The Thr205 phosphorylation site within respiratory syncytial virus matrix (M) protein modulates M oligomerization and virus production.

UNLABELLED: Human respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and pneumonia in infants and the elderly worldwide; however, there is no licensed RSV vaccine or effective drug treatment available. The RSV matrix (M) protein plays key roles in virus assembly and budding, but the protein interactions that govern budding of infectious virus are not known. In this study, we focus on M protein and identify a key phosphorylation site (Thr205) in M that is critical for RSV infectious virus production. Recombinant virus with a nonphosphorylatable alanine (Ala) residue at the site was markedly attenuated, whereas virus with a phosphomimetic aspartate (Asp) resulted in a nonviable virus which could only be recovered with an additional mutation in M (serine to asparagine at position 220), strongly implying that Thr205 is critical for viral infectivity. Experiments in vitro showed that mutation of Thr205 does not affect M stability or the ability to form dimers but implicate an effect on higher-order oligomer assembly. In transfected and infected cells, Asp substitution of Thr205 appeared to impair M oligomerization; typical filamentous structures still formed at the plasma membrane, but M assembly during the ensuing elongation process seemed to be impaired, resulting in shorter and more branched filaments as observed using electron microscopy (EM). Our data thus imply for the first time that M oligomerization, regulated by a negative charge at Thr205, may be critical to production of infectious RSV. IMPORTANCE: We show here for the first time that RSV M's role in virus assembly/release is strongly dependent on threonine 205 (Thr205), a consensus site for CK2, which appears to play a key regulatory role in modulating M oligomerization and association with virus filaments. Our analysis indicates that T205 mutations do not impair M dimerization or viruslike filament formation per se but rather the ability of M to assemble in ordered fashion on the viral filaments themselves. This appears to impact in turn upon the infectivity of released virus rather than on virus production or release itself. Thus, M oligomerization would appear to be a target of interest for the development of anti-RSV agents; further, the recombinant T205-substituted mutant viruses described here would appear to be the first RSV mutants affected in viral maturation to our knowledge and hence of considerable interest for vaccine approaches in the future.

Variation within the CLEC16A gene shows consistent disease association with both multiple sclerosis and type 1 diabetes in Sardinia.

Variation within intron 19 of the CLEC16A (KIAA0350) gene region was recently found to be unequivocally associated with type 1 diabetes (T1D) in genome-wide association (GWA) studies in Northern European populations. A variant in intron 22 that is nearly independent of the intron 19 variant showed suggestive evidence of association with multiple sclerosis (MS). Here, we genotyped the rs725613 polymorphism, representative of the earlier reported associations with T1D within CLEC16A, in 1037 T1D cases, 1498 MS cases and 1706 matched controls, all from the founder, autoimmunity-prone Sardinian population. In these Sardinian samples, allele A of rs725613 is positively associated not only with T1D (odds ratio=1.15, P one-tail=5.1 x 10(-3)) but also, and with a comparable effect size, with MS (odds ratio=1.21, P one-tail 6.7 x 10(-5)). Taken together these data provide evidence of joint disease association in T1D and MS within CLEC16A and underline a shared disease pathway.

[G6PD phenotype and red blood cell sensitivity to the oxidising action of chlorites in drinking water]. / Fenotipo G6PD e sensibilità degli eritrociti all'azione ossidante dei cloriti nelle acque potabili.

Chlorine dioxide is widely used to replace sodium hypochlorite in the disinfection of surface waters for human consumption, in order to avoid or reduce the formation of organohalogenated compounds with mutagenic and carcinogenic activity. However, chlorine dioxide may lead to the formation of by-products, such as chlorites and chlorates, that have an oxidative effect on the blood corpuscled fraction. In this investigation, blood crasis was assessed in relation to the G6PD phenotype and the consumption of tap water, disinfected with chlorine dioxide, or bottled mineral water from non-disinfected underground sources. The results show that the effect of oxidative stress resulting from the uptake of chlorites with drinking water is not additive to the effect due to G6PD deficiency. The observed change in haematological parameters, including those related to the G6PD polymorphism, is always within the normal range. However, it is still possible that more relevant changes would follow exposure to chlorites concentrations greater than that observed in the present study.

The substrate translocation channel of the proteasome.

