Localization of ank1.5 in the sarcoplasmic reticulum precedes that of SERCA and RyR: relationship with the organization of obscurin in developing sarcomeres

Ank1.5 is a muscle-specific isoform of ankyrin1 localized on the sarcoplasmic reticulum (SR) membrane that has been shown to interact with obscurin, a sarcomeric protein. We report here studies on the localization of obscurin and ank1.5 in embryonic and postnatal rodent skeletal muscles. Using two antibodies against epitopes in the N- and C-terminus of obscurin, two distinct patterns of localization were observed. Before birth, the antibodies against the N- and the C-terminus of obscurin stained the Z-disk and M-band, respectively. At the same time, ank1.5 was detected at the Z-disk, rising the possibility that obscurin molecules at M-band may not be able to interact with ank1.5. Localization of ank1.5 at Z-disks in E14 muscle fibers revealed that ank1.5 is among the earliest SR proteins to assemble, since its organization preceded that of other SR proteins, like SERCA and RyR. After birth, the antibody against the N-terminus of obscurin stained the M-band while that against the C-terminus stained both M-bands and the Z-disks. Starting from postnatal day 1, ank1.5 was found at the level of both M-bands and Z-disks. Altogether, from these results we infer that exposure of some obscurin epitopes changes during skeletal muscle development, resulting in distinct, antibody-specific, localization pattern. Why this occurs is not clear, yet these data indicate that the organization of obscurin at different locations in the sarcomere changes during muscle development and that this might affect the interaction with ank1.5.     

A computationally inspired investigation of the solid forms of (R)‐1‐phenylethylammonium‐(S)‐2‐phenylbutyrate

Following the computation of a lattice energy landscape which predicted that there should be more stable, denser forms of (R)‐1‐phenylethylammonium‐(S)‐2‐phenylbutyrate, crystallizations from a range of solvents were performed to search for other polymorphs and investigate the possibility that the known P4₁ structure could be a hydrate. Extensive crystallization experiments from a wide range of solvents gave fine needles or microcrystalline samples. A redetermination of the P4₁ structure by powder X‐ray diffraction located all protons, and in conjunction with other experimental and computational evidence showed that the structure was anhydrous. Evidence for two additional forms was found as mixtures with form I. These include an orthorhombic form, possibly a Z′ = 3 polymorph, and another as yet unidentified form obtained as a minor component from dichloromethane solution. However, both these forms appear to be metastable with respect to form I (P4₁), which is therefore probably the most thermodynamically stable form that can be crystallized from solution under ambient conditions. This determination of the solid state behavior of the less readily crystallized member of the diastereomeric salt system (R)‐1‐phenylethylammonium‐(R/S)‐2‐phenylbutyrate provides a challenge to the theoretical modeling to explain its ideal resolution behavior. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Binding of an ankyrin-1 isoform to obscurin suggests a molecular link between the sarcoplasmic reticulum and myofibrils in striated muscles

Assembly of specialized membrane domains, both of the plasma membrane and of the ER, is necessary for the physiological activity of striated muscle cells. The mechanisms that mediate the structural organization of the sarcoplasmic reticulum with respect to the myofibrils are, however, not known. We report here that ank1.5, a small splice variant of the ank1 gene localized on the sarcoplasmic reticulum membrane, is capable of interacting with a sequence of 25 aa located at the COOH terminus of obscurin. Obscurin is a giant sarcomeric protein of approximately 800 kD that binds to titin and has been proposed to mediate interactions between myofibrils and other cellular structures. The binding sites and the critical aa required in the interaction between ank1.5 and obscurin were characterized using the yeast two-hybrid system, in in vitro pull-down assays and in experiments in heterologous cells. In differentiated skeletal muscle cells, a transfected myc-tagged ank1.5 was found to be selectively restricted near the M line region where it colocalized with endogenous obscurin. The M line localization of ank1.5 required a functional obscurin-binding site, because mutations of this domain resulted in a diffused distribution of the mutant ank1.5 protein in skeletal muscle cells. The interaction between ank1.5 and obscurin represents the first direct evidence of two proteins that may provide a direct link between the sarcoplasmic reticulum and myofibrils.In keeping with the proposed role of obscurin in mediating an interaction with ankyrins and sarcoplasmic reticulum, we have also found that a sequence with homology to the obscurin-binding site of ank1.5 is present in the ank2.2 isoform, which in striated muscles has been also shown to associate with the sarcoplasmic reticulum. Accordingly, a peptide containing the COOH terminus of ank2.2 fused with GST was found to bind to obscurin. Based on reported evidence showing that the COOH terminus of ank2.2 is necessary for the localization of ryanodine receptors and InsP3 receptors in the sarcoplasmic reticulum, we propose that obscurin, through multiple interactions with ank1.5 and ank2.2 isoforms, may assemble a large protein complex that, in addition to a structural function, may play a role in the organization of specific subdomains in the sarcoplasmic reticulum.     

