Analysis of the oligosaccharides on androgen-binding proteins: Implications concerning their role in structure/function relationships

Rat androgen-binding protein (rABP), human testosterone-binding globulin (hTeBG) and rabbit (rb) TeBG are heterodimeric proteins. The source of the heterogeneity arises from the differential glycosylation of a common protein core. This glycosylation results in a heavy subunit (more glycosylation) and a light subunit (less glycosylation). Glycosylation is one factor responsible for multiple charged species seen when rABP, hTeBG, and rbTeBG are analyzed by two-dimensional gel electrophoresis. Enzymatic digestion with the endoglycosidase, peptide: N-glycosidase F indicated that all three proteins have asparagine (Asn)-linked oligosaccharides as their major glycan substituent. Treatment with exoglycosidases provided evidence for terminal sialic acid, galactose and mannose and N-acetylglucosamine residues. About 16–22% of the mass of the heavy subunit and about 8–14% of the mass of the light subunit is contributed by carbohydrate.

Serial lectin chromatography indicated that rABP is glycosylated differently from hTeBG and rbTeBG. About 40% of the rABP contains tri and tetraantennary complex oligosaccharides, while only about 20% of the hTeBG and TeBG from pregnant rabbits contains these types of glycans. About 9% of the TeBG from male rabbits bears these types of oligosaccharides. All of the biantennary complex oligosaccharides on rABP are fucosylated on the chitobiose core, but only 8% of those on hTeBG and none of those on rbTeBG are fucosylated in this manner. All three proteins are glycosylated at more than one site. The data indicate that the proteins may have more than one type of oligosaccharide on them. It is likely that differences in glycosylation are responsible for different physiological roles of the proteins.     

Glutamine synthetase of Chlamydomonas: its role in the control of nitrate assimilation

Work is described which suggests that glutamine synthetase (GS) could play an important and direct regulatory role in the control of NO₃ assimilation by the alga. In both steady-state cells and ones disturbed physiologically by changes in light or nitrogen supply the assimilation of NO₃ appears to be limited by the activity of GS. Moreover although in normal cells NH₃ can completely inhibit NO₃ uptake, promote the deactivation of nitrate reductase (NR) and repress the synthesis of NR and nitrite reductase (NIR), these controls are relaxed in cells in which GS is deactivated by treatment with L-methionine-DL-sulfoximine (MSO). It is proposed that the reversible deactivation of GS may play an important part in the regulation of NO₃ assimilation although it is still not clear whether the enzyme itself or products of its metabolism are responsible.Abbreviations GS glutamine synthetase - GS_s glutamine synthetase, synthetase activity - GS_t glutamine synthetase, transferase activity - NR nitrate reductase - NIR nitrite reductase - GDH glutamate dehydrogenase - CHX cycloheximide - MSO L-methionine-DL-sulfoximine - FAD flavine adenine dinucleotide     

Phase response versus positive and negative division delay in animal cells

The time of median cell division in V79 Chinese hamster cells following high serum pulses was determined for two synchronous cell generations following mitotic selection. Differences in cell cycle time for each pair of pulse and control cultures were computed and plotted as a function of time of serum pulse. This phase response curve for hamster cells with an 8.5 h cell cycle shows a characteristic biphasic pattern. Beginning 0.5 h after mitotic selection, pulses with serum produce delays in the midpoint of the subsequent mitotic waves. Delay is maximum at 1.5 h. Delays give way abruptly to advances at 2.5 h and the amount of advance then decreases as pulses are given between 3 and 5 h into the cycle. At 5 h decreasing advances become delays, with increasing delays due to serum pulses occurring between 5 and 6 h. Delays again give way abruptly to advances at 6 h and again the amount of advance decreases through the late portion of the cycle. Pulses very late in the cycle appear to generate phase delays. This biphasic response to serum is interpreted as an expression of an underlying time-keeping oscillator whose period is nominally of 4 h duration.     

Etiology of two virus diseases of lilac (Syringa vulgaris L.) occurring in Czechoslovakia

Virus origin of lilac ringspot and chlorotic ringspot of lilac was identified serologically by means of double gel diffusion method. The former of these diseases is due to Arabis mosaic virus, the latter to a mixture of Arabis mosaic virus and cherry leaf roll virus. The occurrence of these viruses has been detected for the first time in Czechoslovakia.     

