In vitro histamine H2-antagonist activity of the novel compound HUK 978

Histamine stimulated adenylate cyclase from guinea-pig fundic mucosa and 3H-tiotidine binding in guinea-pig cerebral cortex were used to assess the in-vitro histamine H2-activity of the novel H2-antagonist HUK 978. The results showed that HUK 978 was a more potent H2-antagonist than either cimetidine or ranitidine. HUK 978 was also shown to be devoid of activity at the histamine H1-receptor, the muscarinic receptor and the alpha and beta-adrenergic receptors.     

Structural properties of recombinant nonrepetitive and repetitive parts of major ampullate spidroin 1 from Euprosthenops australis: implications for fiber formation

Spider dragline silk proteins, spidroins, have a tripartite composition; a nonrepetitive N-terminal domain, a central repetitive region built up from many iterated poly-Ala and Gly rich blocks, and a C-terminal nonrepetitive domain. It is generally believed that the repetitive region forms intermolecular contacts in the silk fibers, while precise functions of the terminal domains have not been established. Herein, thermal, pH, and salt effects on the structure and aggregation and/or polymerization of recombinant N- and C-terminal domains, a repetitive segment containing four poly-Ala and Gly rich coblocks, and combinations thereof were studied. The N- and C-terminal domains have mainly alpha-helical structure, and interestingly, both form homodimers. Dimerization of the end domains allows spidroin multimerization independent of the repetitive part. Reduction of an intersubunit disulfide in the C-terminal domain lowers the thermal stability but does not affect dimerization. The repetitive region shows helical secondary structure but appears to lack stable folded structure. A protein composed of this repetitive region linked to the C-terminal domain has a mainly alpha-helical folded structure but shows an abrupt transition to beta-sheet structures upon heating. At room temperature, this protein self-assembles into macroscopic fibers within minutes. The secondary structures of none of the domains are altered by pH or salt. However, high concentrations of phosphate affect the tertiary structure and accelerate the aggregation propensity of the repetitive region. Implications of these results for dragline spidroin behavior in solution and silk fiber formation are discussed.     

Linkage studies with chromosome 17 DNA markers in 45 neurofibromatosis 1 families

A locus for von Recklinghausen neurofibromatosis (NF1) has recently been mapped near the chromosome 17 centromere. We have extended these linkage studies by genotyping 45 NF1 families with three DNA probes known to be linked to the chromosome 17 centromeric region. Of 34 families informative for NF1 and at least one of the three probes, 28 families show no recombinants with the disease gene. These data provide additional support for genetic homogeneity of NF1 and for a primary NF1 locus linked to the chromosome 17 centromere. Among the informative families were 7 families with apparent new NF1 mutations. Our data suggest that these mutations are probably at the chromosome 17 NF1 locus.     

Aging in Mice Reduces the Ability to Sustain Sleep/Wake States

One of the most significant problems facing older individuals is difficulty staying asleep at night and awake during the day. Understanding the mechanisms by which the regulation of sleep/wake goes awry with age is a critical step in identifying novel therapeutic strategies to improve quality of life for the elderly. We measured wake, non-rapid eye movement (NREM) and rapid-eye movement (REM) sleep in young (2–4 months-old) and aged (22–24 months-old) C57BL6/NIA mice. We used both conventional measures (i.e., bout number and bout duration) and an innovative spike-and-slab statistical approach to characterize age-related fragmentation of sleep/wake. The short (spike) and long (slab) components of the spike-and-slab mixture model capture the distribution of bouts for each behavioral state in mice. Using this novel analytical approach, we found that aged animals are less able to sustain long episodes of wakefulness or NREM sleep. Additionally, spectral analysis of EEG recordings revealed that aging slows theta peak frequency, a correlate of arousal. These combined analyses provide a window into the mechanisms underlying the destabilization of long periods of sleep and wake and reduced vigilance that develop with aging.     

A pH-dependent dimer lock in spider silk protein

Spider dragline silk, one of the strongest polymers in nature, is composed of proteins termed major ampullate spidroin (MaSp) 1 and MaSp2. The N-terminal (NT) domain of MaSp1 produced by the nursery web spider Euprosthenops australis acts as a pH-sensitive relay, mediating spidroin assembly at around pH 6.3. Using amide hydrogen/deuterium exchange combined with mass spectrometry (MS), we detected pH-dependent changes in deuterium incorporation into the core of the NT domain, indicating global structural stabilization at low pH. The stabilizing effects were diminished or abolished at high ionic strength, or when the surface-exposed residues Asp40 and Glu84 had been exchanged with the corresponding amides. Nondenaturing electrospray ionization MS revealed the presence of dimers in the gas phase at pH values below—but not above—6.4, indicating a tight electrostatic association that is dependent on Asp40 and Glu84 at low pH. Results from analytical ultracentrifugation support these findings. Together, the data suggest a mechanism whereby lowering the pH to < 6.4 results in structural changes and alteration of charge-mediated interactions between subunits, thereby locking the spidroin NT dimer into a tight entity important for aggregation and silk formation.     

N-terminal nonrepetitive domain common to dragline, flagelliform, and cylindriform spider silk proteins

Spider silk has been extensively studied for its outstanding mechanical properties. Partial intermediate and C-terminal sequences of different spider silk proteins have been determined, and during the past decade also N-terminal domains have been characterized. However, only some of these N-terminal domains have been reported to contain signal peptides, leaving the mechanism whereby they enter the secretory pathway open to speculation. Here we present the sequence of a 394-residue N-terminal region of the Euprosthenops australis major ampullate spidroin 1 (MaSp1). A close comparison with published sequences from other species revealed the presence of N-terminal signal peptides followed by an approximately 130-residue nonrepetitive domain. From secondary structure predictions, helical wheel analysis, and circular dichroism spectroscopy this domain is concluded to contain five alpha-helices and is a conserved constituent of hitherto analyzed dragline, flagelliform, and cylindriform spider silk proteins.     

Systems analysis of primary Sjögren's syndrome pathogenesis in salivary glands identifies shared pathways in human and a mouse model

Bone tissue has an exceptional quality to regenerate to native tissue in response to injury. However, the fracture repair process requires mechanical stability or a viable biological microenvironment or both to ensure successful healing to native tissue. An improved understanding of the molecular and cellular events that occur during bone repair and remodeling has led to the development of biologic agents that can augment the biological microenvironment and enhance bone repair. Orthobiologics, including stem cells, osteoinductive growth factors, osteoconductive matrices, and anabolic agents, are available clinically for accelerating fracture repair and treatment of compromised bone repair situations like delayed unions and nonunions. Preclinical and clinical studies using biologic agents like recombinant bone morphogenetic proteins have demonstrated an efficacy similar or better than that of autologous bone graft in acute fracture healing. A lack of standardized outcome measures for comparison of biologic agents in clinical fracture repair trials, frequent off-label use, and a limited understanding of the biological activity of these agents at the bone repair site have limited their efficacy in clinical applications.