Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

Influence of Deep Ocean Sewage Outfalls on the Microbial Activity of the Surrounding Sediment

The microbial activity near two deep ocean sewage outfalls off the coast of the island of Oahu, Hawaii, was characterized. Water samples and sediment samples to a depth of 4.5 cm were analyzed from an area of approximately 4.5 × 10⁴ m² surrounding the outfalls. Although the effluent water at both sites exhibited heterotrophic activity that was 2 orders of magnitude greater than water from a control site, ambient water samples taken within 1 m of the discharge ports exhibited activity only twice that of the control water. The heterotrophic activity of the outfall sediment was only elevated above that of the control site for surface samples collected within 10 m of the outfall. Likewise, the rates of microbial nucleic acid synthesis and carbon production in the sediment were only elevated immediately adjacent to the outfalls. Total microbial biomass, as determined by the ATP content of the sediment, varied spatially but was generally elevated at the outfall sites. The specific growth rates calculated for the sediment microbial populations, however, were not greater at the outfall sites. At one site the rocks surrounding the diffuser pipe were covered with copious amounts of slime that appeared to be composed entirely of microbial cells and filaments. This microbial mat was extremely active with respect to heterotrophic activity and biomass production. Overall, it appears that the impact of the sewage discharge on the ambient seawater microbiota is slight and that the effect on the sediment microbiota is confined to an area immediately adjacent to the diffuser ports. In the sand itself, the effect is limited to the upper 2 cm at most.     

Heterotrophic Activity Throughout a Vertical Profile of Seawater and Sediment in Halifax Harbor, Canada

The relative heterotrophic activity of marine microorganisms was determined at two sites by the heterotrophic uptake technique throughout the water column, the sediment-water interface, and the surface layer of sediment. In the water column, uptake was greatest at the surface and steadily decreased with depth. The percentage of the substrate that was respired also decreased with depth from 69 to 56%. The activity of the sediment-water interface was several orders of magnitude greater than that of the overlying water and twice that of the sediment immediately below. Hand-collected water samples carefully taken as close as 1 cm from the sediment-water interface had the same characteristically low activity as the bottom few meters of water. Microautoradiography with ³H-labeled glucose, glutamic acid, or thymidine revealed a general decrease in the percentage of active cells with depth from 35 to <1%. The number of active cells in the interface and sediment averaged <10% of the total population. The data indicate that the sediment-water interface is the most active region in this system due to an increased number of active cells rather than an increased percentage of active cells or increased per-cell activity.     

The evolutionary relationships of two families of cottoid fishes of Lake Baikal (East Siberia) as suggested by analysis of mitochondrial DNA

Fragments of mtDNA genes Cyt B, ATPase 6, and ATPase 8 of six cottoid fishes species of Lake Baikal (East Siberia) were amplified and sequenced. In addition mtDNAs of the same fish were subjected to restriction analysis. The data obtained were used to construct phylogenetic trees. The topology of the ATPase tree differs from those of the Res (restriction) and Cyt B trees. Clustering of species within the trees confirms the viewpoint of Taliev (1955, Baicalian Sculpins (Cottoidei)) according to which Baikalian cottoids originate from two ancestral forms. The times of branching obtained do not confirm the existing viewpoint according to which the two golomyankas (Comephorus baicalensis and Comephorus dybowskii) are pre-Baikal (Myocene) relicts: these two species may have originated 1.2–1.8 million years ago in Baikal, and they seem to represent an example of rapid morphological evolution which resulted in the formation of a new family. Correspondence to: S. Ja. Slobodyanuk     

Ouabain-insensitive salt and water movements in duck red cells. II. Norepinephrine stimulation of sodium plus potassium cotransport

