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J L Freeman

Publications and source records attributed to J L Freeman.

At least 55 records · Page 3Linked to original sources
PubMed1998

Regulation of the neutrophil respiratory burst oxidase. Identification of an activation domain in p67(phox).

Superoxide generation by the neutrophil respiratory burst oxidase (NADPH oxidase) can be reconstituted in a cell-free system using flavocytochrome b558 and the cytosolic proteins p47(phox), p67(phox), and Rac. p47(phox) functions as an adaptor protein; it increases the affinity of p67(phox) and Rac in the NADPH oxidase complex, but is not essential when high concentrations of these proteins are used (Freeman, J. L., and Lambeth, J. D. (1996) J. Biol. Chem. 271, 22578-22582), implying that p67(phox) and/or Rac directly regulates enzyme activity. Herein, we describe an activation domain in p67(phox) that is essential for NADPH oxidase activity. A series of C-terminal truncation mutants of p67(phox) showed that residues 211 to the C terminus (residue 526) are not needed for cell-free activity. However, shorter truncations were inactive, pointing to an activation domain within the region spanning residues 199-210. p67(phox) mutated at single amino acid residues within this region showed diminished activity, and p67(phox) V204A was completely inactive. The effects of mutations on activity were independent of p47(phox), and mutations did not affect the binding of p67(phox) to Rac. In the presence of wild-type p67(phox), the V204A mutant was a potent inhibitor of superoxide generation, and inhibition was partially reversed by high concentrations of p67(phox), but not by p47(phox) or Rac. The V204A mutant competed with native p67(phox) for translocation to neutrophil plasma membrane, indicating that p67(phox) V204A assembles to form an inactive complex. The data imply a direct activation of flavocytochrome b558 by an activation domain in p67(phox).

Amino Acid Sequence↗
PubMed1998

Cytoplasmic staining of erbB-2 but not mRNA levels correlates with differentiation in human thyroid neoplasia.

OBJECTIVE: Amplification and overexpression of the erbB-2 proto-oncogene in human carcinomas may have prognostic significance. Its role in thyroid carcinoma is controversial. We investigated human thyroid tumours for erbB-2 gene amplification, activating mutations in the transmembrane domain, quantitative mRNA expression and protein expression. MATERIALS AND METHODS: DNA and mRNA were extracted from 47 morphologically characterized, frozen thyroid tumours including 10 nodular hyperplasias, 3 follicular carcinomas and 34 papillary carcinomas (4 with tall-cell features, 2 with insular and 2 with anaplastic de-differentiation). DNA amplification was analysed by differential PCR. The transmembrane domain of erbB-2 was sequenced in all tumours for activating mutations in position 659. Levels of mRNA expression were determined by competitive mRNA RT-PCR. Immunohistochemistry (IHC) for erbB-2 protein expression in corresponding paraffin-embedded samples was evaluated. RESULTS: Our results showed no DNA amplification of erbB-2. Sequencing of the transmembrane domain of erbB-2 revealed no activating mutations. The level of mRNA expression was variable, 11 papillary carcinomas showing statistically significant elevated mRNA levels compared with corresponding normal thyroid tissue; however, this did not correlate with other indicators of poor prognosis. In contrast to elevated mRNA levels in tumours, the level of protein staining correlated with degree of differentiation, normal and hyperplastic tissue being strongly positive and poorly differentiated tumours negative. CONCLUSION: There are no mutations or amplifications of the erbB-2 gene in human thyroid tumours. Elevated erbB-2 mRNA expression in some thyroid tumours is not associated with clinical features of poor prognosis; however, the significance of the elevated mRNA levels is unclear, as it did not result in protein overexpression. Instead, cytoplasmic erbB-2 protein detection by IHC correlates with differentiation of human thyroid tumours and may be a feature of good prognosis.

Carcinoma, Papillary↗
PubMed1998

The physiologic reservoir of Epstein-Barr virus does not map to upper aerodigestive tissues.

