Investigation of albumin synthesis in rat livers during the perinatal period using immunocytochemistry andin situ hybridization

The immunoreactivity of albumin (ALB) was observed in the hepatocytes of fetal rats on day 18 of gestation, and was especially observable in immature rough endoplasmic reticulum (rER) and Golgi apparatus (GA); by then, a small amount of silver grains of ALB mRNA could already be detected. Just after birth, immunoreactivity of ALB could be observed in fine granules or diffusely in all hepatocytes, and was present in rER and GA. One week after birth immunoreactivity of ALB was observed in all hepatocytes and was visible in developed rER and GA; the grains of ALB mRNA were present in all hepatocytes.     

Relationships of thigh muscle contractile and non-contractile tissue with function, strength, and age in boys with Duchenne muscular dystrophy

The purpose of this study was to assess the contractile and non-contractile content in thigh muscles of patients with Duchenne muscular dystrophy (DMD) and determine the relationship with functional abilities. Magnetic resonance images of the thigh were acquired in 28 boys with DMD and 10 unaffected boys. Muscle strength, timed functional tests, and the Brookes Lower Extremity scale were also assessed. Non-contractile content in the DMD group was significantly greater than in the control group for six muscles, including rectus femoris, biceps femoris-long head and adductor magnus. Non-contractile content in the total thigh musculature assessed by MRI correlated with the Brookes scale (r(s)=0.75) and supine-up test (r(s)=0.68), as well as other functional measures. An age-related specific torque increase was observed in the control group (r(s)=0.96), but not the DMD (r(s)=0.06). These findings demonstrate that MRI measures of contractile and non-contractile content can provide important information about disease progression in DMD.     

Regulation by a chaperone improves substrate selectivity during cotranslational protein targeting

