Physical mapping identifies DXS265 as a useful genetic marker for carrier detection and prenatal diagnosis of X-linked agammaglobulinemia

The gene responsible for X-linked agammaglobulinemia (XLA) has not been identified; however, in the course of genetic linkage studies designed to map the locus more precisely, a number of closely linked polymorphic loci have been identified. These have proved to be useful in identifying carriers and in pre-natal diagnosis of this disease. The DXS178 locus was found to be closest to the XLA locus and has been the most usefully employed probe to date. Using physical mapping techniques, we have identified a previously cloned genetic marker, DXS265, as being situated within 5kb of DXS178. So far, we have found one family that is not informative for DXS178 but that is informative for DXS265; females in this family can now be offered the possibility of carrier determination and pre-natal diagnosis for this life-threatening disease.     

Pain in the Neck and Arm—Diagnosis by History and Examination

The cause of pain in the neck or pain in the arm coming from the neck can be accurately diagnosed by proper clinical examination. This requires knowledge of normal functional anatomy and understanding where pain can originate and what factors initiate the pain. The examination consists of testing for these abnormalities of motion and reproducing the symptoms by these motions.Weighing the history, both of trauma and indications of deep-seated tension or anxiety, against the conditions observed on physical examination, is important to correct diagnosis.Recognizing abnormal neck posture and activities reproducing pain in the neck and arm examination reveals the mechanism of pain. Treatment becomes evident.     

Biochemical and Structural Studies of 6-Carboxy-5,6,7,8-tetrahydropterin Synthase Reveal the Molecular Basis of Catalytic Promiscuity within the Tunnel-fold Superfamily

6-Pyruvoyltetrahydropterin synthase (PTPS) homologs in both mammals and bacteria catalyze distinct reactions using the same 7,8-dihydroneopterin triphosphate substrate. The mammalian enzyme converts 7,8-dihydroneopterin triphosphate to 6-pyruvoyltetrahydropterin, whereas the bacterial enzyme catalyzes the formation of 6-carboxy-5,6,7,8-tetrahydropterin. To understand the basis for the differential activities we determined the crystal structure of a bacterial PTPS homolog in the presence and absence of various ligands. Comparison to mammalian structures revealed that although the active sites are nearly structurally identical, the bacterial enzyme houses a His/Asp dyad that is absent from the mammalian protein. Steady state and time-resolved kinetic analysis of the reaction catalyzed by the bacterial homolog revealed that these residues are responsible for the catalytic divergence. This study demonstrates how small variations in the active site can lead to the emergence of new functions in existing protein folds.     

Evaluation of Vaccines for H5N1 Influenza Virus in Ferrets Reveals the Potential for Protective Single-Shot Immunization

