Biomedical subjects

J C Allen

Publications and source records attributed to J C Allen.

At least 73 records · Page 4Linked to original sources

PubMed1997

Molecular evidence for a vascular smooth muscle H+-K+-ATPase.

Evidence for an H+-K+-adenosinetriphosphatase (HKA) in vascular smooth muscle cells has recently been demonstrated in the rat aorta and a rat smooth muscle cell line (A(7)r(5)) by functional studies. Our laboratory has used reverse transcriptase (RT)-polymerase chain reaction (PCR) and Northern hybridization techniques to define the type of HKA present in canine whole carotid artery and confluent carotid artery cells. PCR primers to detect an HKA message of unknown isoform were designed on the basis of homology to known sequences of gastric, colonic, and toad bladder HKA isoforms and dissimilarity to the Na+-K+-adenosinetriphosphatase isoforms. A PCR product of predicted size was generated from cDNAs of both confluent cells and whole vessel carotid artery that possess 91% nucleotide sequence identity to the canine gastric HKA. Northern hybridization with the PCR product as a probe revealed hybridization to whole vessel carotid artery, cultured confluent carotid artery cell, kidney, and stomach canine total RNA at the known message size for the gastric HKA isoform. These data suggest that vascular smooth muscle cells express an HKA mRNA that may be identical to the gastric isoform.

Amino Acid Sequence↗

PubMed1997

Hypothalamic-pituitary-adrenal function following cranial irradiation.

We assessed the effect of cranial irradiation on hypothalamic-pituitary (HP)-adrenal function in 17 patients (12 females, 5 males) treated with cranial/ craniospinal irradiation for acute leukemia (2 patients) or tumors distant from the hypothalamus and pituitary (8 medulloblastoma, 3 astrocytoma, 3 rhabdomyosarcoma, 1 ependymoma). Estimated doses of radiation (RT) to the HP region ranged from 18 to 72 Gy. Thirteen of seventeen patients were also treated with chemotherapy. Patients were a median of 3.75 years of age (1.5-19 years) at diagnosis and were studied at a median of 5 years (0.1-20 years) after RT. Patients received corticotropin-releasing factor (oCRF, 1 microgram/kg i.v.), and sampling for cortisol and ACTH levels was performed at -15, 0, 15, 30, 60, 90 and 120 min. The-5- and 0-min levels were combined for a standardized baseline value (Base). Cortisol levels at 0, Base, 30 and 120 min, as well as the peak cortisol response, were significantly lower in the patients. Twelve of seventeen patients' peak cortisol levels fell below the normal range. The patients' mean integrated values for cortisol (area under the curve) were not, however, different from controls. The ACTH responses to oCRF did not differ between patients and controls. No relationship was observed between ACTH or cortisol responses and the time elapsed from treatment or dose of HP RT. Further, in 10 of 12 patients, 0-min dehydroepiandrosterone sulfate levels were lower than the expected normal mean levels for age, sex and pubertal status, and in 4 of these 10 patients the values were below the normal range. These data suggest that some patients treated with HP RT may be at risk for adrenal insufficiency.

Adolescent↗

PubMed1997

Insulin acutely inhibits cultured vascular smooth muscle cell contraction by a nitric oxide synthase-dependent pathway.

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.

Animals↗

PubMed1997

De novo 14484 mitochondrial DNA mutation in monozygotic twins discordant for Leber's hereditary optic neuropathy.

Monozygotic twin brothers, clinically discordant for Leber's hereditary optic neuropathy (LHON), had a heteroplasmic point mutation at position 14484 in the mitochondrial DNA that was not detected in their mother. Moreover, the mutation occurred on the rare European haplogroup X, rather than the haplogroup J commonly associated with the 14484 mutation. These data indicate that the 14484 mutation in this family was a new mutation, indicating that it was the de novo occurrence of a common, primary LHON mutation.

