Fine‐scale Population Genetic Structure of Two Dioecious Indian Keystone Species,Ficus hispida and Ficus exasperata (Moraceae)

Although Ficus (Moraceae) is a keystone plant genus in the tropics, providing resources to many frugivorous vertebrates, its population genetic structure, which is an important determinant of its long‐term survival, has rarely been investigated. We examined the population genetic structure of two dioecious fig species (Ficus hispida and Ficus exasperata) in the Indian Western Ghats using co‐dominant nuclear microsatellite markers. We found high levels of microsatellite genetic diversity in both species. The regression slopes between genetic relationship coefficients (f_ij) and spatial distances were significantly negative in both species indicating that, on average, individuals in close spatial proximity were more likely to be related than individuals further apart. Mean parent–offspring distance (σ) calculated using these slopes was about 200 m in both species. This should be contrasted with the very long pollen dispersal distances documented for monoecious Ficus species. Nevertheless, overall population genetic diversity remained large suggesting immigrant gene flow. Further studies will be required to analyze broader scale patterns.     

Site-directed mutagenesis to determine essential residues of ribulose-bisphosphate carboxylase ofRhodospirillum rubrum

Both Lys-166 and His-291 of ribulosebisphosphate carboxylase/oxygenase fromRhodospirillum rubrum have been implicated as the active-site residue that initiates catalysis. To decide between these two candidates, we resorted to site-directed mutagenesis to replace Lys-166 and His-291 with several amino acids. All 7 of the position-166 mutants tested are severely deficient in carboxylase activity, whereas the alanine and serine mutants at position 291 are ∼40% and ∼18% as active as the native carboxylase, essentially ruling out His-291 in theRhodospirillum rubrum carboxylase (and by inference His-298 in the spinach enzyme) as a catalytically essential residue. The ability of some of the mutant proteins to undergo carbamate formation or to bind either ribulosebisphosphate or a transition-state analogue remains largely unimpaired. This implies that Lys-166 is not required for substrate binding; rather, the results corroborate the earlier postulate that Lys-166 functions as an acid-base group in catalysis or in stabilizing a transition state in the reaction pathway.     

Aromaticity at position 37 in human epidermal growth factor is not obligatory for activity.

The third disulfide loop (amino acids 33 to 42) of human epidermal growth factor (hEGF) encompasses the region of highest amino acid conservation among all of the EGF-like family of molecules. The importance of some of these highly conserved residues for the maintenance of biological activity, especially the aromatic amino acid tyrosine at position 37, has until now been considered essential on the basis of previous studies with the EGF-like molecule transforming growth factor alpha. Variants at the Tyr-37 position of hEGF were constructed by site-directed mutagenesis. The substituting amino acids were phenylalanine, histidine, serine, alanine, aspartic acid, arginine, and glycine. The variants were tested for their ability to competitively displace native [125I]hEGF from its receptor and to stimulate the protein-tyrosine kinase activity of the receptor; the order of efficacy of substituting amino acids was Phe greater than His greater than Ser greater than Ala greater than Asp greater than Arg greater than Gly in both assays. All were effective, with no or only moderate reduction in potency, in stimulating the incorporation of [3H]thymidine into acid-insoluble material of quiescent mouse A31 cells. Only Tyr-37----Ala, Tyr-37----Arg and Tyr-37----Gly were slightly less potent in the cell assay. Thus, neither tyrosine nor another aromatic amino acid at position 37 in hEGF is essential for full biological activity.     

Looking for probes of gated channels: studies of the inhibition of glucose and choline transport in erythrocytes

Two seemingly contradictory sets of observations have been made in studies of biological transport, which are essential for our understanding of the transport mechanism: carriers are integral membrane proteins, which span the membrane and are not free to rotate across the membrane; carriers appear to function like a ferryboat, with a substrate binding site moving back and forth from one side of the membrane to the other. To reconcile these facts, it is necessary to postulated gated channels connecting the substrate site with the two membrane surfaces: the channels are arranged so that as one opens the other closes, with the result that the substrate site is alternately accessible from opposite sides of the membrane. Based on these properties, the following distinguishing features of molecules specifically bound in the channels may be predicted: if sufficiently bulky, they inhibit transport; they bind outside the substrate site (though adjacent to it), they bind asymmetrically either to the outward-facing carrier and on the outer surface of the membrane, or to the inward-facing carrier and on the inner surface of the membrane. The asymmetrical inhibition of the glucose and choline transport systems of erythrocytes by various inhibitors is examined, and the behavior in every case is found to conform with these criteria. From the results it may be concluded that the glucose carrier binds cytochalasin B in the inner gated channel and phloretin and tetrathionate in the outer gated channel.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytological Detection of the c25H Deletion Involving the Albino (c) Locus on Chromosome 7 in the Mouse

A deletion of the albino (c) locus on mouse chromosome 7 has been demonstrated using Q- and G-banding methods in a mouse heterozygous for the radiation-induced lethal albino allele, c(25H). The deletion, which is thought to be 1-6 cM long, represents about 7.6% of the length of the metaphase chromosome.     

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.