The quantitative basis for the redistribution of immobile bacterial lipoproteins to division septa

The spatial localisation of proteins is critical for most cellular function. In bacteria, this is typically achieved through capture by established landmark proteins. However, this requires that the protein is diffusive on the appropriate timescale. It is therefore unknown how the localisation of effectively immobile proteins is achieved. Here, we investigate the localisation to the division site of the slowly diffusing lipoprotein Pal, which anchors the outer membrane to the cell wall of Gram-negative bacteria. While the proton motive force-linked TolQRAB system is known to be required for this repositioning, the underlying mechanism is unresolved, especially given the very low mobility of Pal. We present a quantitative, mathematical model for Pal relocalisation in which dissociation of TolB-Pal complexes, powered by the proton motive force across the inner membrane, leads to the net transport of Pal along the outer membrane and its deposition at the division septum. We fit the model to experimental measurements of protein mobility and successfully test its predictions experimentally against mutant phenotypes. Our model not only explains a key aspect of cell division in Gram-negative bacteria, but also presents a physical mechanism for the transport of low-mobility proteins that may be applicable to multi-membrane organelles, such as mitochondria and chloroplasts.     

Magnetic resonance spectroscopy to measure hepatic triglyceride content: prevalence of hepatic steatosis in the general population

Despite the increasing prevalence of nonalcoholic fatty liver disease (NAFLD), the criteria used to diagnose the disorder remain poorly defined. Localized proton magnetic resonance spectroscopy (MRS) accurately measures hepatic triglyceride content (HTGC) but has been used only in small research studies. Here, MRS was used to analyze the distribution of HTGC in 2,349 participants from the Dallas Heart Study (DHS). The reproducibility of the procedure was validated by showing that duplicate HTGC measurements were high correlated (r = 0.99, P < 0.001) and that the coefficient of variation between measurements was low (8.5%). Intake of a high-fat meal did not significantly affect the measurements, and values were similar when measurements were made from the right and left hepatic lobes. To determine the "upper limit of normal" for HTGC, the distribution of HTGC was examined in the 345 subjects from the DHS who had no identifiable risk factors for hepatic steatosis (nonobese, nondiabetic subjects with minimal alcohol consumption, normal liver function tests, and no known liver disease). The 95th percentile of HTGC in these subjects was 5.56%, which corresponds to a hepatic triglyceride level of 55.6 mg/g. With this value as a cutoff, the prevalence of hepatic steatosis in Dallas County was estimated to be 33.6%. Thus MRS provides a sensitive, quantitative, noninvasive method to measure HTGC and, when applied to a large urban US population, revealed a strikingly high prevalence of hepatic steatosis.     

In vitro reconstitution of the spinach chloroplast cytochrome b6 protein from a fusion protein expressed in Escherichia coli

We have developed a strategy for overproduction of spinach apocytochrome b6 as a fusion protein to maltose-binding protein (MBP) in Escherichia coli, using the expression vector pMal-c2. The fusion protein was purified to virtual homogeneity by gel filtration chromatography and the method of insertion of hemes into fusion protein was elaborated. The ambient and low-temperature absorption spectra of the reconstituted cytochrome b6 were similar to those of cytochrome b6 spectra in isolated proteins or cytochrome b6f complexes and are typical for bis-histidine ligated b-type cytochromes. Optical circular dichroism (CD) spectra of the visible region further confirmed the appropriate binding of hemes by the apocytochrome b6 protein. We found that the incorporation of hemes was required for the refolding of the cytochrome b6 protein into the more compact structure found in the native cytochrome protein. Heme staining experiments suggested that the two hemes in the reconstituted cytochrome b6 protein are bound with different affinities. The reconstituted cytochrome b6 protein was cleaved by Xa factor proteolysis from fusion protein and separated for characterization. The procedure presented in this work for reconstitution of hemes into the cytochrome b6 protein should provide an important tool for structure/function studies of membrane-bound cytochrome proteins.     

Measurement of Pancreatic Volume by Abdominal MRI: A Validation Study

Objective

To develop abdominal magnetic resonance imaging (MRI) protocol to measure pancreatic volume in humans and to validate it in large animals.

