Reciprocal regulation of neu tyrosine kinase activity and caveolin-1 protein expression in vitro and in vivo. Implications for human breast cancer

Neu (c-erbB2) is a proto-oncogene product that encodes an epidermal growth factor-like receptor tyrosine kinase. Amplification of wild-type c-Neu and mutational activation of Neu (Neu T) have been implicated in oncogenic transformation of cultured fibroblasts and mammary tumorigenesis in vivo. Here, we examine the relationship between Neu tyrosine kinase activity and caveolin-1 protein expression in vitro and in vivo. Recent studies have suggested that caveolins may function as negative regulators of signal transduction. Our current results show that mutational activation of c-Neu down-regulates caveolin-1 protein expression, but not caveolin-2, in cultured NIH 3T3 and Rat 1 cells. Conversely, recombinant overexpression of caveolin-1 blocks Neu-mediated signal transduction in vivo. These results suggest a reciprocal relationship between c-Neu tyrosine kinase activity and caveolin-1 protein expression. We next analyzed a variety of caveolin-1 deletion mutants to map this caveolin-1-dependent inhibitory activity to a given region of the caveolin-1 molecule. Results from this mutational analysis show that this functional in vivo inhibitory activity is contained within caveolin-1 residues 32-95. In accordance with these in vivo studies, a 20-amino acid peptide derived from this region (the caveolin-1 scaffolding domain) was sufficient to inhibit Neu autophosphorylation in an in vitro kinase assay. To further confirm or refute the relevance of our findings in vivo, we next examined the expression levels of caveolin-1 in mammary tumors derived from c-Neu transgenic mice. Our results indicate that dramatic reduction of caveolin-1 expression occurs in mammary tumors derived from c-Neu-expressing transgenic mice and other transgenic mice expressing downstream effectors of Neu-mediated signal transduction, such as Src and Ras. Taken together, our data suggest that a novel form of reciprocal negative regulation exists between c-Neu and caveolin-1.     

Electromyographic response of the labial muscles during normal liquid swallows using a spoon, a straw, and a cup

PURPOSE: To determine if ethnic and gender differences in smoking (lifetime prevalence and 30-day prevalence) exist among a cohort of Asian, black, Hispanic, and white inner-city adolescents during the 3-year middle school period. METHODS: Students in 22 urban schools completed self-report questionnaires and provided carbon monoxide breath samples at three annual assessments. Chi-square analyses were conducted to test for associations between ethnic group (Asian, black, Hispanic, and white) and smoking and to test for gender differences in smoking within each ethnic group. Additional analyses examined differences in smoking between two Hispanic subgroups (Puerto Rican and Dominican). RESULTS: Ethnicity was associated with lifetime smoking prevalence at all three assessment points but was only associated with 30-day smoking prevalence at the 2-year follow-up. However, there were no differences in smoking between Puerto Rican and Dominican youth. Black girls reported higher lifetime smoking prevalence than black boys at all three assessments. At the 2-year follow-up, Asian boys reported higher lifetime smoking prevalence than Asian girls; Hispanic girls reported higher 30-day prevalence than Hispanic boys. CONCLUSIONS: White and Hispanic adolescents were at higher risk for smoking relative to Asian and black adolescents. With the exception of white youth, gender differences were found within each ethnic group.     

Soluble adhesion molecules in CSF are increased in children with severe head injury

Leukocyte-endothelial adhesion molecules, critical to the development of acute inflammation, are expressed in brain as part of the acute inflammatory response to traumatic brain injury (TBI). We measured the concentrations of the adhesion molecules P-selectin, ICAM-1, E-selectin, L-selectin, and VCAM-1 in ventricular cerebrospinal fluid (CSF) from children with severe TBI (Glasgow coma score < 8) and compared these findings with those from children with bacterial meningitis. P-selectin, an adhesion molecule associated with ischemia/reperfusion, was increased in children with TBI versus meningitis and control. Univariate and multivariate regression analyses demonstrated associations between CSF P-selectin and child abuse and age of < 4 years, and a significant, independent association between CSF intercellular adhesion molecule-1 (ICAM-1) and child abuse. These results are consistent with a specific acute inflammatory component to TBI in children. Future studies of secondary injury mechanisms and therapy after TBI should assess on the roles of P-selectin and ICAM-1 in injury and repair processes in brain after TBI.     

