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© 2014. The 70-kDa heat shock protein (HSP70) is known to protect the brain from injury through multiple mechanisms. We investigated the effect of pharmacological HSP70 induction in experimental traumatic brain injury (TBI). 3-month-old male C57/B6 mice were given 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) intraperitoneally (IP, 2. mg/kg) or intracerebroventricularly (ICV, 1. μg/kg) to determine whether HSP70 could be induced in the brain. Mice were subjected to TBI via cortical controlled impact, and were treated with 17-AAG (or vehicle) IP according to one of two treatment regimens: (1) 2. mg/kg at the time of injury, (2) a total of three doses (4. mg/kg) at 2 and 1. d prior to TBI and again at the time of injury. Brains were assessed for HSP70 induction, hemorrhage volume at 3. d, and lesion size at 14. d post-injury. Immunohistochemistry showed that both IP and ICV administration of 17-AAG increased HSP70 expression primarily in microglia and in a few neurons by 24. h but not in astrocytes. 17-AAG induced HSP70 in injured brain tissue as early as 6. h, peaking at 48. h and largely subsiding by 72. h after IP injection. Both treatment groups showed decreased hemorrhage volume relative to untreated mice as well as improved neurobehavioral outcomes. These observations indicate that pharmacologic HSP70 induction may prove to be a promising treatment for TBI.

The early growth response-1 (Egr-1) is crucial in many cell regulatory processes related to the progression of human cancers. Its overexpression has been demonstrated in variable human cancers and may have prognostic significance. The aims of this current study were to evaluate whether Egr-1 affects invasive and oncogenic phenotypes of human gastric cancer cells, and to examine the relationships between its expression and various clinicopathological parameters, including survival in human gastric cancer patients. We investigated the biologic role of Egr-1 in tumor cell behavior by using a small interfering RNA in human gastric cancer cell lines, AGS and TMK1. The expression of Egr-1 by reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry was investigated in human gastric cancer tissues. The knockdown of Egr-1 suppressed tumor cell migration and invasion in AGS and TMK1 cells. Egr-1 expression was significantly increased in human gastric cancer and metastatic lymph node tissues compared to the normal gastric mucosa and non-metastatic lymph node tissues. Positive expression of Egr-1 was significantly associated with tumor size, depth of invasion, lymph node metastasis, tumor stage and poor survival. These results indicate that Egr-1 is associated with human gastric cancer progression through the alteration of tumor cell behavior, such as migration and invasion. Egr-1 expression may help in predicting the clinical outcomes of human gastric cancer patients.

Traumatic brain injury (TBI) causes disruption of the blood brain barrier (BBB) leading to hemorrhage which can complicate an already catastrophic illness. Matrix metalloproteinases (MMPs) involved in the breakdown of the extracellular matrix may lead to brain hemorrhage. We explore the contribution of the 70 kDa heat shock protein (Hsp70) to outcome and brain hemorrhage in a model of TBI. Male, wildtype (Wt), Hsp70 knockout (Ko) and transgenic (Tg) mice were subjected to TBI using controlled cortical impact (CCI). Motor function, brain hemorrhage and lesion size were assessed at 3, 7 and 14 days. Brains were evaluated for the effects of Hsp70 on MMPs.In Hsp70 Tg mice, CCI led to smaller brain lesions, decreased hemorrhage and reduced expression and activation of MMPs compared to Wt. CCI also significantly decreased right-biased swings and corner turns in the Hsp70 Tg mice. Conversely, Hsp70 Ko mice had significantly increased lesion size, worsened brain hemorrhage and increased expression and activation of MMPs with worsened behavioral outcomes compared to Wt. Hsp70 is protective in experimental TBI. To our knowledge, this is the direct demonstration of brain protection by Hsp70 in a TBI model. Our data demonstrate a new mechanism linking TBI-induced hemorrhage and neuronal injury to the suppression of MMPs by Hsp70, and support the development of Hsp70 enhancing strategies for the treatment of TBI. © 2013 Elsevier Inc.

Intact protein masses can be measured directly from immobilized pH gradient (IPG) isoelectric focusing (IEF) gels loaded with mammalian and prokaryotic samples, as demonstrated here with murine macrophage and Methanosarcina acetivorans cell lysates. Mass accuracy and resolution is improved by employing instruments which decouple the desorption event from mass measurement; e.g., quadrupole time-of-flight instruments. MALDI in-source dissociation (ISD) is discussed as a means to pursue top-down sequencing for protein identification. Methods have been developed to enzymatically digest all proteins in an IEF gel simultaneously, leaving the polyacrylamide gel attached to its polyester support. By retaining all gel pieces and their placement relative to one another, sample handling and tracking are minimized, and comparison to 2-D gel images is facilitated. MALDI-MS and MS/MS can then be performed directly from dried, matrix-treated IPG strips following whole-gel trypsin digestion, bottom-up methodology. Side-to-side proteomics, highlighting the link between virtual and classical 2-D gel electrophoresis, is introduced to describe a method whereby intact masses are measured from one side (the IEF gel), while proteins are identified based on analyses performed from the other side (the SDS-PAGE gel).