Differential consequences of lateral and central fluid percussion brain injury on receptor coupling in rat hippocampus

We have identified alterations in the responses of muscarinic and metabotropic receptors in rat hippocampus that persist for at least 15 days after central fluid percussion injury. This study compares the effect of lateral fluid percussion and central fluid percussion on these responses. Moderate injury was obtained by displacement and deformation of the brain within the closed cranial cavity using a fluid percussion device positioned either centrally or laterally. Carbachol and (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD)-stimulated polyphosphoinositide (PPI) hydrolysis was assayed in hippocampus from injured and sham-injured controls at 15 days following injury. At 15 days after central fluid percussion traumatic brain injury (TBI), the response to carbachol was enhanced by 30% and the response to trans-ACPD was enhanced by 75% compared to sham-injured animals. At 15 days after lateral fluid percussion TBI the response to trans-ACPD was enhanced by 40% both ipsilateral and contralateral to the side of injury. In contrast, the response to carbachol was enhanced by 29% contralateral to the side of injury but was diminished by 12% ipsilateral to the side of injury. Cresyl violet staining shows no hippocampal cell death after central fluid percussion injury or on the side contralateral to lateral fluid percussion injury but on the ipsilateral side cell death was identified in hippocampal area CA3. Thus, abnormal hippocampal cell signaling through the phosphoinositide pathway occurs in the absence of cell death and may contribute to cognitive impairment.     

Physiologic correlates to running performance in pre-pubertal distance runners

In adults, four major variables have been shown to be associated with success in distance running performance: submaximal oxygen consumption (running economy), peak oxygen consumption (Peak VO2), ventilatory threshold (VT) and fractional utilisation (FU). The primary aim of this study was to describe the relationship between the 3000 m race times of run-trained prepubertal boys to these four variables. Thirteen male run-trained pre-pubertal boys (age 11.7 +/- 1.1 yrs, mean +/- SD), volunteered to take part in a 3000 m time trial and laboratory assessment, consisting of treadmill running at four submaximal speeds (8, 9.6, 11.2 and 12.8 km.h-1) as well as a peak VO2 test. The group demonstrated a heterogeneous array of peak VO2 data. A high level of association (p < 0.05) was found between mass-relative peak VO2 and 3000 m time trial results (r = -0.83). In addition ventilatory threshold expressed as %peak VO2, VO2 at VT and estimated velocity at VT was also highly related to 3000 m time trial (r = -0.78, -0.77 and -0.77) respectively. Fractional utilisation (%peak VO2) was significantly (p < 0.05) associated with race time at the final two submaximal running speeds only (11.2 and 12.8 km.h-1) (r = 0.61 and 0.67, respectively). Respiratory Exchange Ratio (RER) was also found to be significantly (p < 0.05) associated with 3000 m race time at 11.2 and 12.8 km.h-1. Overall peak VO2 appeared to be the single most important factor associated with success at 3000 m.     

Fibrolamellar carcinoma of the liver: composition of the extracellular matrix and expression of cell-matrix and cell-cell adhesion molecules

We have analyzed the composition of the tumor stroma and the expression of cell-matrix and cell-cell adhesion molecules in 11 cases of fibrolamellar carcinoma of the liver (FLC), in comparison with 34 cases of hepatocellular carcinoma and 8 cases of focal nodular hyperplasia. Fibrolamellar carcinoma was characterized by the presence of large amounts of tenascin in tumor stroma and by the scarce expression of basement membrane components at the contact of neoplastic clusters. Like normal hepatocytes, neoplastic cells constantly expressed the alpha1 integrin chain, lacked the beta4 integrin chain, and coexpressed E-cadherin and the hepatocyte N-related cadherin. Abnormalities in the expression of cell adhesion molecules, including altered cadherin expression, alphaV integrin chain induction, and CD44 expression, were detected in the majority of cases. The composition of the tumor stroma and the pattern of expression of cell adhesion molecules in fibrolamellar carcinoma were reminiscent of those observed in grade III and grade IV hepatocellular carcinomas. Our results therefore show that, despite its slow local growth and good prognosis, fibrolamellar carcinoma expresses many characteristics usually associated with clinically aggressive malignancies. Further studies are needed to identify the factors responsible for the apparent dissociation between clinical behavior and biological characteristics in this tumor.     

