Functional reconstitutional of the human epidermal growth factor receptor system in Xenopus oocytes

We have expressed the human EGF receptor (hEGF-R) in Xenopus oocytes by injecting mRNA synthesized in vitro using SP6 vectors containing receptor cDNAs. Each oocyte could express over 1 x 10(10) receptors of a single affinity class and these were able to bind and rapidly internalize EGF. Occupancy resulted in receptor tyrosine autophosphorylation, downregulation, and release of intracellular calcium. Occupied receptors also rapidly induced meiotic maturation in stage VI oocytes. Receptors lacking tyrosine kinase activity bound EGF normally, but did not downregulate or induce any biological responses. The rate of oocyte maturation was proportional to hEGF-R occupancy and was significantly faster than progesterone-induced maturation at nanomolar EGF concentrations. Mutant hEGF-R truncated at residue 973 displayed identical phenotypes in both mammalian cells and oocytes in that they were defective in their ability to release intracellular calcium, undergo ligand induced internalization and receptor downregulation. However, these receptors were fully capable of inducing oocyte maturation. The remarkable retention of specific biological activities of different hEGF-R in the context of oocytes suggests that this receptor system interacts with generally available cellular components that have been conserved during evolution. In addition, it suggests that cell surface tyrosine kinase activity may play an important role in regulating resumption of the cell cycle.     

Peptide binding to HLA-DR1: a peptide with most residues substituted to alanine retains MHC binding

Major histocompatibility complex (MHC) glycoproteins play an important role in the development of an effective immune response. An important MHC function is the ability to bind and present 'processed antigens' (peptides) to T cells. We show here that the purified human class II MHC molecule, HLA-DR1, binds peptides that have been shown to be immunogenic in vivo. Detergent-solubilized HLA-DR1 and a papain-cleaved form of the protein lacking the transmembrane and intracellular regions have similar peptide binding properties. A total of 39 single substitutions were made throughout an HLA-DR1 restricted hemagglutinin epitope and the results determine one amino acid in this peptide which is crucial to binding. Based on this analysis, a synthetic peptide was designed containing two residues from the original hemagglutinin epitope embedded in a chain of polyalanine. This peptide binds to HLA-DR1, indicating that the majority of peptide side chains are not required for high affinity peptide binding.     

PERSPECTIVE: INDIRECT MATE CHOICE,COMPETITION FOR MATES,AND COEVOLUTION OF THE SEXES

When Darwin first proposed the possibility of sexual selection, he identified two mechanisms, male competition for mates and female choice of mates. Extending this classification, we distinguish two forms of mate choice, direct and indirect. This distinction clarifies the relationship between Darwin's two mechanisms and, furthermore, indicates that the potential scope for sexual selection is much wider than thus far realized. Direct mate choice, the focus of most research on sexual selection in recent decades, requires discrimination between attributes of individuals of the opposite sex. Indirect mate choice includes all other behavior or morphology that restricts an individual's set of potential mates. Possibilities for indirect mate choice include advertisement of fertility or copulation, evasive behavior, aggregation or synchronization with other individuals of the same sex, and preferences for mating in particular locations. In each of these cases, indirect mate choice sets the conditions for competition among individuals of the opposite sex and increases the chances of mating with a successful competitor. Like direct mate choice, indirect mate choice produces assortative mating. As a consequence, the genetic correlation between alleles affecting indirect choice and those affecting success in competition for mates can produce self-accelerating evolution of these complementary features of the sexes. The broad possibilities for indirect mate choice indicate that sexual selection has more pervasive influences on the coevolution of male and female characteristics than previously realized.     

Engineering dynamics of growth factors and other therapeutic ligands

Peptide growth factors and other receptor-binding cytokine ligands are of interest in contemporary molecular health care approaches in applications such as wound healing, tissue regeneration, and gene therapy. Development of effective technologies based on operation of these regulatory molecules requires an ability to deliver the ligands to target cells in a reliable and well-characterizable manner. Quantitative information concerning the fate of peptide ligands within tissues is necessary for adequate interpretation of experimental observations at the tissue level and for truly rational engineering design of ligand-based therapies. To address this need, we are undertaking efforts to elucidate effects of key molecular and cellular parameters on temporal and spatial distribution of cytokines in cell population and cell/matrix systems. In this article we summarize some of our recent findings on dynamics of growth factor depletion by cellular endocytic trafficking, growth factor transport through cellular matrices, and growth factor production and release by autocrine cell systems. (c) 1996 John Wiley & Sons, Inc.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Mechanisms of immune activation of human immunodeficiency virus in monocytes/macrophages.

