The determination of fresh organic carbon weight from formaldehyde preserved macrofaunal samples

Methodologies are presented whereby the fresh organic carbon weight of formaldehyde preserved macrofaunal samples may be estimated. Length-organic carbon weight regressions were determined for the four numerically dominant bivalves in Narragansett Bay, Rhode Island (Nucula annulata, Yoldia limatula, Mulinia lateralis, and Pandora gouldiana) and one commercially important, but less abundant species (Mercenaria mercenaria). Constants were determined to convert the dry weight of preserved softbodied organisms (polychaetes, oligochaetes, amphipods, etc.) to fresh (unpreserved) organic carbon weight. These results can be used by investigators studying the energetics of benthic communities similar to those in Narragansett Bay.     

Tissue specific expression of p422 protein, a putative lipid carrier, in mouse adipocytes

The differentiation of 3T3-L1 preadipocytes leads to the expression of a new protein, p422, and its mRNA. This protein has 70% and 20-30% amino acid sequence homology to myelin P2 and the fatty acid binding proteins of liver and intestine, respectively. Investigation of the distribution in mouse tissues of p422 protein by immunoblotting and of p422 mRNA by cDNA hybridization indicates that they are expressed only in adipose tissue. Liver and intestinal fatty acid binding protein mRNA's were not detectable in mouse adipose tissue or in 3T3-L1 adipocytes. It is suggested that p422 functions as an adipocyte fatty acid binding protein.     

The genomic organization of dispersed tRNA and 5 S RNA genes in Xenopus laevis

The 5 S DNAs and several tDNAs of Xenopus laevis reside primarily in large clusters of tandem repeating units. We have discovered that a substantial number of these genes, along with portions of their adjacent spacer sequences, are also located in dispersed genomic locations apart from the major clusters. This was accomplished by "null-digesting" total genomic DNA with restriction enzymes that do not cut within the X. laevis tDNA or 5 S DNA major repeats. The tDNA and 5 S DNA main clusters therefore remain intact and can be easily separated on gels from the dispersed tDNAs and 5 S DNAs present as low molecular weight restriction fragments. Probing these smaller fragments with different portions of the major repeats has revealed that many of the dispersed genes are organized differently from the corresponding tDNAs and 5 S DNAs of the primary clusters. Some of the fragments containing dispersed genes are actually present in multiple copies. In addition, many tDNA null-digestion fragments contain more than one type of tRNA coding region. One set of "dispersed" tDNAs actually comprises a tandemly arranged minor tDNA family which has retained the same repeat length (3.18 kb) as the major tDNA family, but has a substantially different organization. There is significant population polymorphism in the organization of the dispersed tDNAs and 5 S DNAs. Dispersed genes that appear to be derived from clusters of tandem repeats ("orphons") have been described for several gene families in invertebrates. The occurrence of this phenomenon in vertebrates as well, suggests that such dispersed genes may be a general feature of all eukaryotic genomes.     

The in vitro cell fusion of embryonic chick muscle without DNA synthesis

A system has been developed for the in vitro development of chick skeletal muscle monolayers, in which a burst of synchronous fusion occurs, such that some 40% of the spindle-shaped cells fuse in a 10-hr period. Cells inhibited from synthesizing DNA by ara-C do fuse, but at a later time than the normal burst. If ara-C is added to cultures 6 hr or more before the normal fusion time, fusion is delayed, but no delay results when the drug is added after this time. A medium change will delay the fusion if done 4 hr or more before fusion, but gives no delay if done later. Cells grown in conditioned medium fuse some 10 hr earlier than controls, even in the presence of ara-C, as do cultures prepared at higher than normal cell densities. The data suggest that muscle cell fusion is independent of DNA synthesis in vitro, but depends upon a modification of the culture medium to a sufficient degree required for initiating the synthetic program for fusion.     

Morphogenetic studies of a genetically controlled tumor-like condition in Lycopersicon hybrids

Summary A dominant tumor-like condition recently isolated in a tomato hybrid is described from the viewpoint of morphogenesis. Tumors, consisting of masses of enlarged parenchymatous cells, generally appear on the ventral surface of the third leaf and the subsequently formed leaves of the hybrid derivatives. These outgrowths do not differentiate into teratomas. Abortive floral buds develop under greenhouse conditions and the tumorous plants are much dwarfed compared to the normal segregants of the same population. the same tumor genotype behaves differently under the field conditions: it grows and blossoms like the normal plants, setting fruits with viable seeds, and tumors fail to develop. Thus, tumor expression and general morphology of the tumor plants are greatly modified by environmental conditions.Tissue culture studies employing a variety of media have shown that tissues excised from tumor-producing plants are not autonomous with respect to growth hormones, nor are tissues from the non-tumorous segregants. Nevertheless, tissues from tumor and non-tumor genotypes show different growth requirements. Tumor and non-tumor tomatoes can thus be distinguished on the basis of in vitro growth responses, a result consistent with their different genetic constitution. Differentiation of buds or roots was not observed in either type of tissue.Abbreviations used GA gibberellic acid - IAA 3-indoleacetic acid - 2.4-D 2.4-dichlorophenoxyacetic acid Work Supported by National Science Foundation Grant GB-3198 and funds from The Cairncrest Foundation.     