The ribosome exit site is a crowded environment where numerous factors contact nascent polypeptides to influence their folding, localization, and quality control. Timely and accurate selection of nascent polypeptides into the correct pathway is essential for proper protein biogenesis. To understand how this is accomplished, we probe the mechanism by which nascent polypeptides are accurately sorted between the major cotranslational chaperone trigger factor (TF) and the essential cotranslational targeting machinery, signal recognition particle (SRP). We show that TF regulates SRP function at three distinct stages, including binding of the translating ribosome, membrane targeting via recruitment of the SRP receptor, and rejection of ribosome-bound nascent polypeptides beyond a critical length. Together, these mechanisms enhance the specificity of substrate selection into both pathways. Our results reveal a multilayered mechanism of molecular interplay at the ribosome exit site, and provide a conceptual framework to understand how proteins are selected among distinct biogenesis machineries in this crowded environment.Proper protein biogenesis is a prerequisite for the maintenance of a functional proteome. Accumulating data indicate that this process begins at the ribosome exit site, where many protein biogenesis machineries can interact and gain access to the nascent polypeptide. This includes chaperones (1–5) such as trigger factor (TF) (1, 4, 6, 7), Hsp70, and the nascent polypeptide-associated complex (8–13); modification enzymes (10, 14–16) such as N-acetyl transferase, methionine aminopeptidase, and arginyl transferase; protein-targeting and translocation machineries such as signal recognition particle (SRP) (17–20), SecA (21), the SecYEG (or Sec61p) (22, 23) and YidC translocases (24, 25), and the ribosome-bound quality control complex (26–30). Engagement of these factors with nascent polypeptides influences their folding, assembly, localization, processing, and quality control. Within seconds after the nascent polypeptide emerges from the ribosomal exit tunnel, it must engage the correct set of factors and thus commit to the proper biogenesis pathway. How this is accomplished in the crowded environment at the ribosome exit site is an emerging question. In this work, we address this question by deciphering how nascent proteins are selected between two major protein biogenesis machineries in bacteria, SRP and TF.SRP is a universally conserved ribonucleoprotein complex responsible for the cotranslational targeting of proteins to the eukaryotic endoplasmic reticulum (ER), or the bacterial plasma membrane (31). SRP recognizes ribosome-nascent chain complexes (termed RNC or cargo) carrying strong signal sequences and delivers them to the SecYEG or YidC translocation machinery on the target membrane. SRP binds RNC via two interactions: a helical N domain in the SRP54 protein (called Ffh in bacteria) binds the ribosomal protein L23, and a methionine-rich M domain binds hydrophobic signal sequences on nascent proteins as they emerge from the translating ribosome (Fig. 1A). Both SRP and SRP receptor (called FtsY in bacteria) also contain a conserved NG domain, comprised of a GTPase (guanosine 5′-triphosphate hydrolase) G domain and the N domain, whose direct interaction mediates the delivery of cargo to the target membrane.Open in a separate windowFig. 1.TF binds to SRP-occupied RNCs and weakens SRP binding. (A) Schematic depiction of the FRET assay to measure RNC–SRP binding. Green dot denotes Cm (donor); red dot denotes BODIPY FL (acceptor). (B) N-terminal sequences of the different substrates used in this study. Bold highlights the hydrophobic core of the signal sequences. Asterisk denotes the position where the amino acid is replaced by the Cm dye. (C and D) Equilibrium titrations for RNC–SRP binding in the presence of increasing TF concentration (indicated as increasing shades of red). The data were fitted to Eq. S2 and yielded the following parameters. (C) Apparent K_d values for RNC_FtsQ binding of 1.1 nM, 1.5 nM, 9.2 nM, and 16.6 nM and FRET end points of 0.54, 0.35, 0.29, and 0.17, respectively, with 0 µM, 1 µM, 5 µM, and 30 µM TF present. (D) Apparent K_d values for RNC_phoA binding of 17.2 nM, 21.1 nM, 30.3 nM, 28.3 nM, 31.5 nM, 104.5 nM, 106.3 nM, and 131.9 nM and FRET end points of 0.40, 0.41, 0.39, 0.29, 0.21, 0.19, 0.09, and 0.08, respectively, with 0 µM, 0.1 µM, 0.2 µM, 0.5 µM, 1 µM, 2 µM, 5 µM, and 10 µM TF present. (E) Summary of the effect of TF on apparent RNC–SRP binding affinity with the different substrates. The red dashed line denotes the cellular SRP concentration. Error bars are shown but may not be visible. Error bars are SDs from two to three measurements.Biophysical analyses (32–34) showed that membrane targeting is a two-step process in which SRP and FtsY first associate via their N domains to form a transient early intermediate (31, 32, 35). GTP (guanosine 5′-triphosphate)-driven rearrangements then bring the G domains of both proteins into close contact, giving a stable closed complex (36, 37). This rearrangement also exposes a membrane-binding helix of FtsY and thus is coupled to the membrane targeting of cargo (38). Importantly, SRP•FtsY assembly contributes extensively to the fidelity of SRP (39). The initial recognition of RNC by SRP is insufficient to reject suboptimal cargos bearing weak signal sequences (40, 41). Instead, a correct cargo strongly stabilizes the otherwise labile early intermediate and thus accelerates formation of the SRP•FtsY closed complex over 10³-fold, whereas suboptimal cargos provide much less stimulation (34, 40, 42). This enables rapid delivery of the correct cargos to the target membrane and provides kinetic discrimination against suboptimal cargos (Fig. S1).TF is a major cotranslational chaperone in bacteria, with an estimated cellular concentration of 50–80 µM (6). With a dissociation constant (K_d) of ∼1 µM for ribosomes (43), TF is bound to virtually every ribosome in the cell. Like SRP, TF contacts the ribosome via the L23 and L29 proteins near the ribosome exit site (3, 5, 44). Also analogous to SRP, TF preferentially interacts with hydrophobic sequences on the nascent polypeptide (1, 2, 4, 45, 46), mediated by a large concave surface rich in hydrophobic residues (1, 3–6). Despite these similarities with SRP, TF directs substrate proteins to distinct biogenesis pathways: It exhibits synthetic lethality with DnaK/J and facilitates the productive folding of cytosolic proteins (1, 4, 7, 9, 11). It also interacts with a subset of secretory and outer membrane proteins and interfaces with the posttranslational SecA/B pathway (8, 10, 12–14).SRP and TF are two distinct biogenesis pathways that a nascent protein must commit to. This raises intriguing questions: How do these two factors, which have overlapping substrate preferences, compete and/or collaborate at the ribosome exit site? How are nascent proteins sorted between them and committed to the correct pathway in a timely and accurate manner? Extensive past work to address these questions has led to different (and sometimes contradictory) models, including (i) TF and SRP compete for binding to the RNC (10, 15, 16, 18); (ii) TF and SRP can bind to the same RNC simultaneously (17, 19–21); (iii) FtsY rejects TF from SRP-bound ribosomes (17); and (iv) TF preferentially occupies longer nascent chains (13, 45–47) and, by inference, SRP preferentially binds short nascent chains. A unifying model that reconciles all these observations and explains how nascent chains on the ribosome are selected by TF or SRP is still lacking. Most importantly, most of the previous studies have focused on the initial binding of SRP or TF to the nascent polypeptide, which may not be the step at which nascent proteins are committed to their respective biogenesis pathways.In this work, we used high-resolution biochemical and biophysical analyses to investigate the interplay between TF and SRP at the ribosome exit site in molecular detail. We show that TF regulates SRP function by three distinct mechanisms, which together enhance the ability of the SRP pathway to reject suboptimal substrates. Our results establish a comprehensive and cohesive model that explains previous observations, delineates the complex interplay between protein biogenesis factors at the ribosome exit site, and provides a conceptual foundation to understand how timely and accurate selection of substrates is achieved in this crowded environment.     

