Identification of commercial hake and grenadier species by proteomic analysis of the parvalbumin fraction

Analysis of parvalbumin fractions through proteomic methodologies allowed the differential classification of ten commercial, closely related species of the family Merlucciidae. Muscle extracts from nine hake species of the genus Merluccius including two subspecies of Merluccius australis (australis and polylepsis) and one grenadier species Macruronus novaezelandiae with two populations (novaezelandiae and magellanicus) were evaluated by 2-DE and MALDI-TOF MS. 2-DE demonstrated that the species tested displayed a low intra-specific degree of polymorphism and the isoform patterns were noticeably species-specific. MALDI-TOF mass fingerprints showed clear differences in the pattern of peptides produced by tryptic digestion between the Merluccius and the Macruronus, making the genus differentiation possible. In addition, a selective peptide mass present in the spectra from certain hakes allowed its classification in two groups: Euro-African and American hakes. Besides, some specific masses allowed a clearly individual identification for M. bilinearis, M. australis polylepsis, M. australis australis, M. productus, M. paradoxus and M. polli, while the rest of the hake species can be grouped in two clusters, comprising M. hubbsi and M. gayi in one and M. merluccius and M. capensis in the other.     

Low temperature-induced systems failure in Escherichia coli: insights from rescue by cold-adapted chaperones

The growth of Escherichia coli cells is impaired at temperatures below 21 degrees C and stops at 7.5 degrees C; however, growth of a transgenic strain producing the cold-adapted chaperones Cpn60 and Cpn10 from the psychrophilic bacterium Oleispira antarctica is good at low temperatures. The E. coli cpn(+) transgene offers a novel opportunity for examining the essential protein for cell viability at low temperatures. By screening a large-scale protein map (proteome) of cells of K-12 and its Cpn(+) transgene incubated at 4 degrees C, we identified 22 housekeeping proteins involved in systems failure of E. coli when confronted with low temperature. Through co-immunoprecipitation of Cpn60, Northern blot, and in vitro refolding, we systematically identified that protein-chaperone interactions are key determinants of their protein functions at low temperatures. Furthermore, chromosomal gene deletion experiments suggest that the mechanism of cold-induced systems failure in E. coli is cold-induced inactivation of the GroELS chaperonins and the resulting failure to refold cold-inactivated Dps, ClpB, DnaK and RpsB proteins. These findings: (1) indicate the potential importance of chaperones in cold sensitivity, cold adaptation and cold tolerance in cellular systems, and (2) suggest the identity of a few key cold-sensitive chaperone-interacting proteins that get inactivated and ultimately cause systems failure in E. coli cells at low temperatures.     

Simple purification of immunoglobulins from whey proteins concentrate

We have developed a new protocol with only two steps for purification of immunoglobulins (Ig) from a protein concentrate of whey. Following this protocol, we have an 80% recovery of immunoglobulins, fairly pure. The purification was achieved by eliminating the BSA, via a strong adsorption on DEAE-agarose. Full desoprtion of the other serum proteins could be achieved without contamination with BSA. Thus, a protein solution containing only Ig and very small proteins (e.g., beta-lactoglobulins and alpha-lactalbumin) was obtained. Offering this protein mixture to a lowly activated aminated support, only Ig adsorbed on the support. It has been shown that BSA is able to interact with other proteins (including Ig and lactalbumins). This ability to form complexes with other proteins prevented the success of the direct adsorption of Ig on this mildly activated support, even although Ig should be the largest protein presented in dairy whey.     