Identification of a peptide of the guanosine triphosphate binding site within brain glutamate dehydrogenase isoproteins using 8-azidoguanosine triphosphate

Photoaffinity labeling with [gamma-32P]8N3GTP (8-azidoguanosine triphosphate) was used to identify the guanine binding peptides of the GTT binding site within two types of glutamate dehydrogenase isoproteins (GDH I and GDH II) isolated from bovine brain. 8N3GTP, without photolysis, mimicked the inhibitory properties of GTP on GDH I and GDH II activities. Saturation of photoinsertion of GDH isoproteins revealed an apparent Kd of 8 microM (GDH I) and 24 microM (GDH II) for [gamma-32P]8N3GTP. Ion exchange and reversed-phase high-performance liquid chromatography (HPLC) were used to isolate photolabel-containing peptides generated with trypsin. This identified a portion of the guanine binding domain within the GTP binding site is the region containing the sequence I-S-G-A-S-E-X-D-I-V-H-S-A-L-A-Y-T-M E-R (GDH I) and I-S-G-A-S-E-X-D-I-V-H-S-G-L-A-Y-T-M-E-R (GDH II). The symbol X indicates a position for which no phenylthiohydantoin-amino acid could be assigned. The missing residue, however, can be designated as a photolabeled lysine since the sequences including the lysine residue in question have a complete identity with those of the other GDH species known. Also, trypsin was unable to cleave the photolabeled peptide at this site. Photolabeling of these peptides was prevented by the presence of GTP during photolysis, while other nucleotides could not reduce the amount of photoinsertion as effectively as GTP. These results demonstrate selectivity of the photoprobe for the GTP binding site and suggest that the peptide identified using the photoprobe is located in the GTP binding domain of the brain GDH isoproteins.     

Inhibition of intratracheal lung cancer development by systemic delivery of E1A

Amplification or overexpression of HER-2/neu in human lung cancer has been correlated with poor prognosis and chemoresistance. We have previously reported that the adenovirus type 5 early region 1A (E1A) gene product can suppress HER-2/neu-mediated transformation phenotypes through inhibition of HER-2/neu expression. To find an efficient way to treat HER-2/neu-overexpressing lung cancer with E1A, a replication-deficient adenovirus containing the E1A gene, Ad.E1A(+), was used to transduce E1A into HER-2/neu-overexpressing and low expressing human lung cancer cell lines. Tumour cell growth in vitro and colony formation in soft agarose were greatly inhibited by Ad.E1A(+) transduction in HER-2/neu-overexpressing lung cancer cell lines. In HER-2/neu low expressing cell lines, E1A could not inhibit cell growth in vitro but could reduce the colony formation ability in soft agarose, indicating different effects of E1A in these two types of cancer cells. To test the therapeutic efficacy of E1A to lung cancer by systemic delivery in vivo, tumor-bearing mice were established by intratracheal injection of lung cancer cells and treated by i.v. tail injections of Ad.E1A(+). As a result, Ad.E1A(+) suppressed HER-2/neu overexpression and inhibited intratracheal lung cancer growth. However, no significant tumor suppression effect of Ad.E1A(+) was observed in mice bearing HER-2/neu low expressing cell line when the same therapeutic procedure was followed. Thus, we conclude that systemic delivery of Ad.E1A(+) can efficiently achieve therapeutic effect in HER-2/neu-overexpressing lung cancer in vivo.     

