Identification of minor fimbrial subunits involved in biosynthesis of K88 fimbriae.

The nucleotide sequences of the genes faeF, faeH, faeI, and faeJ encoding K88 minor fimbrial subunits were determined. Analysis of the primary structure of the gene products revealed that all four proteins are synthesized with an amino-terminal signal sequence. The molecular masses of the mature FaeF, FaeH, FaeI, and FaeJ proteins were calculated to be 15,161, 25,461, 24,804, and 25,093 Da, respectively. FaeH, FaeI, and FaeJ showed significant homology with FaeG, the major fimbrial subunit of K88 fimbriae. Mutations in the respective genes were constructed. Analysis of the mutants showed that the minor fimbrial subunits FaeF and FaeH play an essential role in the biogenesis but not in the adhesive properties of the K88 fimbriae. Mutations in faeI or faeJ had no significant effect on K88 production or adhesive capacity. Specific antisera against FaeF and FaeH were raised by immunization with hybrid Cro-LacZ-FaeF and Cro-LacZ-FaeH proteins. Immunoblotting and immunoelectron microscopy revealed that FaeF and FaeH are located in or along the K88 fimbrial structure.     

The linkage of Hb Valletta [α2β287(F3)Thr→Pro] and Hb F-Malta-I [α2 Gγ2117(G19)His→Arg] in the Maltese population

Summary We have identified a new stable abnormal hemoglobin called Hb Valletta, which is characterized by a ThrPro substitution at position 87 of the chain. This mutation was found to be linked to that of the chain variant Hb F-Malta-I with a HisArg mutation at position 117 of the ^G chain. Both variants were detected in the blood samples of 34 Maltese and two Italian new-born babies with isoelectrofocusing and reversed phase high performance liquid chromatography. Similar analyses of cord blood from 388 additional Maltese newborns failed to identify either one of these two variants. Additional analyses of 353 Maltese adults (including 39 -thalassemia heterozygotes) resulted in the detection of two adult Hb Valletta heterozygotes. Dot-blot hybridization analyses of amplified DNA with a probe specific for the ^G-F-Malta-I variant showed that both also carried that mutation. These results show close linkage of the mutant forms of the ^G- and -globin genes, 27–28 kb apart, and a failure to identify chromosomes with either the Hb F-Malta-I mutation alone or with the Hb Valletta mutation alone, indicating a low recombination frequency.     

Evidence for a role of virulent human immunodeficiency virus (HIV) variants in the pathogenesis of acquired immunodeficiency syndrome: studies on sequential HIV isolates.

Sequential human immunodeficiency virus (HIV) isolates, recovered from a panel of longitudinally collected peripheral blood mononuclear cells obtained from 20 initially asymptomatic HIV-seropositive homosexual men, were studied for differences in replication rate, syncytium-inducing capacity, and host range. Eleven individuals remained asymptomatic; nine progressed to acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC) at the time point at which the last HIV isolate was obtained. In 16 individuals, only non-syncytium-inducing (NSI) isolates, with a host range restricted to mononuclear cells, were observed. From four individuals, high-replicating, syncytium-inducing (SI) isolates that could be transmitted to the H9, RC2A, and U937 cell lines were recovered. From two of these four individuals, SI isolates were obtained throughout the observation period. In the two others, a transition from NSI to SI HIV isolates was observed during the period of study. Three of these four individuals developed ARC or AIDS 9 to 15 months after the first isolation of an SI isolate. With the exception of the two individuals in whom a transition from NSI to SI isolates was observed, within a given individual the replication rate of sequential HIV isolates was constant. A significant correlation was found between the mean replication rate of isolates obtained from an individual and the rate of CD4+ cell decrease observed in this individual. In individuals with low-replicating HIV isolates, no significant CD4+ cell loss was observed. In contrast, recovery of high-replicating isolates, in particular when these were SI isolates, was associated with rapid decline of CD4+ cell numbers and development of ARC or AIDS. These findings indicate that variability in the biological properties of HIV isolates is one of the factors influencing the course of HIV infection.     

Evidence for subsites in the galectins involved in sugar binding at the nonreducing end of the central galactose of oligosaccharide ligands: sequence analysis, homology modeling and mutagenesis studies of hamster galectin-3

