 20).Recently, we demonstrated that this is indeed the case: ubiquitin can exert significant effects on a substrate’s energy landscape depending on the site of ubiquitination and the identity of the substrate protein. Moreover, these changes can have direct consequences for proteasomal processing (21). By examining the energetic effects of native, isopeptide-linked ubiquitin attachment to three different sites within the small protein barstar from Bacillus amyloliquefaciens, we found that ubiquitin attached at either lysine 2 or lysine 60 destabilizes the protein both globally and via subglobal fluctuations, and we thus refer to these residues as sensitive sites. By contrast, ubiquitination at lysine 78 produces little effect on the energy landscape (21), and we therefore term it a nonsensitive site. Another study found that ubiquitin, appended through a nonnative linkage, can destabilize a folded substrate as measured by changes in the midpoints for thermally induced unfolding transitions (22).Ubiquitination at the two sensitive sites in barstar increases the population of partially unfolded, high-energy states on the landscape sufficient for proteasomal engagement and degradation. Ubiquitination at the single nondestabilizing site does not allow for proteasomal degradation. These results suggest that ubiquitin-mediated destabilization can reveal an obligate unstructured region in substrates that otherwise lack such a region. Furthermore, ubiquitination at sensitive sites results in more rapid degradation of these barstar variants when a proteasome-engageable unstructured tail is fused to their C termini (21).This previous work clearly demonstrates that ubiquitin-mediated changes to the protein landscape can play an important role in proteasomal selectivity and processing; it did not, however, uncover the molecular mechanisms through which these site-specific effects arise. Here, we interrogate the molecular mechanisms of ubiquitin-induced changes for these same single-lysine variants of barstar. We investigate differences in the intrinsic dynamics of these regions within barstar and differences in how the protein responds to ubiquitination at these individual sites. We employed two sets of complementary approaches: 1) NMR and HDX-MS (hydrogen–deuterium exchange mass spectrometry) to characterize the equilibrium conformational fluctuations of the substrate protein in the presence and absence of ubiquitin and 2) molecular dynamics (MD) simulations to track the position of every atom in barstar, in the presence and absence of ubiquitin, starting from its native conformation over the timescale of microseconds.We find that ubiquitination has only subtle effects on the native structure of barstar. Ubiquitination at the sensitive sites, however, selectively increases fluctuations that expose barstar’s C terminus. While both of the sensitive sites arise in regions of barstar with relatively high conformational flexibility, the observed destabilization appears to occur through different thermodynamic mechanisms. By contrast, ubiquitination at the nonsensitive site has a protective effect on barstar’s C terminus. Thus, the effects of ubiquitination at each site are highly dependent on the local context. This mechanistic understanding of the site-specific effects of ubiquitination should aid in developing predictive models of the energetic consequences of individual ubiquitination events and also of the ways in which aberrant lysine targeting leads to disease (23–25).     

Expression and functional role of urokinase-type plasminogen activator receptor in normal and acute leukaemic cells

Urokinase-type plasminogen activator receptor (UPA-R-CD87) is a GPI-anchored membrane protein which promotes the generation of plasmin on the surface of many cell types, probably facilitating cellular extravasation and tissue invasion. A flow cytometric quantitative analysis of expression levels for UPA-R was performed on fresh blast cells from patients with acute myeloid leukaemia (AML, n = 74), acute lymphoblastic leukaemia (ALL, n = 24), and biphenotypic leukaemia (BAL, n = 3) using two CD87 monoclonal antibodies (McAbs) (3B10 and VIM5). Peripheral blood and bone marrow (BM) cells from 15 healthy adults served as controls. Using 3B10 McAb, UPA-R was expressed (>99%) by blood monocytes, neutrophils, and BM myelomonocytic precursors in controls, whereas resting T and B lymphocytes, and CD34⁺ cells were UPA-R negative. We also attempted to clarify whether UPA-R has a role in mediating neutrophil functions. Oriented locomotion induced by different chemotaxins and lysozyme release by granules stimulated with fMLP or PMA were significantly decreased when UPA-R was neutralized by CD87 McAb. In contrast, the anti-UPA-R McAb had no effect on superoxide anion generation of normal neutrophils. Blasts from AML showed a heterogenous pattern of expression for the UPA-R McAbs, with reactivity strictly dependent on FAB subtype. The highest UPA-R expression was seen in the M5 group: all patients tested (n = 20) showed strong positivity for the UPA-R McAb whereas only 12% (3/24) of ALL patients were CD87 positive, and 2/3 of BAL patients showed a dim expression for CD87. The number of receptors expressed by blast cells in 6/74 (8.1%) AML patients was higher than those of normal samples; in addition, since co-expression of UPA-R and CD34 was not found in normal haemopoietic cells, it may be postulated that CD87 can be used alone (when overexpressed) or in combination with CD34 for the detection of minimal residual disease. Results also indicated that patients with UPA-receptors >12 × 10³ ABC/cell, irrespective of FAB subtype, had a greater tendency for cutaneous and tissue infiltration and a higher frequency of chromosome abnormalities, thus suggesting the concept that cellular UPA-R content positively correlates with the invasive potential of AML cells. The combination of higher UPA-R positivity, abnormalities of chromosome 11, and M5 FAB morphology may identify a peculiar subset of AML, characterized by a more aggressive clinical course.     

Molecular and cellular effects of antisickling concentrations of alkylureas

Alkylureas are capable of inhibiting sickling in vitro and the gelation of solutions of hemoglobin S at concentrations between 0.05 and 0.1 M with increasing effectiveness that is directly proportional to the length of the alkyl chain (butyl greater than propyl greater than ethyl greater than methyl). 6The inhibitory effect is independent of pH between 6.5 and 7.5 and is a process driven by entropy. The alkylureas at concentrations of 0.1 M have minimal effects on several erythrocyte functions. Oxygen equilibria, osmotic fragility, reduced glutathione content, and glutathione reductase activity are totally unaffected, while pyruvic kinase activity is decreased only by butylurea by about 20%, and glucose-6-phosphate dehydrogenase activity is decreased progressively to a maximum of 30% in direct proportion to the length of the alkyl chain. Alkylureas not only inhibit sickling but are also capable of desickling erythrocytes that have been maintained in the deoxygenated state. They have little effect on several erythrocyte functions at antisickling concentrations, but their toxicity must be evaluated before they can be examined as potential therapeutic agents for the treatment or prevention of acute episodes in sickle cell anemia.