The human Epstein-Barr herpesvirus (EBV) has distinct oncogenic potential, but with over 90% of the adult world population infected, malignancy is a rare outcome of carrier status. However, EBV's association with over half of Hodgkin's and non-Hodgkin's lymphomas as well as several solid tumors, notably nasopharyngeal carcinoma, makes EBV-linked malignancies one of the largest single cancer entities. EBV is a B-lymphotropic virus, well controlled by surveillant T cells in immunocompetent hosts. To determine the presence and site of principal virus reservoirs is a likely prerequisite for understanding the etiology of EBV-associated tumors. Its near 100% association with nasopharyngeal carcinoma led to postulates that the upper aerodigestive tract tissue may be common sites of persistent latent or low-grade replicating infection. Using a protocol designed to avoid viral crosscontamination, the authors employed polymerase chain reaction to detect genomic EBV DNA sequences in 231 biopsies from different mucosal sites in the upper aerodigestive tract, as well as from salivary gland tissue and neck nodes in individuals not suspected to have EBV-related malignancy. Only two samples, one from oral cavity mucosa and one from parotid gland tissue, were positive for EBV. The observation that oropharyngeal tissue is not the principal EBV reservoir has mechanistic implications for the development of EBV-positive tumors in that locale.

Adolescent↗
PubMed1998

The effect of bipolar electrocautery on peripheral nerves.

Although bipolar cautery was designed to minimize trauma to the central nervous system, little is known about the effects of bipolar cautery on peripheral nerve tissue. This experiment was designed to study the effect of direct bipolar cautery on a peripheral nerve and the muscles innervated by that nerve. Lewis rats (n = 200) were assigned to five different groups: control, sham, and three cautery groups (duration of either 0.5, 1.0, or 1.5 seconds). The hind limb tibial nerves were isolated in the sham group and isolated and cauterized in the cautery groups. Assessments performed at 2 hours, 2 weeks, 4 weeks, and 8 weeks postoperatively included isometric contractile function studies of both a fast- and a slow-twitch muscle, muscle weights, and nerve histology/morphometry. Significant muscle weight loss and reduced muscle function were found in the cautery groups at 2, 4, and 8 weeks (p < 0.05). Histologically, the nerves of the cautery groups showed nerve damage consistent with Sunderland's type 4 nerve injury when examined at 2 weeks and showed nerve regeneration at 4 and 8 weeks. Both the fast-twitch muscle and the shorter duration cautery were associated with faster recovery relative to the slow-twitch muscles and longer duration cautery, respectively. Bipolar cautery, as administered to rat tibial nerves in this experiment, is associated with a significant injury to the nerve and loss of function of the muscles innervated by the nerve.

Animals↗
PubMed1998

Distinct multiple RET/PTC gene rearrangements in multifocal papillary thyroid neoplasia.

Rearrangements involving the RET protooncogene have been implicated in the development of papillary thyroid carcinoma (PC). Transgenic mice, expressing thyroid-targeted RET/PTC-1, develop PC; but the clinical significance of this oncogene remains uncertain. We examined the expression of RET/PTC-1, -2, and -3 in human thyroid microcarcinomas and clinically evident PC to determine its role in early stage vs. developed PC and to examine the diversity of RET/PTC in multifocal disease. RNA was extracted from paraffin-embedded microcarcinomas and clinically evident PCs; the results obtained from paraffin-embedded tissue were confirmed on RNA from corresponding snap-frozen tissue of clinically evident PCs. RT and PCR was performed using primers for RET/PTC-1, -2, and -3; PGK-1 (the housekeeping gene) analysis was used to ensure integrity of the RNA and efficiency of the RT reaction. PCR products were resolved by gel electrophoresis, and Southern hybridization was performed with RET/PTC-1, -2, and -3 probes. A polyclonal antibody to the carboxyterminus of RET was used for immunohistochemistry on paraffin sections. Thirty-nine occult papillary thyroid microcarcinomas from 21 patients were analyzed. Of the 30 tumors (77%) positive for RET/PTC rearrangements, 12 were positive for RET/PTC-1, 3 for RET/ PTC-2, 6 for RET/PTC-3, and 9 for multiple RET/PTC oncogenes. In clinically evident tumors, 47% had RET/PTC rearrangements. Immunohistochemistry demonstrated close correlation with RT-PCR-derived findings. RET/PTC expression is highly prevalent in microcarcinoma and occurs more frequently than in clinically evident PC (P < 0.005). Multifocal disease, identified in 17 of the 21 patients, exhibited identical RET/PTC rearrangements within multiple tumors in only 2 patients; the other 15 patients had diverse rearrangements in individual tumors. Our results indicate that RET/PTC oncogene rearrangements may play a role in early-stage papillary thyroid carcinogenesis, but they seem to be less important in determining progression to clinically-evident disease. In multifocal disease, the diversity of RET/PTC profiles, in the majority of cases, suggests that individual tumors arise independently in a background of genetic or environmental susceptibility.