Catecholamines induce net salt and water movements in duck red cells incubated in isotonic solutions. The rate of this response is approximately three times greater than a comparable effect observed in 400 mosmol hypertonic solutions in the absence of hormone (W.F. Schmidt and T. J. McManus. 1977 a.J. Gen. Physiol. 70:59-79. Otherwise, these two systems share a great many similarities. In both cases, net water and salt movements have a marked dependence on external cation concentrations, are sensitive to furosemide and insensitive to ouabain, and allow the substitution of rubidium for external potassium. In the presence of ouabain, but the absence of external potassium (or rubidium), a furosemide-sensitive net extrusion of sodium against a large electrochemical gradient can be demonstrated. When norepinephrine-treated cells are incubated with ouabain and sufficient external sodium, the furosemide-sensitive, unidirectional influxes of both sodium and rubidium are half- maximally saturated at similar rubidium concentrations; with saturating external rubidium, the same fluxes are half-maximal at comparable levels of external sodium. In the absence of sodium, a catecholamine-stimulated, furosemide-sensitive influx of rubidium persists. In the absence of rubidium, a similar but smaller component of sodium influx can be seen. We interpret these results in terms of a cotransport model for sodium plus potassium which is activated by hypertonicity or norepinephrine. When either ion is absent from the incubation medium, the system promotes an exchange-diffusion type of movement of the co-ion into the cells. In the absence of external potassium, net movement of potassium out of the cell leads to a coupled extrusion of sodium against its electrochemical gradient.     

Morphological characterization of small cells resulting from nutrient starvation of a psychrophilic marine vibrio.

Upon starvation, Ant-300, a psychrophilic marine vibrio, was observed to decrease in size and change in shape from a rod to a coccus. After 3 weeks of starvation 50% of the starved population was able to pass through a filter with a pore size of 0.4 mum. Electron microscopy of thin sections of the small cells revealed normal cell structure except for an enlarged periplasmic space. When inoculated into a fresh medium, starved cells growth without a significant lag and regained "normal" size and shape within 48 h.     

In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

Here, we describe a fast, easy-to-use, and sensitive method to profile in-depth structural micro-heterogeneity, including intricate N-glycosylation profiles, of monoclonal antibodies at the native intact protein level by means of mass spectrometry using a recently introduced modified Orbitrap Exactive Plus mass spectrometer. We demonstrate the versatility of our method to probe structural micro-heterogeneity by describing the analysis of three types of molecules: (1) a non-covalently bound IgG4 hinge deleted full-antibody in equilibrium with its half-antibody, (2) IgG4 mutants exhibiting highly complex glycosylation profiles, and (3) antibody-drug conjugates. Using the modified instrument, we obtain baseline separation and accurate mass determination of all different proteoforms that may be induced, for example, by glycosylation, drug loading and partial peptide backbone-truncation. We show that our method can handle highly complex glycosylation profiles, identifying more than 20 different glycoforms per monoclonal antibody preparation and more than 30 proteoforms on a single highly purified antibody. In analyzing antibody-drug conjugates, our method also easily identifies and quantifies more than 15 structurally different proteoforms that may result from the collective differences in drug loading and glycosylation. The method presented here will aid in the comprehensive analytical and functional characterization of protein micro-heterogeneity, which is crucial for successful development and manufacturing of therapeutic antibodies     

Association between virus-specific T-cell responses and plasma viral load in human immunodeficiency virus type 1 subtype C infection

Virus-specific T-cell immune responses are important in restraint of human immunodeficiency virus type 1 (HIV-1) replication and control of disease. Plasma viral load is a key determinant of disease progression and infectiousness in HIV infection. Although HIV-1 subtype C (HIV-1C) is the predominant virus in the AIDS epidemic worldwide, the relationship between HIV-1C-specific T-cell immune responses and plasma viral load has not been elucidated. In the present study we address (i) the association between the level of plasma viral load and virus-specific immune responses to different HIV-1C proteins and their subregions and (ii) the specifics of correlation between plasma viral load and T-cell responses within the major histocompatibility complex (MHC) class I HLA supertypes. Virus-specific immune responses in the natural course of HIV-1C infection were analyzed in the gamma interferon (IFN-gamma)-enzyme-linked immunospot assay by using synthetic overlapping peptides corresponding to the HIV-1C consensus sequence. For Gag p24, a correlation was seen between better T-cell responses and lower plasma viral load. For Nef, an opposite trend was observed where a higher T-cell response was more likely to be associated with a higher viral load. At the level of the HLA supertypes, a lower viral load was associated with higher T-cell responses to Gag p24 within the HLA A2, A24, B27, and B58 supertypes, in contrast to the absence of such a correlation within the HLA B44 supertype. The present study demonstrated differential correlations (or trends to correlation) in various HIV-1C proteins, suggesting (i) an important role of the HIV-1C Gag p24-specific immune responses in control of viremia and (ii) more rapid viral escape from immune responses to Nef with no restraint of plasma viral load. Correlations between the level of IFN-gamma-secreting T cells and viral load within the MHC class I HLA supertypes should be considered in HIV vaccine design and efficacy trials.     