Continuity of Visual and Auditory Rhythms Influences Sensorimotor Coordination

People often coordinate their movement with visual and auditory environmental rhythms. Previous research showed better performances when coordinating with auditory compared to visual stimuli, and with bimodal compared to unimodal stimuli. However, these results have been demonstrated with discrete rhythms and it is possible that such effects depend on the continuity of the stimulus rhythms (i.e., whether they are discrete or continuous). The aim of the current study was to investigate the influence of the continuity of visual and auditory rhythms on sensorimotor coordination. We examined the dynamics of synchronized oscillations of a wrist pendulum with auditory and visual rhythms at different frequencies, which were either unimodal or bimodal and discrete or continuous. Specifically, the stimuli used were a light flash, a fading light, a short tone and a frequency-modulated tone. The results demonstrate that the continuity of the stimulus rhythms strongly influences visual and auditory motor coordination. Participants'' movement led continuous stimuli and followed discrete stimuli. Asymmetries between the half-cycles of the movement in term of duration and nonlinearity of the trajectory occurred with slower discrete rhythms. Furthermore, the results show that the differences of performance between visual and auditory modalities depend on the continuity of the stimulus rhythms as indicated by movements closer to the instructed coordination for the auditory modality when coordinating with discrete stimuli. The results also indicate that visual and auditory rhythms are integrated together in order to better coordinate irrespective of their continuity, as indicated by less variable coordination closer to the instructed pattern. Generally, the findings have important implications for understanding how we coordinate our movements with visual and auditory environmental rhythms in everyday life.     

T-wave Oversensing with Inappropriate Therapy in Remote Monitoring

T-wave oversensing can cause inappropriate implantable cardioverter-defibrillator (ICD) therapies that are difficult to correct. Remote monitoring allows follow-up of ICD patients without visiting the hospital and can help in early detection of any malfunctions. We describe the case of a patient who experienced inappropriate antitachycardia pacing therapy due to T-wave oversensing; the problem was promptly detected by remote monitoring and corrected by device reprogramming.     

Vaccination reduces simian-human immunodeficiency virus sequence reversion through enhanced viral control

It has been suggested that vaccination prior to infection may direct the mutational evolution of human immunodeficiency virus type 1 (HIV-1) to a less fit virus, resulting in an attenuated course of disease. The present study was initiated to explore whether prior immunization might prevent the reversion of the virus to the wild-type form. Mamu-A*01 monkeys were vaccinated to generate a cytotoxic T-lymphocyte response to the immunodominant Gag p11C epitope and were then challenged with a cloned pathogenic CXCR4-tropic simian-human immunodeficiency virus (SHIV) expressing a mutant Gag p11C sequence (Δp11C SHIV). The epitopic and extraepitopic compensatory mutations introduced into gag of Δp11C SHIV resulted in attenuated replicative capacity and eventual reversions to the wild-type Gag p11C sequence in naïve rhesus monkeys. However, in vaccinated rhesus monkeys, no reversions of the challenge virus were observed, an effect that may have been a consequence of significantly decreased viral replication rather than a redirection of the mutational evolution of the virus. These findings highlight the multifactorial pressures that affect the evolution of primate immunodeficiency viruses.CD8⁺ cytotoxic T-lymphocyte (CTL) responses are important for controlling replication of human immunodeficiency virus type 1 (HIV-1) in humans and simian immunodeficiency virus (SIV) in rhesus monkeys (6, 15, 19, 25, 32, 37, 39-41). However, the accumulation of mutations in dominant epitopes of these viruses can undermine this immune control (1, 8, 13, 18, 28). It has been proposed that a preexisting memory-specific CTL response elicited by vaccination prior to HIV-1/SIV infection might change the epitope specificity or the mutational pattern of the infecting virus (9). It is also possible that vaccine-induced cellular immunity might diminish the level of virus replication in individuals following infection and in doing so decrease the rate of accumulation of viral mutations and the likelihood of emergence of viruses that can escape CTL recognition.Our laboratory has previously described a rare SHIV-89.6P escape virus that contains a mutation in the dominant Mamu-A*01-restricted Gag p11C C-M (CTPYDINQM) epitope (3, 4). The emergence of this viral variant was associated with an increase in viral load and the eventual death of the previously vaccinated rhesus monkey 798. Analysis of the escape virus demonstrated a threonine-to-isoleucine mutation at amino acid position 47 (T47I) of the SIV capsid protein, which corresponds to position 2 of the Gag p11C epitope. This T47I mutation abrogated binding to the Mamu-A*01 class I molecule, allowing the virus to escape from recognition by the dominant epitope-specific CTL population (4). In addition to the T47I mutation, a downstream isoleucine-to-valine (I71V) substitution was found to be required for the viability of the escape virus in vitro (12, 29, 30, 42).The present studies were initiated to study the effects of prior vaccination on Gag p11C sequence reversion by infecting monkeys with a simian-human immunodeficiency virus (SHIV) clone containing the gag mutations found in the escape virus that evolved in monkey 798. We first explored the effects of these mutations in vivo by infecting naïve Mamu-A*01⁺ rhesus monkeys with a cloned SHIV (Δp11C SHIV) containing both the Gag p11C T47I mutation and the downstream I71V compensatory substitutions. We then determined whether vaccination prior to infection could generate a cellular immune response that might alter the expected pattern of virus mutation in the immunodominant Mamu-A*01-restricted Gag p11C epitope of Δp11C SHIV.     

