Neonatal adaptation of energy and protein metabolism

During the last decade, the development of "bedside" investigative methods, including indirect calorimetry, nutritional balance and stable isotope techniques, have given a new insight into energy and protein metabolism in the neonates. Neonates and premature infants especially, create an unusual opportunity to study the metabolic adaptation to extrauterine life because their physical environment can be controlled, their energy intake and energy expenditure can be measured and the link between their protein metabolism and the energetics of their postnatal growth can be assessed with accuracy. Thus, relatively abstract physiological concepts such as the postnatal timecourse of heat production, energy cost of growth, energy cost of physical activity, thermogenic effect of feeding, efficiency of protein gain, metabolic cost of protein gain and protein turnover have been quantified. These results show that energy expenditure and heat production rates increase postnatally from average values of 40 kcal/kgxday during the first week to 60 kcal/kgxday in the third week. This increase parellels nutritional intakes as well as the rate of weight gain. The thermogenic effect of feeding and the physical activity are relatively low and account only for an average of 5% each of the total heat production. The cost of protein turnover is the highest energy demanding process. The fact that nitrogen balance becomes positive within 72 hours after birth places the newborn in a transitional situation of dissociated balance between energy and protein metabolism: dry body mass and fat decrease while there is a gain in protein and increase in supine length. This particular situation ends during the second postnatal week and soon thereafter the rate of weight gain matches the statural growth. The goals of the following review are to summarize recent data on the physiological aspects of energy and protein metabolism directly related to the extrauterine adaptation, to describe experimental approaches which recently were adapted to the newborns in order to get "bedside results" and to discuss how far these results can help everyday's neonatal practice.

Clara cell protein and surfactant protein B in garbage collectors and in wastewater workers exposed to bioaerosols

OBJECTIVES: Inhalation of bioaerosols has been hypothesised to cause "toxic pneumonitis" that should increase lung epithelial permeability at the bronchioloalveolar level. Serum Clara cell protein (CC16) and serum surfactant protein B (SPB) have been proposed as sensitive markers of lung epithelial injury. This study was aimed at looking for increased lung epithelial permeability by determining CC16 and SPB in workers exposed to bioaerosols from wastewater or garbage. METHODS: Subjects (778 wastewater, garbage and control workers; participation 61%) underwent a medical examination, lung function tests [American Thoracic Society (ATS) criteria], and determination of CC16 and SPB. Symptoms of endotoxin exposure and several potential confounders (age, gender, smoking, kidney function, obesity) were looked for. Results were examined with multiple linear or logistic regression. RESULTS: Exposure to bioaerosols increased CC16 concentration in the wastewater workers. No effect of exposure on SPB was found. No clue to work-related respiratory diseases was found. CONCLUSIONS: The increase in CC16 in serum supports the hypothesis that bioaerosols cause subclinical "toxic pneumonitis", even at low exposure. [Authors]

Sustained activation and tumor targeting of NKT cells using a CD1d-anti-HER2-scFv fusion protein induce antitumor effects in mice

Invariant NKT (iNKT) cells are potent activators of DCs, NK cells, and T cells, and their antitumor activity has been well demonstrated. A single injection of the high-affinity CD1d ligand alpha-galactosylceramide (alphaGalCer) leads to short-lived iNKT cell activation followed, however, by long-term anergy, limiting its therapeutic use. In contrast, we demonstrated here that when alphaGalCer was loaded on a recombinant soluble CD1d molecule (alphaGalCer/sCD1d), repeated injections led to sustained iNKT and NK cell activation associated with IFN-gamma secretion as well as DC maturation in mice. Most importantly, when alphaGalCer/sCD1d was fused to a HER2-specific scFv antibody fragment, potent inhibition of experimental lung metastasis and established s.c. tumors was obtained when systemic treatment was started 2-7 days after the injection of HER2-expressing B16 melanoma cells. In contrast, administration of free alphaGalCer at this time had no effect. The antitumor activity of the CD1d-anti-HER2 fusion protein was associated with HER2-specific tumor localization and accumulation of iNKT, NK, and T cells at the tumor site. Targeting iNKT cells to the tumor site thus may activate a combined innate and adaptive immune response that may prove to be effective in cancer immunotherapy

The NF-κB signaling protein Bcl10 regulates actin dynamics by controlling AP1 and OCRL-bearing vesicles.