Adolescent↗

PubMed1997

Binding of retinoids to beta-lactoglobulin isolated by bioselective adsorption.

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.

Adsorption↗

PubMed1997

Binding of vitamin D and cholesterol to beta-lactoglobulin.

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.

Adsorption↗

PubMed1997

Immunogenicity of casein phosphopeptides derived from tryptic hydrolysis of beta-casein.

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.

Animals↗

PubMed1997

A phase I study of high-dose tamoxifen for the treatment of refractory malignant gliomas of childhood.

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.

Administration, Oral↗

PubMed1996

Hyperfractionated craniospinal radiotherapy and adjuvant chemotherapy for children with newly diagnosed medulloblastoma and other primitive neuroectodermal tumors.

PURPOSE: This single-institution Phase III study conducted from 1989 to 1995 evaluates the feasibility of a multimodality protocol combining hyperfractionated craniospinal radiotherapy (HFRT) followed by adjuvant chemotherapy in 23 patients with newly diagnosed primitive neuroectodermal tumors (PNET) arising in the central nervous system. METHODS AND MATERIALS: All 23 patients had a histologically confirmed PNET and were over 3 years of age at diagnosis. The eligibility criteria for PNET patients with cerebellar primaries (medulloblastoma) included either a high T stage (T3b or 4) or high M stage (M1-3). All patients with noncerebellar primaries were eligible regardless of T or M stage. The median age of the 23 patients was 9 years (mean 3-25); 11 were female. The primary tumor arose in the cerebellum in 19. Of these medulloblastoma patients, 15 had high T stages (T3b or T4) with large locally invasive tumors and no evidence of metastases (M0), constituting Group 1. Thirteen (86%) of these patients had gross total resections. Four other medulloblastoma patients had both high T and high M stages, constituting Group 2. Group 3 consisted of four other patients with exocerebellar primaries (two brain, one brain stem, and one cauda equina), three of whom were M3. Hyperfractionated radiotherapy was administered within 4 weeks of surgery. Twice-daily 1-Gy fractions were administered separated by 4-6 h. The total dose to the primary intracranial tumor and other areas of measurable intracranial disease was 72 Gy. The prophylactic craniospinal axis dose was 36 Gy, and boosts of 44-56 Gy were administered to metastatic spinal deposits. Following radiotherapy, monthly courses of multiagent chemotherapy were administered sequentially (cyclophosphamide-vincristine followed by cisplatin-etoposide followed by carboplatin-vincristine) for a total of 9 months. RESULTS: All patients completed radiotherapy as planned. Only three patients lost >10% of their body weight. One patient had clinically apparent radiation-induced esophagitis. The mean white blood count (WBC) nadir was 2.5/dl, and hematologic recovery occurred in all within 4 weeks of completing HFRT without the need of granulocyte-colony-stimulating factor. Two patients refused adjuvant chemotherapy, 3 patients experienced tumor progression during chemotherapy, and 2 of 18 remaining patients could not tolerate the full 9 months owing to hematologic toxicity. Of the 15 patients (93%) in Group 1, 14 remain in continuous remission for a median of 78 months, and none have died. Two of four patients in Group 2 are in continuous remission at 67 and 35 months, and two died at 18 and 30 months. One of the two patients in Group 2 who died refused adjuvant chemotherapy and developed tumor progression in the bone marrow. None of the three patients in Group 3 with evaluable disease (M3) had a complete response to therapy, and eventually all four died of progressive or recurrent disease. CONCLUSION: This multimodality protocol is feasible in the short term, and long-term monitoring of neurocognitive and neuroendocrine effects are in progress. Excellent long-term disease control has been achieved for medulloblastoma patients with high T stages who were M0 at diagnosis (Group 1), the majority of whom had gross total resections. This group has a progression-free survival of 95% after a median period of follow-up of 6.5 years. Alternative treatment strategies must be developed for patients with high M stages, as five of seven patients died of progressive or recurrent disease.

