Stimulation of hexose uptake in rat thymic lymphocytes by phorbol ester. A role for Ca2+ and Na+H+ exchange?

The tumor promoter 12-0-tetradecanoyl phorbol-13-acetate (TPA) stimulates hexose uptake into rat thymocytes. This study explores two possible messengers of this stimulation: changes in cytosolic [Ca2+], and activation of the Na+/H+ antiport. The cytosolic level of Ca2+, determined by the fluorescence of quin-2, was elevated by TPA, and this rise required extracellular Ca2+. In contrast, stimulation of hexose uptake was still observed in Ca2+ -free media even when cytoplasmic [Ca2+] was buffered with quin-2. TPA also raised the cytoplasmic pH, presumably through activation of the Na+/H+ exchange. However, replacement of extracellular Na+ by N-methylglucamine+ or choline+ which prevents the cytoplasmic alkanization did not prevent stimulation of hexose uptake by TPA. Moreover, amiloride, at concentrations that inhibit Na+/H+ exchange in these cells, did not interfere with stimulation of hexose uptake by TPA. In conclusion, stimulation of hexose uptake by phorbol ester in rat thymocytes does not appear to be mediated by changes in cytosolic free Ca2+ or in the activity of the Na+/H+ antiport.     

A novel strategy for regulated expression of a cytotoxic gene.

The tetracycline (Tet) transactivator system is a powerful promoter system to control gene expression. However, expression of a cytotoxic gene in this system has been limited due to the lethal effect caused by low levels of basal expression of the toxic gene. In this report, we describe a novel strategy to express a toxic gene using the Tet system. The barstar gene is placed downstream of a minimal promoter and the barnase gene downstream of the tetracycline responsive element minimal promoter. When barnase is expressed at a basal level, its toxicity in human cell culture is offset by the similar basal level expression of barstar. However, when the barnase expression is induced with the transactivator protein, its overproduction leads to cell death. Therefore, this strategy allows cytotoxicity to be effectively regulated by tetracycline.     

The Effect of Ochre Suppression on Meiosis and Ascospore Formation in Saccharomyces

The effect of altered tyrosyl-tRNAs on the developmental process of sporulation was examined. Mutations in eight independent loci resulting in tyrosine-inserting nonsense suppressor were tested for their effects on sporulation. Different levels of inhibition were found ranging from SUP3-omicron, which caused the greatest reduction of sporulation (7-17% of wild type), to SUP11-omicron which caused no reduction in sporulation. Since the SUP3-omicron mutation exhibited the greatest effect, it was studied in detail. Although SUP3-omicron is a dominant nonsense suppressor, its effect on sporulation is recessive. Expression of the sporulation deficiency is dependent upon the stage of transfer from glucose growth medium (i.e., log, early stationary, etc.) to sporulation medium. SUP3-omicron/SUP3-omicron diploid cells transferred from log or early stationary phase are capable of sporulation, whereas cells transferred after early stationary phase (i.e., after adaptation to respiration) exhibit poor sporulative ability. Sporulation events were examined under restrictive conditions to observe those events completed by SUP3-omicron/SUP3-omicron diploids. The early events of sporulation occur in these cells. Later events are completed by progressively fewer cells. Premeiotic DNA synthesis occurred in approximately 40% of the cells, nuclear segregation occurred in 20%, and finally, only 2% formed asci. The fact that fewer late-sporulation events occur under restrictive conditions can be explained by increased efficiency of suppression.     

Adaptation of mouse leukemic cells (L5178Y) to anisotonic media : II. Cell growth

Mouse lymphoma cells (L5178Y) exposed to hypertonic media for 1 h behave as osmometers, but in hypotonic media, after initial swelling, they shrink back to normal volume and maintain it for long periods of time. The lower limit of osmolarity at which this “volume adaptation” will occur lies between 140 and 185 mosM. The “volume adaptation” is associated with a loss of cellular K⁺ probably due to a transient increase in K⁺ permeability and to loss of associated anions and osmotically obliged water. Partial dissipation of the large gradient of K⁺ between cells and medium by pre-exposure to ouabain or to K⁺-free medium results in a diminished capacity to adapt. After the shrinking phase is completed, a new steady state is established with a reduced cellular K⁺ content, normal Na⁺, normal K⁺-permeability, and a reduced activity of the Na⁺ − K⁺ transport system. When adapted cells are returned to normal medium, an initial shrinking is followed by a re-swelling to normal size, associated with a gain in K⁺ content, presumably due to the return to normal activity of the Na⁺ − K⁺ transport system.     

Quantification of angiogenesis in estrogen receptor-positive and negative breast carcinoma

Background

The objective of this study was to evaluate angiogenesis according to CD34 antigen expression in estrogen receptor (ER)-positive and negative breast carcinomas.

Methods

This study comprised 64 cases of infiltrating ductal carcinoma in postmenopausal women divided into two groups: Group A: ER-positive, n = 35; and Group B: ER-negative, n = 29. The anti-CD34 monoclonal antibody was used as a marker for endothelial cells. Microvessel count was carried out in 10 fields per slide using a 40× objective lens (magnification 400×). Statistical analysis of the data was performed using Student's t-test (p < 0.05).

Results

The mean number of vessels stained with the anti-CD34 antibody in the estrogen receptor-positive and negative tumors was 23.51 ± 1.15 and 40.24 ± 0.42, respectively. The number of microvessels was significantly greater in the estrogen receptor-negative tumors (p < 0.001).

Conclusion

ER-negative tumors have significantly greater CD34 antigen expression compared to ER-positive tumors.