Carcinoma, Papillary↗
PubMed1998

Geographic variations in breast cancer mortality: do higher rates imply elevated incidence or poorer survival?

OBJECTIVES: Mortality rates from breast cancer are approximately 25% higher for women in the northeastern United States than for women in the South or West. This study examined the hypothesis that the elevation is due to decreased survival rather than increased incidence. METHODS: Data on breast cancer incidence, treatment, and mortality were reviewed. RESULTS: The elevated mortality in the Northeast is apparent only in older women. For women aged 65 years and older, breast cancer mortality is 26% higher in New England than in the South, while incidence is only 3% higher. Breast cancer mortality for older women by state correlates poorly with incidence (r = 0.28). CONCLUSIONS: Those seeking to explain the excess breast cancer mortality in the Northeast should assess survival and should examine differences in cancer control practices that affect survival.

Adult↗
PubMed1998

Facial nerve reconstruction: outcome and failures.

OBJECTIVE: Our study was conducted to assess the outcome of reconstruction of the facial nerve after various resections for parotid neoplasms. METHOD: A 10-year retrospective analysis of 350 primary parotid neoplasms (of which 70 were malignant) and 10 recurrent benign neoplasms involving facial nerve resection in 19 and 2 patients, respectively. Four patients had total resection of the facial nerve and reconstruction with a five-branch assemblage using standard microtechniques. Four patients had reconstruction of the orbital branch only. Twelve patients, of whom three also had a midface branch reconstructed, had reconstruction of the ramus mandibularis. One patient had a direct anastomosis of a partial resection of the main trunk of the nerve. Objective assessment of muscle activity was carried out in the distribution of the reconstructed nerve with a minimum of 18 months follow-up. RESULTS: All four patients with total facial nerve reconstruction had excellent tone in repose. None had purposeful focused movement, and all had mass movement; however, all had good eye closure. None of the patients with mandibular branch grafting alone had useful return of function, but those who had concomitant midface grafting had good return of function of the reconstructed branches. All patients who had orbital grafting had appropriate eye closure. CONCLUSIONS: Facial nerve grafting is effective to rehabilitate gaps in the pes anserinus, even if patients undergo postoperative radiotherapy. Total nerve grafting provides good tone in repose and eye closure, which justifies the procedure. Single-branch grafting is mandatory for the orbital branch, but one can question the use of a single-branch graft for the mandibular branch.

Adult↗
PubMed1997

Rac binding to p67(phox). Structural basis for interactions of the Rac1 effector region and insert region with components of the respiratory burst oxidase.

Activation of the respiratory burst oxidase involves the assembly of the membrane-associated flavocytochrome b558 with the cytosolic components p47(phox), p67(phox), and the small GTPase Rac. Herein, the interaction between Rac and p67(phox) is explored using functional and physical methods. Mutually facilitated binding (EC50) of Rac1 and p67(phox) within the NADPH oxidase complex was demonstrated using steady state kinetic methods measuring NADPH-dependent superoxide generation. Direct binding of Rac1 and Rac2 to p67(phox) was shown using a fluorescent analog of GTP (methylanthraniloyl guanosine-5'-[beta,gamma-imido]triphosphate) bound to Rac as a reporter group. An increase in the methylanthraniloyl fluorescence was seen with added p67(phox) but not p47(phox), and the emission maximum shifted from 445 to 440 nm. Rac1 and Rac2 bound to p67(phox) with a 1:1 stoichiometry and with Kd values of 120 and 60 nM, respectively. Mutational studies (Freeman, J., Kreck, M., Uhlinger, D. J., and Lambeth, J. D. (1994) Biochemistry 33, 13431-13435; Freeman, J. L., Abo, A., and Lambeth, J. D. (1996) J. Biol. Chem. 271, 19794-19801) previously identified two regions in Rac1 that are important for activity: the "effector region" (residues 26-45) and the "insert region" (residues 124-135). Proteins mutated in the effector region (Rac1(N26H), Rac1(I33N), and Rac1(D38N)) showed a marked increase in both the Kd and the EC50, indicating that mutations in this region affect activity by inhibiting Rac binding to p67(phox). Insert region mutations (Rac1(K132E) and L134R), while showing markedly elevated EC50 values, bound with normal affinity to p67(phox). The structure of Rac1 determined by x-ray crystallography reveals that the effector region and the insert region are located in defined sectors on the surface of Rac1. A model is discussed in which the Rac1 effector region binds to p67(phox), the C terminus binds to the membrane, and the insert region interacts with a different protein component, possibly cytochrome b558.

