Faster cytotoxicity with age: Increased perforin and granzyme levels in cytotoxic CD8 + T cells boost cancer cell elimination

A variety of intrinsic and extrinsic factors contribute to the altered efficiency of CTLs in elderly organisms. In particular, the efficacy of antiviral CD8⁺ T cells responses in the elderly has come back into focus since the COVID‐19 pandemic outbreak. However, the exact molecular mechanisms leading to alterations in T cell function and the origin of the observed impairments have not been fully explored. Therefore, we investigated whether intrinsic changes affect the cytotoxic ability of CD8⁺ T cells in aging. We focused on the different subpopulations and time‐resolved quantification of cytotoxicity during tumor cell elimination. We report a surprising result: Killing kinetics of CD8⁺ T cells from elderly mice are much faster than those of CD8⁺ T cells from adult mice. This is true not only in the total CD8⁺ T cell population but also for their effector (T_EM) and central memory (T_CM) T cell subpopulations. TIRF experiments reveal that CD8⁺ T cells from elderly mice possess comparable numbers of fusion events per cell, but significantly increased numbers of cells with granule fusion. Analysis of the cytotoxic granule (CG) content shows significantly increased perforin and granzyme levels and turns CD8⁺ T cells of elderly mice into very efficient killers. This highlights the importance of distinguishing between cell‐intrinsic alterations and microenvironmental changes in elderly individuals. Our results also stress the importance of analyzing the dynamics of CTL cytotoxicity against cancer cells because, with a simple endpoint lysis analysis, cytotoxic differences could have easily been overlooked.     

Impairing L-Threonine Catabolism Promotes Healthspan through Methylglyoxal-Mediated Proteohormesis

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Interaction of Shc with Grb2 regulates association of Grb2 with mSOS.

The adapter protein Shc has been implicated in Ras signaling via many receptors, including the T-cell antigen receptor (TCR), B-cell antigen receptor, interleukin-2 receptor, interleukin-3 receptor, erythropoietin receptor, and insulin receptor. Moreover, transformation via polyomavirus middle T antigen is dependent on its interaction with Shc and Shc tyrosine phosphorylation. One of the mechanisms of TCR-mediated, tyrosine kinase-dependent Ras activation involves the simultaneous interaction of phosphorylated Shc with the TCR zeta chain and with a second adapter protein, Grb2. Grb2, in turn, interacts with the Ras guanine nucleotide exchange factor mSOS, thereby leading to Ras activation. Although it has been reported that in fibroblasts Grb2 and mSOS constitutively associate with each other and that growth factor stimulation does not alter the levels of Grb2:mSOS association, we show here that TCR stimulation leads to a significant increase in the levels of Grb2 associated with mSOS. This enhanced Grb2:mSOS association, which occurs through an SH3-proline-rich sequence interaction, is regulated through the SH2 domain of Grb2. The following observations support a role for Shc in regulating the Grb2:mSOS association: (i) a phosphopeptide corresponding to the sequence surrounding Tyr-317 of Shc, which displaces Shc from Grb2, abolished the enhanced association between Grb2 and mSOS; and (ii) addition of phosphorylated Shc to unactivated T cell lysates was sufficient to enhance the interaction of Grb2 with mSOS. Furthermore, using fusion proteins encoding different domains of Shc, we show that the collagen homology domain of Shc (which includes the Tyr-317 site) can mediate this effect. Thus, the Shc-mediated regulation of Grb2:mSOS association may provide a means for controlling the extent of Ras activation following receptor stimulation.     

Evidence for a requirement for both phospholipid and phosphotyrosine binding via the Shc phosphotyrosine-binding domain in vivo.

The adapter protein Shc is a critical component of mitogenic signaling pathways initiated by a number of receptors. Shc can directly bind to several tyrosine-phosphorylated receptors through its phosphotyrosine-binding (PTB) domain, and a role for the PTB domain in phosphotyrosine-mediated signaling has been well documented. The structure of the Shc PTB domain demonstrated a striking homology to the structures of pleckstrin homology domains, which suggested acidic phospholipids as a second ligand for the Shc PTB domain. Here we demonstrate that Shc binding via its PTB domain to acidic phospholipids is as critical as binding to phosphotyrosine for leading to Shc phosphorylation. Through structure-based, targeted mutagenesis of the Shc PTB domain, we first identified the residues within the PTB domain critical for phospholipid binding in vitro. In vivo, the PTB domain was essential for localization of Shc to the membrane, as mutant Shc proteins that failed to interact with phospholipids in vitro also failed to localize to the membrane. We also observed that PTB domain-dependent targeting to the membrane preceded the PTB domain's interaction with the tyrosine-phosphorylated receptor and that both events were essential for tyrosine phosphorylation of Shc following receptor activation. Thus, Shc, through its interaction with two different ligands, is able to accomplish both membrane localization and binding to the activated receptor via a single PTB domain.     