As part of influenza pandemic preparedness, policy decisions need to be made about how best to utilize vaccines once they are manufactured. Since H5N1 avian influenza virus has the potential to initiate the next human pandemic, isolates of this subtype have been used for the production and testing of prepandemic vaccines. Clinical trials of such vaccines indicate that two injections of preparations containing adjuvant will be required to induce protective immunity. However, this is a working assumption based on classical serological measures only. Examined here are the dose of viral hemagglutinin (HA) and the number of inoculations required for two different H5N1 vaccines to achieve protection in ferrets after lethal H5N1 challenge. Ferrets inoculated twice with 30 μg of A/Vietnam/1194/2004 HA vaccine with AlPO₄, or with doses as low as 3.8 μg of HA with Iscomatrix (ISCOMATRIX, referred to as Iscomatrix herein, is a registered trademark of CSL Limited) adjuvant, were completely protected against death and disease after H5N1 challenge, and the protection lasted at least 15 months. Cross-clade protection was also observed with both vaccines. Significantly, complete protection against death could be achieved with only a single inoculation of H5N1 vaccine containing as little as 15 μg of HA with AlPO₄ or 3.8 μg of HA with Iscomatrix adjuvant. Ferrets vaccinated with the single-injection Iscomatrix vaccines showed fewer clinical manifestations of infection than those given AlPO₄ vaccines and remained highly active. Our data provide the first indication that in the event of a future influenza pandemic, effective mass vaccination may be achievable with a low-dose “single-shot” vaccine and provide not only increased survival but also significant reduction in disease severity.The emergence in 2004 and continued persistence of highly pathogenic H5N1 influenza A virus in bird populations is justifiably considered a potential pandemic threat (19). The virus has become endemic in many areas of the world and has demonstrated an ability to infect humans through transmission from poultry, thus far with limited human-to-human spread (26). Of great concern is that the case fatality rate for H5N1 infection of humans is reported to be >60%, compared to 0.1% for the 1957 and 1968 pandemics and 2 to 3% for the 1918 pandemic, which together resulted in at least 50 million deaths (14, 20). For these reasons, the development of strategies to minimize the impact if the virus mutates to acquire efficient human-to-human spread is essential.Vaccination is considered the best method to ultimately control an influenza pandemic and should be implemented as soon as the pandemic strain is identified and vaccines produced (9, 23). To maximize coverage, pandemic vaccines will need to be available rapidly and will have to include the minimal dose of antigen to achieve solid immunity. This poses several major problems. One is that the human population is predominantly immunologically naive to the emerging subtype of virus, and so very large numbers of people will need to be protected as quickly as possible, which will place a huge demand on vaccine supply. The use of an adjuvant to lower the dose of antigen required (8) may ameliorate this problem to some degree, but there are few adjuvants that are suitable for human use, particularly those in ready supply in the event of a pandemic. In addition, we have little understanding of what levels and what type of immunity will provide protection from death or severe disease due to H5N1 infection (19).Clinical trials with candidate H5N1 vaccines have been initiated with traditional virus preparations (egg-grown whole or detergent-disrupted “split” virions) and alternative vaccine strategies (recombinant protein, live-attenuated, and adjuvant-containing vaccines) (24). Using split virus alone, high amounts of antigen, containing 90 μg of hemagglutinin (HA), given twice, were required to elicit what is considered to be a protective antibody response in ca. 50% of subjects (25). Adjuvants, such as those based on aluminum salts (3) or the oil-in-water adjuvants MF59 (2, 17, 22) and ASO3 (13, 21), have provided considerable antigen dose reduction, but in all clinical trials and preclinical animal evaluation to date, two doses of vaccine have been required to achieve what is considered to be adequate anti-HA antibody levels or protection, respectively (8, 24).One aim of the present study was to determine how suitable the ferret model is for making assumptions about human responsiveness to influenza vaccination. To do this, we evaluated in ferrets the same H5N1 pandemic vaccines, formulated with or without AlPO₄ adjuvant, that had been examined in phase 1 and II randomized trials in healthy adults (18). We then sought to compare whether the responses to these vaccines were protective against lethal H5N1 challenge and whether the protective effects could be achieved with less antigen by using the more potent saponin-based Iscomatrix (ISCOMATRIX, referred to as Iscomatrix herein, is a registered trademark of CSL Limited) adjuvant. The Iscomatrix adjuvant has been shown to be safe and well tolerated in humans and to induce strong and long-lived antibody and cytotoxic T-cell responses in both humans and animal studies (7). Finally, the encouraging results with these adjuvants led us to examine whether protection from severe disease and death could be achieved after only a single injection of the H5N1 vaccines.     

A Charged Second-Site Mutation in the Fusion Peptide Rescues Replication of a Mutant Avian Sarcoma and Leukosis Virus Lacking Critical Cysteine Residues Flanking the Internal Fusion Domain