Animals↗
PubMed1997

Prognostic features in tall cell papillary carcinoma and insular thyroid carcinoma.

Tall cell papillary carcinoma (TCPC) and insular carcinoma (IC) are variants of thyroid carcinoma that are considered to be more aggressive than well differentiated papillary or follicular carcinoma. To determine the clinical significance of these diagnoses, we evaluated 65 patients with these tumors. There were 30 TCPCs, 27 ICs, and 8 ICs or TCPCs with focal anaplastic carcinoma (FAC). Forty-two patients (27 TCPCs, 14 ICs, and 1 FAC) are alive and free of disease. Nine patients with IC are alive with distant metastases. Ten patients (2 TCPCs, 2 ICs, and 7 FACs) died of disease. Univariate analysis of disease-free interval determined that, as for all thyroid carcinomas, patient age, tumor size, extrathyroidal extension, and lymph node metastases were significant for prognosis. ICs did significantly worse than TCPCs. Focal anaplastic dedifferentiation predicted a worse prognosis. Multivariate analysis for disease-free interval showed age, number of lymph node metastases, and tumor type to be significant. Analysis of the same factors for prediction of mortality showed that TCPC and IC were not significantly different. These data suggest that TCPC is less aggressive than IC, which often results in disseminated disease. Focal AC predicts poor survival.

Adult↗
PubMed1996

Membrane association of Rac is required for high activity of the respiratory burst oxidase.

NADPH-dependent superoxide generation can be reconstituted in a cell-free system using recombinant cytosolic factors (p47-phox, p67-phox, and Rac) plus flavocytochrome b558. Rac1 and Rac2 are closely related small GTPases, differing primarily in the C-terminal 10 residues where Rac1 but not Rac2 contains a polybasic sequence. In their nonisoprenylated forms, Rac1 was highly effective in reconstituting NADPH oxidase activity (low EC50, high Vmax), whereas Rac2 was only minimally effective (high EC50, low Vmax). In contrast, low concentrations of isoprenylated Rac1 and Rac2 both supported high rates of superoxide generation. Like full length Rac2, truncated forms of both Rac1 and Rac2 in which the C-terminal 10 residues were eliminated were poorly activating, pointing to the C terminus of Rac1 as a determinant of activity. Mutation of single positively charged residues in the C terminus of nonisoprenylated Rac1 markedly reduced its ability to support superoxide generation, affecting both its EC50 and the Vmax. In contrast, mutation or truncation of the C terminus failed to affect the activation of PAK, a Rac-regulated protein kinase. The EC50 for Rac1 increased with increasing salt concentrations, whereas that of Rac2 was independent of salt, implicating the involvement of electrostatic forces for the former. Using flavocytochrome b558 reconstituted into phosphatidylcholine vesicles, the EC50 for Rac1 but not Rac2 decreased (increased binding) when an acidic phospholipid (phosphatidylinositol) was present, supporting a role for the Rac1 polybasic C terminus in binding to the membrane. A model in which Rac must associate simultaneously both with p67-phox and with the membrane to activate the NADPH oxidase can account for the above observations.

Amino Acid Sequence↗
PubMed1996

NADPH oxidase activity is independent of p47phox in vitro.