SCID-Hu mouse as a model for human lung HIV-1 infection

HIV induces a multi-organ infection with a dual tropism for both lymphocytes and monocytes/macrophages. The lung is a target both for HIV infection and HIV-related opportunistic infections. The SCID mouse has provided the opportunity to develop a small animal model for HIV infection. However, HIV-1 infection of the human fetal thymus and liver (SCID Liv/Thy) implanted in these mice occurred only after direct intraimplant injection of HIV-1 and the resultant HIV-1 infection was restricted to the human thymus. Here we report that human foetal lung can develop in SCID Liv/Thy mice resulting in the development of normal human alveolar and bronchiolar lung compartments which can be productively infected with cell-free HIV-1 virus, leading to a systemic and bifocal infection. This SCID-Hu model should be useful for studying AIDS physiopathology, human viruses with lung tropism and for helping to define gene therapy protocols in lung human cells in vivo.     

Salivary receptors for recombinant fimbrillin of Porphyromonas gingivalis

Fimbriae are considered important in the adherence and colonization of Porphyromonas gingivalis in the oral cavity. It has been demonstrated that purified fimbriae bind to whole human saliva adsorbed to hydroxyapatite (HAP) beads, and the binding appears to be mediated by specific protein-protein interactions. Recently, we expressed the recombinant fimbrillin protein (r-Fim) of P. gingivalis corresponding to amino acid residues 10 to 337 of the native fimbrillin (A. Sharma, H.T. Sojar, J.-Y. Lee, and R.J. Genco, Infect. Immun. 61:3570-3573, 1993). We examined the ability of individual salivary components to promote the direct attachment of r-Fim to HAP beads. Purified r-Fim was radiolabeled with 125I and incubated with HAP beads which were coated with saliva or purified individual salivary components. Whole, parotid, and submandibular-sublingual salivas increased the binding of 125I-r-Fim to HAP beads. Submandibular-sublingual saliva was most effective in increasing the binding of 125I-r-Fim to HAP beads (1.8 times greater than that to uncoated HAP beads). The binding of 125I-r-Fim to HAP beads coated with acidic proline-rich protein 1 (PRP1) or statherin was four and two times greater, respectively, than that to uncoated HAP beads. PRP1 and statherin molecules were also found to bind 125I-r-Fim in an overlay assay. The binding of intact P. gingivalis cells to HAP beads coated with PRP1 or statherin was also enhanced, by 5.4 and 4.3 times, respectively, over that to uncoated HAP beads. The interactions of PRP1 and statherin with 125I-r-Fim were not inhibited by the addition of carbohydrates or amino acids. PRP1 and statherin in solution did not show inhibitory activity on 125I-r-Fim binding to HAP beads coated with PRP1 or statherin. These results suggest that P. gingivalis fimbriae bind strongly through protein-protein interactions to acidic proline-rich protein and statherin molecules which coat surfaces.     

Effects of green tea and black tea on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone bioactivation, DNA methylation, and lung tumorigenesis in A/J mice

Previous studies in our laboratory showed that decaffeinated green tea and black tea extracts inhibited 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced tumorigenicity in A/J female mice. In order to understand the mechanism of the inhibitory action, we examined the effects of decaffeinated green tea, black tea, and tea components on the metabolic activation of NNK in vitro and in vivo in this animal model. When added to incubation mixtures containing mouse lung microsomes, decaffeinated green tea and black tea extracts and their fractions, at concentrations up to 0.4 mg/ml, inhibited NNK oxidation and NNK-induced DNA methylation. Among the tea components examined, (-)-epigallocatechin-3-gallate was the most potent inhibitor with 50% inhibitory concentrations of about 0.12 mM for both NNK oxidation and DNA methylation. At these concentrations, (-)-epigallocatechin-3-gallate inhibited the catalytic activities of several P450 enzymes and was more potent against P450 1A and 2B1 than 2E1. When decaffeinated green or black tea extracts were given to female A/J mice as the sole source of drinking fluid before an i.p. injection of NNK (100 mg/kg body weight), a statistically significant inhibition of lung DNA methylation, however, was not observed, although a significant reduction in lung tumor multiplicity was observed. The results suggest that, although inhibition of the metabolic activation of NNK and the subsequent DNA alkylation by tea extracts can be demonstrated in vitro, this mechanism may not be important for the inhibitory action of tea against lung tumorigenesis.