The protein Bcl10 contributes to adaptive and innate immunity through the assembly of a signaling complex that plays a key role in antigen receptor and FcR-induced NF-κB activation. Here we demonstrate that Bcl10 has an NF-κB-independent role in actin and membrane remodeling downstream of FcR in human macrophages. Depletion of Bcl10 impaired Rac1 and PI3K activation and led to an abortive phagocytic cup rich in PI(4,5)P(2), Cdc42, and F-actin, which could be rescued with low doses of F-actin depolymerizing drugs. Unexpectedly, we found Bcl10 in a complex with the clathrin adaptors AP1 and EpsinR. In particular, Bcl10 was required to locally deliver the vesicular OCRL phosphatase that regulates PI(4,5)P(2) and F-actin turnover, both crucial for the completion of phagosome closure. Thus, we identify Bcl10 as an early coordinator of NF-κB-mediated immune response with endosomal trafficking and signaling to F-actin remodeling.

Phosphorylation of the Na,K-ATPase alpha-subunit by protein kinase A and C in vitro and in intact cells. Identification of a novel motif for PKC-mediated phosphorylation.

Na,K-ATPase is a potential target for regulatory phosphorylation by protein kinase A and C (PKA and PKC). To identify the phosphorylation sites, we have mutated the alpha 1-subunit of Bufo marinus in a highly conservative PKA and in 20 different PKC consensus sequences. The mutants were expressed in Xenopus oocytes and their phosphorylation capacity tested in homogenates upon stimulation of PKA or PKC. While serine 943 (Ser-943) was identified as a unique target site for PKA, none of the PKC consensus serine or threonine residues are implicated in PKC phosphorylation. Controlled trypsinolysis of phosphorylated alpha-subunits of various purified enzyme preparations and of alpha/beta complexes from oocyte homogenates revealed that PKC phosphorylation was exclusively associated with the N terminus. A fusion protein containing the first 32 amino acids of the Bufo alpha-subunit was phosphorylated in vitro and serine and threonine residues (Thr-15 and Ser-16) in this region were identified by site-directed mutagenesis as the PKC phosphorylation sites. Finally, the Bufo alpha-subunit was phosphorylated by protein kinases in transfected COS-7 cells. In intact cells, PKA stimulation induced phosphorylation exclusively on Ser-943 and PKC stimulation mainly on Thr-15 and Ser-16, which are contained in a novel PKC phosphorylation motif.

Energy-nitrogen balances and protein turnover in small and appropriate for gestational age low birthweight infants.

The aim of the present study was to compare, under the same nursing conditions, the energy-nitrogen balance and the protein turnover in small for gestational age (SGA) and appropriate for gestational age (AGA) low birthweight infants. We compared 8 SGA's (mean +/- s.d.: gestational age 35 +/- 2 weeks, birthweight 1520 +/- 330 g) to 11 AGA premature infants (32 +/- 2 weeks, birthweight 1560 +/- 240 g). When their rate of weight gain was above 15 g/kg/d (17.6 +/- 3.0 and 18.2 +/- 2.6 g/kg/d, mean postnatal age 18 +/- 10 and 20 +/- 9 d respectively) they were studied with respect to their metabolizable energy intake, their energy expenditure, their energy and protein gain and their protein turnover. Energy balance was assessed by the difference between metabolizable energy and energy expenditure as measured by indirect calorimetry. Protein gain was calculated from the amount of retained nitrogen. Protein turnover was estimated by a stable isotope enrichment technique using repeated nasogastric administration of 15N-glycine for 72 h. Although there was no difference in their metabolizable energy intakes (110 +/- 12 versus 108 +/- 11 kcal/kg/d), SGA's had a higher rate of resting energy expenditure (64 +/- 8 versus 57 +/- 8 kcal/kg/d, P less than 0.05). Protein gain and composition of weight gain was very similar in both groups (2.0 +/- 0.4 versus 2.1 +/- 0.4 g protein/kg/d; 3.5 +/- 1.1 versus 3.3 +/- 1.4 g fat/kg/d in SGA's and AGA's respectively). However, the rate of protein synthesis was significantly lower in SGA's (7.7 +/- 1.6 g/kg/d) as compared to AGA's (9.7 +/- 2.8 g/kg/d; P less than 0.05). It is concluded that SGA's have a more efficient protein gain/protein synthesis ratio since for the same weight and protein gains, SGA's show a 20 per cent slower protein turnover. They might therefore tolerate slightly higher protein intakes. Postconceptional age seems to be an important factor in the regulation of protein turnover.

Protein-protein interactions at the adrenergic receptors.

