Extracellular calcium influx stimulates metalloproteinase cleavage and secretion of heparin-binding EGF-like growth factor independently of protein kinase C

The phorbol ester, tetradecanoyl-phorbol 13-acetate (TPA), stimulates rapid proteolytic processing of the transmembrane, pro- form of heparin-binding epidermal growth factor-like growth factor (HB-EGF) at cell surfaces, suggesting the involvement of protein kinase C (PKC) isoforms in the HB-EGF secretion mechanism. To test this possibility, we expressed a chimeric protein, consisting of proHB-EGF fused to placental alkaline phosphatase (AP) near the amino terminus of processed HB-EGF, in NbMC-2 prostate epithelial cells. The proHB-EGF-AP chimera localized to plasma membranes and functioned as a diphtheria toxin receptor. Secreted HB-EGF-AP bound to heparin and exhibited potent growth factor activity. The presence of the AP moiety allowed highly quantitative measurements of cleavage-secretion responses of proHB-EGF to extracellular stimuli. As expected, rapid secretion of HB-EGF-AP was induced in a time- and dose-dependent manner by TPA. However, this was also observed with the Ca²⁺ionophore, ionomycin, suggesting the involvement of extracellular Ca²⁺ ions in the secretion mechanism. Ionomycin-induced secretion was inhibited by extracellular calcium chelation but not by the PKC inhibitors, GF109203X, staurosporine, or chelerythrine. The TPA-mediated secretion effect was inhibited by staurosporine, GF109203X, and by pretreatment with TPA, but not by calcium chelation. A small secretion response was induced by thapsigargin, which releases Ca²⁺ from intracellular stores, but this was completely eliminated by extracellular calcium chelation. Ionomycin- and TPA-induced HB-EGF-AP secretion was not dependent on the presence of the proHB-EGF cytoplasmic domain and was specifically inhibited by the metalloproteinase inhibitors 1,10-phenanthroline and tissue inhibitor of metalloproteinase-1 (TIMP-1). These data demonstrate that extracellular Ca²⁺ influx activates a membrane-associated metalloproteinase to process proHB-EGF by a pathway that does not require PKC. J. Cell. Biochem. 69:143–153, 1998. © 1998 Wiley-Liss, Inc.     

Phylogeny, diversification rates and species boundaries of Mesoamerican firs (Abies, Pinaceae) in a genus-wide context

The genus Abies is distributed discontinuously in the temperate and subtropical montane forests of the northern hemisphere. In Mesoamerica (Mexico and northern Central America), modern firs originated from the divergence of isolated mountain populations of migrating North American taxa. However, the number of ancestral species, migratory waves and diversification speed of these taxa is unknown. Here, variation in repetitive (Pt30204, Pt63718, and Pt71936) and non-repetitive (rbcL, rps18-rpl20 and trnL-trnF) regions of the chloroplast genome was used to reconstruct the phylogenetic relationships of the Mesoamerican Abies in a genus-wide context. These phylogenies and two fossil-calibrated scenarios were further employed to estimate divergence dates and diversification rates within the genus, and to test the hypothesis that, as in many angiosperms, conifers may exhibit accelerated speciation rates in the subtropics. All phylogenies showed five main clusters that mostly agreed with the currently recognized sections of Abies and with the geographic distribution of species. The Mesoamerican taxa formed a single group with species from southwestern North America of sections Oiamel and Grandis. However, populations of the same species were not monophyletic within this group. Divergence of this whole group dated back to the late Paleocene and the early Miocene depending on the calibration used, which translated in very low diversification rates (r_0.0 = 0.026-0.054, r_0.9 = 0.009-0.019 sp/Ma). Such low rates were a constant along the entire genus, including both the subtropical and temperate taxa. An extended phylogeographic analysis on the Mesoamerican clade indicated that Abies flinckii and A. concolor were the most divergent taxa, while the remaining species (A. durangensis, A. guatemalensis, A. hickelii, A. religiosa and A. vejari) formed a single group. Altogether, these results show that divergence of Mesoamerican firs coincides with a model of environmental stasis and decreased extinction rate, being probably prompted by a series of range expansions and isolation-by-distance.     

