Faculty Bibliography

Insulin acutely decreases contractile agonist-induced Ca2+ influx and contraction in endothelium-free cultured vascular smooth muscle (VSM) cells, but the mechanism is not known. Since it has been reported that insulin-induced vasodilation in humans is linked to nitric oxide synthase activity, we wished to determine whether insulin inhibits Ca2+ influx and contraction of cultured vascular smooth muscle cells by a nitric oxide synthase-dependent pathway. Primary cultures of endothelial cell-free VSM cells from canine femoral artery were preincubated with and without 1 nmol/L insulin for 30 minutes, and the 5-minute production of cGMP was measured. Insulin alone did not affect cGMP production, but in the presence of 10(-5) mol/L serotonin insulin stimulated cGMP production by 60%. N(G)-monomethyl-L-arginine (0.1 mmol/L), an inhibitor of nitric oxide synthase, inhibited the conversion of arginine to citrulline by these cells, blocked insulin-stimulated cGMP production, and blocked the inhibition by insulin of 5-hydroxytryptamine (5-HT)-stimulated Mn+2 (a Ca2+ surrogate) influx and contraction. Insulin did not affect contraction of VSM cells grown under conditions designed to deplete the cells of tetrahydrobiopterin, an essential cofactor of nitric oxide synthase. These studies demonstrate that insulin acutely inhibits 5-HT-stimulated Ca2+ influx and contraction of endothelium-free cultured VSM cells by a nitric oxide synthase-dependent mechanism.

The immunogenicity of phosphopeptides derived from tryptic hydrolysis of beta-casein (CN) was investigated in a rat model system. The titers of specific immunoglobulin (Ig)G and IgE antibodies made in response to intraperitoneal sensitization to beta-CN, casein phosphopeptides, and skim milk proteins were examined using indirect and amplified indirect ELISA, respectively. Serum IgG antibodies from rats injected with beta-CN were significantly more reactive to beta-CN, casein phosphopeptides, and skim milk proteins coated on microtiter plate wells than were the IgG antibodies generated in rats that had been subjected to other treatments. A significant difference in titers because of the time of sampling (14 or 21 d postinjection) was noted for IgE but not for IgG. Rats that were injected with casein phosphopeptides did not produce IgG antibodies that crossreacted with either skim milk proteins or beta-CN. Specific antibody levels for the IgE class rarely exceeded those of unimmunized controls. The findings suggest that immunogenicity of the phosphopeptides was reduced compared with that of native beta-CN and skim milk proteins.

BACKGROUND: Primary hypothyroidism is a common sequela of craniospinal radiotherapy in the treatment of pediatric brain tumors. METHODS: The authors compared the incidence of primary hypothyroidism after hyperfractionated radiotherapy (HFRT) (n = 14 patients) versus conventionally fractionated radiotherapy (CRT) (n = 34 patients) in a group of pediatric patients with medulloblastoma/primitive neuroectodermal tumors (MB/PNET). RESULTS: The mean age at the time of tumor diagnosis was 7.9 years in the HFRT group and 8.4 years in the CRT group. The patients were followed for a mean of 4.6 years (HFRT) and 8.3 years (CRT) after diagnosis. Mean radiation doses to the thyroid were similar in both radiotherapy groups (29 gray [Gy] [HFRT] vs. 24 Gy [CRT]). Approximately 14% of the HFRT and 62% of the CRT patients developed primary hypothyroidism within a similar period after irradiation (3.2 years [HFRT] vs. 3.0 years [CRT]). Analysis by cumulative incidence function demonstrated a significant difference in the risk of developing thyroid dysfunction between these two groups of patients (P = 0.02). CONCLUSIONS: The current study findings suggest that the use of HFRT in the treatment of pediatric patients with MB/PNET is associated with a lower risk of these patients developing primary hypothyroidism

Recent studies have indicated that the proliferation of malignant gliomas is in part dependent on excessive activation of protein kinase C (PKC)-mediated pathways. Conversely, inhibiting PKC may provide a novel approach for blocking glioma growth. The antiestrogen tamoxifen, a moderately potent PKC inhibitor, has been shown in vitro to block the proliferation of malignant glioma cell lines at concentrations several-fold higher than those typically attained during the treatment of breast cancer; such serum concentrations may be achieved with doses > 40 mg/m2 b.i.d. The safety and efficacy of these high doses for producing disease control in patients with malignant gliomas has recently been noted anecdotally, although a rigorous study of this agent has been lacking. To address this issue, we examined the safety and efficacy of high-dose tamoxifen in a series of children with malignant gliomas that had progressed after conventional therapy. An initial group was treated with 60 mg/m2 p.o. b.i.d. and a second group with 100 mg/m2 b.i.d. Steady-state serum tamoxifen and metabolite levels were measured in most patients. Toxicity with the regimen was minimal; two patients treated at the higher dose required reduction to the lower dose because of asymptomatic prolongation of the QT interval on an electrocardiogram. Although none of the patients exhibited clear-cut tumor regression, 4 of 14 patients had stabilization of previously progressive disease for at least 3 months; the longest survivor lived for 17 months after beginning tamoxifen. The moderate efficacy of this agent in otherwise end-stage disease coupled with its low toxicity and the relative ease of oral administration provides a rationale for proceeding with larger studies of this agent in patients with malignant gliomas, possibly as a means for potentiating the effects of conventional chemotherapeutic agents, which to date have shown limited efficacy in the treatment of these tumors