The adrenergic receptors are among the best characterized G protein-coupled receptors (GPCRs) and knowledge on this receptor family has provided several important paradigms about GPCR function and regulation. One of the most recent paradigms initially supported by studies on adrenergic receptors is that both βarrestins and G proteincoupled receptors themselves can act as scaffolds binding a variety of proteins and this can result in growing complexity of the receptor-mediated cellular effects. In this review we will briefly summarize the main features of βarrestin binding to the adrenergic receptor subtypes and we will review more in detail the main proteins found to selectively interact with distinct AR subtype. At the end, we will review the main findings on oligomerization of the AR subtypes.

Hypertension differentially affects the expression of the gap junction protein connexin43 in cardiac myocytes and aortic smooth muscle cells.

Electrical and mechanical coupling of myocytes in heart and of smooth muscle cells in the aortic wall is thought to be mediated by intercellular channels aggregated at gap junctions. Connexin43 (Cx43) is one of the predominant membrane proteins forming junctional channels in the cardiovascular system. This study was undertaken to assess its expression during experimental hypertension. Rats were made hypertensive by clipping one renal artery (two-kidney, one-clip renal hypertension) or by administering deoxycorticosterone and salt (DOCA-salt hypertension). After four weeks, rats from both models showed a similar increase in intra-arterial mean blood pressure, as well as in the thickness of both aorta and heart walls. Northern blot analysis showed that, compared to controls, hypertensive rats expressed twice more Cx43 in aorta, but not in heart. These results suggest that localized mechanical forces induced by hypertension are major tissue-specific regulators of Cx43 expression.

Hepatitis C virus-linked mitochondrial dysfunction promotes hypoxia-inducible factor 1 alpha-mediated glycolytic adaptation.

Hepatitis C virus (HCV) infection induces a state of oxidative stress by affecting mitochondrial-respiratory-chain activity. By using cell lines inducibly expressing different HCV constructs, we showed previously that viral-protein expression leads to severe impairment of mitochondrial oxidative phosphorylation and to major reliance on nonoxidative glucose metabolism. However, the bioenergetic competence of the induced cells was not compromised, indicating an efficient prosurvival adaptive response. Here, we show that HCV protein expression activates hypoxia-inducible factor 1 (HIF-1) by normoxic stabilization of its alpha subunit. In consequence, expression of HIF-controlled genes, including those coding for glycolytic enzymes, was significantly upregulated. Similar expression of HIF-controlled genes was observed in cell lines inducibly expressing subgenomic HCV constructs encoding either structural or nonstructural viral proteins. Stabilization and transcriptional activation of HIF-1alpha was confirmed in Huh-7.5 cells harboring cell culture-derived infectious HCV and in liver biopsy specimens from patients with chronic hepatitis C. The HCV-related HIF-1alpha stabilization was insensitive to antioxidant treatment. Mimicking an impairment of mitochondrial oxidative phosphorylation by treatment of inducible cell lines with oligomycin resulted in stabilization of HIF-1alpha. Similar results were obtained by treatment with pyruvate, indicating that accumulation of intermediate metabolites is sufficient to stabilize HIF-1alpha. These observations provide new insights into the pathogenesis of chronic hepatitis C and, possibly, the HCV-related development of hepatocellular carcinoma.

Microtubule-associated protein-2 immunoreactivity: a useful tool in the differential diagnosis of low-grade neuroepithelial tumors.

Complex and variable morphological phenotypes pose a major challenge to the histopathological classification of neuroepithelial tumors. This applies in particular for low-grade gliomas and glio-neuronal tumors. Recently, we and others have identified microtubule-associated protein-2 (MAP2) as an immunohistochemical marker expressed in the majority of glial tumors. Characteristic cell morphologies can be recognized by MAP2 immunoreactivity in different glioma entities, i.e., process sparse oligodendroglial versus densely ramified astrocytic elements. Here, we describe MAP2-immunoreactivity patterns in a large series of various neuroepithelial tumors and related neoplasms (n = 960). Immunohistochemical analysis led to the following conclusions: (1) specific pattern of MAP2-positive tumor cells can be identified in 95% of glial neoplasms; (2) ependymal tumors do not express MAP2 in their rosette-forming cell component; (3) tumors of the pineal gland as well as malignant embryonic tumors are also characterized by abundant MAP2 immunoreactivity; (4) virtually no MAP2 expression can be observed in the neoplastic glial component of glio-neuronal tumors, i.e. gangliogliomas; (5) malignant glial tumor variants (WHO grade III or IV) exhibit different and less specific MAP2 staining patterns compared to their benign counterparts (WHO grade I or II); (6) with the exception of melanomas and small cell lung cancers, MAP2 expression is very rare in metastatic and non-neuroepithelial tumors; (7) glial MAP2 expression was not detected in 56 non-neoplastic lesions. These data point towards MAP2 as valuable diagnostic tool for pattern recognition and differential diagnosis of low-grade neuroepithelial tumors.

