Faculty Bibliography

OBJECTIVES: To observe the effect of ultrashortwave (USW) therapy on nerve regeneration after acellular nerve allografts(ANA) repairing the sciatic nerve gap of rats and discuss its acting mechanisms. METHODS: Sixteen Wistar rats weighing 180-220 g were randomly divided into four groups with four rats in each group: normal control group; acellular group (ANA, treated by hypotonic-chemical detergent, was applied for bridging a 10 mm-long sciatic nerve defect); USW group (After 24 h of ANA repairing the sciatic nerve gap, low dose USW was administrated for 7 min, once a day, 20 times a course of treatment, three courses of treatment in all); and autografts group. 12 weeks after operation, a series of examinations was performed, including electrophysiological methods, the restoring rate of tibialis anterior muscle wet weight, histopathological observation (myelinated nerve number, myelin sheath thickness, and axon diameter), vascular endothelial growth factor (VEGF) mRNA expression of spinal cord, and muscle at injury site, and analyzed statistically. RESULTS: Compared to acellular nerve allografts alone, USW therapy can increase nerve conductive velocity, the restoring rate of tibialis anterior muscle wet weight, myelinated nerve number, axon diameter, VEGF mRNA expression of spinal cord, and muscle at injury site, the difference is significant. There were no differences between USW group and autografts group except myelin sheath thickness. CONCLUSIONS: USW therapy can promote nerve axon regeneration and Schwann cells proliferation after ANA repairing the sciatic nerve gap of rats, the upregulation of VEGF mRNA expression of spinal cord and muscle may play an important role.

Transforming growth factor-beta1 (TGF-beta1) has potent physiologic and pathologic effects on a variety of cell types at subnanomolar concentrations. Platelets contain 40 times as much TGF-beta1 as other cells and secrete it as an inactive (latent) form in complex with latency-associated peptide (LAP), which is disulfide bonded via Cys33 to latent TGF-beta binding protein 1 (LTBP-1). Little is known about how latent TGF-beta1 becomes activated in vivo. Here we show that TGF-beta1 released from platelets or fibroblasts undergoes dramatic activation when subjected to stirring or shear forces, providing a potential mechanism for physiologic control. Thiol-disulfide exchange appears to contribute to the process based on the effects of thiol-reactive reagents and differences in thiol labeling of TGF-beta1 before and after stirring or shear. Activation required the presence of LTBP, as TGF-beta1 contained in complex with only LAP could not be activated by stirring when studied as either a recombinant purified protein complex or in the platelet releasates or sera of mice engineered to contain an LAP C33S mutation. Release and activation of latent TGF-beta1 in vivo was demonstrated in a mouse model 5 minutes after thrombus formation. These data potentially provide a novel mechanism for in vivo activation of TGF-beta1.

We identify a new class of decidable hybrid automata: namely, parallel compositions of semi-algebraic o-minimal automata. The class we consider is fundamental to hierarchical modeling in many exemplar systems, both natural and engineered. Unfortunately, parallel composition, which is an atomic operator in such constructions, does not preserve the decidability of reachability. Luckily, this paper is able to show that when one focuses on the composition of semi-algebraic o-minimal automata, it is possible to translate the decidability problem into a satisfiability problem over formulae involving both real and integer variables. While in the general case such formulae would be undecidable, the particular format of the formulae obtained in our translation allows combining decidability results stemming from both algebraic number theory and first-order logic over (?, 0, 1, +,*, <) to yield a novel decidability algorithm. From a more general perspective, this paper exposes many new open questions about decidable combinations of real/integer logics.

All common negative stains are salts of heavy metals. To remedy several technical defects inherent in the use of heavy metal compounds, this study investigates whether salts of the light metals sodium, magnesium, and aluminum can function as negative stains. Screening criteria require aqueous solubility at pH 7.0, formation of a smooth amorphous layer upon drying, and transmission electron microscope imaging of the 87-A (8.7-nm) lattice periodicity in thin catalase crystals. Six of 23 salts evaluated pass all three screens; detection of the protein shell in ferritin macromolecules indicates that light metal salts also provide negative staining of single particle specimens. Appositional contrast is less than that given by heavy metal negative stains; image density can be raised by increasing electron phase contrast and by selecting salts with phosphate or sulfate anions, thereby adding strong scattering from P or S atoms. Low-dose electron diffraction of catalase crystals negatively stained with 200 mM magnesium sulfate shows Bragg spots extending out to 4.4 A. Future experimental use of sodium phosphate buffer and magnesium sulfate for negative staining is anticipated, particularly in designing new cocktail (multicomponent) negative stains able to support and protect protein structure to higher resolution levels than are currently achieved.