T cell stimulation remodels the latently HIV-1 infected cell population by differential activation of proviral chromatin

The reservoir of latently HIV-1 infected cells is heterogeneous. To achieve an HIV-1 cure, the reservoir of activatable proviruses must be eliminated while permanently silenced proviruses may be tolerated. We have developed a method to assess the proviral nuclear microenvironment in single cells. In latently HIV-1 infected cells, a zinc finger protein tethered to the HIV-1 promoter produced a fluorescent signal as a protein of interest came in its proximity, such as the viral transactivator Tat when recruited to the nascent RNA. Tat is essential for viral replication. In these cells we assessed the proviral activation and chromatin composition. By linking Tat recruitment to proviral activity, we dissected the mechanisms of HIV-1 latency reversal and the consequences of HIV-1 production. A pulse of promoter-associated Tat was identified that contrasted to the continuous production of viral proteins. As expected, promoter H3K4me3 led to substantial expression of the provirus following T cell stimulation. However, the activation-induced cell cycle arrest and death led to a surviving cell fraction with proviruses encapsulated in repressive chromatin. Further, this cellular model was used to reveal mechanisms of action of small molecules. In a proof-of-concept study we determined the effect of modifying enhancer chromatin on HIV-1 latency reversal. Only proviruses resembling active enhancers, associated with H3K4me1 and H3K27ac and subsequentially recognized by BRD4, efficiently recruited Tat upon cell stimulation. Tat-independent HIV-1 latency reversal of unknown significance still occurred. We present a method for single cell assessment of the microenvironment of the latent HIV-1 proviruses, used here to reveal how T cell stimulation modulates the proviral activity and how the subsequent fate of the infected cell depends on the chromatin context.     

PcoB is a defense outer membrane protein that facilitates cellular uptake of copper

Copper (Cu) is one of the most abundant trace metals in all organisms, involved in a plethora of cellular processes. Yet elevated concentrations of the element are harmful, and interestingly prokaryotes are more sensitive for environmental Cu stress than humans. Various transport systems are present to maintain intracellular Cu homeostasis, including the prokaryotic plasmid‐encoded multiprotein pco operon, which is generally assigned as a defense mechanism against elevated Cu concentrations. Here we structurally and functionally characterize the outer membrane component of the Pco system, PcoB, recovering a 2.0 Å structure, revealing a classical β‐barrel architecture. Unexpectedly, we identify a large opening on the extracellular side, linked to a considerably electronegative funnel that becomes narrower towards the periplasm, defining an ion‐conducting pathway as also supported by metal binding quantification via inductively coupled plasma mass spectrometry and molecular dynamics (MD) simulations. However, the structure is partially obstructed towards the periplasmic side, and yet flux is permitted in the presence of a Cu gradient as shown by functional characterization in vitro. Complementary in vivo experiments demonstrate that isolated PcoB confers increased sensitivity towards Cu. Aggregated, our findings indicate that PcoB serves to permit Cu import. Thus, it is possible the Pco system physiologically accumulates Cu in the periplasm as a part of an unorthodox defense mechanism against metal stress. These results point to a previously unrecognized principle of maintaining Cu homeostasis and may as such also assist in the understanding and in efforts towards combatting bacterial infections of Pco‐harboring pathogens.     

Amazonian Anthrosols Support Similar Microbial Communities that Differ Distinctly from Those Extant in Adjacent, Unmodified Soils of the Same Mineralogy

We compared the microbial community composition in soils from the Brazilian Amazon with two contrasting histories; anthrosols and their adjacent non-anthrosol soils of the same mineralogy. The anthrosols, also known as the Amazonian Dark Earths or terra preta, were managed by the indigenous pre-Colombian Indians between 500 and 8,700 years before present and are characterized by unusually high cation exchange capacity, phosphorus (P), and calcium (Ca) contents, and soil carbon pools that contain a high proportion of incompletely combusted biomass as biochar or black carbon (BC). We sampled paired anthrosol and unmodified soils from four locations in the Manaus, Brazil, region that differed in their current land use and soil type. Community DNA was extracted from sampled soils and characterized by use of denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism. DNA bands of interest from Bacteria and Archaea DGGE gels were cloned and sequenced. In cluster analyses of the DNA fingerprints, microbial communities from the anthrosols grouped together regardless of current land use or soil type and were distinct from those in their respective, paired adjacent soils. For the Archaea, the anthrosol communities diverged from the adjacent soils by over 90%. A greater overall richness was observed for Bacteria sequences as compared with those of the Archaea. Most of the sequences obtained were novel and matched those in databases at less than 98% similarity. Several sequences obtained only from the anthrosols grouped at 93% similarity with the Verrucomicrobia, a genus commonly found in rice paddies in the tropics. Sequences closely related to Proteobacteria and Cyanobacteria sp. were recovered only from adjacent soil samples. Sequences related to Pseudomonas, Acidobacteria, and Flexibacter sp. were recovered from both anthrosols and adjacent soils. The strong similarities among the microbial communities present in the anthrosols for both the Bacteria and Archaea suggests that the microbial community composition in these soils is controlled more strongly by their historical soil management than by soil type or current land use. The anthrosols had consistently higher concentrations of incompletely combusted organic black carbon material (BC), higher soil pH, and higher concentrations of P and Ca compared to their respective adjacent soils. Such characteristics may help to explain the longevity and distinctiveness of the anthrosols in the Amazonian landscape and guide us in recreating soils with sustained high fertility in otherwise nutrient-poor soils in modern times.