Materials and Methods

We performed abdominal MRI in eight mini-pigs using a clinical 3T MRI system. We used consecutive parallel abdominal slices, covering the entire pancreas to calculate pancreatic volume. Following MRI, animals were sacrificed, the pancreas was removed, and the volume of the pancreas was measured by water displacement. We used the same MRI protocol to measure pancreatic volume in 21 humans. To assess reproducibility of in vivo measurement we repeated MRI pancreas volume evaluation within 24 hours in additional five humans.

Results

In mini-pigs the measurements of pancreatic volume by MRI and by water displacement were almost identical (R² = 0.9867; p<0.0001). In humans the average pancreas volume was 72.7+/−4.5 ml, range from 35.0 to 105.5 ml. This result is in strong agreement with results of previous large postmortem and computed tomography (CT) studies. Repeated measurements of pancreatic volume in humans were highly reproducible. Pancreatic volume measured in vivo was negatively correlated with age, body fat mass, pancreatic TG levels, and visceral fat mass.

Conclusions

These initial results are highly encouraging and our protocol for pancreatic volume estimation in vivo may prove useful in obesity research to follow in vivo changes of pancreatic volume and structure during time course of obesity and type 2 diabetes development.     

Nuclear factor-kappa B localization and function within intrauterine tissues from term and preterm labor and cultured fetal membranes

Background  

The objective of this study was to quantify the nuclear localization and DNA binding activity of p65, the major transactivating nuclear factor-kappa B (NF-kappaB) subunit, in full-thickness fetal membranes (FM) and myometrium in the absence or presence of term or preterm labor.     

Affinity resins containing enzymatically resistant mRNA cap analogs--a new tool for the analysis of cap-binding proteins

Cap-binding proteins have been routinely isolated using m⁷GTP-Sepharose; however, this resin is inefficient for proteins such as DcpS (scavenger decapping enzyme), which interacts not only with the 7-methylguanosine, but also with the second cap base. In addition, DcpS purification may be hindered by the reduced resin capacity due to the ability of DcpS to hydrolyze m⁷GTP. Here, we report the synthesis of new affinity resins, m⁷GpCH₂pp- and m⁷GpCH₂ppA-Sepharoses, with attached cap analogs resistant to hydrolysis by DcpS. Biochemical tests showed that these matrices, as well as a hydrolyzable m⁷GpppA-Sepharose, bind recombinant mouse eIF4E^(28-217) specifically and at high capacity. In addition, purification of cap-binding proteins from yeast extracts confirmed the presence of all expected cap-binding proteins, including DcpS in the case of m⁷GpCH₂pp- and m⁷GpCH₂ppA-Sepharoses. In contrast, binding studies in vitro demonstrated that recombinant human DcpS efficiently bound only m⁷GpCH₂ppA-Sepharose. Our data prove the applicability of these novel resins, especially m⁷GpCH₂ppA-Sepharose, in biochemical studies such as the isolation and identification of cap-binding proteins from different organisms.     

Chemical sympathectomy alters regulation of body weight during prolonged ICV leptin infusion

To assess the importance of the sympathetic nervous system in regulating body weight during prolonged leptin infusion, we evaluated food intake, body weight, and physical activity in conscious, unrestrained rats. Initial studies illustrated that prolonged intracerebroventricular (ICV) infusion of leptin enhanced substrate oxidation so that adipose tissue lipid stores were completely ablated, and muscle triglyceride and liver glycogen stores were depleted. After neonatal chemical sympathectomy, changes in weight and food intake were compared in groups of sympathectomized (SYM) and control (CON) adult animals during ICV infusion of leptin. CON animals lost 60 +/- 9 g over 10 days vs. 25 +/- 3 g in the SYM animals when food intake was matched between the two groups. Greater weight loss despite similar energy intake points to an important role of the sympathetic nervous system in stimulating energy expenditure during ICV leptin infusion by increasing the resting metabolic rate, since no differences in physical activity were observed between CON and SYM groups. In conclusion, activation of the SNS by leptin increases energy expenditure by augmenting the resting metabolic rate.