Structural requirements of peptide hormone binding for peptide-potentiated self-association of bovine neurophysin II

Site-specific, truncated, and sequence-simplified analogs of the hormone [Arg8]vasopressin were investigated for the relationship between their abilities to recognize immobilized bovine neurophysin and to promote neurophysin self-association. Peptide binding to neurophysin was measured quantitatively by analytical high performance affinity chromatography on immobilized bovine neurophysin II. Neurophysin self-association, measured as binding of soluble to immobilized neurophysin, was promoted (made higher affinity) by soluble peptide hormone and its analogs, with the effect of particular peptides being proportional to their binding affinities for neurophysin. Sequence-redesigned peptides able to recognize neurophysin, including dipeptide amides, were able to potentiate the self-association to the same extent as the natural hormone when tested at concentrations adjusted to effect equal degrees of saturation of neurophysin. The relationship between peptide affinity to neurophysin and the potentiation of self-association suggests that the latter is directly dependent on the former and can occur even with limited segments of hormone sequence. The data fit best to a model in which hormone binding and self-association surfaces of neurophysin are separate and linked through the neurophysin molecule to produce cooperativity (hormone-promoted self-association). Given that only limited structural elements of hormone are required for promoting self-association, the results fit less well with models in which cooperativity requires that hormone make dimer-stabilizing contacts with both self-associating subunits of neurophysin simultaneously.     

Age-related changes in rat adipose tissue in response to fasting: protein, lactate and pyruvate levels

1. The evolution with age of the metabolic response of adipose tissue to fasting has been investigated in two groups of rats, at different ages, fed and fasted. 2. The protein tissue content increases in response to fasting in young rats but not in adult-old ones. This indicates a lower lipomobilizing response to starvation with increasing age. 3. In contrast to young rats, the adult rat lactate is not increased by fasting while pyruvate is increased. 4. As a result of lactate and pyruvate variations, NAD/NADH is also changed: after fasting it decreases in young rats, while it increases in older rats. 5. Absolute NAD level is decreased by fasting both in young and older rats. 6. These data provide experimental support for the decreased sensitivity of fat pads from older animals to stimuli affecting lipolysis.     

Molecular Typing of Lung Adenocarcinoma on Cytological Samples Using a Multigene Next Generation Sequencing Panel

Identification of driver mutations in lung adenocarcinoma has led to development of targeted agents that are already approved for clinical use or are in clinical trials. Therefore, the number of biomarkers that will be needed to assess is expected to rapidly increase. This calls for the implementation of methods probing the mutational status of multiple genes for inoperable cases, for which limited cytological or bioptic material is available. Cytology specimens from 38 lung adenocarcinomas were subjected to the simultaneous assessment of 504 mutational hotspots of 22 lung cancer-associated genes using 10 nanograms of DNA and Ion Torrent PGM next-generation sequencing. Thirty-six cases were successfully sequenced (95%). In 24/36 cases (67%) at least one mutated gene was observed, including EGFR, KRAS, PIK3CA, BRAF, TP53, PTEN, MET, SMAD4, FGFR3, STK11, MAP2K1. EGFR and KRAS mutations, respectively found in 6/36 (16%) and 10/36 (28%) cases, were mutually exclusive. Nine samples (25%) showed concurrent alterations in different genes. The next-generation sequencing test used is superior to current standard methodologies, as it interrogates multiple genes and requires limited amounts of DNA. Its applicability to routine cytology samples might allow a significant increase in the fraction of lung cancer patients eligible for personalized therapy.     