Prostaglandin E2 gel In ripening of cervix in induction of labour

A study was done in 75 patients who underwent induction of labour with Prostaglandin E2 gel. All these patients had an unripe cervix. The commonest indications were post-datism, intrauterine growth retardation and pregnancy-induced hypertension. All patients were primigravidas with singleton pregnancy and beyond 35 weeks of pregnancy. The mean Bishop score at the time of instillation was less than three. The improvement of another 2-3 points within six hours and by 7-8 points within 12 hours was found after instillation of the gel. 92% of the patients went into spontaneous labour and 8% required reinstillation. The incidence of failed induction was 1.33%. The mean duration of latent phase was 10.34 hours. Induction delivery time was 16.43 hours. 68.1% patients required augmentation of labour and 31.9% did not require augmentation of labour with oxytocin drip. The incidence of vaginal delivery was 81.33% and that of caesarean section was 17.33%. The commonest indication of caesarean section was foetal distress.     

Investigation of albumin synthesis in rat livers during the perinatal period using immunocytochemistry andin situ hybridization

The immunoreactivity of albumin (ALB) was observed in the hepatocytes of fetal rats on day 18 of gestation, and was especially observable in immature rough endoplasmic reticulum (rER) and Golgi apparatus (GA); by then, a small amount of silver grains of ALB mRNA could already be detected. Just after birth, immunoreactivity of ALB could be observed in fine granules or diffusely in all hepatocytes, and was present in rER and GA. One week after birth immunoreactivity of ALB was observed in all hepatocytes and was visible in developed rER and GA; the grains of ALB mRNA were present in all hepatocytes. This study was presented at the 25th Annual Meeting of the Clinical Electron Microscopy Society of Japan, Matsumoto, September 28–30, 1993.     

T2 mapping provides multiple approaches for the characterization of muscle involvement in neuromuscular diseases: a cross‐sectional study of lower leg muscles in 5–15‐year‐old boys with Duchenne muscular dystrophy

Skeletal muscles of children with Duchenne muscular dystrophy (DMD) show enhanced susceptibility to damage and progressive lipid infiltration, which contribute to an increase in the MR proton transverse relaxation time (T₂). Therefore, the examination of T₂ changes in individual muscles may be useful for the monitoring of disease progression in DMD. In this study, we used the mean T₂, percentage of elevated pixels and T₂ heterogeneity to assess changes in the composition of dystrophic muscles. In addition, we used fat saturation to distinguish T₂ changes caused by edema and inflammation from fat infiltration in muscles. Thirty subjects with DMD and 15 age‐matched controls underwent T₂‐weighted imaging of their lower leg using a 3‐T MR system. T₂ maps were developed and four lower leg muscles were manually traced (soleus, medial gastrocnemius, peroneal and tibialis anterior). The mean T₂ of the traced regions of interest, width of the T₂ histograms and percentage of elevated pixels were calculated. We found that, even in young children with DMD, lower leg muscles showed elevated mean T₂, were more heterogeneous and had a greater percentage of elevated pixels than in controls. T₂ measures decreased with fat saturation, but were still higher (P < 0.05) in dystrophic muscles than in controls. Further, T₂ measures showed positive correlations with timed functional tests (r = 0.23–0.79). The elevated T₂ measures with and without fat saturation at all ages of DMD examined (5–15 years) compared with unaffected controls indicate that the dystrophic muscles have increased regions of damage, edema and fat infiltration. This study shows that T₂ mapping provides multiple approaches that can be used effectively to characterize muscle tissue in children with DMD, even in the early stages of the disease. Therefore, T₂ mapping may prove to be clinically useful in the monitoring of muscle changes caused by the disease process or by therapeutic interventions in DMD. Copyright © 2012 John Wiley & Sons, Ltd.     

Structural analysis and thermal behavior of diopside–fluorapatite–wollastonite-based glasses and glass–ceramics

Glass–ceramics in the diopside (CaMgSi₂O₆)–fluorapatite (Ca₅(PO₄)₃F)–wollastonite (CaSiO₃) system are potential candidates for restorative dental and bone implant materials. The present study describes the influence of varying SiO₂/CaO and CaF₂/P₂O₅ molar ratio on the structure and thermal behavior of glass compositions in the CaO–MgO–SiO₂–P₂O₅–Na₂O–CaF₂ system. The structural features and properties of the glasses were investigated by nuclear magnetic resonance (NMR), infrared spectroscopy, density measurements and dilatometry. Sintering and crystallization behavior of the glass powders were studied by hot-stage microscopy and differential thermal analysis, respectively. The microstructure and crystalline phase assemblage in the sintered glass powder compacts were studied under non-isothermal heating conditions at 825 °C. X-ray diffraction studies combined with the Rietveld-reference intensity ratio (R.I.R) method were employed to quantify the amount of amorphous and crystalline phases in the glass–ceramics, while scanning electron microscopy was used to shed some light on the microstructure of resultant glass–ceramics. An increase in CaO/SiO₂ ratio degraded the sinterability of the glass powder compacts, resulting in the formation of akermanite as the major crystalline phase. On the other hand, an increase in P₂O₅/CaF₂ ratio improved the sintering behavior of the glass–ceramics, while varying the amount of crystalline phases, i.e. diopside, fluorapatite and wollastonite.