Converting enzyme-independent release of tumor necrosis factor α and IL-1β from a stimulated human monocytic cell line in the presence of activated neutrophils or purified proteinase 3

Two important cytokines mediating inflammation are tumor necrosis factor α (TNFα) and IL-1β, both of which require conversion to soluble forms by converting enzymes. The importance of TNFα-converting enzyme and IL-1β-converting enzyme in the production of circulating TNFα and IL-1β in response to systemic challenges has been demonstrated by the use of specific converting enzyme inhibitors. Many inflammatory responses, however, are not systemic but instead are localized. In these situations release and/or activation of cytokines may be different from that seen in response to a systemic stimulus, particularly because associations of various cell populations in these foci allows for the exposure of procytokines to the proteolytic enzymes produced by activated neutrophils, neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (Cat G). To investigate the possibility of alternative processing of TNFα and/or IL-1β by neutrophil-derived proteinases, immunoreactive TNFα and IL-1β release from lipopolysaccharide-stimulated THP-1 cells was measured in the presence of activated human neutrophils. Under these conditions, TNFα and IL-1β release was augmented 2- to 5-fold. In the presence of a specific inhibitor of NE and PR3, enhanced release of both cytokines was largely abolished; however, in the presence of a NE and Cat G selective inhibitor, secretory leucocyte proteinase inhibitor, reduction of the enhanced release was minimal. This finding suggested that the augmented release was attributable to PR3 but not NE nor Cat G. Use of purified enzymes confirmed this conclusion. These results indicate that there may be alternative pathways for the production of these two proinflammatory cytokines, particularly in the context of local inflammatory processes.

The essential protein encoded by the UL31 gene of herpes simplex virus 1 depends for its stability on the presence of UL34 protein

To pursue an earlier observation that the protein encoded by the UL34 gene binds to intermediate chain of dynein, we constructed a series of mutants from which sequences encoding the entire protein (ΔUL34) or amino-terminal [UL34Δ(3–119)] or carboxyl-terminal [UL34Δ(245–275)] domains were deleted. The mutant lacking the sequence encoding the carboxyl-terminal domain grew in all cell lines tested. The two other mutants replicated only in cell type-dependent manner and poorly. Rescue of ΔUL34 mutant with a fragment that does not encompass the UL31 ORF restored wild-type phenotype. UL34 protein interacts physically with UL31, and the UL31 deletion mutant appears to have a phenotype similar to that of UL34 deletion mutant. Experiments designed to determine whether the phenotypes of the deletion mutants have a common base revealed that cells infected with the ΔUL34 mutant accumulate UL31 RNA but not the corresponding protein. The UL31 protein accumulated, however, to near wild-type virus-infected cell levels in cells infected with ΔUL34 mutant and treated with the MG132 proteosomal inhibitor at 6 h after infection. This is evidence that the stability of an essential viral protein requires the presence of another protein. The observation raises the bar for identification of gene function on the basis of analyses of the phenotype of mutants in which the gene has been deleted or rendered inoperative.

Structure of a biological oxygen sensor: A new mechanism for heme-driven signal transduction

The FixL proteins are biological oxygen sensors that restrict the expression of specific genes to hypoxic conditions. FixL’s oxygen-detecting domain is a heme binding region that controls the activity of an attached histidine kinase. The FixL switch is regulated by binding of oxygen and other strong-field ligands. In the absence of bound ligand, the heme domain permits kinase activity. In the presence of bound ligand, this domain turns off kinase activity. Comparison of the structures of two forms of the Bradyrhizobium japonicum FixL heme domain, one in the “on” state without bound ligand and one in the “off” state with bound cyanide, reveals a mechanism of regulation by a heme that is distinct from the classical hemoglobin models. The close structural resemblance of the FixL heme domain to the photoactive yellow protein confirms the existence of a PAS structural motif but reveals the presence of an alternative regulatory gateway.

Thyroid hormone receptor β-dependent expression of a potassium conductance in inner hair cells at the onset of hearing

To elucidate the role of thyroid hormone receptors (TRs) α1 and β in the development of hearing, cochlear functions have been investigated in mice lacking TRα1 or TRβ. TRs are ligand-dependent transcription factors expressed in the developing organ of Corti, and loss of TRβ is known to impair hearing in mice and in humans. Here, TRα1-deficient (TRα1−/−) mice are shown to display a normal auditory-evoked brainstem response, indicating that only TRβ, and not TRα1, is essential for hearing. Because cochlear morphology was normal in TRβ−/− mice, we postulated that TRβ regulates functional rather than morphological development of the cochlea. At the onset of hearing, inner hair cells (IHCs) in wild-type mice express a fast-activating potassium conductance, IK,f, that transforms the immature IHC from a regenerative, spiking pacemaker to a high-frequency signal transmitter. Expression of IK,f was significantly retarded in TRβ−/− mice, whereas the development of the endocochlear potential and other cochlear functions, including mechanoelectrical transduction in hair cells, progressed normally. TRα1−/− mice expressed IK,f normally, in accord with their normal auditory-evoked brainstem response. These results establish that the physiological differentiation of IHCs depends on a TRβ-mediated pathway. When defective, this may contribute to deafness in congenital thyroid diseases.