Transformation of pecan and regeneration of transgenic plants

A gene transfer system developed for walnut (Juglans regia L.) was successfully applied to pecan (Carya illinoensis [Wang] K. Koch). Repetitively embryogenic somatic embryos derived from open-pollinated seed of Elliott, Wichita, and Schley were co-cultivated with Agrobacterium strain EHA 101/pCGN 7001, which contains marker genes for beta-glucuronidase activity and resistance to kanamycin. Several modifications of the standard walnut transformation techniques were tested, including a lower concentration of kanamycin and a modified induction medium, but these treatments had no measurable effect on efficiency of transformation. Nineteen of the 764 viable inoculated embryos produced transgenic subclones; 13 of these were from the line Elliott6, 3 from Schley5/3, and 3 from Wichita9. Transgenic embryos of Wichita9 germinated most readily and three subclones were successfully micropropagated. Three transgenic plants of one of these subclones were obtained by grafting the tissue cultured shoots to seedling pecan rootstock in the greenhouse. Gene insertion, initially detected by GUS activity, was confirmed by detection of integrated T-DNA sequences using Southern analysis.     

Insulin/IGF-I Signaling Pathways Enhances Tumor Cell Invasion through Bisecting GlcNAc N-glycans Modulation. An Interplay with E-Cadherin

Changes in glycosylation are considered a hallmark of cancer, and one of the key targets of glycosylation modifications is E-cadherin. We and others have previously demonstrated that E-cadherin has a role in the regulation of bisecting GlcNAc N-glycans expression, remaining to be determined the E-cadherin-dependent signaling pathway involved in this N-glycans expression regulation. In this study, we analysed the impact of E-cadherin expression in the activation profile of receptor tyrosine kinases such as insulin receptor (IR) and IGF-I receptor (IGF-IR). We demonstrated that exogenous E-cadherin expression inhibits IR, IGF-IR and ERK 1/2 phosphorylation. Stimulation with insulin and IGF-I in MDA-MD-435 cancer cells overexpressing E-cadherin induces a decrease of bisecting GlcNAc N-glycans that was accompanied with alterations on E-cadherin cellular localization. Concomitantly, IR/IGF-IR signaling activation induced a mesenchymal-like phenotype of cancer cells together with an increased tumor cell invasion capability. Altogether, these results demonstrate an interplay between E-cadherin and IR/IGF-IR signaling as major networking players in the regulation of bisecting N-glycans expression, with important effects in the modulation of epithelial characteristics and tumor cell invasion. Here we provide new insights into the role that Insulin/IGF-I signaling play during cancer progression through glycosylation modifications.     

Transgenic Fluorescent Plasmodium cynomolgi Liver Stages Enable Live Imaging and Purification of Malaria Hypnozoite-Forms

A major challenge for strategies to combat the human malaria parasite Plasmodium vivax is the presence of hypnozoites in the liver. These dormant forms can cause renewed clinical disease after reactivation through unknown mechanisms. The closely related non-human primate malaria P. cynomolgi is a frequently used model for studying hypnozoite-induced relapses. Here we report the generation of the first transgenic P. cynomolgi parasites that stably express fluorescent markers in liver stages by transfection with novel DNA-constructs containing a P. cynomolgi centromere. Analysis of fluorescent liver stages in culture identified, in addition to developing liver-schizonts, uninucleate persisting parasites that were atovaquone resistant but primaquine sensitive, features associated with hypnozoites. We demonstrate that these hypnozoite-forms could be isolated by fluorescence-activated cell sorting. The fluorescently-tagged parasites in combination with FACS-purification open new avenues for a wide range of studies for analysing hypnozoite biology and reactivation.     

Risk Perception and Risk-Taking Behaviour during Adolescence: The Influence of Personality and Gender

This study investigated the influence of personality characteristics and gender on adolescents’ perception of risk and their risk-taking behaviour. Male and female participants (157 females: 116 males, aged 13–20) completed self-report measures on risk perception, risk-taking and personality. Male participants perceived behaviours as less risky, reportedly took more risks, were less sensitive to negative outcomes and less socially anxious than female participants. Path analysis identified a model in which age, behavioural inhibition and impulsiveness directly influenced risk perception, while age, social anxiety, impulsiveness, sensitivity to reward, behavioural inhibition and risk perception itself were directly or indirectly associated with risk-taking behaviour. Age and behavioural inhibition had direct relationships with social anxiety, and reward sensitivity was associated with impulsiveness. The model was representative for the whole sample and male and female groups separately. The observed relationship between age and social anxiety and the influence this may have on risk-taking behaviour could be key for reducing adolescent risk-taking behaviour. Even though adolescents may understand the riskiness of their behaviour and estimate their vulnerability to risk at a similar level to adults, factors such as anxiety regarding social situations, sensitivity to reward and impulsiveness may exert their influence and make these individuals prone to taking risks. If these associations are proven causal, these factors are, and will continue to be, important targets in prevention and intervention efforts.     