Drug failure during HIV-1 treatment. New perspectives in monitoring drug resistance

Since the discovery of 3'-azido-3'deoxthymidine (zidovudine) as an effective antiretroviral agent against human immunodeficiency virus type 1 (HIV-1), drug therapy has been widely used in the treatment of AIDS. To date, new combination therapies have significantly altered the longterm prognosis for HIV-infected patients showing a reduction of plasma viral load, associated with clinical and immunological recovery. Nevertheless, in various circumstances treatment can fail for several reasons, such as patient noncompliance with the therapeutic regimen, suboptimal antiviral drug concentrations, drug pharmacokinetics, and virus resistance to one or more drugs. Virus drug resistance is the most important factor contributing to the failure of antiretroviral therapy. Since some evidence indicates that viral resistance and treatment failure are closely linked, this brief review explores the routine determination of drug resistance and its importance to shed more light on the meaning of mutations correlated to drug resistance.     

Improving fluorescence-based assays for the in vitro analysis of cell adhesion and migration

Cell adhesion and cell migration are two primary cellular phenomena to be approached in vitro in order to allow for the effective dissection of the individual events and the unravelling of their underlying molecular mechanisms. The use of assays dedicated to the analysis of cell adhesion and migration in vitro also affords an efficient way of conducting larger basic and applied research screenings of the conditions affecting these processes and are potentially exploitable in the context of routine tests in the biological and medical fields. Therefore, there is a substantial interest in devicing more rationale such assays and major contributions in this direction have been provided by the advent of procedures based on fluorescent cell tagging. In this article we describe three fluorescence-based model assays for the qualitative and quantitative assessment of cell adhesion and cell locomotion in static and dynamic conditions. The assays are easily performed, accurate and reproducible, and can be automatized for high-throughput screenings of cell behavior in vitro. Performance of the assays involves the use of certain dedicated disposable accessories, which are commercially available, and a few instruments that, due to their versatility, can be regarded as constituents of a more generic laboratory setup.     

Efficient HPLC method for the determination of nicarbazin, as dinitrocarbanilide in broiler liver

A simple, fast and reliable HPLC-UV method has been developed for the determination of dinitrocarbanilide residues in broiler liver. Liver samples (2 g) were extracted with two portions of acetonitrile (10 and 5 ml), defatted with hexane and evaporated to dryness under nitrogen. Extracts were reconstituted in acetonitrile-water (70/30, v/v, 500 microl), loaded onto C18 solid phase (SPE) cartridges and eluted with acetonitrile-water (70/30, v/v, 2.5 ml) into clean test-tubes. Extracts were evaporated to dryness and reconstituted in acetonitrile-water (80/20, v/v, 500 microl). An aliquot of the extract was assayed by high performance liquid chromatography (HPLC) with UV detection at 350 nm. The method was validated according to EU guidelines using liver tissues fortified at levels of 100, 200 and 300 microg/kg, with dinitrocarbanilide. The decision limit (CC(alpha)) and the detection capability (CC(beta)) were calculated from the within laboratory repeatability data to be 228 and 266 microg/kg, respectively. The mean recovery was typically >70% and the limits of quantitation was 12.5 microg/kg (based on the lowest standard on the calibration curve).     

A case of granulomatous skin lesions caused by Mycobacterium marinum in the Campania region

The diagnosis of cutaneous Mycobacterium marinum infection is frequently presumptive, as detection by conventional methods is difficult. We describe a patient with granulomatous skin lesions on the right dorsal hand and forearm. Histological examinations were presumptive for mycobacterium lesions. We identified Mycobacterium marinum directly in the patient's lesional skin biopsy combining polymerase chain reaction (PCR) amplification using Mycobacterium genus-specific primers, and subsequent restriction enzyme analysis enabling identification to the species level. The symptoms were no longer present after specific therapy, thereby confirming the initial diagnosis.     