A model of the carbohydrate recognition domain CRD, residues 111-245, of hamster galectin-3 has been made using homology modeling and dynamics minimization methods. The model is based on the known x-ray structures of bovine galectin-1 and human galectin-2. The oligosaccharides NeuNAc-alpha2,3-Gal-beta1,4-Glc and GalNAc-alpha1, 3- [Fuc-alpha1,2]-Gal-beta1,4-Glc, known to be specific high-affinity ligands for galectin-3, as well as lactose recognized by all galectins were docked in the galectin-3 CRD model structure and a minimized binding conformation found in each case. These studies indicate a putative extended carbohydrate-binding subsite in the hamster galectin- 3 involving Arg139, Glu230, and Ser232 for NeuNAc-alpha2,3-; Arg139 and Glu160 for fucose-alpha1,2-; and Arg139 and Ile141 for GalNAc-alpha1,3- substituents on the primary galactose. Each of these positions is variable within the whole galectin family. Two of these residues, Arg139 and Ser232, were selected for mutagenesis to probe their importance in this newly identified putative subsite. Residue 139 adopts main-chain dihedral angles characteristic of an isolated bridge structural feature, while residue 232 is the C-terminal residue of beta- strand-11, and is followed immediately by an inverse gamma-turn. A systematic series of mutant proteins have been prepared to represent the residue variation present in the aligned sequences of galectins-1, - 2, and -3. Minimized docked models were generated for each mutant in complex with NeuNAc-alpha2,3-Gal-beta1,4-Glc, GalNAc-alpha1, 3-[Fuc- alpha1,2]-Gal-beta1,4- Glc, and Gal-beta1,4-Glc. Correlation of the computed protein-carbohydrate interaction energies for each lectin- oligosaccharide pair with the experimentally determined binding affinities for fetuin and asialofetuin or the relative potencies of lactose and sialyllactose in inhibiting binding to asiolofetuin is consistent with the postulated key importance of Arg139 in recognition of the extended sialylated ligand.      

Competition and facilitation in multispecies plant-herbivore systems of productive environments

We develop a multispecies plant-herbivore model to explore how plant competition for light and the selectivity of herbivores affect abundance patterns of plants and herbivores along productivity gradients. The model considers a small and a tall plant species, a generalist herbivore, and a selective herbivore. The selective herbivore feeds only on the small plant species. In the absence of the generalist herbivore, the tall plant species becomes increasingly dominant with increasing productivity, and the small plant and its selective herbivore disappear. The model shows that generalist herbivores can facilitate selective herbivores by suppressing competition for light. This favours the small plant species, and thereby the selective herbivores. The model predictions are qualitatively consistent with field studies of multispecies plant-herbivore systems.     

RECRUITMENT OF BENTHIC MICROCYSTIS (CYANOPHYCEAE) TO THE WATER COLUMN: INTERNAL BUOYANCY CHANGES OR RESUSPENSION?1

In some lakes, large amounts of the potentially toxic cyanobacterium Microcystis overwinter in the sediment. This overwintering population might inoculate the water column in spring and promote the development of dense surface blooms of Microcystis during summer. In the Dutch Lake Volkerak, we found photochemically active Microcystis colonies in the sediment throughout the year. The most vital colonies originated from shallow sediments within the euphotic zone. We investigated whether recruitment of Microcystis colonies from the sediment to the water column was an active process, through production of gas vesicles or respiration of carbohydrate ballast. We calculated net buoyancy, as an indication of relative density, using the amounts and densities of the major cell constituents (carbohydrates, proteins, and gas vesicles). Carbohydrate content of benthic Microcystis cells was very low throughout the year. Buoyancy changes of benthic Microcystis were mostly a result of changes in gas vesicle volume. Before the summer bloom, net buoyancy and the amount of buoyant colonies in the sediment did not change. Therefore, recruitment of Microcystis from the sediment does not seem to be an active process regulated by internal buoyancy changes. Instead, our observations indicate that attachment of sediment particles to colonies plays an important part in the buoyancy state of benthic colonies. Therefore, we suggest that recruitment of Microcystis is more likely a passive process resulting from resuspension by wind‐induced mixing or bioturbation. Consequently, shallow areas of the lake probably play a more important role in recruitment of benthic Microcystis than deep areas.     

Nucleotide sequence analysis of the human salivary protein genes HIS1 and HIS2, and evolution of the STATH/HIS gene family

Human histatins are a family of low-M(r), neutral to very basic, histidine-rich salivary polypeptides. They probably function as part of the nonimmune host defense system in the oral cavity. A 39-kb region of DNA containing the HIS1 and HIS2 genes was isolated from two human genomic phage libraries as a series of overlapping clones. The nucleotide sequences of the HIS1 gene and part of the HIS2(1) gene were determined. The transcribed region of HIS1 spans 8.5 kb and contains six exons and five introns. The HIS1 and HIS2(1) genes exhibit 89% overall sequence identity, with exon sequences exhibiting 95% identity. The two loci probably arose by a gene duplication event approximately 15-30 Mya. The HIS1 sequence data were also compared with that of STATH. Human statherin is a low-M(r) acidic phosphoprotein that acts as an inhibitor of precipitation of calcium phosphate salts in the oral cavity. The HIS1 and STATH genes show nearly identical overall gene structures. The HIS1 and STATH loci exhibit 77%-81% sequence identity in intron DNA and 80%-88% sequence identity in noncoding exons but only 38%-43% sequence identity in the protein-coding regions of exons 4 and 5. These unusual data suggest that HIS1, HIS2, and STATH belong to a single gene family exhibiting accelerated evolution between the HIS and STATH coding sequences.