Influence of vanadate on glycolysis,intracellular sodium,and pH in perfused rat hearts

Vanadium compounds have been shown to cause a variety of biological and metabolic effects including inhibition of certain enzymes, alteration of contractile function, and as an insulin like regulator of glucose metabolism. However, the influence of vanadium on metabolic and ionic changes in hearts remains to be understood. In this study we have examined the influence of vanadate on glucose metabolism and sodium transport in isolated perfused rat hearts. Hearts were perfused with 10 mM glucose and varying vanadate concentrations (0.7100 M) while changes in high energy phosphates (ATP and phosphocreatine (PCr)), intracellular pH, and intracellular sodium were monitored using 31P and 23Na NMR spectroscopy. Tissue lactate, glycogen, and (Na+, K+)-ATPase activity were also measured using biochemical assays. Under baseline conditions, vanadate increased tissue glycogen levels two fold and reduced (Na+, K+)-ATPase activity. Significant decreases in ATP and PCr were observed in the presence of vanadate, with little change in intracellular pH. These changes under baseline conditions were less severe when the hearts were perfused with glucose, palmitate and b-hydroxybutyrate. During ischemia vanadate did not limit the rise in intracellular sodium, but slowed sodium recovery on reperfusion. The presence of vanadate during ischemia resulted in attenuation of acidosis, and reduced lactate accumulation. Reperfusion in the presence of vanadate resulted in a slower ATP recovery, while intracellular pH and PCr recovery was not affected. These results indicate that vanadate alters glucose utilization and (Na+, K+)-ATPase activity and thereby influences the response of the myocardium to an ischemic insult.     

In vitro adventitious shoot formation from leaf cultures of Clerodendrum inerme (L) Gaertn.

In vitro adventitious shoots (about 28) of Clerodendrum inerme were regenerated from leaf segments on MS medium containing BA (4 mg/L). These shoots developed directly from the leaf explants without callusing after 5 weeks. Leaf explant when cultured in MS medium containing BA (2 mg/L) and NAA (0.5 mg/L) developed compact callus that became nodular and regenerated shoots (about 50) after 5 weeks. The in vitro developed shoots were rooted in MS medium supplemented with IAA (2 mg/L). The hardened plantlets were successfully established in the field with 90% survival.     

Higher‐order assemblies of oligomeric cargo receptor complexes form the membrane scaffold of the Cvt vesicle

Selective autophagy is the mechanism by which large cargos are specifically sequestered for degradation. The structural details of cargo and receptor assembly giving rise to autophagic vesicles remain to be elucidated. We utilize the yeast cytoplasm‐to‐vacuole targeting (Cvt) pathway, a prototype of selective autophagy, together with a multi‐scale analysis approach to study the molecular structure of Cvt vesicles. We report the oligomeric nature of the major Cvt cargo Ape1 with a combined 2.8 Å X‐ray and negative stain EM structure, as well as the secondary cargo Ams1 with a 6.3 Å cryo‐EM structure. We show that the major dodecameric cargo prApe1 exhibits a tendency to form higher‐order chain structures that are broken upon interaction with the receptor Atg19 in vitro. The stoichiometry of these cargo–receptor complexes is key to maintaining the size of the Cvt aggregate in vivo. Using correlative light and electron microscopy, we further visualize key stages of Cvt vesicle biogenesis. Our findings suggest that Atg19 interaction limits Ape1 aggregate size while serving as a vehicle for vacuolar delivery of tetrameric Ams1.     

Water quality studies on Buckingham Canal (Madras,India) — A discriminant analysis

Stepwise multiple discriminant analysis was applied to a two year water quality monitoring study of the Buckingham Canal at Madras, India. The variables were divided into

1. physical and chemical
2. pollutants; and
3. biological

to test which group is efficient in discriminating stations which differ in water quality. Biological variables obtained higher loadings on the discriminant functions than the other two groups. The derived discriminant functions were tested for their discriminating power through the classification phase with the same raw data. The \lsHit and Misses\rs tables of classification indicate that 81.660% of the samples were correctly classified by the biological variables, against only 46.660% of the samples in the case of physical and chemical variables and 48.33% in the case of pollutants.     

Ion pairs in alpha helices

A survey of 47 globular proteins was made to determine the probability of occurrence of ion pairs separated by 1,2,3,... and 8 residues in the alpha helices. As a control, the probability of occurrence of like charged pairs was also determined. The survey showed that ion pairs of the type i,i +/- 3 and i, i +/- 4 are the most predominant. Such a preference was not observed for like charged pairs. The observed frequency of ion pairs is significantly greater than their expected frequency. The normalized frequencies of occurrence of the ion pairs were also found to increase generally with the helix length. These results indicate that the ion pairs may contribute to the stability of solvent-exposed alpha helices. Since the stabilization of protein secondary structure enhances the stability of protein tertiary structure, these results may throw light on the mechanism of protein folding.