Phenotypic analysis of random hns mutations differentiate DNA-binding activity from properties of fimA promoter inversion modulation and bacterial motility

H-NS is a major Escherichia coli nucleoid-associated protein involved in bacterial DNA condensation and global modulation of gene expression. This protein exists in cells as at least two different isoforms separable by isoelectric focusing. Among other phenotypes, mutations in hns result in constitutive expression of the proU and fimB genes, increased fimA promoter inversion rates, and repression of the flhCD master operon required for flagellum biosynthesis. To understand the relationship between H-NS structure and function, we transformed a cloned hns gene into a mutator strain and collected a series of mutant alleles that failed to repress proU expression. Each of these isolated hns mutant alleles also failed to repress fimB expression, suggesting that H-NS-specific repression of proU and fimB occurs by similar mechanisms. Conversely, alleles encoding single amino acid substitutions in the C-terminal DNA-binding domain of H-NS resulted in significantly reduced affinity for DNA yet conferred a wild-type fimA promoter inversion frequency, indicating that the mechanism of H-NS activity in modulating promoter inversion is independent of DNA binding. Furthermore, two specific H-NS amino acid substitutions resulted in hypermotile bacteria, while C-terminal H-NS truncations exhibited reduced motility. We also analyzed H-NS isoform composition expressed by various hns mutations and found that the N-terminal 67 amino acids were sufficient to support posttranslational modification and that substitutions at positions 18 and 26 resulted in the expression of a single H-NS isoform. These results are discussed in terms of H-NS domain organization and implications for biological activity.     

Photoperiodic effects on gonadotropin-releasing hormone (GnRH) content and the GnRH-immunoreactive neuronal system of male Siberian hamsters

Despite profound photoperiodic differences in circulating gonadotropin levels, consistent differences in the GnRH system have not been observed in Siberian hamsters (Phodopus sungorus) housed chronically in short or long days. During the transition from short to long days, however, male hamsters exhibit a transient increase in the number of cells expressing prepro-GnRH mRNA on the morning of the second long day. Here, we present two experiments designed to examine whether or not this change in mRNA level is associated with changes in GnRH protein synthesis. In the first experiment, we used RIA to measure GnRH content in preoptic area-mediobasal hypothalamic homogenates. We observed a significant increase in GnRH protein levels on the morning of the second long day relative to short- and long-day controls. The latter two groups did not differ from one another. In the second experiment, we used immunocytochemistry to quantify GnRH cell number in the various treatment groups. GnRH-immunoreactive (-ir) cell number did not increase significantly after long-day transfer relative to that in short-day controls; however, both of these groups had significantly more GnRH-ir neurons than long-day controls. We hypothesize that during the transition from short to long days, male Siberian hamsters experience a transient increase in GnRH production in a stable population of neurons. When GnRH secretion subsequently increases on long days, peptide content within neuronal cell bodies declines, leading to a decrease in the number of immunoreactive neurons detected. The rapid response of the hypothalamo-pituitary-gonadal axis in Siberian hamsters to a change in day length defines a narrow temporal window in which to identify the physiological, cellular, and molecular mechanisms mediating the photoperiodic regulation of reproduction.     

Influence of Drying Conditions on the Hazelnut Quality. III. Browning

ABSTRACT

The influence of drying conditions on browning in hazelnut (Corylus avellana L.) has been studied by means of color measurement. Samples of unshelled and shelled hazelnuts were dried under six air temperatures (30. 40, 50, 60, 70 and 80°C). The drying experiments were done with forced air circulation, and two varieties of hazelnut were investigated, Negret and Pauetet. CiELab system was used to evaluate changes in total color (δE^?), lightness (L^?), chroma (C^?) and hue angle (H^?) on dried hazelnuts. Browning of hazelnut was observed to occur a1 all temperatures studied. The rate of pigment formation was determined from the δE' values with zero-order kinetic model, and temperature dependence was described by the Arrhenius equation with an activation energy (E_a) varying from 28 to 39 kJlmol. Negret variety showed the highest rate of browning.     