The entry process of the avian sarcoma and leukosis virus (ASLV) family of retroviruses requires first a specific interaction between the viral surface (SU) glycoproteins and a receptor on the cell surface at a neutral pH, triggering conformational changes in the viral SU and transmembrane (TM) glycoproteins, followed by exposure to low pH to complete fusion. The ASLV TM glycoprotein has been proposed to adopt a structure similar to that of the Ebola virus GP2 protein: each contains an internal fusion peptide flanked by cysteine residues predicted to be in a disulfide bond. In a previous study, we concluded that the cysteines flanking the internal fusion peptide in ASLV TM are critical for efficient function of the ASLV viral glycoproteins in mediating entry. In this study, replication-competent ASLV mutant subgroup A [ASLV(A)] variants with these cysteine residues mutated were constructed and genetically selected for improved replication capacity in chicken fibroblasts. Viruses with single cysteine-to-serine mutations reverted to the wild-type sequence. However, viruses with both C9S and C45S (C9,45S) mutations retained both mutations and acquired a second-site mutation that significantly improved the infectivity of the genetically selected virus population. A charged-amino-acid second-site substitution in the TM internal fusion peptide at position 30 is preferred to rescue the C9,45S mutant ASLV(A). ASLV(A) envelope glycoproteins that contain the C9,45S and G30R mutations bind the Tva receptor at wild-type levels and have improved abilities to trigger conformational changes and to form stable TM oligomers compared to those of the C9,45S mutant glycoprotein.All retroviruses have envelope glycoproteins that interact with a receptor protein on the cell surface to initiate entry (18, 36). The viral glycoprotein is synthesized as a precursor polyprotein consisting of the surface (SU) glycoprotein, which contains the domains that bind with the cellular receptor, and the transmembrane (TM) glycoprotein, which tethers the protein to the viral surface and contains the domains responsible for fusion of the viral and cellular membranes (32). After synthesis, the precursor viral glycoproteins form trimers through the interaction of the TM domains. The SU and TM domains are then cleaved by a cellular protease, forming a mature, metastable complex capable of mediating viral entry. A specific receptor protein interaction with the SU domain of the mature Env is required to initiate a conformational change in the trimer, separating the globular SU domains to allow the TM glycoproteins to form a structure that projects the fusion peptide toward the target membrane. Two domains in TM, the N-terminal heptad repeat and the C-terminal heptad repeat, are critical for the formation of the extended structure (13, 31)}. The fusion peptide is thought to interact with a target membrane irreversibly, forming an extended prehairpin TM oligomer structure anchored in both the viral and target membranes (35). The cooperation of several of these extended prehairpin TM oligomer structures is most likely required to complete fusion. The viral and target membranes are brought into close proximity when the C-terminal heptad repeats fold back into grooves formed by the N-terminal heptad repeats, forming presumably the most stable TM structure, the six-helix bundle (6HB). Fusion of the membranes proceeds through the initial mixing of the outer lipid leaflets, hemifusion, followed by initial fusion pore formation, pore widening, and the completion of fusion. The 6HB may undergo some additional structural rearrangement in order to bring the fusion peptide and membrane-spanning domain of TM into close proximity to form the final trimeric hairpin structure (22, 24, 33).Until recently, the triggering of class I virus fusion proteins was thought to occur by one of two mechanisms (13, 35, 36). In one mechanism, the viral glycoproteins interact with receptors on the cell surface, resulting in the trafficking of the virion into an endocytic compartment, followed by the triggering of structural rearrangements in the viral glycoproteins to initiate fusion by exposure to low pH (e.g., influenza virus hemagglutinin [HA]). In a second entry mechanism, the interaction of the viral glycoproteins with receptors on the cell surface in a neutral pH environment triggers the structural rearrangements in the viral glycoproteins directly, initiating viral entry. Retroviruses predominately employ the second entry mechanism, although two cellular protein receptors may be required to complete the conformational changes in the viral glycoproteins necessary to complete entry (e.g., human immunodeficiency virus type 1). However, the entry process of the avian sarcoma and leukosis virus (ASLV) family of retroviruses demonstrates a third entry mechanism for the action of class I virus fusion proteins (25). ASLV entry requires both a specific interaction between the viral glycoproteins and receptors at the cell surface at neutral pH, triggering initial conformational changes in the viral glycoproteins, and a subsequent exposure to low pH to complete fusion (2, 3, 22-24).The fusion peptides of ASLVs are not at the N terminus of the cleaved TM, as in all other retroviral TM proteins, but in a proposed internal loop (TM residues 22 to 37) flanked by two cysteine residues (residues C9 and C45) (Fig. ​(Fig.1).1). The ASLV TM glycoprotein has been proposed to adopt a structure similar to that of the Ebola virus GP2 protein: both contain an internal fusion peptide flanked by cysteine residues predicted to be in a disulfide bond (10). Other viruses contain internal fusion peptides also predicted to be in looped structures (35). In a study to determine if the cysteines that flank the ASLV fusion peptide are required for function, mutant ASLV Env proteins were constructed with one or both of these cysteines changed to serine (C9S, C45S, or C9S C45S [C9,45S]) (8). The mutant subgroup A ASLV [ASLV(A)] Env proteins were expressed, processed, and incorporated into virions at levels similar to those of wild-type (WT) ASLV(A) Env. The mutant and WT ASLV(A) Env proteins bound the Tva receptor with similar affinities. However, murine leukemia virus (MLV) virions pseudotyped with the mutant Envs were ∼500-fold less infectious (titer, ∼2 × 10³ inclusion-forming units [IFU]/ml) than MLV virions pseudotyped with WT ASLV(A) Env (titer, ∼1 × 10⁶ IFU/ml). The ability of the mutant Envs to mediate cell fusion was also greatly impaired compared to that of WT ASLV(A) Env in a cell-cell fusion assay. We concluded that the cysteines flanking the internal fusion peptide in ASLV TM are critical for efficient function of the ASLV viral glycoproteins in mediating entry. In a recent study, the cysteines flanking the fusion peptide region were shown to be critical for the lipid mixing stage of fusion (6).Open in a separate windowFIG. 1.Schematic representations of the ASLV-based RCASBP retroviral vector and the major domains of the envelope glycoproteins. The RCASBP(A)AP replication-competent vector contains a subgroup A env and a reporter gene coding for heat-stable AP. The hypervariable domains (vr1, vr2, hr1, hr2, and vr3) of the SU glycoprotein, the proteolytic cleavage site, the putative fusion peptide region (shaded box), and the membrane-spanning domain (MSD) of the TM glycoprotein are shown schematically. The first 45 residues of the TM glycoprotein are shown for wild-type subgroup A Env (WT) and for the three mutants tested in this study, with either a substitution of serine for the cysteine at position 9 in TM (C9S), a substitution of serine for the cysteine at position 45 in TM (C45S), or both substitutions (C9,45S). The complete sequence of the ASLV(A) WT TM glycoprotein is shown, with the fusion peptide region, N-terminal and C-terminal heptad repeat regions (N-alpha-helix; C-alpha helix), and membrane-spanning domain indicated.Very little is known about the structures of fusion peptides in the context of full-length, trimeric, viral glycoproteins upon interaction with target membranes. Also, natural membrane targets contain a variety of lipid and protein compositions in an asymmetrical organization that is difficult to reproduce experimentally (27). In addition, little is known about how fusion proteins with internal fusion peptide regions interact with target membranes or the possible conformational changes that might be required to complete the fusion process (19, 20). In this study, replication-competent ASLV(A) viruses containing the C9S, C45S, or C9,45S mutations were constructed and genetically selected for improved replication in chicken fibroblasts in order to further explore the importance of these cysteines for proper TM function. Viruses with single cysteine-to-serine mutations reverted to the WT sequence. However, viruses with both the C9S and the C45S mutation retained both mutations and acquired a second-site mutation that significantly enhanced the infectivity of the genetically selected virus population. Unexpectedly, the selected second-site mutation was a charged residue located in the middle of the hydrophobic fusion peptide within TM.     