MgATP-dependent and MgATP-independent [3H]noradrenaline release from perforated synaptosomes both use N-ethylmaleimide-sensitive fusion protein

In streptolysin-O (SLO)-perforated rat brain cortical synaptosomes, Ca2+-induced [3H]noradrenaline (3H-NA) release began with a phase lasting about 1 min that did not depend on MgATP. Subsequent release became increasingly MgATP-dependent. The first phase involved release from previously "primed" synaptic vesicles. MgATP-dependent release, on the other hand, was release from unprimed vesicles that needed to be primed by ATP hydrolysis before they could be fused with the presynaptic membrane. Vesicle depriming was detected by observing that the initial release decreased when the synaptosomes were perforated and incubated for 2 min in the absence of MgATP before increasing Ca2+ to promote release. One millimolar N-ethylmaleimide (NEM) inhibited both MgATP-dependent and MgATP-independent release at all times of incubation (0.5-5 min), and inhibition by NEM was partially reversed at short (0.5 min) and longer (5 min) times by adding intact N-ethylmaleimide sensitive fusion protein (NSF) to the perforated synaptosomes. Polyclonal antibodies against the N-terminal domain of NSF produced dose-dependent inhibition of Ca2+-induced 3H-NA release. This inhibition occurred in both early and late release phases and was highly significant at early times if the perforated synaptosomes were preincubated for 2 min with anti-NSF. These results indicate participation of NSF both after vesicular fusion, probably for separation of SNARE proteins in v/t-SNARE complexes before endocytosis, and, surprisingly, after docking, possibly to maintain vesicles in a primed state and reverse depriming during regulated secretion.     

Fungal beta-tubulin, expressed as a fusion protein, binds benzimidazole and phenylcarbamate fungicides

Benzimidazoles are important antitubulin agents used in veterinary medicine and plant disease control. Resistance is a practical problem correlated with single amino acid changes in beta-tubulin and is often linked to greater sensitivity to phenylcarbamates. This negative cross-resistance creates opportunities for durable antiresistance strategies. Attempts to understand the molecular basis of benzimidazole resistance have been hampered by the inability to purify tubulin from filamentous fungi. We have overcome some of these problems by expressing beta-tubulin as a fusion with a maltose binding protein. This fusion protein is soluble, and we confirm for the first time using a gel filtration assay that benzimidazoles indeed bind to beta-tubulin. This binding is reduced by the mutation Glu198-->Gly198, which also confers resistance. Binding of phenylcarbamates is the complete opposite, reflecting their biological activity and the negative cross-resistance. This suggests that the fungicide binding sites fold correctly in the fusion protein.     

Conformational behavior of the HAV-VP3(110-121) peptidic sequence and synthetic analogs in membrane environments studied by CD and computational methods

The present study was undertaken to examine the structural features that may be important to explain the immunogenicity of the (110-121) peptide sequence (FWRGDLVFDFQV) of VP3 capsid protein of hepatitis A virus. A conformational analysis of the preferred conformations by CD and molecular mechanics was carried out. Present results suggest that the interaction with liposomes as biomembrane model induces and stabilizes the amphipathic beta-structure of the peptide. To study the contribution of amino acid replacements at the RGD tripeptide as well as the influence of the peptide chain length on peptide conformation, solid-phase peptide synthesis of several peptide analogs was carried out and the peptide conformation was studied using CD spectroscopy. The results show that the RGD sequence is necessary to induce the beta-structure in the presence of liposomes.     

An epithelial exclusion technique using the CO2 laser for the treatment of periodontal defects

When treating osseous defects associated with periodontitis, the healed result is a compromised regeneration of the attachment apparatus from epithelial downgrowth. This article demonstrates a laser ablation technique for excluding the epithelium from contacting the root surface of the periodontal wound. In accordance with the principles of guided tissue regeneration, the epithelium should be excluded for at least 30 days after surgical therapy. A series of case reports demonstrate the technique and the 6-month results that can be obtained using this approach. The regenerated tissue is confirmed through reentry procedures and radiographs.     

Familial partial epilepsy with variable foci: a new partial epilepsy syndrome with suggestion of linkage to chromosome 2

Traditional T2-based imaging techniques are geared toward imaging long-T2 species. Traditional techniques are, therefore, not optimal in clinical situations where the information of interest lies in the short-T2 species. T2-selective RF excitation (TELEX) is a technique for obtaining a T2-based contrast that highlights short-T2 values while suppressing long-T2 values-opposite to traditional T2 contrast. Previously, TELEX has been demonstrated qualitatively to highlight only very short-T2 values (T2 approximately 0.001 s). When applied to longer T2 values (T2 > or = 0.01 s), TELEX becomes sensitive to deltaB0 non-uniformities. This restricts its application to problems in which the T2 of interest is very short. In this study, TELEX is characterized quantitatively. Furthermore, a bandwidth broadening scheme is developed that reduces the deltaB0 sensitivity of TELEX. This permits the technique to be applied to longer T2 values. The capabilities and limitations of a practical implementation of TELEX are discussed.     