Improvement of liver cancer detection with simultaneous assessment of circulating levels of free alpha-fetoprotein (AFP) and AFP-IgM complexes

We assessed the presence of alpha-fetoprotein (AFP) complexed with IgM (AFP-IgM IC) in serum of patients affected by hepatocellular carcinoma (HCC), cirrhosis and chronic hepatitis as well as in healthy subjects by means of a dedicated ELISA assay. The amount of AFP-IgM IC was expressed in arbitrary units (AU) on a reference standard curve. Free AFP (FAFP) levels were determined in parallel in each sample by means of an automated immunoassay system. The mean serum concentration of AFP-IgM IC was significantly higher in HCC patients (mean +/- SD: 1378.3 +/- 2935.7 AU/mL) than in cirrhotic patients (129.8 +/- 261.4 AU/mL) and in patients with chronic hepatitis (80.9 +/- 168.9 AU/mL) (p < 0.01). HCC patients had FAFP values above the 20 ng/mL cutoff in 44% of cases (22/50) and AFP-IgM IC values above the 120 AU/mL cutoff in 60% of cases (30/50). The occurrence of the free and IgM-complexed form of circulating AFP did not overlap, and 82% of patients (41/50) were positive for at least one marker. The results indicate that AFP-IgM IC is a complementary serological marker to FAFP and that the combination of these biomarkers may be useful in the diagnosis of liver cancer.     

Prevention of systemic lupus erythematosus in MRL/lpr mice by administration of an immunoglobulin-binding peptide

Systemic lupus erythematosus (SLE) is a multisystem chronic inflammatory disease of unknown etiology that affects many organs, including the kidney. The presence of multiple autoantibodies and other immunological abnormalities point to basic defects in immunoregulatory controls that normally maintain self-tolerance. The deposition on kidney tissue of autoantibodies as immune complexes (ICs) through the interaction with Fc-receptor gamma-chains is thought to trigger an inflammatory response typical of SLE, leading to glomerulonephritis. Using combinatorial chemistry approaches, we have identified a peptide able to bind to immunoglobulins and to interfere with Fcgamma-receptor recognition. Administration of this peptide to MRL/lpr mice, an animal model used to study SLE, resulted in a remarkable enhancement of the survival rate (80%) compared to placebo-treated animals (10%). Consistent with this was a significant reduction of proteinuria, a clinical sign of SLE. Kidney histological examination of treated animals confirmed the preservation of tissue integrity and a remarkable reduction in IC deposition. These results support the role of Fcgamma receptors in SLE pathogenesis and open new avenues for the development of drugs to treat autoimmune disorders.     

A synthetic ligand for IgA affinity purification

We reported previously that TG19318, a synthetic ligand deduced from the screening of combinatorial libraries, displays specific and selective recognition properties for immunoglobulins of the G class and can be used conveniently for affinity chromatography purification of monoclonal and polyclonal antibodies. In this study we have extended the ligand characterization, examining its ability to bind IgA from cell culture supernatants and from IgG-deprived serum. Affinity columns prepared by immobilizing TG19318 on Sepharose allowed convenient one-step purification of monoclonal IgA directly from crude feedstocks, in high yield and with full recovery of immunoreactivity. Optimal column adsorption occurred with phosphate buffer at neutral pH, while elution of adsorbed IgA could be accomplished by a buffer pH change to acidic or basic conditions. Column capacity was close to 7 mg IgA/ml support.     

The effects of seed availability on habitat use by a specialist seed predator

Space-use patterns of seed predators are strongly affected by spatiotemporal variation in the abundance of different tree seeds, their major food source. However, most studies have measured relationships between overall food availability and space use, and there are few cases where effects of different food resources have been explored. We studied the effects of two food resources, Norway spruce and silver fir seeds, on space and habitat use in red squirrel in a subalpine conifer forest from 2000 to 2006. Fir seeds disperse in the autumn of the year they are produced, spruce the following spring. We estimated spruce and fir seed availability within individual home ranges and monitored home-range size using radiotelemetry. Males had larger home ranges than females and the sexes responded differently to variation in food and density. Spruce seed availability negatively affected home-range and core-area sizes of males in spring–summer. Space use was not affected by fir seed availability. Squirrels positively selected spruce for foraging and spruce was always preferred over fir. Our results showed that spruce, but not fir, affected space and habitat use of squirrels, suggesting they do not behaviourally respond to early seed dispersal in fir.