Use of colony-based bacterial strain typing for tracking the fate of Lactobacillus strains during human consumption

Background  

The Lactic Acid Bacteria (LAB) are important components of the healthy gut flora and have been used extensively as probiotics. Understanding the cultivable diversity of LAB before and after probiotic administration, and being able to track the fate of administered probiotic isolates during feeding are important parameters to consider in the design of clinical trials to assess probiotic efficacy. Several methods may be used to identify bacteria at the strain level, however, PCR-based methods such as Random Amplified Polymorphic DNA (RAPD) are particularly suited to rapid analysis. We examined the cultivable diversity of LAB in the human gut before and after feeding with two Lactobacillus strains, and also tracked the fate of these two administered strains using a RAPD technique.     

Mitochondrial matters of the brain: mitochondrial dysfunction and oxidative status in epilepsy

Epilepsy is a neurological disorder characterized by spontaneous, recurrent and paroxysmal cerebral discharge, clinically leading to persistent alterations in function and morphology of neurons. Oxidative stress is one of possible mechanisms in the pathogenesis of epilepsy. Oxidative stress resulting from mitochondrial dysfunction gradually disrupts the intracellular calcium homeostasis, which modulates neuronal excitability and synaptic transmission making neurons more vulnerable to additional stress, and leads to neuronal loss in epilepsy. In addition, the high oxidative status is associated with the severity and recurrence of epileptic seizure. Hence, treatment with antioxidants is critically important in epileptic patients through scavenging the excessive free radicals to protect the neuronal loss. In this review, we reviewed the recent findings that focus on the role for antioxidants in prevention of mitochondrial dysfunction and the correlation between oxidative status and disease prognosis in patients with epilepsy.