Autosomal recessive inheritance of erythrokeratoderma variabilis

Erythrokeratoderma variabilis is a rare genodermatosis conventionally regarded as autosomal dominant in inheritance. We describe the clinical features and light and electron microscopic findings in two affected siblings born to unaffected parents and suggest an autosomal recessive mode of inheritance in this family. We also briefly review the literature on this disorder.     

Roles of transmembrane prolines and proline-induced kinks of the lutropin/choriogonadotropin receptor

The lutropin/choriogonadotropin receptor is a seven-helix transmembrane (TM) receptor. A unique feature of TM helices is the content of Pro, which generally is absent in alpha helices of globular proteins. Because Pro disrupts helices and introduces a approximately 26 degrees kink, it has been speculated that Pro plays a crucial role in the structure of TM helices, exoloops, and cytoloops of TM receptors. To examine the roles of the five TM Pros of the lutropin/choriogonadotropin receptor, these residues were individually substituted. Mutant receptors were examined for surface expression, hormone binding, and cAMP induction. Surface expression was monitored after introducing the flag epitope into the receptors. Flag epitopes slightly affected cAMP induction but not hormone binding or surface expression of receptors as monitored by immunofluorescence microscopy and 125I-anti-flag antibody. The results indicate that Pro479 in TM 4 and Pro598 in TM 7 play important yet contrasting roles. Pro479 is crucial for hormone binding at the cell surface but not after solubilization of the receptor. This is more likely due to the Pro side chain than the Pro-induced kink. Pro598 is important for surface expression. The kinks of Pro463 of TM 4, Pro562 of TM 6, or Pro591 of TM 7 are not important because the substitution of Phe for these residues did not significantly impact surface expression, hormone binding, and cAMP induction.     

An extracellular proteolytic cascade promotes neuronal degeneration in the mouse hippocampus

Mice lacking the serine protease tissue plasminogen activator (tPA) are resistant to excitotoxin-mediated hippocampal neuronal degeneration. We have used genetic and cellular analyses to study the role of tPA in neuronal cell death. Mice deficient for the zymogen plasminogen, a known substrate for tPA, are also resistant to excitotoxins, implicating an extracellular proteolytic cascade in degeneration. The two known components of this cascade, tPA and plasminogen, are both synthesized in the mouse hippocampus. tPA mRNA and protein are present in neurons and microglia, whereas plasminogen mRNA and protein are found exclusively in neurons. tPA-deficient mice exhibit attenuated microglial activation as a reaction to neuronal injury. In contrast, the microglial response of plasminogen-deficient mice was comparable to that of wild-type mice, suggesting a tPA-mediated, plasminogen-independent pathway for activation of microglia. Infusion of inhibitors of the extracellular tPA/plasmin proteolytic cascade into the hippocampus protects neurons against excitotoxic injury, suggesting a novel strategy for intervening in neuronal degeneration.     

Ykt6p, a prenylated SNARE essential for endoplasmic reticulum-Golgi transport

Vesicular transport between secretory compartments requires specific recognition molecules called SNAREs. Here we report the identification of three putative SNAREs, p14 (Sft1p), p28 (Gos1p), and a detailed characterization of p26 (Ykt6p). All three were originally isolated as interacting partners of the cis Golgi target membrane-associated SNARE Sed5p, when Sec18p (yeast NSF) was inactivated. YKT6 is an essential gene that codes for a novel vesicle-associated SNARE functioning at the endoplasmic reticulum-Golgi transport step in the yeast secretory pathway. Depletion of Ykt6p results in the accumulation of the p1 precursor (endoplasmic reticulum form) of the vacuolar enzyme carboxypeptidase Y and morphological abnormalities consistent with a defect in secretion. Membrane localization of Ykt6p is essential for protein function and is normally mediated by isoprenylation. However, replacement of the isoprenylation motif with a bona fide transmembrane anchor results in a functional protein confirming that membrane localization, but not isoprenylation per se, is required for function. Ykt6p and its homologues are highly conserved from yeast to human as demonstrated by the functional complementation of the loss of Ykt6p by its human counterpart. This is the first example of a human SNARE protein functionally replacing a yeast SNARE. This observation implies that the specific details of the vesicle targeting code, like the genetic code, are conserved in evolution.