Origin of a chloroplast protein importer

During evolution, chloroplasts have relinquished the majority of their genes to the nucleus. The products of transferred genes are imported into the organelle with the help of an import machinery that is distributed across the inner and outer plastid membranes. The evolutionary origin of this machinery is puzzling because, in the putative predecessors, the cyanobacteria, the outer two membranes, the plasma membrane, and the lipopolysaccharide layer lack a functionally similar protein import system. A 75-kDa protein-conducting channel in the outer envelope of pea chloroplasts, Toc75, shares ≈22% amino acid identity to a similarly sized protein, designated SynToc75, encoded in the Synechocystis PCC6803 genome. Here we show that SynToc75 is located in the outer membrane (lipopolysaccharide layer) of Synechocystis PCC6803 and that SynToc75 forms a voltage-gated, high conductance channel with a high affinity for polyamines and peptides in reconstituted liposomes. These findings suggest that a component of the chloroplast protein import system, Toc75, was recruited from a preexisting channel-forming protein of the cyanobacterial outer membrane. Furthermore, the presence of a protein in the chloroplastic outer envelope homologous to a cyanobacterial protein provides support for the prokaryotic nature of this chloroplastic membrane.

The reversible condensation and expansion of the rotavirus genome

Understanding the structural organization of the genome is particularly relevant in segmented double-stranded RNA viruses, which exhibit endogenous transcription activity. These viruses are molecular machines capable of repeated cycles of transcription within the intact capsid. Rotavirus, a major cause of infantile gastroenteritis, is a prototypical segmented double-stranded RNA virus. From our three-dimensional structural analyses of rotavirus examined under various chemical conditions using electron cryomicroscopy, we show here that the viral genome exhibits a remarkable conformational flexibility by reversibly changing its packaging density. In the presence of ammonium ions at high pH, the genome condenses to a radius of ≈180 Å from ≈220 Å. Upon returning to physiological conditions, the genome re-expands and fully maintains its transcriptional properties. These studies provide further insights into the genome organization and suggest that the observed isometric and concentric nature of the condensation is due to strong interactions between the genome core and the transcription enzymes anchored to the capsid inner surface. The ability of the genome to condense beyond what is normally observed in the native virus indicates that the negative charges on the RNA in the native state may be only partially neutralized. Partial neutralization may be required to maintain appropriate interstrand spacing for templates to move around the enzyme complexes during transcription. Genome condensation was not observed either with increased cation concentrations at normal pH or at high pH without ammonium ions. This finding indicates that the observed genome condensation is a synergistic effect of hydroxyl and ammonium ions involving disruption of protein–RNA interactions that perhaps facilitate further charge neutralization and consequent reduction in the interstrand spacing.

Brain responses associated with consciousness of breathlessness (air hunger)

Little is known about the physiological mechanisms subserving the experience of air hunger and the affective control of breathing in humans. Acute hunger for air after inhalation of CO2 was studied in nine healthy volunteers with positron emission tomography. Subjective breathlessness was manipulated while end-tidal CO2- was held constant. Subjects experienced a significantly greater sense of air hunger breathing through a face mask than through a mouthpiece. The statistical contrast between the two conditions delineated a distributed network of primarily limbic/paralimbic brain regions, including multiple foci in dorsal anterior and middle cingulate gyrus, insula/claustrum, amygdala/periamygdala, lingual and middle temporal gyrus, hypothalamus, pulvinar, and midbrain. This pattern of activations was confirmed by a correlational analysis with breathlessness ratings. The commonality of regions of mesencephalon, diencephalon and limbic/paralimbic areas involved in primal emotions engendered by the basic vegetative systems including hunger for air, thirst, hunger, pain, micturition, and sleep, is discussed with particular reference to the cingulate gyrus. A theory that the phylogenetic origin of consciousness came from primal emotions engendered by immediate threat to the existence of the organism is discussed along with an alternative hypothesis by Edelman that primary awareness emerged with processes of ongoing perceptual categorization giving rise to a scene [Edelman, G. M. (1992) Bright Air, Brilliant Fire (Penguin, London)].