Serotonin transporter: gene, genetic disorders, and pharmacogenetics

The highly evolutionarily conserved serotonin transporter (SERT) regulates the entire serotoninergic system and its receptors via modulation of extracellular fluid serotonin concentrations. Differences in SERT expression and function produced by three SERT genes and their variants show associations with multiple human disorders. Screens of DNA from patients with autism, ADHD, bipolar disorder, and Tourette's syndrome have detected signals in the chromosome 17q region where SERT is located. Parallel investigations of SERT knockout mice have uncovered multiple phenotypes that identify SERT as a candidate gene for additional human disorders ranging from irritable bowel syndrome to obesity. Replicated studies have demonstrated that the SERT 5'-flanking region polymorphism SS genotype is associated with poorer therapeutic responses and more frequent serious side effects during treatment with antidepressant SERT antagonists, namely, the serotonin reuptake inhibitors (SRIs).     

Left ventricular hypertrophy is prevalent in Sprague-Dawley rats

Unrecognized cardiovascular abnormalities may confound the interpretation of research data collected using rats. However, although SPF rat colonies are screened for microbes and kept under standardized environmental conditions, their cardiovascular status is largely unknown. We recently performed surgery on anesthetized 80-d-old Sprague-Dawley rats and observed a high mortality that could not be attributed to the procedures or preceding treatments. Upon necropsy, cardiomyopathy was readily apparent in a substantial proportion of these rats. To further evaluate the nature of this condition, we evaluated the histology and morphology of hearts from both Sprague-Dawley and Lewis rats. Compared with Lewis rats, Sprague-Dawley rats had greater left ventricular wall thickness and larger cardiomyocyte cell size. Severe left ventricle hypertrophy was present in 38% of young adult Sprague-Dawley rats. These findings may have implications for research models that use Sprague-Dawley rats.     

Body temperature variation controls pre-mRNA processing and transcription of antiviral genes and SARS-CoV-2 replication

Antiviral innate immunity represents the first defense against invading viruses and is key to control viral infections, including SARS-CoV-2. Body temperature is an omnipresent variable but was neglected when addressing host defense mechanisms and susceptibility to SARS-CoV-2 infection. Here, we show that increasing temperature in a 1.5°C window, between 36.5 and 38°C, strongly increases the expression of genes in two branches of antiviral immunity, nitric oxide production and type I interferon response. We show that alternative splicing coupled to nonsense-mediated decay decreases STAT2 expression in colder conditions and suggest that increased STAT2 expression at elevated temperature induces the expression of diverse antiviral genes and SARS-CoV-2 restriction factors. This cascade is activated in a remarkably narrow temperature range below febrile temperature, which reflects individual, circadian and age-dependent variation. We suggest that decreased body temperature with aging contributes to reduced expression of antiviral genes in older individuals. Using cell culture and in vivo models, we show that higher body temperature correlates with reduced SARS-CoV-2 replication, which may affect the different vulnerability of children versus seniors toward severe SARS-CoV-2 infection. Altogether, our data connect body temperature and pre-mRNA processing to provide new mechanistic insight into the regulation of antiviral innate immunity.     

The chronic lymphatic leukemia lymphocyte: Correlation of functional,metabolic, and surface immunoglobulin characteristics

This study determined the correlation between the functional capacity of chronic lymphatic leukemia lymphocytes as determined by their response to nonspecific mitogens with their glucose metabolism and surface immunoglobulin characteristics. A majority of patients (12) were found to have lymphocytes with impaired transformation to both PHA and pokeweed mitogens. These cells also had impaired glucose metabolism in unstimulated cultures and failed to have the striking increase in glucose metabolism in response to mitogens which is characteristic of normal lymphocytes. Most of these lymphocytes had IgM surface immunoglobulins. However, we were not able to demonstrate surface immunoglobulins on the lymphocytes of one patient in this group. Two patients were found to have lymphocytes with normal lymphoblastic transformation to PHA and impaired transformation to pokeweed suggesting cells of T origin. The glucose metabolism of these lymphocytes were less impaired in unstimulated cultures than those of the other patients and had a striking increment in glucose metabolism in response to PHA similar to normal lymphocytes. Unexpectedly, these lymphocytes were found to have IgG on their surface suggesting cells of B origin. These results indicate that there may be two groups of CLL patients with clinically similar disease in whom the functional and metabolic characteristics of the lymphocytes are distinct and that the surface immunoglobulin characteristic of lymphocytes may not always predict their functional characteristic.