MAR elements as tools to increase protein production by CHO cells

One of the major hurdles of isolating stable, inducible or constitutive high-level producer cell lines is the time-consuming selection, analysis and characterization of the numerous clones required to identify one with the desired characteristics. Various boundary elements, matrix attachment regions, and locus control regions were screened for for their ability to augment the expression of heterologous genes in CHO and other cells. The 5'-matrix-attachment region (MAR) of the chicken lysozyme gene was found to significantly increase stable gene expression, in culture dishes and in bioreactors. These MAR elements can be easily combined with various existing expression systems, as they can be added in trans (i.e. on a separate plasmid) in co-transfections with previously constructed expression vectors. Using cell population analysis, we found that the use of the MAR increases the proportion of high-producing CHO cell clones, thus reducing the number of cell lines that need to be screened while increasing maximal productivity. Random cDNA cloning and sequencing indicated that over 12% of the ESTs correspond to the transgene. Thus, productivity is no longer limited by transcriptional events in such MAR-containing cell lines. The identification of small and more convenient active MAR portions will also be summarized. Finally, we will show examples of how MAR elements can be combined with short term expression to increase the simultaneous synthesis of many proteins in parallel by CHO cells. Overall, we conclude that the MAR sequence is a versatile tool to increase protein expression in short and long term production processes.

Plasmodium vivax antigen discovery based on alpha-helical coiled coil protein motif.

Protein α-helical coiled coil structures that elicit antibody responses, which block critical functions of medically important microorganisms, represent a means for vaccine development. By using bioinformatics algorithms, a total of 50 antigens with α-helical coiled coil motifs orthologous to Plasmodium falciparum were identified in the P. vivax genome. The peptides identified in silico were chemically synthesized; circular dichroism studies indicated partial or high α-helical content. Antigenicity was evaluated using human sera samples from malaria-endemic areas of Colombia and Papua New Guinea. Eight of these fragments were selected and used to assess immunogenicity in BALB/c mice. ELISA assays indicated strong reactivity of serum samples from individuals residing in malaria-endemic regions and sera of immunized mice, with the α-helical coiled coil structures. In addition, ex vivo production of IFN-γ by murine mononuclear cells confirmed the immunogenicity of these structures and the presence of T-cell epitopes in the peptide sequences. Moreover, sera of mice immunized with four of the eight antigens recognized native proteins on blood-stage P. vivax parasites, and antigenic cross-reactivity with three of the peptides was observed when reacted with both the P. falciparum orthologous fragments and whole parasites. Results here point to the α-helical coiled coil peptides as possible P. vivax malaria vaccine candidates as were observed for P. falciparum. Fragments selected here warrant further study in humans and non-human primate models to assess their protective efficacy as single components or assembled as hybrid linear epitopes.

Evolution of the correlation between expression divergence and protein divergence in mammals.

Divergence of protein sequences and gene expression patterns are two fundamental mechanisms that generate organismal diversity. Here, we have used genome and transcriptome data from eight mammals and one bird to study the positive correlation of these two processes throughout mammalian evolution. We demonstrate that the correlation is stable over time and most pronounced in neural tissues, which indicates that it is the result of strong negative selection. The correlation is not driven by genes with specific functions and may instead best be viewed as an evolutionary default state, which can nevertheless be evaded by certain gene types. In particular, genes with developmental and neural functions are skewed toward changes in gene expression, consistent with selection against pleiotropic effects associated with changes in protein sequences. Surprisingly, we find that the correlation between expression divergence and protein divergence is not explained by between-gene variation in expression level, tissue specificity, protein connectivity, or other investigated gene characteristics, suggesting that it arises independently of these gene traits. The selective constraints on protein sequences and gene expression patterns also fluctuate in a coordinate manner across phylogenetic branches: We find that gene-specific changes in the rate of protein evolution in a specific mammalian lineage tend to be accompanied by similar changes in the rate of expression evolution. Taken together, our findings highlight many new aspects of the correlation between protein divergence and expression divergence, and attest to its role as a fundamental property of mammalian genome evolution.