The core particle (CP) of the yeast proteasome is composed of four heptameric rings of subunits arranged in a hollow, barrel-like structure. We have found that the CP is autoinhibited by the N-terminal tails of the outer (alpha) ring subunits. Crystallographic analysis showed that deletion of the tail of the alpha3 subunit opens a channel into the proteolytically active interior chamber of the CP, thus derepressing peptide hydrolysis. In the latent state of the particle, the tails prevent substrate entry by imposing topological closure on the CP. Inhibition by the alpha subunit tails is relieved upon binding of the regulatory particle to the CP to form the proteasome holoenzyme. Opening of the CP channel by assembly of the holoenzyme is regulated by the ATPase domain of Rpt2, one of 17 subunits in the RP. Thus, open-channel mutations in CP subunits suppress the closed-channel phenotype of an rpt2 mutant. These results identify a specific mechanism for allosteric regulation of the CP by the RP.

Properties of 3H-MPTP binding sites in human blood platelets.

Our study demonstrates that 3H-1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (3H-MPTP) specifically binds to platelet membrane sites in humans. This specific, high affinity and saturable binding has properties similar to those of 3H-MPTP binding to rat and monkey brain, with a higher affinity. Deprenyl, a specific inhibitor of MAO type B enzyme, was the most potent drug in displacing 3H-MPTP from platelet binding sites. Platelets are considered a good model for central aminergic neurons and are very rich with MAO enzymatic activity, exclusively of type B. Our findings support previous evidence indicating a correspondence between 3H-MPTP binding sites and MAO-B enzyme. Moreover the presence of 3H-MPTP binding sites on human platelets suggests the use of this peripheral tissue as a simple model to study at least partially the mechanisms of neurotoxic action of MPTP.

[Anti-HLA antibodies in nephropathic patients]. / Rilevazione di anticorpi anti HLA con nuova metodica citofluorimetrica in una casistica di pazienti nefropatici candidati al trapianto.

BACKGROUND: The presence of anti-human leukocyte antigen (HLA) alloantibodies in nephropathic patients is due to immunogenic stimuli such as transfusions, pregnancies, and transplantations. These stimuli can be highlighted using a classic aspecific serologic technique, such as complement-dependent cytotoxicity (CDC) or using more recent and specific techniques, such as cytofluorimetrics or enzyme linked immunoabsorbant assay (ELISA). Because the presence of anti-HLA preformed antibodies is linked to the largest incidence of both acute and chronic rejection, it seems appropriate to re-evaluate that data obtained using aspecific classic serological analysis techniques by using the more specific cytofluorimetric technique. To aid in the possible prevention of ant-HLA antibody formation, it is also appropriate to analyze the influence of immunogenic stimuli on the development of these antibodies. METHODS: We studied 116 patients (37 women and 79 men). Anti-HLA antibodies were detected using microlymphotoxic technique after separation of B and T lymphocytes. This separation was obtained using magnetic balls. We used a 30-cell panel. We also used a recent cytofluorimetric test (Flow Pra screening; One Lambda Inc., 21001 Kittridge St., Canoga Park, California, U.S.A.) with a panel of micrograins covered with class I and class II purified antigens. Statistical analysis was performed using chi-square analysis or Fischer s exact test. For each test, sensibility, specificity, and positive and negative value were measured. RESULTS: Among 33 patients testing positive using the classic CDC-PRA technique (17 positive for B-lymphocytes and 16 positive for both B and T lymphocytes), using cytometry, 25 were positive for anti-HLA-specific antibodies (10 among the B lymphocyte-positive patients and 15 among the B + T lymphocyte-positive patients). Two patients were shown positive only using the cytofluorimetric method. Of the 27 patients positive at cytometry, 18 were positive for class I and class II, 4 for class I, and 5 for class II. FLOW-PRA screening results were less sensitive and more specific. The results obtained by the two methods are comparable(p<0.0001). The immunogenic stimuli found responsible for immunization were: transfusion in 10 of 25 patients, pregnancies in 3/9 patients, transplant in 4/8 patients, and different immunogenic stimuli in 10/12 patients. The results were not statistically significant (p>0.05). CONCLUSIONS: Data show that positivity for B lymphocytes obtained using CDC-PRA is not always linked to the development of anti-HLA antibodies, whereas positivity for B+T lymphocytes, obtained using CDC -PRA, is often linked to specific antibody development. Immune response is more often directed against class I and II antibodies. The specific detection of HLA antibodies using the cytofluorimetric method allows us to identify patients at risk for rejection, and it suggests that red cells should be filtrated to prevent anti-HLA immunization secondary to transfusion in transplantation candidates.