Human immunodeficiency virus type 1 subtype C molecular phylogeny: consensus sequence for an AIDS vaccine design?

An evolving dominance of human immunodeficiency virus type 1 subtype C (HIV-1C) in the AIDS epidemic has been associated with a high prevalence of HIV-1C infection in the southern African countries and with an expanding epidemic in India and China. Understanding the molecular phylogeny and genetic diversity of HIV-1C viruses may be important for the design and evaluation of an HIV vaccine for ultimate use in the developing world. In this study we analyzed the phylogenetic relationships (i) between 73 non-recombinant HIV-1C near-full-length genome sequences, including 51 isolates from Botswana; (ii) between HIV-1C consensus sequences that represent different geographic subsets; and (iii) between specific isolates and consensus sequences. Based on the phylogenetic analyses of 73 near-full-length genomes, 16 "lineages" (a term that is used hereafter for discussion purposes and does not imply taxonomic standing) were identified within HIV-1C. The lineages were supported by high bootstrap values in maximum-parsimony and neighbor-joining analyses and were confirmed by the maximum-likelihood method. The nucleotide diversity between the 73 HIV-1C isolates (mean value of 8.93%; range, 2.9 to 11.7%) was significantly higher than the diversity of the samples to the consensus sequence (mean value of 4.86%; range, 3.3 to 7.2%, P < 0.0001). The translated amino acid distances to the consensus sequence were significantly lower than distances between samples within all HIV-1C proteins. The consensus sequences of HIV-1C proteins accompanied by amino acid frequencies were presented (that of Gag is presented in this work; those of Pol, Vif, Vpr, Tat, Rev, Vpu, Env, and Nef are presented elsewhere [http://www.aids.harvard.edu/lab_research/concensus_sequence.htm]). Additionally, in the promoter region three NF-kappa B sites (GGGRNNYYCC) were identified within the consensus sequences of the entire set or any subset of HIV-1C isolates. This study suggests that the consensus sequence approach could overcome the high genetic diversity of HIV-1C and facilitate an AIDS vaccine design, particularly if the assumption that an HIV-1C antigen with a more extensive match to the circulating viruses is likely to be more efficacious is proven in efficacy trials.     

The diageotropica mutation alters auxin induction of a subset of the Aux/IAA gene family in tomato

The diageotropica (dgt) mutation has been proposed to affect either auxin perception or responsiveness in tomato plants. It has previously been demonstrated that the expression of one member of the Aux/IAA family of auxin-regulated genes is reduced in dgt plants. Here, we report the cloning of ten new members of the tomato Aux/IAA family by PCR amplification based on conserved protein domains. All of the gene family members except one (LeIAA7) are expressed in etiolated tomato seedlings, although they demonstrate tissue specificity (e.g. increased expression in hypocotyls vs. roots) within the seedling. The wild-type auxin-response characteristics of the expression of these tomato LeIAA genes are similar to those previously described for Aux/IAA family members in Arabidopsis. In dgt seedlings, auxin stimulation of gene expression was reduced in only a subset of LeIAA genes (LeIAA5, 8, 10, and 11), with the greatest reduction associated with those genes with the strongest wild-type response to auxin. The remaining LeIAA genes tested exhibited essentially the same induction levels in response to the hormone in both dgt and wild-type hypocotyls. These results confirm that dgt plants can perceive auxin and suggest that a specific step in early auxin signal transduction is disrupted by the dgt mutation.