Changes of beta-endorphin and somatostatin concentrations in different hypothalamic areas of female rats after chronic starvation

To study the effect of starvation on hypothalamic beta-endorphin and somatostatin (SRIF) concentrations in relation to starvation induced anestrus, groups of 8 rats were fed 50% of their normal daily chow consumption. Rats were sacrificed after 4, 8, 12, and 16 days during diestrus or anestrus. beta-endorphin concentrations decreased in the preoptic suprachiasmatic area (0.52 +/- 0.13 vs 0.21 +/- 0.05 ng/mg tissue wet weight) and increased in the posterior hypothalamus (0.31 +/- 0.06 vs 0.57 +/- 0.11 ng/mg) after 4 days of starvation. No significant change occurred in the arcuate nucleus or in the median eminence. On day 8 and 12 of starvation, beta-endorphin was unaltered in all areas compared to controls. Vaginal smears showed constant diestrus in a significant number of rats (5 out of 8) after 12 days. beta-endorphin concentrations in the arcuate nuclei of these rats were significantly reduced on day 16 (1.00 +/- 0.33 vs 0.30 +/- 0.11 ng/mg). The SRIF levels changed only in the median eminence with increased concentrations on day 12 (45.2 +/- 8.4 vs 79.5 +/- 14.8 ng/mg). At this time serum levels of luteinizing hormone (LH), prolactin (PRL), and growth hormone (GH) were significantly reduced. The results indicate that changes in hypothalamic beta-endorphin accompany the events leading to starvation induced anestrus.     

Novel Bacillus thuringiensis binary insecticidal crystal proteins active on western corn rootworm, Diabrotica virgifera virgifera LeConte

A new family of insecticidal crystal proteins was discovered by screening sporulated Bacillus thuringiensis cultures for oral activity against western corn rootworm (WCR) larvae. B. thuringiensis isolates PS80JJ1, PS149B1, and PS167H2 have WCR insecticidal activity attributable to parasporal inclusion bodies containing proteins with molecular masses of ca. 14 and 44 kDa. The genes encoding these polypeptides reside in apparent operons, and the 14-kDa protein open reading frame (ORF) precedes the 44-kDa protein ORF. Mutagenesis of either gene in the apparent operons dramatically reduced insecticidal activity of the corresponding recombinant B. thuringiensis strain. Bioassays performed with separately expressed, biochemically purified 14- and 44-kDa polypeptides also demonstrated that both proteins are required for WCR mortality. Sequence comparisons with other known B. thuringiensis insecticidal proteins failed to reveal homology with previously described Cry, Cyt, or Vip proteins. However, there is evidence that the 44-kDa polypeptide and the 41.9- and 51.4-kDa binary dipteran insecticidal proteins from Bacillus sphaericus are evolutionarily related. The 14- and 44-kDa polypeptides from isolates PS80JJ1, PS149B1, and PS167H2 have been designated Cry34Aa1, Cry34Ab1, and Cry34Ac1, respectively, and the 44-kDa polypeptides from these isolates have been designated Cry35Aa1, Cry35Ab1, and Cry35Ac1, respectively.     

Impact of antigen presentation on TCR modulation and cytokine release: implications for detection and sorting of antigen-specific CD8+ T cells using HLA-A2 wild-type or HLA-A2 mutant tetrameric complexes