Bimolecular Complementation Defines Functional Regions of Herpes Simplex Virus gB That Are Involved with gH/gL as a Necessary Step Leading to Cell Fusion

Herpes simplex virus (HSV) entry into cells requires four membrane glycoproteins: gD is the receptor binding protein, and gB and gH/gL constitute the core fusion machinery. Crystal structures of gD and its receptors have provided a basis for understanding the initial triggering steps, but how the core fusion proteins function remains unknown. The gB crystal structure shows that it is a class III fusion protein, yet unlike other class members, gB itself does not cause fusion. Bimolecular complementation (BiMC) studies have shown that gD-receptor binding triggers an interaction between gB and gH/gL and concurrently triggers fusion. Left unanswered was whether BiMC led to fusion or was a by-product of it. We used gB monoclonal antibodies (MAbs) to block different aspects of these events. Non-virus-neutralizing MAbs to gB failed to block BiMC or fusion. In contrast, gB MAbs that neutralize virus blocked fusion. These MAbs map to three functional regions (FR) of gB. MAbs to FR1, which contains the fusion loops, and FR2 blocked both BiMC and fusion. In contrast, MAbs to FR3, a region involved in receptor binding, blocked fusion but not BiMC. Thus, FR3 MAbs separate the BiMC interaction from fusion, suggesting that BiMC occurs prior to fusion. When substituted for wild-type (wt) gB, fusion loop mutants blocked fusion and BiMC, suggesting that loop insertion precedes BiMC. Thus, we postulate that each of the gB FRs are involved in different aspects of the path leading to fusion. Upon triggering by gD, gB fusion loops are inserted into target lipid membranes. gB then interacts with gH/gL, and this interaction is eventually followed by fusion.Entry of herpes simplex virus (HSV) into cells requires four viral glycoproteins, gB, gD, gH, and gL, plus one of several cell receptors, either herpesvirus entry mediator (HVEM), nectin-1, or 3-OST (45). Crystal structures and other studies have documented that receptor binding triggers conformational changes to gD that trigger the downstream events leading to fusion (10, 11, 18, 26, 28, 52). Moreover, when HSV receptor-bearing cells are transfected with expression plasmids for glycoproteins gB, gD, gH, and gL, the cells fuse to form multinucleated giant cells or syncytia (39, 48). However, the precise series of events that take place after receptor binding have not yet been fully elucidated. What we do know is that both gB and a heterodimer of gH/gL constitute the core fusion machinery that is conserved and required for the fusion step of entry of all herpesviruses (18, 26, 30, 46, 49).Thus far, we know the crystal structure of one form of the gB ectodomain of HSV type 1 (HSV-1) (19). This protein has the characteristics of a fusion protein and is a charter member of the class III group of viral fusion proteins (4). Others in this class include Epstein-Barr virus gB, vesicular stomatitis virus (VSV) G, and baculovirus gp64 (5, 22, 41). Like VSV G and gp64, gB has two putative fusion loops at the base of each protomer of the crystallized trimer. Single-amino-acid mutations in many of the hydrophobic residues of the putative fusion loops of gB ablate its ability to function in cell-cell fusion assays (16, 17). Moreover, these mutants are unable to complement the entry of a gB-null virus (16). Finally, the ectodomains of these mutants, unlike wild-type protein, failed to coassociate with liposomes, indicating that the putative fusion loops do insert into membranes (16, 17). Recently, it was shown that several of these mutants are also defective for fusion events involved in virus egress (51). Together, these studies provide compelling evidence that HSV gB functions as a fusion protein and that the fusion loops are critical for this function. However, unlike VSV G and baculovirus gp64, gB does not function on its own in entry but, rather, requires the participation of gH/gL. In the absence of crystallographic data for gH/gL, it is not yet clear what role it plays in herpesvirus fusion. In a previous study, we used bimolecular complementation (BiMC) to examine protein-protein interactions that occur among the viral glycoproteins during fusion (1). A similar study was carried out by Avitabile et al. (2). The BiMC assay is based on the