Polarity of transport of 2-deoxy-D-glucose and D-glucose by cultured renal epithelia (LLC-PK1).

At least two types of glucose transporter exist in cultured renal epithelial cells, a Na(+)-glucose cotransporter (SGLT), capable of interacting with D-glucose but not 2-deoxy-D-glucose (2dglc) and a facilitated transporter (GLUT) capable of interacting with both D-glucose and 2dglc. In order to examine the polarity of transport in cultured renal epithelia, 2dglc and D-glucose uptakes were measured in confluent cultures of LLC-PK1 cells grown on collagen-coated filters that permitted access of medium to both sides of the monolayer. The rates of basolateral uptake of both 1 mM glucose (Km 3.6 mM) and 1 mM 2dglc (Km 1.5 mM) were greater than apical uptake rates and the (apical-to-basolateral)/(basolateral-to-apical) flux ratio was high for glucose (9.4) and low for 2dglc (0.8), thus, confirming the lack of interaction of 2dglc with the apical SGLT. Specific glucose transport inhibitor studies using phlorizin, phloretin and cytochalasin B confirmed the polarised distribution of SGLT and GLUT in LLC-PK1 cells. Basolateral sugar uptake could be altered by addition of insulin (1 mU/ml) which increased 2dglc uptake by 72% and glucose uptake by 50% and by addition of 20 mM glucose to the medium during cell culture which decreased 2dglc uptake capacity at confluence by 30%. During growth to confluence, 2dglc uptake increased to a maximum, then decreased at the time of confluence, coincident with a rise in uptake capacity for alpha-methyl-D-glucoside, a hexose that interacts only with the apical SGLT. It was concluded that the non-metabolisable sugar 2dglc was a useful, specific probe for GLUT in LLC-PK1 cells and that GLUT was localised at the basolateral membrane after confluence.     

Leghemoglobin-like sequences in the DNA of four actinorhizal plants

Summary A cloned cDNA partial copy of a soybean leghemoglobin mRNA was used to probe genomic DNA of four species of actinorhizal plants. Southern blot hybridization revealed the presence of sequences with homology to the leghemoglobin probe in DNA from Alnus glutinosa, Casuarina glauca, Ceanothus americanus and Elaeagnus pungens. The hybridization patterns of the restriction fragments revealed some fragment size conservation between the DNA of soybean and the DNA of four actinorhizal plants which are taxonomically unrelated to soybean or to each other. The results presented here indicate that globin gene sequences are much more widely distributed in the plant kingdom than has previously been thought. Furthermore, if sequence conservation is actually as high as the restriction fragment patterns suggest, the evolution of the DNA surrounding the globin sequences has been highly constrained.     

Molecular analysis of actinorhizal symbiotic systems: Progress to date

The application of molecular tools to questions related to the genetics, ecology and evolution of actinorhizal symbiotic systems has been especially fruitful during the past two years. Host plant phylogenies based on molecular data have revealed markedly different relationships among host plants than have previously been suspected and have contributed to the development of new hypotheses on the origin and evolution of actinorhizal symbiotic systems. Molecular analyses of host plant gene expression in developing nodules have confirmed the occurrence of nodulin proteins and in situ hybridization techniques have been successfully adapted to permit the study of the spatial and temporal patterns of gene expression within actinorhizal nodules. The use of heterologous probes in combination with nucleotide sequence analysis have allowed a number of nif genes to be mapped on the Frankia chromosome which will ultimately contribute to the development of hypotheses related to nif gene regulation in Frankia. The use of both 16S and 23S rDNA nucleotide sequences has allowed the construction of phylogenetic trees that can be tested for congruence with symbiotic characters. In addition the development of Frankia-specific gene probes and amplification primers have contributed to studies on the genetic diversity and distribution of Frankia in the soil.     

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.      

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.      

Bioaccumulation and depuration of enteroviruses by the soft-shelled clam, Mya arenaria.

Low levels of feces-associated natural virus, simulating virus numbers estimated to exist in moderately polluted shellfish-growing waters, were used to evaluate the effectiveness of depuration as a virus depletion procedure in soft-shell clams. Depuration effectiveness depended upon the numbers of virus bioaccumulated and whether virus was solids associated. Virus uptake was greatest when viruses were solids associated and pollution levels were equivalent or greater than those likely to be found in grossly polluted growing waters. Virtually all bioaccumulated feces-associated natural virus was deposited within either the hepatopancreas or siphon tissues. Viruses usually were eliminated within a 24- to 48-h depuration period. Dependence upon depuration of clams to elimate health hazards of virus etiology involved a risk factor not measureable in the study. The greatest reduction of health risks would come from the routine depuration of clams harvested from growing waters of good sanitary quality.     

In situ measurement of filtering rates of the salp, Thalia democratica, on phytoplankton and bacteria

Salps were gently captured in a perspex chamber by a SCUBA diver,who then injected a suspension of ¹⁴C-labeled phytoplanktonand ³H-labeled bacteria into the chamber. After 0.5 h incubationin situ, filtering rates were estimated from incorporation ofthe two isotopes, and expressed as a function of each salp'ssize. Both bacteria and phytoplankton were grazed by Thalia,the latter at higher rates. Weight-specific grazing rates increasedwith increasing size of salp. Estimated hourly rations rangedfrom 1% of bodily C for small Thalia feeding only on phytoplanktonto 8% of bodily C for large Thalia feeding on all particulateorganic C. The method gave repeatable results for Thalia, butwas unsatisfactory for the pteropod, Cavolinia. ¹Present address: Institute of Marine Resources, A-018, Universityof California, San Diego, Scripps Institution of Oceanography,La Jolla, CA 92093, USA.     

Isolation and characterization of beta- and gamma-caseins from horse milk.

Three groups of casein components were isolated from horse milk. Group I is almost insoluble at acid and neutral pH, and is rather heterogeneous on alkaline gels with or without sodium dodecyl sulphate. Group II shows strong similarity to beta-casein from other species, as concluded from its amino acid composition and its N- and C-terminal sequences. This group consists of five electrophoretically distinguishable forms, all containing ester phosphate groups but no carbohydrate. Group III is composed of C-terminal fragments of the beta-like (group II) fraction and probably arises from the action of a plasmin-like enzyme present in horse milk. It does not contain phosphate or carbohydrate. Homology of this group with bovine gamma-caseins is demonstrated. Both beta- and gamma-like caseins are more soluble at 4 degrees C than at room temperature.