Chains of magnetite crystals in the meteorite ALH84001: Evidence of biological origin

The presence of magnetite crystal chains, considered missing evidence for the biological origin of magnetite in ALH84001 [Thomas-Keprta, K. L., Bazylinski, D. A., Kirschvink, J. L., Clemett, S. J., McKay, D. S., Wentworth, S. J., Vali, H., Gibson, E. K., Jr., & Romanek, C. S. (2000) Geochim. Cosmochim. Acta 64, 4049–4081], is demonstrated by high-power stereo backscattered scanning electron microscopy. Five characteristics of such chains (uniform crystal size and shape within chains, gaps between crystals, orientation of elongated crystals along the chain axis, flexibility of chains, and a halo that is a possible remnant of a membrane around chains), observed or inferred to be present in magnetotactic bacteria but incompatible with a nonbiological origin, are shown to be present. Although it is unlikely that magnetotactic bacteria were ever alive in ALH84001, decomposed remains of such organisms could have been deposited in cracks in the rock while it was still on the surface on Mars.

The Notch ligand Jagged1 is required for inner ear sensory development

Within the mammalian inner ear there are six separate sensory regions that subserve the functions of hearing and balance, although how these sensory regions become specified remains unknown. Each sensory region is populated by two cell types, the mechanosensory hair cell and the supporting cell, which are arranged in a mosaic in which each hair cell is surrounded by supporting cells. The proposed mechanism for creating the sensory mosaic is lateral inhibition mediated by the Notch signaling pathway. However, one of the Notch ligands, Jagged1 (Jag1), does not show an expression pattern wholly consistent with a role in lateral inhibition, as it marks the sensory patches from very early in their development—presumably long before cells make their final fate decisions. It has been proposed that Jag1 has a role in specifying sensory versus nonsensory epithelium within the ear [Adam, J., Myat, A., Roux, I. L., Eddison, M., Henrique, D., Ish-Horowicz, D. & Lewis, J. (1998) Development (Cambridge, U.K.) 125, 4645–4654]. Here we provide experimental evidence that Notch signaling may be involved in specifying sensory regions by showing that a dominant mouse mutant headturner (Htu) contains a missense mutation in the Jag1 gene and displays missing posterior and sometimes anterior ampullae, structures that house the sensory cristae. Htu/+ mutants also demonstrate a significant reduction in the numbers of outer hair cells in the organ of Corti. Because lateral inhibition mediated by Notch predicts that disruptions in this pathway would lead to an increase in hair cells, we believe these data indicate an earlier role for Notch within the inner ear.

14-3-3 protein is a regulator of the mitochondrial and chloroplast ATP synthase

Mitochondrial and chloroplast ATP synthases are key enzymes in plant metabolism, providing cells with ATP, the universal energy currency. ATP synthases use a transmembrane electrochemical proton gradient to drive synthesis of ATP. The enzyme complexes function as miniature rotary engines, ensuring energy coupling with very high efficiency. Although our understanding of the structure and functioning of the synthase has made enormous progress in recent years, our understanding of regulatory mechanisms is still rather preliminary. Here we report a role for 14-3-3 proteins in the regulation of ATP synthases. These 14-3-3 proteins are highly conserved phosphoserine/phosphothreonine-binding proteins that regulate a wide range of enzymes in plants, animals, and yeast. Recently, the presence of 14-3-3 proteins in chloroplasts was illustrated, and we show here that plant mitochondria harbor 14-3-3s within the inner mitochondrial-membrane compartment. There, the 14-3-3 proteins were found to be associated with the ATP synthases, in a phosphorylation-dependent manner, through direct interaction with the F1 β-subunit. The activity of the ATP synthases in both organelles is drastically reduced by recombinant 14-3-3. The rapid reduction in chloroplast ATPase activity during dark adaptation was prevented by a phosphopeptide containing the 14-3-3 interaction motif, demonstrating a role for endogenous 14-3-3 in the down-regulation of the CFoF1 activity. We conclude that regulation of the ATP synthases by 14-3-3 represents a mechanism for plant adaptation to environmental changes such as light/dark transitions, anoxia in roots, and fluctuations in nutrient supply.