Norepinephrine-deficient mice harbor a disruption of the gene for dopamine-beta-hydroxylase (DBH-KO). Corticotropin-releasing hormone knockout mice (CRH-KO) have markedly reduced HPA activity. The aim of the present work was to study how deficiency of DBH and CRH would affect tyrosine hydroxylase (TH), DBH, and phenylethanolamine N-methyltransferase (PNMT) gene expression and protein levels in the adrenal medulla (AM) and stellate ganglia (SG) of control and stressed mice. Both in AM and SG, single immobilization significantly increased TH and DBH mRNA and protein levels both in wild-type (WT) and CRH-KO mice. On the other hand, the stress-triggered increase in PNMT mRNA and protein levels seen in WT mice was absent in CRH-KO mice. DBH-KO mice are more sensitive to stress but survive a single 2 h restraint stress in a tube. The increase in TH mRNA levels induced by restraint stress in WT was not observed in DBH-KO mice. PNMT mRNA and especially PNMT protein levels were significantly elevated in AM of DBH-KO mice. In SG of DBH-KO mice, TH mRNA levels were not affected; however, PNMT gene expression was highly elevated. Thus, disruption of the DBH gene surprisingly blocks the stress-induced elevation of TH mRNA levels in AM but increases PNMT gene expression in both AM and SG. Our data indicate that adrenergic signaling is required for stress-induced increase in TH mRNA and that this signaling restrains stress-induced increase in PNMT mRNA. They also confirm that the HPA system plays a crucial role in the stress-induced regulation of PNMT gene expression.

Egr1, a transcription factor rapidly induced by various stimuli including stress, can elevate transcription of genes for the catecholamine biosynthetic enzymes TH and PNMT. To examine if Egr1 also regulates dopamine beta-hydroxylase (DBH) gene expression, PC12 cells were transfected with expression vector for full length or truncated inactive Egr1 and various DBH promoter-driven luciferase constructs. While Egr1 elevated TH promoter activity, DBH promoter activity was reduced. The reduction occurred as early as 4 h and reached maximal inhibition 16-40 h after transfection. Egr1 also reduced the expression of endogenous DBH mRNA and the induction of DBH promoter activity by cAMP. These effects were not observed with truncated Egr1 lacking the DNA binding domain. The first 247, but not 200, nucleotides of DBH promoter are sufficient for this suppression. Several putative Egr1 motifs were identified, and mutagenesis showed that the motif at -227/-224 is required. Binding of Egr1 to this region of the DBH promoter was verified by chromatin immunoprecipitation and electrophoretic mobility shift assays. This study demonstrates that DBH promoter contains at least one functional Egr1 motif; and indicates, for the first time, that Egr1 can play an inhibitory role in regulation of DBH gene transcription.

Classically, upon hypothalamic stimulation, adrenocorticotropic hormone (ACTH) is released from the pituitary and acts on melanocortin 2 receptors (MC2R) in the adrenal cortex, stimulating glucocorticoid synthesis and release. Our earlier studies suggested that ACTH might have a direct effect on sympathetic ganglia. To analyze further the involvement of ACTH in regulation of gene expression of norepinephrine (NE) biosynthetic enzymes, we examined the effect of bilateral adrenalectomy (ADX) of Sprague-Dawley male rats. Fourteen days post-ADX, as expected, plasma ACTH was elevated, and levels of tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH) and MC2R mRNAs in superior cervical ganglia (SCG), and TH mRNA in locus coeruleus (LC) were increased compared with sham-operated animals. To determine effect of pulsatile elevation of ACTH, corticosterone pellets were implanted to ADX rats. Similar to immobilization (IMO) stress ACTH injections to these animals caused a rise in ACTH in plasma and triggered elevation of TH and DBH mRNAs in SCG and in LC with single and repeated daily injections, and MC2R mRNA in SCG with single injections. To study the effect of ACTH in isolated cells, primary cultures of rat SCG were transfected with TH and DBH promoter constructs and treated with ACTH. In agreement with the in vivo data, ACTH elevated their promoter activities similar to levels triggered by cyclic AMP analog. ACTH in the human SK-N-SH neuroblastoma cells increased TH and DBH promoter activity and endogenous DBH mRNA levels. The results show that ACTH can have a direct effect on transcription and gene expression of NE biosynthetic enzymes even without contribution of adrenal hormones.

Stress triggers changes in gene expression mediating important adaptive and maladaptive responses. The full repertoire of genes whose expression in the adrenal medulla is altered by stress has not been previously determined. In this study, gene profiling (RAE 230 2.0 Affymetrix) was applied to elucidate global changes in gene expression in adrenal medulla of rats exposed to 2-h immobilization (IMO) stress once or repeatedly for 6 consecutive days. The number of transcripts significantly (P < 0.01) altered with single IMO (651 up, 487 down) was more than with repeated IMO (370 up, 195 down). The annotated transcripts were further analyzed and categorized. The largest numbers of changes were in mRNA levels in the transcription factor and cell signaling categories. Robust changes were also observed in transcripts related to growth factors, apoptosis, neurosecretion/neuropeptides, heat shock proteins, structural proteins, chemokines, cytokines, metabolism/lipid-metabolism, and proteases. Many (>80%) were uniquely induced by single IMO. About half of transcripts changed by repeated IMO were also responsive to single IMO. Pathway analysis was applied to identify direct interactions and common targets among gene products altered by single and repeated IMO. In this paper, we briefly describe the most pronounced changes observed, with emphasis on those that may provide new insight into the common and distinct mechanisms whereby the adrenal medulla responses to a first encounter with stress compared to repeated exposure to the same stressor.