Molecular characterization of a high A2 beta thalassemia by direct sequencing of single strand enriched amplified genomic DNA

Two families, one of Anglo-Saxon-Dutch descent, and the other, West Indian black, have an atypical beta thalassemia characterized by an unusually high level of Hb A2 in the heterozygous state. Restriction endonuclease mapping showed a deletion of about 1.35 kilobase (kb) in the 5' region of the beta globin gene. Direct sequencing of a specific region of genomic DNA amplified by a new modification of the polymerase chain reaction defined the deletion to be 1,393 base pairs (bp) and to be the same in both families. The deletion extends from 485 bp 5' to the mRNA CAP site to the middle of the second intervening sequence. This deletion, together with three others previously described that remove the 5' end of the beta gene but leave the delta gene intact, are all associated with unusually high levels of Hb A2 in the heterozygous state.     

Astrocytes enhance glioblastoma growth

Glioblastoma (GBM) is a deadly disease with a need for deeper understanding and new therapeutic approaches. The microenvironment of glioblastoma has previously been shown to guide glioblastoma progression. In this study, astrocytes were investigated with regard to their effect on glioblastoma proliferation through correlative analyses of clinical samples and experimental in vitro and in vivo studies. Co-culture techniques were used to investigate the GBM growth enhancing potential of astrocytes. Cell sorting and RNA sequencing were used to generate a GBM-associated astrocyte signature and to investigate astrocyte-induced GBM genes. A NOD scid GBM mouse model was used for in vivo studies. A gene signature reflecting GBM-activated astrocytes was associated with poor prognosis in the TCGA GBM dataset. Two genes, periostin and serglycin, induced in GBM cells upon exposure to astrocytes were expressed at higher levels in cases with high “astrocyte signature score”. Astrocytes were shown to enhance glioblastoma cell growth in cell lines and in a patient-derived culture, in a manner dependent on cell–cell contact and involving increased cell proliferation. Furthermore, co-injection of astrocytes with glioblastoma cells reduced survival in an orthotopic GBM model in NOD scid mice. In conclusion, this study suggests that astrocytes contribute to glioblastoma growth and implies this crosstalk as a candidate target for novel therapies.     

Increased levels of Homer1b/c and Shank1a in the post-synaptic density of spinal dorsal horn neurons are associated with neuropathic pain in rats

Activity-dependent plasticity in the spinal dorsal horn may underlie the development of neuropathic pain following peripheral nerve injury. In this study we examined whether the expression and loss of behavioral signs of neuropathic pain were associated with changes in the content of the scaffolding proteins Homer and Shank in the post-synaptic density (PSD) of the spinal dorsal horn. In animals exhibiting thermal hyperalgesia and differential weight-bearing behavior 7 days after loose ligation of the sciatic nerve the levels of Homer1b/c and Shank1a were significantly greater than in control, uninjured or sham-operated animals. These greater levels were specifically a reflection of increases in the injured, ipsilateral and not contralateral dorsal horn. In contrast, there were no differences in the PSD content of Homer1b/c and Shank1a in the dorsal horn of control or sham-operated animals and ligated animals in which the thermal hyperalgesia and differential weight-bearing behavior had disappeared 28 days after the loose ligation. These data revealed a close association between the expression and loss of allodynia and hyperalgesia with changes in the levels of Homer1b/c and Shank1a in the spinal dorsal horn. The reversible shift in the content of scaffolding proteins in the PSD may have important implications for the development of injury-elicited neuropathic pain.     

Chronic exposure to lead acetate affects the development of protein kinase C activity and the distribution of the PKCgamma isozyme in the rat hippocampus.

This study has examined the effect of chronic inorganic lead exposure on phospholipid-dependent protein kinase C (PKC) activity, and the distribution of its alpha (alpha), beta II (betaII), gamma (gamma), and zeta (zeta) isozymes in subcellular fractions of the developing rat hippocampus. Dams were exposed to either 0 or 1000 ppm lead acetate in their drinking water for one week and mated. Offspring were exposed to lead in utero, via lactation, and directly in the drinking water after weaning. The offspring were sacrificed at postnatal days 1 (P1), 8 (P8), 15 (P15), and 29 (P29). PKC activity was determined in the post-synaptosomal supernatant (PSS) and synaptosomal (P-2) membrane fractions by an in vitro assay using histone as the phosphate acceptor. The selected PKC isozymes were detected by immunoblotting techniques. In control animals, PKC activity (pmole/min/mg total protein) in both subcellular fractions substantially increased between P1 and P8. In chronically exposed rats exhibiting clinically relevant blood lead concentrations, this marked increase in PKC activity on P8 was significantly attenuated in both subcellular fractions. On this postnatal day, the amount of immunodetectable PKC gamma was significantly higher in the synaptosomal membrane fraction of lead-exposed rats. Other isozymes were unaffected. These results imply that in lead-exposed animals the PKC gamma isozyme was inactive even though it was associated with the membrane. These results also suggest that prolonged exposure to the heavy metal attenuated PKC activity at an important developmental time to potentially adversely affect normal hippocampal function.