The neutrophil superoxide generating NADPH oxidase is activated by the assembly of cytosolic protein components with a membrane-associated flavocytochrome. The activity can be reconstituted in vitro using purified cytosolic factors p47(phox), p67(phox), and Rac plus the phospholipid-reconstituted flavocytochrome b558. Here, we demonstrate that activity is reconstituted in the absence of p47(phox) when high concentrations of p67(phox) and Rac are used. Vmax values were the same in the presence or absence of p47(phox), yet p47(phox) increases the affinity of both p67(phox) and Rac for the oxidase complex by nearly 2 orders of magnitude. p67(phox)-(1-246), a truncated form of the protein which eliminates SH3 domains involved in binding to p47(phox), also supports superoxide generation, both in the presence and absence of p47(phox), providing further evidence for p47(phox) independent activity. In the absence of p47(phox), p67(phox)-(1-246) binds to the NADPH oxidase complex 3-fold more tightly than does native p67(phox), indicating that the C terminus contains a region which masks binding to the oxidase complex. Results indicate that p47(phox) does not play a direct role in regulating electron transfer. Rather, its function is to serve as an adaptor protein to enhance the assembly of the other cytosolic components with the flavocytochrome and possibly to unmask a binding region in the N terminus of p67(phox) by binding to its C-terminal domains. p67(phox) and/or Rac play a more direct role in regulating electron transfer.

Arachidonic Acid↗
PubMed1996

Rac "insert region" is a novel effector region that is implicated in the activation of NADPH oxidase, but not PAK65.

The small GTPase Rac assembles with the cytosolic p47(phox) and p67(phox) and the membrane-associated flavocytochrome b558 to form the multicomponent respiratory burst oxidase. Mutation of amino acids in a region of Rac (residues 26-45), homologous to an effector region in Ras, was previously shown to interfere with Rac binding to the oxidase. Herein we have elucidated an additional region in Rac involved in regulating oxidase activity. Rho family small GTPases contain a 12-amino acid "insert" region (residues 124-135) that is not present in Ras. Point mutations in and deletion of this region were constructed and used for in vitro studies of the activation of PAK65 and NADPH oxidase. Apparent binding constants (based on EC50 values) of the mutant Rac proteins for the oxidase are at least 13-25-fold higher than for wild-type Rac. Mutations in the insert region versus the 26-45 effector region resulted in distinct kinetic consequences, pointing to different roles for these two protein regions: mutations in the insert region but not the 26-45 effector region resulted in an increase in the EC50 for p67(phox). Although mutations in the 26-45 amino acid effector region showed markedly diminished activation of both PAK and the NADPH oxidase, insert region mutations did not affect activation of PAK. We propose that the combinatorial use of the 26-45 effector region and the insert region provides the Rho family GTPases with versatility in their specificity for several downstream targets.

Amino Acid Sequence↗
PubMed1996

Prevalence of activating ras mutations in morphologically characterized thyroid nodules.

Ras proteins are signal-transducing proteins that share common properties with membrane-anchored G proteins. Mutations at codon 12/13 or codon 61 alter GTP-binding or GTPase activity, respectively. Such activating mutations are present in nearly 30-50% of various malignancies including colon, breast, and lung carcinomas. There are conflicting data regarding the prevalence of ras mutations in the thyroid and their possible pathogenetic role in the different tumor types. To address this question, we examined 45 morphologically characterized thyroid carcinomas, adenomas, and hyperplastic nodules using a highly sensitive single-stranded conformation polymorphism (SSCP) approach combined with DNA-sequencing. DNA from cell lines with known mutations served as controls. A G to A H13 codon substitution replacing an Asp for a Gly residue was detected in 1 papillary carcinoma. Although no H12 or H61 codon substitutions were identified, 2 discrete alterations were identified in codons H17 and 22. No N12/13 codon substitutions were identified. N61 codon substitutions of A to G resulting in a Gly to Arg substitution were detected in 2 papillary carcinomas; the same mutation was also found in one follicular adenoma. Interestingly, K12/13 and K61 ras mutations were not present in any of the tumors examined. These data establish a low prevalence of mutations in all ras gene family members in human thyroid neoplasms. This difference from neoplasms of other organs may explain the relatively indolent biologic behavior of many thyroid tumors and supports an alternate early genetic mutation that is more characteristic of these neoplasms.

Adult↗