Soluble MHC class I molecules loaded with antigenic peptides are available either to detect and to enumerate or, alternatively, to sort and expand MHC class I-restricted and peptide-reactive T cells. A defined number of MHC class I/peptide complexes can now be implemented to measure T cell responses induced upon Ag-specific stimulation, including CD3/CD8/zeta-chain down-regulation, pattern, and quantity of cytokine secretion. As a paradigm, we analyzed the reactivity of a Melan-A/MART-1-specific and HLA-A2-restricted CD8(+) T cell clone to either soluble or solid-phase presented peptides, including the naturally processed and presented Melan-A/MART-1 peptide AAGIGILTV or the peptide analog ELAGIGILTV presented either by the HLA-A2 wild-type (wt) or mutant (alanineright arrowvaline aa 245) MHC class I molecule, which reduces engagement of the CD8 molecule with the HLA-A2 heavy chain. Soluble MHC class I complexes were used as either monomeric or tetrameric complexes. Soluble monomeric MHC class I complexes, loaded with the Melan-A/MART-1 peptide, resulted in CD3/CD8 and TCR zeta-chain down-regulation, but did not induce measurable cytokine release. In general, differences pertaining to CD3/CD8/zeta-chain regulation and cytokine release, including IL-2, IFN-gamma, and GM-CSF, were associated with 1) the format of Ag presentation (monomeric vs tetrameric MHC class I complexes), 2) wt vs mutant HLA-A2 molecules, and 3) the target Ag (wt vs analog peptide). These differences are to be considered if T cells are exposed to recombinant MHC class I Ags loaded with peptides implemented for detection, activation, or sorting of Ag-specific T cells.     

Structures of single‐layer β‐sheet proteins evolved from β‐hairpin repeats

Free‐standing single‐layer β‐sheets are extremely rare in naturally occurring proteins, even though β‐sheet motifs are ubiquitous. Here we report the crystal structures of three homologous, single‐layer, anti‐parallel β‐sheet proteins, comprised of three or four twisted β‐hairpin repeats. The structures reveal that, in addition to the hydrogen bond network characteristic of β‐sheets, additional hydrophobic interactions mediated by small clusters of residues adjacent to the turns likely play a significant role in the structural stability and compensate for the lack of a compact hydrophobic core. These structures enabled identification of a family of secreted proteins that are broadly distributed in bacteria from the human gut microbiome and are putatively involved in the metabolism of complex carbohydrates. A conserved surface patch, rich in solvent‐exposed tyrosine residues, was identified on the concave surface of the β‐sheet. These new modular single‐layer β‐sheet proteins may serve as a new model system for studying folding and design of β‐rich proteins.     

Gene expression response to misfolded protein as a screen for soluble recombinant protein

Proper protein folding is key to producing recombinant proteins for structure determination. We have examined the effect of misfolded recombinant protein on gene expression in Escherichia coli. Comparison of expression patterns indicates a unique set of genes responding to translational misfolding. The response is in part analogous to heat shock and suggests a translational component to the regulation. We have further utilized the expression information to generate reporters responsive to protein misfolding. These reporters were used to identify properly folded recombinant proteins and to create soluble domains of insoluble proteins for structural studies.     

Altered olfactory receptor neuron responsiveness in rare Ostrinia nubilalis males attracted to the O. furnacalis pheromone blend

Three percent of E-strain Ostrinia nubilalis males fly upwind in response to the Ostrinia furnacalis pheromone blend [a 40:60 ratio of (E)-12-tetradecenyl acetate to (Z)-12-tetradecenyl acetate (E12-14:OAc to Z12-14:OAc)], in addition to their own pheromone blend [a 99:1 ratio of (E)-11-tetradecenyl acetate to (Z)-11-tetradecenyl acetate) (E11-14:OAc to Z11-14:OAc)]. We assessed the olfactory receptor neuron (ORN) responses of these behaviorally "rare" males versus those of normal males. For the three ORNs housed within each sensillum, we tested responsiveness to Z12-14:OAc, E12-14:OAc, Z11-14:OAc, E11-14:OAc, and the behavioral antagonist (Z)-9-tetradecenyl acetate (Z9-14:OAc). Z11-14:OAc, E11-14:OAc, and Z9-14:OAc stimulated ORNs exhibiting distinct small, large, and medium spike sizes, respectively. For rare and normal males, both Z12-14:OAc and E12-14:OAc usually elicited responses from the largest-spiking ORN. In many ORNs of normal males, Z12-14:OAc or E12-14:OAc stimulated the smaller-spiking ORN that is responsive to Z11-14:OAc. In rare males, detectable ORN responses from the smaller-spiking ORN in response to Z12- and E12-14:OAc were virtually non-existent. These differences in ORN tuning in rare males will tend to create an ORN firing ratio between the large- and small-spiking ORNs in response to the O. furnacalis blend that is similar to that elicited by the O. nubilalis blend.