Monocytes/macrophages (M/M) are the major host of human immunodeficiency virus (HIV) in solid tissues. However, blood monocytes are nonpermissive for HIV infection, indicating that M/M activation or differentiation is necessary for HIV replication. Since M/M are activated during immune responses, we investigated the effect of T-cell activation on HIV expression in M/M derived from peripheral blood of HIV-infected individuals. Previously, we reported that coculture of monocytes from HIV-infected donors with T cells and mitogens resulted in M/M differentiation and HIV expression. Production of HIV by M/M from infected donors required direct contact between monocytes and T cells (for the first 24 h), and the response to alloantigens, but not mitogens, was restricted to HLA-DR. In this study, we found that HIV was more readily recovered from M/M of asymptomatic HIV seropositive donors (69%) than from M/M of symptomatic donors (57%). Viral antigens (e.g., inactivated herpes simplex virus) could initiate the immune response and HIV expression. The ability of noninfected T cells to activate HIV expression in M/M and observations that treatments of M/M with antibodies to deplete T cells did not reduce HIV expression suggested that the monocytes were endogenously infected. To define the aspects of immune activation specifically involved in initiating HIV expression in M/M, interactions of M/M and T cells and participation of cytokines were investigated. The T cell which activated M/M was CD4+ CD8-. Fixed allogeneic cells are known to induce T-cell activation but were not able to serve as antigen for M/M differentiation, suggesting that M/M may need to function as antigen-presenting cells to receive the signal to differentiate and express HIV. Blocking of M/M-T-cell interaction with antibodies directed against LFA-1 or interleukin-1 prevented HIV expression. However, inhibition of later stages of T-cell activation, such as blocking of interleukin-2 receptors, did not diminish HIV expression in M/M. Consistent with the requirement for cell-cell contact between M/M and T cells, a variety of cytokines were unable to initiate HIV replication in M/M. The ability of T cells to induce cellular differentiation and HIV replication in M/M in vitro suggests that initiation of an immune response to an antigen, such as an opportunistic pathogen, could be a mechanism by which HIV disseminates to tissues in vivo.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

d-Alanine metabolism in the lucinid clam Lucinoma aequizonata

The chemoautotrophic symbiont-bearing clam Lucinoma aequizonata contains very high levels of free d-alanine in all tissues. The possible sources for this amino acid and its involvement in the clams' metabolism were investigated. Very low levels of d-alanine (generally below 1 mol·l^-1) were measured in the sediment porewaters from the habitat of the clams. Experiments with ¹⁴C-labeled tracers demonstrate an active metabolism of d-alanine in the clams rather than a role as inert waste product. d-alanine is metabolized at about 0.12 mol·g fw^-1·h^-1. Label from aspartate, but not glucose and CO₂, is incorporated into d-alanine. Incubation with labeled d-alanine did not result in formation of radioactive l-alanine. Tests for alanine racemase (EC 5.1.1.1) and d-amino acid oxidase (EC 1.4.3.3.) did not show activity in either gill, i.e. symbiont and host, or foot tissue. d-Alanine amino transferase (EC 2.6.1.b.) was demonstrated in gill and foot tissues. Two sources for d-alanine are proposed: a degradation of cell walls of symbiotic bacteria and production by the host using a d-specific alanine transaminase.Abbreviations aa amino acid(s) - fw fresh weight - HPLC high-performance liquid chromatography - MBH methyl benzethonium hydroxyde - NAC N-acetyl-l-cysteine - OPA ortho-phthaldialdehyde - TCA tricarbonic acid     

Somatostatin inhibits adrenergic and cholinergic neurotransmission in smooth muscle.

Somatostatin reduced the response to field stimulation in the guinea pig ileum and reduced the spontaneous contractions in the rabbit jejunum, an effect that was blocked by tetrodotoxin. Somatostatin also inhibited field stimulated alpha adrenergic contractions in the rat vas deferens and rabbit ear artery. However, the responses to direct application of either acetylcholine in the ileum or to norepinephrine in the ear artery or vas deferens were not affected by somatostatin. These results strongly suggest that somatostatin inhibits neuronal release of cholinergic and adrenergic transmitter substances in smooth muscle.