Pallister-Hall syndrome is a human developmental disorder that is inherited in an autosomal dominant pattern. The phenotypic features of the syndrome include hypothalamic hamartoma, polydactyly, imperforate anus, laryngeal clefting, and other anomalies. Here we describe the clinical characterisation of a family with 22 affected members and the genetic mapping of the corresponding locus. Clinical, radiographic, and endoscopic evaluations showed that this disorder is a fully penetrant trait with variable expressivity and low morbidity. By analysing 60 subjects in two families using anonymous STRP markers, we have established linkage to 7p13 by two point analysis with D7S691 resulting in a lod score of 7.0 at theta = 0, near the GLI3 locus. Deletions and translocations in GLI3 are associated with the Greig cephalopolysyndactyly syndrome. Although Greig cephalopolysyndactyly syndrome has some phenotypic overlap with Pallister-Hall syndrome, these two disorders are clinically distinct. The colocalisation of loci for these distinct phenotypes led us to analyse GLI3 for mutations in patients with Pallister-Hall syndrome. We have previously shown GLI3 mutations in two other small, moderately affected families with Pallister-Hall syndrome. The linkage data reported here suggest that these larger, mildly affected families may also have mutations in GLI3

A retrospective review of all British Army patients presenting to the Queen Elizabeth Military Hospital with a diagnosis of Hodgkin's disease between 1977 and 1987 was performed, with a minimum follow-up of 7 years. The results were similar to those reported in other (similar) series, and the changes in investigations and treatment observed during this period matched those occurring in wider clinical practice through close co-operation with a recognised Cancer Centre. Despite their comparative rarity, Army patients with Hodgkin's disease receive modern, effective treatment.

beta-Lactoglobulin was isolated directly from acidic whey by bioselective adsorption on N-retinyl-Celite, yielding preparations of > or = 96% purity. Interactions of these preparations with vitamin D2, vitamin D3, ergosterol, cholesterol, and 7-dehydrocholesterol were examined by following changes in the fluorescence spectra. Both the excitation and emission spectra indicated that energy was transferred between the tryptophanyl residues of the protein and the chromophore of the ligand. Analyses of the fluorescence changes that occurred upon titration of beta-LG with the various ligands allowed determination of the dissociation constant for the complex and the number of moles bound per mole of protein. The affinity for vitamin D2 (dissociation constant of 4.91 nM) was 10-fold higher than that of the other compounds, except for ergosterol, which was 5-fold larger than the others. Also, the affinity was 10-fold higher than that typically reported for the retinoids. Furthermore, the value obtained for the number of moles bound per mole of protein was 2 mol.mol-1 for each of the ligands examined in this study; it has been well established that all of the retinoids are bound with a stoichiometry of 1.0. These results suggest that beta-LG may be a better carrier of vitamin D than of vitamin A.

Binding of the retinoids, all-trans-retinol, all-trans-retinal, all-trans-retinyl acetate, and all-trans-retinoic acid, to beta-lactoglobulin (LG) (96% purity) that had been prepared by bioselective adsorption on N-retinyl-Celite was determined from changes in the fluorescence quenching (332 nm) of the protein tryptophanyl residues. High affinity binding of all of these compounds occurred at pH 7.0, and the apparent dissociation constant ranged from 1.7 to 3.6 x 10(-8) M. Furthermore, a stoichiometry of 1.0 mol.mol-1 of protein was obtained for each case, indicating that all of the sites in the protein preparation were available. When beta-LG in whey protein isolate (57.4% beta-LG) was studied, a stoichiometry of 0.65 to 0.82 mol.mol-1 of protein was obtained, indicating that a large number of the sites already had bound lipid or that the protein had been denatured. As the pH was lowered toward 5.15, the affinity decreased about fourfold, but the stoichiometry of binding was unchanged. Far UV circular dichroism spectra indicated that the secondary structure of the protein was not significantly affected by ligand binding; however, the near UV spectra were changed, indicating that the flexibility of tryptophanyl residues decreased. The latter effect is consistent with the change in fluorescence quenching and suggests that a tryptophan is in the binding site.

The optimum treatment of nonresectable low-grade gliomas of childhood remains undecided. There has been increased interest in the use of chemotherapy for young children, but little information concerning the long-term efficacy of such treatment. Seventy-eight children with a mean age of 3 years (range 3 months-16 years) who had newly diagnosed, progressive low-grade gliomas were treated with combined carboplatin and vincristine chemotherapy. The patients were followed for a median of 30 months from diagnosis, with 31 patients followed for more than 3 years. Fifty-eight children had diencephalic tumors, 12 had brainstem gliomas, and three had diffuse leptomeningeal gliomas. Forty-four (56%) of 78 patients showed an objective response to treatment. Progression-free survival rates were 75 +/- 6% at 2 years and 68 +/- 7% at 3 years. There was no statistical difference in progression-free survival rates between children with neurofibromatosis Type 1 and those without the disease (2-year, progression-free survival 79 +/- 11% vs. 75 +/- 6%, respectively). The histological subtype of the tumor, its location, and its maximum response to chemotherapy did not have an impact on the duration of disease control. The only significant prognostic factor was age: children 5 years old or younger at the time of treatment had a 3-year progression-free survival rate of 74 +/- 7% compared with a rate of 39 +/- 21% in older children (p < 0.01). Treatment with carboplatin and vincristine is effective, especially in younger children, in controlling newly diagnosed progressive low-grade gliomas