     

Patterns of DON and DOC Leaching Losses Across a Natural N Availability Gradient in Temperate Hardwood Forests

Dissolved organic nitrogen (DON) is a potentially significant vector of N loss from forest ecosystems that has been characterized as an “N leak.” Although the term “leak” suggests a lack of regulation, it is clear DON losses are a function of biological and physicochemical processes that influence its production and retention across the landscape. In this study, we investigated how soil processes that influence DON cycling impact ecosystem patterns of DON loss in five northern hardwood forests that spanned a gradient of N availability, tree species composition, and moisture–edaphic characteristics. We collected soil leachate from the forest floor and at 15 and 100 cm soil depths and related solution chemistry to its physical environment. We found that DON losses were a function of ecosystem N status and increased modestly with soil N stock. We also found a unimodal pattern of DOC/DON losses across the gradient driven by low DOC/DON in the lowest N availability stand, likely due to the interaction between strongly sorbing DOM inputs from C-rich, oak-derived leaf litter with highly sorptive soils. We suggest DOM losses from forests depend on interactions between soil solution input chemistry from the forest floor, which reflects changes in tree species composition across the landscape, and soil sorptive processes where organic compounds are dynamically exchanged between solid and dissolved phases. These results emphasize the need to understand how fine-scale processes can interact to shape ecosystem patterns of DOM loss.     

Loss of callose in the stigma papillae does not affect the Brassica self-incompatibility phenotype

As part of the Brassicaceae self-incompatibility response, callose is deposited in the stigma papillar cells. To determine if callose plays an important role in the rejection of incompatible pollen by the stigma, transgenic Brassica napus. L. plants were produced which express the tobacco β-1,3-glucanase cDNA (the enzyme which degrades callose) in the stigma papillae. Using aniline blue fluorescence, little or no callose was detected in the papillar cells of transgenic stigmas. However, the self-incompatibility system appeared to be unaffected based on the lack of pollen tube growth and the subsequent lack of seed set. The transgene had no effect on compatible pollinations. Thus, while callose deposition is associated with the B. napus self-incompatibility response, it is not required for the rejection of incompatible pollen. Received: 14 March 1997 / Accepted: 15 April 1997     

Sml1p is a dimer in solution: characterization of denaturation and renaturation of recombinant Sml1p

Sml1p is a small 104-amino acid protein from Saccharomyces cerevisiae that binds to the large subunit (Rnr1p) of the ribonucleotide reductase complex (RNR) and inhibits its activity. During DNA damage, S phase, or both, RNR activity must be tightly regulated, since failure to control the cellular level of dNTP pools may lead to genetic abnormalities, such as genome rearrangements, or even cell death. Structural characterization of Sml1p is an important step in understanding the regulation of RNR. Until now the oligomeric state of Sml1p was unknown. Mass spectrometric analysis of wild-type Sml1p revealed an intermolecular disulfide bond involving the cysteine residue at position 14 of the primary sequence. To determine whether disulfide bonding is essential for Sml1p oligomerization, we mutated the Cys14 to serine. Sedimentation equilibrium measurements in the analytical ultracentrifuge show that both wild-type and C14S Sml1p exist as dimers in solution, indicating that the dimerization is not a result of a disulfide bond. Further studies of several truncated Sml1p mutants revealed that the N-terminal 8-20 residues are responsible for dimerization. Unfolding/refolding studies of wild-type and C14S Sml1p reveal that both proteins refold reversibly and have almost identical unfolding/refolding profiles. It appears that Sml1p is a two-domain protein where the N-terminus is responsible for dimerization and the C-terminus for binding and inhibiting Rnr1p activity.     

A protease-activatable luminescent biosensor and reporter cell line for authentic SARS-CoV-2 infection

Efforts to define serological correlates of protection against COVID-19 have been hampered by the lack of a simple, scalable, standardised assay for SARS-CoV-2 infection and antibody neutralisation. Plaque assays remain the gold standard, but are impractical for high-throughput screening. In this study, we show that expression of viral proteases may be used to quantitate infected cells. Our assays exploit the cleavage of specific oligopeptide linkers, leading to the activation of cell-based optical biosensors. First, we characterise these biosensors using recombinant SARS-CoV-2 proteases. Next, we confirm their ability to detect viral protease expression during replication of authentic virus. Finally, we generate reporter cells stably expressing an optimised luciferase-based biosensor, enabling viral infection to be measured within 24 h in a 96- or 384-well plate format, including variants of concern. We have therefore developed a luminescent SARS-CoV-2 reporter cell line, and demonstrated its utility for the relative quantitation of infectious virus and titration of neutralising antibodies.     

Mechanism of osmotic activation of Na+/H+ exchange in rat thymic lymphocytes

The activity of the Na+/H+ exchange system of rat thymic lymphocytes was determined by means of intracellular (pHi) and extracellular pH (pH0) measurements. In isotonic media, the antiport is virtually quiescent at physiological pHi (7.0-7.1), but is greatly activated by cytoplasmic acidification. At normal pHi, the antiport can also be activated by osmotic shrinking. Osmotic activation occurs after a delay of 20-30 s and is reversed several minutes after iso-osmolarity is restored. The mechanism of activation was analyzed by comparing the kinetic parameters of transport in resting (isotonic) and hyperosmotically stressed cells. The affinities of the external substrate site for Na+ and H+ are not altered in shrunken cells. In contrast, the Hi+ sensitivity of the antiport (which is largely dictated by an allosteric modifier site) was increased, which accounted for the activation. The concentration of free cytoplasmic Ca2+ [( Ca2+]i) increased after osmotic shrinking. This increase was dependent on the presence of extracellular Ca2+ and Na+ and was blocked by inhibitors of Na+/H+ exchange, which suggests that it is a consequence, rather than the cause, of the activation of the antiport. It is concluded that the shift in the pHi dependence of the modifier site of the Na+/H+ antiport is the primary event underlying the regulatory volume increase that follows osmotic shrinkage.