Structures, Assays and Receptors for Locust Adipokinetic Hormones*

This review is concerned mainly with the adipokinetic hormones (AKHs) of locusts: their molecular conformations, actions and functions and the development of microfiltration assays in vitro. The physiological significance of having multiple hormones with overlapping actions whose efficacy changes during development is discussed in relation to the possibility that these reflect variations in populations of receptors and/or the pharmacokinetics of the peptides. The involvement of second messengers in the transduction mechanism of AKHs is reviewed, and we describe hormone-induced changes of intracellular calcium in single dispersed fat body cells. The structure activity relationships of the three locust AKHs and a number of analogues with variations at the N- and C-termini are discussed. A number of areas are identified where there are gaps in our understanding of these hormones, and some of these will be the focus of our future research.     

Epithelial cell distribution and abundance in rhesus monkey airways during postnatal lung growth and development.

Lung development is both a pre- and postnatal process. Although many lung diseases have their origins in early childhood, few quantitative data are available on the normal growth and differentiation of both the conducting airways and the airway epithelium during the postnatal period. We examined rhesus monkey lungs from five postnatal ages: 4-6 days and 1, 2, 3, and 6 mo. Airways increase significantly in both length and circumference as monkeys increase significantly in body weight from 5 days to 6 mo. In this study we asked: as basement membrane surface area increases, does the epithelial cell organization change? To answer this question, we quantified total epithelial cell mass using high-resolution light micrographs and morphometric techniques on sections from defined airway regions: trachea, proximal intrapulmonary bronchus (generations 1 or 2), and distal intrapulmonary bronchus (generations 6-8). Epithelial thickness decreased in the smaller, more distal, airways compared with trachea but did not change with age in the trachea and proximal bronchus. The volume fraction of all cell types measured did not change significantly. Ciliated cells in the distal bronchus and goblet cells in the trachea both decreased in abundance with increasing age. Overall, the epithelial cell populations changed little in terms of mass or relative abundance to each other during this period of active postnatal lung growth. Regarding the proximal conducting airway epithelium, we conclude that 1) the steady-state abundance is tightly regulated to keep the proportion of cell types constant, and 2) establishment of these cell types occurs before 4-6 days postnatal age. We conclude that growth of the proximal airways occurs primarily in length and lags behind that of the lung parenchyma.     

Responses of cultured rat trigeminal ganglion neurons to bitter tastants

The initial steps in taste and olfaction result from the activation by chemical stimuli of taste receptor cells (TRCs) and olfactory receptor neurons (ORNs). In parallel with these two pathways is the chemosensitive trigeminal pathway whose neurons terminate in the oral and nasal cavities and which are activated by many of the same chemical stimuli that activate TRCs and ORNs. In a recent single unit study we investigated the responses of rat chorda tympani and glossopharnygeal neurons to a variety of bitter-tasting alkaloids, including nicotine, yohimbine, quinine, strychnine and caffeine, as well as capsaicin, the pungent ingredient in hot pepper. Here we apply many of these same compounds to cultured rat trigeminal ganglion (TG) neurons and measure changes in intracellular calcium [Ca2+]i to determine whether TG neurons will respond to these same compounds. Of the 89 neurons tested, 34% responded to 1 mM nicotine, 7% to 1 mM caffeine, 5% to 1 mM denatonium benzoate, 22% to 1 mM quinine hydrochloride, 18% to 1 mM strychnine and 55% to 1 microM capsaicin. These data suggest that neurons from the TG respond to the same bitter-tasting chemical stimuli as do TRCs and are likely to contribute information sent to the higher CNS regarding the perception of bitter/irritating chemical stimuli.      

MUCILAGE-PRODUCING CELLS IN THE SEED COAT OF PLANTAGO OVATA: DEVELOPMENTAL FINE STRUCTURE

As the ovule of Plantago ovata matures into a seed its epidermal cells are transformed from undifferentiated parenchyma to thin-walled containers of almost pure mucilage. During this process the volume of the cells increases 60–80 fold, and the protoplast degenerates to a remnant. Rapid cell expansion begins with pollination and is accompanied by an increase in the size of the nucleus and nucleolus, a change in the random arrangement of ribosomes, a decrease in the thickness of cell walls, and synthesis of starch. Deposition of mucilage inside vacuoles and between the plasma membrane and cell wall accompanies a marked increase in the number and size of Golgi vesicles. Histochemical evidence using the thiocarbohydrazide-osmium vapor method shows polysaccharide to be present within Golgi vesicles while they are still attached to the Golgi apparatus. Mucilage deposition is associated with further cell expansion, separation of the protoplast from the cell wall, fusion of vacuoles and extra protoplasmic space, and the disappearance of starch.     