The voltage-dependent conductances of rat neocortical layer I neurons

Whole cell patch-clamp techniques were used to study voltage-dependent sodium (Na+), calcium (Ca2+), and potassium (K+) conductances in acutely isolated neurons from cortical layer I of adult rats. Layer I cells were identified by means of gamma-aminobutyric acid (GABA) immunocytochemistry. Positive stainings for the Ca2+-binding protein calretinin in a subset of cells, indicated the presence of Cajal-Retzius (C-R) cells. All investigated cells displayed a rather homogeneous profile of voltage-dependent membrane currents. A fast Na+ current activated at about -45 mV, was half-maximal steady-state inactivated at -66.6 mV, and recovery from inactivation followed a two-exponential process (tau1 = 8.4 ms and tau2 = 858.8 ms). Na+ currents declined rapidly with two voltage-dependent time constants, reaching baseline current after some tens of milliseconds. In a subset of cells (< 50%) a constant current level of < 65 pA remained at the end of a 90 ms step. A transient outward current (Ifast) activated approximately -40 mV, declined rapidly with a voltage-insensitive time constant (tau approximately 350 ms) and was relatively insensitive to tetraethylammonium (TEA, 20 mM). Ifast was separated into two components based on their sensitivity to 4-aminopyridine (4-AP): one was blocked by low concentrations (40 microM) and a second by high concentrations (6 mM). After elimination of Ifast by a conditioning prepulse (50 ms to -50 mV), a slow K+ current (I(KV)) could be studied in isolation. I(KV) was only moderately affected by 4-AP (6 mM), while TEA (20 mM) blocked most (> 80%) of the current. I(KV) activated at about -40 mV, declined monoexponentially in a voltage-dependent manner (tau approximately 850 ms at -30 mV), and revealed an incomplete steady-state inactivation. In addition to Ifast and I(KV), indications of a Ca2+-dependent outward current component were found. When Na+ currents, Ifast, and I(KV) were blocked by tetrodotoxin (TTX, 1 microM), 4-AP (6 mM) and TEA (20 mM) an inward current carried by Ca2+ was found. Ca2+ currents activated at depolarized potentials at about -30 mV, were completely blocked by 50 microM cadmium (Cd2+), were sensitive to verapamil (approximately 40% block by 10 microM), and were not affected by nickel (50 microM). During current clamp recordings, isolated layer I neurons displayed fast spiking behaviour with short action potentials (approximately 2 ms, measured at half maximal amplitude) of relative small amplitude (approximately 83 mV, measured from the action potential threshold).     

Molecular ordering of interfacially localized tryptophan analogs in ester- and ether-lipid bilayers studied by 2H-NMR

Perdeuterated indole-d6 and N-methylated indole-d6 were solubilized in lamellar liquid crystalline phases composed of either 1,2-diacyl-glycero-3-phosphocholine (14:0)/water or 1,2-dialkyl-glycero-3-phosphocholine(14:0/water. The molecular ordering of the tryptophan analogs was determined from deuteron quadrupole splittings observed in 2H-NMR spectra on macroscopically aligned lipid bilayers. NMR spectra were recorded with the bilayers oriented perpendicular to or parallel with the external magnetic field, and the values of the splittings differed by a factor of 2 between these distinct orientations, indicating fast rotational motion of the molecules about an axis parallel to the bilayer normal. In all cases the splittings were found to decrease with increasing temperature. Relatively large splittings were observed in all systems, demonstrating that the tryptophans partition into a highly anisotropic environment. Solubilization most likely occurs at the lipid/water interface, as indicated by 1H-NMR chemical shift studies. The 2H-NMR spectra obtained for each analog were found to be rather similar in ester and ether lipids, but with smaller splittings in the ether lipid under similar conditions. The difference was slightly less for the indole molecule. Furthermore, in both lipid systems the positions of the splittings from indole were different from those of N-methyl indole. The results suggest that 1) the tryptophan analogs are solubilized in the interfacial region of the lipid bilayer, 2) the behavior may be modulated by hydrogen bonding in the case of indole, and 3) hydrogen bonding with the lipid carbonyl groups is not likely to play a major role in the solubilization of single indole molecules in the ester lipid bilayer interface.