[Spinal analgesia and perioperative low molecular weight heparin (LMWH) prophylaxis of thrombosis. Safety aspect]. / Znieczulenie zewnatrzoponowe i podpajeczynówkowe a okolooperacyjna profilaktyka przeciwzakrzepowa niskoczasteczkowa heparyna. Aspekt bezpieczenstwa.

For many gynecological surgery patients belonging to deep vein thrombosis (DVT) high-risk group the analgesia of choice is regional spinal analgesia. Perioperatively LMWH--Fraxiparine was administered to 426 gynecological surgery patients and to 113 caesarean section patients. The first dose 7500 ICU s.c. was administered 2 hours before operation and consecutive ones every 24 hours for 5 to 7 days. The drug didn't cause any anaesthesia complications like enhanced bleeding after lumbar punction. It was emphasised in the discussion that in choosing this kind of prophylaxis certain conditions should be fulfilled in order to avoid spinal hematoma.

[Appendiceal abscess in the third gestational trimester of pregnancy, complications pre and postoperatively]. / Zapalenie wyrostka robaczkowego w trzecim trymestrze ciazy, powiklania przed i pooperacyjne.

Delayed surgical intervention connected with misdiagnosis of preterm labour and urinary tract infection caused in gravida 3 in 34th gestational week appendiceal abscess, septic shock, stillbirth by cesarean section, necessity of hysterectomy, recidivism of multi peritoneal and pleural abscesses. Although the patient was rescued the retrospective pro memoria considerations of our procedure are regarded.

Keepers at the final gates: regulatory complexes and gating of the proteasome channel.

The proteolytic active sites of the 26S proteasome are sequestered within the central chamber of its 20S catalytic core particle. Access to this chamber is through a narrow channel defined by the outer alpha subunits. Free proteasome 20S core particles are found in an autoinhibited state in which the N-termini of neighboring alpha subunits are anchored by an intricate lattice of interactions blocking access to the channel. Entry of substrates into proteasomes can be enhanced by attachment of activators or regulatory particles. An important part of this activation is channel gating; regulatory particles rearrange the blocking residues to form an open pore and promote substrate entry into the proteolytic chamber. Interestingly, some substrates can open the entrance themselves and thus facilitate their own destruction. In this review, we will discuss the mechanisms proposed for channel gating and the interactions required to maintain stable closed and open conformations.

Occupational medicine content of Oregon family physician practices.

BACKGROUND: Little has been published in the medical literature about the occupational medicine content of family practice. Little is known about the educational interventions needed for family physicians to improve the care they provide to patients suffering from occupational-related disorders. METHODS: A questionnaire based on a curriculum in occupational medicine proposed by a subcommittee of the Education Committee of the American Academy of Family Physicians for the guidance of family practice residency directors was sent to a random sample of 100 Oregon family physicians from a total of approximately 570 active practicing members of the Oregon Academy of Family Physicians. Ninety-three completed questionnaires were returned. RESULTS: Occupational medicine constituted a significant part (14 percent) of the practices of Oregon family physicians. The respondents rated management of chronic disability, disability determination, repetitive trauma disorders, and legal issues as the most important occupational medicine problems in their practices. These issues were also those about which they thought they needed additional training. CONCLUSIONS: Education in occupational medicine for family physicians must be better tailored to fit their needs and their priorities. The responses to this survey of practicing family physicians in Oregon suggest that more training in the areas of chronic disability management, disability determination, the management of repetitive trauma disorders, and legal aspects of occupational disorders is needed.

Effective administration of recombinant tissue plasminogen activator (rt-PA) during resuscitation of a post partum patient with massive pulmonary embolism.

Severe pulmonary embolism with cardiac arrest occurred half an hour after the 5th spontaneous delivery in a 34-year-old multipara. Standard resuscitation for 30 minutes remained ineffective. Only after Actilyse rt-PA infusion the patient's state improved. She regained consciousness on the third day. After 32 days of hospital treatment the patient was discharged in a generally good condition. Today, 5 years after the described event her state of health is very good.

A gated channel into the proteasome core particle.

The core particle (CP) of the yeast proteasome is composed of four heptameric rings of subunits arranged in a hollow, barrel-like structure. We report that the CP is autoinhibited by the N-terminal tails of the outer (alpha) ring subunits. Crystallographic analysis showed that deletion of the tail of the alpha 3-subunit opens a channel into the proteolytically active interior chamber of the CP, thus derepressing peptide hydrolysis. In the latent state of the particle, the tails prevent substrate entry by imposing topological closure on the CP. Inhibition by the alpha-subunit tails is relieved upon binding of the regulatory particle to the CP to form the proteasome holoenzyme.