Buoyant Density Studies of Chloroplast and Nuclear Deoxyribonucleic Acid from Control and 3-Amino-1,2,4-Triazole-treated Wheat Seedlings, Triticum vulgare

The isolation of chloroplast and nuclear DNA from dark- and light-grown, control- and 3-amino-1,2,4-triazole-treated wheat seedlings, Triticum vulgare, is described. Contrary to a previous report, we found that chloroplast and nuclear DNA had similar buoyant densities (1.702 grams per cubic centimeter) and that they could not be resolved by buoyant density centrifugation in CsCl. Difference in renaturation behavior of the chloroplast and nuclear DNA was used as the criterion for distinguishing one from the other. Only chloroplast DNA readily renatured whereas nuclear DNA renatured only slightly. Light-grown, 3-amino-1,2,4-triazole-treated plants were found to lack detectable quantities of chloroplast DNA whereas treated, dark-grown plants contained plastid DNA. We suggest that 3-amino-1,2,4-triazole affects the accumulation of chloroplast DNA by inhibiting the formation of chloroplast membranes, enzymes, and pigments.     

Flexibility of KorA,a plasmid-encoded,global transcription regulator,in the presence and the absence of its operator

The IncP (Incompatibility group P) plasmids are important carriers in the spread of antibiotic resistance across Gram-negative bacteria. Gene expression in the IncP-1 plasmids is stringently controlled by a network of four global repressors, KorA, KorB, TrbA and KorC interacting cooperatively. Intriguingly, KorA and KorB can act as co-repressors at varying distances between their operators, even when they are moved to be on opposite sides of the DNA. KorA is a homodimer with the 101-amino acid subunits, folding into an N-terminal DNA-binding domain and a C-terminal dimerization domain. In this study, we have determined the structures of the free KorA repressor and two complexes each bound to a 20-bp palindromic DNA duplex containing its consensus operator sequence. Using a combination of X-ray crystallography, nuclear magnetic resonance spectroscopy, SAXS and molecular dynamics calculations, we show that the linker between the two domains is very flexible and the protein remains highly mobile in the presence of DNA. This flexibility allows the DNA-binding domains of the dimer to straddle the operator DNA on binding and is likely to be important in cooperative binding to KorB. Unexpectedly, the C-terminal domain of KorA is structurally similar to the dimerization domain of the tumour suppressor p53.     

Three-dimensional dynamic structure of the liquid-ordered domain in lipid membranes as examined by pulse-EPR oxygen probing

Membranes made of dimyristoylphosphatidylcholine and cholesterol, one of the simplest paradigms for the study of liquid ordered-disordered phase separation, were investigated using a pulse-EPR spin-labeling method in which bimolecular collision of molecular oxygen with the nitroxide spin label is measured. This method allowed discrimination of liquid-ordered, liquid-disordered, and solid-ordered domains because the collision rates (OTP) differ in these domains. Furthermore, the oxygen transport parameter (OTP) profile across the bilayer provides unique information about the three-dimensional dynamic organization of the membrane domains. First, the OTP in the bilayer center in the liquid-ordered domain was comparable to that in the liquid-disordered domain without cholesterol, but the OTP near the membrane surface (up to carbon 9) was substantially smaller in the ordered domain, i.e., the cholesterol-based liquid-ordered domain is ordered only near the membrane surface, still retaining high levels of disorder in the bilayer center. This property may facilitate lateral mobility in ordered domains. Second, in the liquid-disordered domain, the domains with ~5 mol % cholesterol exhibited higher OTP than those without cholesterol, everywhere across the membrane. Third, the transmembrane OTP profile in the liquid-ordered domain that contained 50 mol % cholesterol dramatically differed from that which contained 27 mol % cholesterol.     

The crystal structure of the SIV gp41 ectodomain at 1.47 A resolution.

Cell membrane fusion by human (HIV) and simian (SIV) immunodeficiency viruses is mediated by the envelope glycoproteins gp120 and gp41. Although the precise mechanism of the fusion process is unknown, the ectodomain of gp41 is thought to undergo dramatic rearrangement from its prefusogenic state. To elucidate this process further, the crystal structure of the SIV gp41 ectodomain (residues 27-149) was determined at 1.47 A resolution and is reported herein. It is the most accurate and complete structure of a retroviral gp41 ectodomain determined to date. The rod-like trimeric structure of SIV gp41 comprises three parallel N-terminal alpha-helices assembled as a coiled coil in the center with three antiparallel C-terminal alpha-helices packed on the outside connected by highly flexible loops. Portions of the loops in all three monomers are crystallographically disordered and could not be accurately modeled. The core of the structure is similar (but not identical) to those of smaller HIV/SIV gp41 segments previously determined by X-ray crystallography with root mean square deviations in main chain atoms of less than 1.0 A. The crystal structure differs more substantially from the reported NMR solution structure of the identical SIV construct. The mechanisms of viral fusion and the inhibition by peptides are discussed in the context of the three-dimensional structure.