Establishment of functional human pituitary tumor cell cultures

Five primary human pituitary tumor cell cultures were initiated from adenoma fragments obtained from patients with prolactin-secreting adenomas and acromegaly. Functional cell cultures were maintained and propagated in monolayer or suspension culture for up to 9 months. Optimal cell viability and growth were achieved using Ham's F10 medium enriched with 20% fetal bovine serum, although cells from a patient with acromegaly also grew in serum-free, defined, hormone-containing medium. Bromocriptine (100 ng/ml) did not alter the growth curve of replicating cells derived from a patient with acromegaly. These cells initially secreted 5.5 micrograms human growth hormone/10(6) cells, and hormone production diminished after 6 wk. Prolactin secretion by cells derived from prolactinomas (0.5 to 1.3 micrograms/10(6) cells/24 h) was stimulated by thyrotropin-releasing hormone (10 ng/ml) in two of the cultures. Both dopamine (10 ng/ml) and nickel chloride (1 mM) suppressed PRL secretion. These studies demonstrate that responsive human pituitary tumor cell cultures can be initiated and maintained.     

Polyunsaturated fatty acid status and neurodevelopment: A summary and critical analysis of the literature

The rationale for randomized trials designed to measure the effects of variable docosahexaenoic acid (DHA) status on neurodevelopment in human infants came from earlier studies of neurodevelopment in animals that were deficient in DHA owing to diets low in alpha-linolenic acid. The session on neurodevelopment looked at the results of these animal studies and discussed outcomes that appear to be analogous in human infants with variable DHA status. Presentations focused mainly on measures of development that may be attributed to more specific developmental domains (e.g., visual attention, recognition memory, problem-solving), some of which have been shown to be affected by long-chain polyunsaturated fatty acid (LCPUFA) status. This paper derives from discussions that took place during the session and reviews subsequent developments in this area. Although more difficult to interpret, global measures of infant development (e.g., the Bayley Scales of Infant Development, and Brunet-Lezine) can only suggest a relationship to specific developmental domains, but they have been applied in some randomized trials of LCPUFA and infant development. Those results are also summarized here.     

Development of an In Situ Cancer Vaccine via Combinational Radiation and Bacterial‐Membrane‐Coated Nanoparticles

Neoantigens induced by random mutations and specific to an individual's cancer are the most important tumor antigens recognized by T cells. Among immunologically “cold” tumors, limited recognition of tumor neoantigens results in the absence of a de novo antitumor immune response. These “cold” tumors present a clinical challenge as they are poorly responsive to most immunotherapies, including immune checkpoint inhibitors (ICIs). Radiation therapy (RT) can enhance immune recognition of “cold” tumors, resulting in a more diversified antitumor T‐cell response, yet RT alone rarely results in a systemic antitumor immune response. Therefore, a multifunctional bacterial membrane‐coated nanoparticle (BNP) composed of an immune activating PC7A/CpG polyplex core coated with bacterial membrane and imide groups to enhance antigen retrieval is developed. This BNP can capture cancer neoantigens following RT, enhance their uptake in dendritic cells (DCs), and facilitate their cross presentation to stimulate an antitumor T‐cell response. In mice bearing syngeneic melanoma or neuroblastoma, treatment with BNP+RT results in activation of DCs and effector T cells, marked tumor regression, and tumor‐specific antitumor immune memory. This BNP facilitates in situ immune recognition of a radiated tumor, enabling a novel personalized approach to cancer immunotherapy using off‐the‐shelf therapeutics.     

Male-produced aggregation pheromone ofCarpophilus mutilatus (Coleoptera: Nitidulidae)

Males ofCarpophilus mutilatus Erichson produce an aggregation pheromone to which both sexes respond. The pheromone includes two hydrocarbon components, (3E,5E,7E)-5-ethyl-7-methyl-3,5,7-undecatriene (1) and (3E,5E,7E)-6-ethyl-4-methyl-3,5,7-decatriene (2). These were emitted in a 101 ratio and in a total amount of ca. 5 ng per feeding male per day. All tested doses of1 and2, from 0.03 to 30 ng, were more attractive than controls in wind-tunnel tests, but there was no evidence of synergism between these trienes. Dramatic synergism between the pheromone and a food-type coattractant occurred in the field, however. In a date garden in southern California, traps with a combination of synthetic1 and fermenting whole-wheat bread dough attracted 22 times more beetles than dough by itself and 295 times more than1 by itself. Volatile collections from males also contained three oxygenated compounds that were absent from females. One of these was tetradecanal (ca. 5 ng per male per day), but the structures of the other two are presently undetermined (0.8 and 1.1 ng per male per day). No function for these was demonstrated. One compound originating in the artificial diet, 2-phenylethanol, was particularly attractive in the wind-tunnel bioassay, as was the chromatographic solvent, methanol.     

Features of calmodulin that are important in the activation of the catalytic subunit of phosphorylase kinase

Calmodulin (CaM) is an integral subunit, called delta, of the phosphorylase kinase hexadecamer, and the activity of the isolated catalytic gamma-subunit of the kinase is stimulated by CaM. We report here the first analysis of functionally important features of CaM for activation of the gamma-subunit. A set of genetically engineered CaMs, in which acidic residues in each of the four E-helices of the "EF-hands" were changed to basic lysine residues, was used to probe the relative importance of charge features in each domain of CaM. The maximal activation of the isolated gamma-subunit was diminished by all of the charge reversal mutations. The gamma-subunit was especially sensitive to reversals in the second and third E-helix of CaM (residues 45-47 and 82-84), the latter being present in the central helix. The results suggest the functional importance of electrostatics in the interactions between the delta-subunit (CaM) and the catalytic gamma-subunit of phosphorylase kinase, which is similar to results obtained with CaM-dependent myosin light chain kinase (MLCK) from chicken gizzard and CaM-dependent protein kinase II (CaMPK-II). However, novel features of the interaction between CaM and the gamma-subunit of phosphorylase kinase are the significant contribution of electrostatics throughout the CaM molecule, including residues in both halves and on more than one face of CaM, and the lack of a major effect of the CaM mutations on substrate kinetic parameters, unlike the effects observed with MLCK and CaMPK-II. These results are consistent with a model in which the delta-subunit (CaM) of phosphorylase kinase interacts with an extended region or multiple regions of the gamma-subunit and suggest that the mechanism of CaM activation of the gamma-subunit may have features that are distinct from those of MLCK and CaMPK-II.     

Exploring protein-ligand recognition with Binding MOAD

We have recently announced the largest database of protein-ligand complexes, Binding MOAD (Mother of All Databases). After the August 2004 update, Binding MOAD contains 6816 complexes. There are 2220 protein families and 3316 unique ligands. After searching 6000+ crystallography papers, we have obtained binding data for 1793 (27%) of the complexes. We have also created a non-redundant set of complexes with only one complex from each protein family; in that set, 630 (28%) of the unique complexes have binding data. Here, we present information about the data provided at the Binding MOAD website. We also present the results of mining Binding MOAD to map the degree of solvent exposure for binding sites. We have determined that most cavities and ligands (70-85%) are well buried in the complexes. This fits with the common paradigm that a large degree of contact between the ligand and protein is significant in molecular recognition. GoCAV and the GoCAV viewer are the tools we created for this study. To share our data and make our online dataset more useful to other research groups, we have integrated the viewer into the Binding MOAD website (www.BindingMOAD.org).