observation that N- and C-terminal fragments of green fluorescent protein (GFP) (and derivatives such as enhanced yellow fluorescent protein [EYFP]) do not spontaneously reconstitute a functional fluorophore (20, 29, 40). However, the codons for each half can be appended to the genes for two interacting proteins (23, 24). When these are cotransfected, an interaction between the two proteins of interest brings the two halves of the fluorophore in close enough contact to restore fluorescence.When HSV receptor-bearing cells, such as B78H1 cells that are engineered to express nectin-1, are transfected with plasmids that express gB, gD, gH, and gL, they undergo cell-cell fusion (13, 15, 27, 31, 48). When gD is omitted, no fusion occurs. We found that fusion of these transfected cells could be triggered by addition of a soluble form of gD (the gD ectodomain). We then used this approach to examine interactions between gB and gH/gL during cell fusion (1). Therefore, we tagged gB with the C-terminal half of EYFP and gH with the N-terminal half. When plasmids bearing these forms were cotransfected into C10 cells along with a plasmid for untagged gL, no fusion occurred, but importantly, no BiMC occurred. However, when we added gD306, cells began to fuse within 10 min, and all of the syncytia that formed exhibited bright EYFP fluorescence indicative of BiMC. We concluded that gD triggers both fusion and a physical interaction between gB and gH/gL. However, these experiments did not separate these two events, so we were unable to determine if the interaction preceded fusion or merely was a by-product of it.The purpose of this study was to determine if the gB-gH/gL interaction is essential for fusion and if it occurs prior to fusion. We focused on gB because its structure is known and we have a panel of well-characterized monoclonal antibodies (MAbs) to gB. Our approach was to determine which of these MAbs, if any, could block fusion and also block the interaction with gH/gL. We also examined the effect of mutations to the fusion loops of gB on its interaction with gH/gL. We previously mapped these MAbs to four functional regions (FR) of gB, three of which were resolved in the crystal structure (6, 19). Of these, FR1 contains the fusion loops, FR2 is in the center of the gB structure with no known function, and FR3 is at in the crown of the protein and may be involved in binding to cells (7). Our rationale was that if the interaction between gB and gH/gL is important for fusion, then it should not be blocked by nonneutralizing anti-gB MAbs. At the same time, we thought that some neutralizing MAbs might not only block fusion but also block BiMC. We found that neutralizing MAbs to FR1 and FR2 inhibited both BiMC and fusion. In contrast, we found that neutralizing MAbs that map to FR3 blocked fusion but failed to block the interaction between gB and gH/gL, thereby dissociating the two events. Finally, we found that gB mutants with changes in the fusion loops that were fusion negative were also unable to bind to gH/gL. The latter results suggest that insertion of gB into the target membrane precedes its interaction with gH/gL.     

The chromatin-remodeling factor CHD4 coordinates signaling and repair after DNA damage

In response to ionizing radiation (IR), cells delay cell cycle progression and activate DNA repair. Both processes are vital for genome integrity, but the mechanisms involved in their coordination are not fully understood. In a mass spectrometry screen, we identified the adenosine triphosphate–dependent chromatin-remodeling protein CHD4 (chromodomain helicase DNA-binding protein 4) as a factor that becomes transiently immobilized on chromatin after IR. Knockdown of CHD4 triggers enhanced Cdc25A degradation and p21^Cip1 accumulation, which lead to more pronounced cyclin-dependent kinase inhibition and extended cell cycle delay. At DNA double-strand breaks, depletion of CHD4 disrupts the chromatin response at the level of the RNF168 ubiquitin ligase, which in turn impairs local ubiquitylation and BRCA1 assembly. These cell cycle and chromatin defects are accompanied by elevated spontaneous and IR-induced DNA breakage, reduced efficiency of DNA repair, and decreased clonogenic survival. Thus, CHD4 emerges as a novel genome caretaker and a factor that facilitates both checkpoint signaling and repair events after DNA damage.