The pathogenic fungus Cryptococcus neoformans expresses two functional GDP-mannose transporters with distinct expression patterns and roles in capsule synthesis

Cryptococcus neoformans is a fungal pathogen that is responsible for life-threatening disease, particularly in the context of compromised immunity. This organism makes extensive use of mannose in constructing its cell wall, glycoproteins, and glycolipids. Mannose also comprises up to two-thirds of the main cryptococcal virulence factor, a polysaccharide capsule that surrounds the cell. The glycosyltransfer reactions that generate cellular carbohydrate structures usually require activated donors such as nucleotide sugars. GDP-mannose, the mannose donor, is produced in the cytosol by the sequential actions of phosphomannose isomerase, phosphomannomutase, and GDP-mannose pyrophosphorylase. However, most mannose-containing glycoconjugates are synthesized within intracellular organelles. This topological separation necessitates a specific transport mechanism to move this key precursor across biological membranes to the appropriate site for biosynthetic reactions. We have discovered two GDP-mannose transporters in C. neoformans, in contrast to the single such protein reported previously for other fungi. Biochemical studies of each protein expressed in Saccharomyces cerevisiae show that both are functional, with similar kinetics and substrate specificities. Microarray experiments indicate that the two proteins Gmt1 and Gmt2 are transcribed with distinct patterns of expression in response to variations in growth conditions. Additionally, deletion of the GMT1 gene yields cells with small capsules and a defect in capsule induction, while deletion of GMT2 does not alter the capsule. We suggest that C. neoformans produces two GDP-mannose transporters to satisfy its enormous need for mannose utilization in glycan synthesis. Furthermore, we propose that the two proteins have distinct biological roles. This is supported by the different expression patterns of GMT1 and GMT2 in response to environmental stimuli and the dissimilar phenotypes that result when each gene is deleted.     

Residue Gly1326 of the N-type calcium channel alpha 1B subunit controls reversibility of omega-conotoxin GVIA and MVIIA block

We recently reported that amino acid residues contained within a putative EF hand motif in the domain III S5-H5 region of the alpha(1B) subunit affected the relative barium:calcium permeability of N-type calcium channels (Feng, Z. P., Hamid, J., Doering, C., Jarvis, S. E., Bosey, G. M., Bourinet, E., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 5726-5730). Since this region partially overlaps with residues previously implicated in block of the channel by omega-conotoxin GVIA, we assessed the effects of mutations in the putative EF hand domain on channel block by omega-conotoxin GVIA and the structurally related omega-conotoxin MVIIA. Both of the toxins irreversibly block the activity of wild type alpha(1B) N-type channels. We find that in addition to previously identified amino acid residues, residues in positions 1326 and 1332 are important determinants of omega-conotoxin GVIA blockade. Substitution of residue Glu(1332) to arginine slows the time course of development of block. Point mutations in position Gly(1326) to either arginine, glutamic acid, or proline dramatically decrease the time constant for development of the block. Additionally, in the G1326P mutant channel activity was almost completely recovered following washout. A qualitatively similar result was obtained with omega-conotoxin MVIIA, suggesting that common molecular determinants underlie block by these two toxins. Taken together the data suggest that residue Gly(1326) may form a barrier, which controls the access of peptide toxins to their blocking site within the outer vestibule of the channel pore and also stabilizes the toxin-channel interaction.     

EGF-like domain calcium affinity modulated by N-terminal domain linkage in human fibrillin-1

Calcium binding epidermal growth factor-like domains (cbEGFs) are present in many extracellular proteins, including fibrillin-1, Notch-3, protein S, factor IX and the low density lipoprotein (LDL) receptor, which perform a diverse range of functions. Genetic mutations that cause amino acid changes within these proteins have been linked to the Marfan syndrome (MFS), CADASIL, protein S deficiency, haemophilia B and familial hypercholesterolaemia, respectively. A number of these mutations disrupt calcium binding to cbEGFs, emphasising the critical functional role of calcium in these proteins.We have determined the calcium binding affinity of two sites within a cbEGF pair (cbEGF12-13) from human fibrillin-1 using two-dimensional nuclear magnetic resonance (NMR) and fluorescence techniques. Fibrillin-1 is a mosaic protein containing 43 cbEGF domains, mainly arranged as tandem repeats. Our results show that the cbEGF13 site in the cbEGF12-13 pair possesses the highest calcium affinity of any cbEGF investigated from fibrillin-1. A comparative analysis of these and previously reported calcium binding data from fibrillin-1 demonstrate that the affinity of cbEGF13 is enhanced more than 70-fold by the linkage of an N-terminal cbEGF domain. In contrast, comparison of calcium binding by cbEGF32 in isolation relative to when linked to a transforming growth factor beta-binding protein-like domain (TB6-cbEGF32) reveals that the same enhancement is not observed for this heterologous domain pair. Taken together, these results indicate that fibrillin-1 cbEGF Ca2+ affinity can be significantly modulated by the type of domain which is linked to its N terminus. The cbEGF12-13 pair is located within the longest contiguous section of cbEGFs in fibrillin-1, and a number of mutations in this region are associated with the most severe neonatal form of MFS. The affinities of cbEGF domains 13 and 14 in this region are substantially higher than in the C-terminal region of fibrillin-1. This increased affinity may be important for fibrillin assembly into 10-12 nm connective tissue microfibrils and/or may contribute to the biomechanical properties of the microfibrillar network.