Antibody to HIV-1 Tat protein, a key molecule in HIV-1 pathogenesis. A brief review

In the last few years, literature reports have unequivocally established that the 86-101 aminoacid Tat protein, essential for an efficient viral replication, can be actively secreted by infected cells. The contribution of extracellular Tat to the progression of viral infection is underlined by the ability of neutralizing anti Tat antibody to reduce the viral load in vitro and possibly also in vivo. Considering that at least some of the effect of Tat protein seem to be the consequence of an autocrine loop and that anti Tat antibody is an efficient inhibitor of viral replication, it is reasonable to suppose that extracellular Tat play a functional role in HIV-1 infection and that HIV antibody may interfere with a possible Tat driven pathogenesis. This review explores the meaning of anti Tat antibody in vitro and in vivo and its importance to shed more light on viral pathogenesis and the recent development of Tat containing vaccine.     

A Region Containing a Proline-Rich Motif Targets sGi2 to the Golgi Apparatus

The central function of heterotrimeric GTP-binding proteins (G proteins) is the transduction of extracellular signals, via membrane receptors, leading to the activation of intracellular effectors. In addition to being associated with the plasma membrane, the α subunits of some of these proteins have also been localized in intracellular compartments. The mRNA of the G-protein inhibitory α subunit 2 (G_αi2) encodes two proteins, G_αi2 and sG_i2, by an alternative splicing mechanism. sG_i2 differs from G_αi2 in the C-terminal region and localizes in the Golgi in contrast to the plasma membrane localization of G_αi2. In this paper we show that the sequence specific to sG_i2 can direct the Golgi localization of other G_αi subunits, but not of the stimulatory subunit G_αs or of a secreted protein. This indicates that, in addition to the sG_i2 C-terminus, sequences located elsewhere in the protein are required to determine the Golgi localization. Inside the sG_i2 C-terminal region we have identified a 14-amino-acid proline-rich motif which specifies the Golgi localization. Finally, we show that the sG_i2 subunit, once activated, leaves the Golgi to be localized in the endoplasmic reticulum.     

The Growing Trend of Moderate Preterm Births: An Ecological Study in One Region of Brazil

Background

Preterm birth is a serious public health problem, as it is linked to high rates of neonatal and child morbidity and mortality, with Brazil listed among the countries with the ten highest numbers of premature births. Nonetheless, knowledge is scarce regarding prematurity and associated factors in mid-sized cities. The objective of this study was to analyze the trend of preterm births and associated factors in a municipality located in the state of Paraná, Brazil.

Methods

This was an ecological time series study of births recorded into the Live Birth Information System for residents of Maringá, Paraná, Brazil, between 2000 and 2013. The polynomial regression model was used for trend analysis of preterm birth, characteristics of the mother, gestation and delivery, and newborn. The association with preterm birth was analyzed using odds ratio (OR).

Results

A total of 61,634 live births were analyzed, of which 5,632 were preterm births. Prematurity increased from 7.9% in 2000 to 11.2% in 2013 –an average increase of 0.54% per year (r² = 0.93)–with a growing share of moderate preterm births (32 to <37 weeks), which rose from 7.0% in 2000 to 9.7% in 2013. Between 2011 and 2013, multiple pregnancy (OR = 16.64; CI = 13.24–20.92), inadequate number of prenatal visits (OR = 2.81; CI = 2.51–3.15), Apgar score below 7 at 1 (OR = 4.07; CI = 3.55–4.67) and 5 minutes (OR = 10.88; CI = 7.71–15.36), low birth weight (OR = 38.75; CI = 33.72–44.55) and congenital malformations (OR = 3.18; CI = 2.14–4.74) were associated with preterm birth. A growing trend was observed for multiple pregnancies, with an average annual increase of 0.32% (r² = 0.90), as well as for C-section birth (2.38% yearly increase). Of all newborn characteristics, Apgar score below 7 at 5 minutes (-0.19% per year) and low birth weight (-1.43%) decreased, whereas congenital malformations rose (0.20% per year).

Conclusions

Efforts are required to prevent premature delivery, particularly during the moderate period, as well as greater care during the prenatal period towards expectant mothers bearing multiple pregnancies, birth defects, in addition to reducing C-section birth as it may be linked to preterm birth.