Rethinking the role of phosducin: Light-regulated binding of phosducin to 14-3-3 in rod inner segments

Phosducin (Pd), a small protein found abundantly in photoreceptors, is widely assumed to regulate light sensitivity in the rod outer segment through interaction with the heterotrimeric G protein transducin. But, based on histochemistry and Western blot analysis, Pd is found almost entirely in the inner segment in both light and dark, most abundantly near the rod synapse. We report a second small protein, 14-3-3, in the rod with a similar distribution. By immunoprecipitation, phospho-Pd is found to interact with 14-3-3 in material from dark-adapted retina, and this interaction is markedly diminished by light, which dephosphorylates Pd. Conversely, unphosphorylated Pd binds to inner segment G protein(s) in the light. From these results and reported functions of 14-3-3, we have constructed a hypothesis for the regulation of light sensitivity at the level of rod synapse. By dissociating the Pd/14-3-3 complex, light enables both proteins to function in this role.

Rhesus monkeys know when they remember

Humans are consciously aware of some memories and can make verbal reports about these memories. Other memories cannot be brought to consciousness, even though they influence behavior. This conspicuous difference in access to memories is central in taxonomies of human memory systems but has been difficult to document in animal studies, suggesting that some forms of memory may be unique to humans. Here I show that rhesus macaque monkeys can report the presence or absence of memory. Although it is probably impossible to document subjective, conscious properties of memory in nonverbal animals, this result objectively demonstrates an important functional parallel with human conscious memory. Animals able to discern the presence and absence of memory should improve accuracy if allowed to decline memory tests when they have forgotten, and should decline tests most frequently when memory is attenuated experimentally. One of two monkeys examined unequivocally met these criteria under all test conditions, whereas the second monkey met them in all but one case. Probe tests were used to rule out “cueing” by a wide variety of environmental and behavioral stimuli, leaving detection of the absence of memory per se as the most likely mechanism underlying the monkeys' abilities to selectively decline memory tests when they had forgotten.

Planetary exploration in the time of astrobiology: Protecting against biological contamination

These are intriguing times in the exploration of other solar-system bodies. Continuing discoveries about life on Earth and the return of data suggesting the presence of liquid water environments on or under the surfaces of other planets and moons have combined to suggest the significant possibility that extraterrestrial life may exist in this solar system. Similarly, not since the Viking missions of the mid-1970s has there been as great an appreciation for the potential for Earth life to contaminate other worlds. Current plans for the exploration of the solar system include constraints intended to prevent biological contamination from being spread by solar-system exploration missions.

The structures of anthranilate synthase of Serratia marcescens crystallized in the presence of (i) its substrates, chorismate and glutamine, and a product, glutamate, and (ii) its end-product inhibitor, l-tryptophan

The crystal structure of anthranilate synthase (AS) from Serratia marcescens, a mesophilic bacterium, has been solved in the presence of its substrates, chorismate and glutamine, and one product, glutamate, at 1.95 Å, and with its bound feedback inhibitor, tryptophan, at 2.4 Å. In comparison with the AS structure from the hyperthermophile Sulfolobus solfataricus, the S. marcescens structure shows similar subunit structures but a markedly different oligomeric organization. One crystal form of the S. marcescens enzyme displays a bound pyruvate as well as a putative anthranilate (the nitrogen group is ambiguous) in the TrpE subunit. It also confirms the presence of a covalently bound glutamyl thioester intermediate in the TrpG subunit. The tryptophan-bound form reveals that the inhibitor binds at a site distinct from that of the substrate, chorismate. Bound tryptophan appears to prevent chorismate binding by a demonstrable conformational effect, and the structure reveals how occupancy of only one of the two feedback inhibition sites can immobilize the catalytic activity of both TrpE subunits. The presence of effectors in the structure provides a view of the locations of some of the amino acid residues in the active sites. Our findings are discussed in terms of the previously described AS structure of S. solfataricus, mutational data obtained from enteric bacteria, and the enzyme's mechanism of action.

Functional switch between motor tracts in the presence of the mAb IN-1 in the adult rat

Fine finger and hand movements in humans, monkeys, and rats are under the direct control of the corticospinal tract (CST). CST lesions lead to severe, long-term deficits of precision movements. We transected completely both CSTs in adult rats and treated the animals for 2 weeks with an antibody that neutralized the central nervous system neurite growth inhibitory protein Nogo-A (mAb IN-1). Anatomical studies of the rubrospinal tracts showed that the number of collaterals innervating the cervical spinal cord doubled in the mAb IN-1- but not in the control antibody-treated animals. Precision movements of the forelimb and fingers were severely impaired in the controls, but almost completely recovered in the mAb IN-1-treated rats. Low threshold microstimulation of the motor cortex induced a rapid forelimb electromyography response that was mediated by the red nucleus in the mAb IN-1 animals but not in the controls. These findings demonstrate an unexpectedly high capacity of the adult central nervous system motor system to sprout and reorganize in a targeted and functionally meaningful way.