Adolescent↗

PubMed1996

Abortion beliefs.

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Abortion, Induced↗

PubMed1996

Phase II evaluation of topotecan for pediatric central nervous system tumors.

BACKGROUND: Topotecan is a topoisomerase I inhibitor that has good penetration across the blood-brain barrier and significant antitumor activity against human brain tumor xenografts. In a Phase I trial in children with refractory cancer, topotecan was well tolerated when administered as a 24-hour infusion. The maximum tolerated dose was 5.5 mg/m2 and the dose-limiting toxicity was myelosuppression. This Phase II study of topotecan was performed to assess the activity of topotecan against childhood brain tumors. METHODS: Forty-five children with either a previously treated primary brain tumor that was refractory to standard therapy, or an untreated brain stem glioma or glioblastoma multiforme, received topotecan administered as a 24-hour intravenous infusion every 21 days. The initial dose was 5.5 mg/m2 with escalation to 7.5 mg/m2 on the second and subsequent doses in patients who did not experience dose-limiting toxicity. RESULTS: There were no complete or partial responses in the patients with high grade glioma (n=9), medulloblastoma (n=9), or brain stem glioma (n=14). One of 2 patients with a low grade glioma had a partial response lasting more than 17 months; 3 patients with a brain stem glioma had stable disease for 12 to 28 weeks; and 1 patient with a malignant neuroepithelial tumor and 1 patient with an optic glioma had stable disease for 41 weeks and 22 weeks, respectively. Dose escalation from 5.5 mg/m2 to 7.5 mg/m2 was well tolerated in the first 11 patients enrolled on this study who had not received prior craniospinal radiation therapy. The starting dose was subsequently increased to 7.5 mg/m2 for patients without prior craniospinal radiation. CONCLUSIONS: Topotecan administered as a 24-hour infusion every 21 days is inactive in high grade gliomas, medulloblastomas, and brain stem tumors.

Adolescent↗

PubMed1996

Migration of brain tumor cells on extracellular matrix proteins in vitro correlates with tumor type and grade and involves alphaV and beta1 integrins.

An important contributor to the malignancy of brain tumors is their ability to infiltrate the brain. Extracellular matrix molecules and cell adhesion molecules on cell surfaces play key roles in cell migration. In the present study, we used reaggregates of dissociated cells from freshly excised human brain tumors to analyze the migration of cells from human brain tumors of different types and grades on many different adhesion proteins adsorbed to glass substrates. Proteins were chosen based on their presence in normal or neoplastic nervous tissue, and included the extra-cellular matrix molecules fibronectin, collagens, fibrinogen, laminin, tenascin-C, thrombospondin, and the neuron-glia cell adhesion molecule, Ng-CAM. Cells from astrocytomas (n = 24) migrated on a variety of substrates, in contrast to cells from primitive neuroectodermal tumors cells (n=6), which only migrated well on laminin, fibronectin, or type IV collagen but not on the other substrates. Typically, migrating cells from astrocytomas of all grades had long, slender processes, were usually bipolar, and their cell bodies did not spread well on any substrate. Although there was variability in the migration of cells from astrocytomas of the same grade, cells from high-grade astrocytomas tended to migrate more extensively (42.3 +/- 4.7 micrometers/16 h: n = 16) than cells from lower grade astrocytomas (28.9 +/- 3.9 micrometers/16 h; P = 0.07; n = 8); the most striking differences were observed for collagen substrates, on which cells from lower grade astrocytomas migrated at very low levels (7.6 +/- 2 .6 micrometers/16 h) and cells from high-grade astrocytomas at higher levels (24.4 +/- 5.2 micrometers;P = 0.01). In contrast to primary cells from glioblastomas (n = 13), glioblastoma cell lines (n = 10) consistently spread on various substrates and migrated at high levels (69.5 +/- 7.6 versus 46.4 +/-5.7 micrometers/16 h; P = 0.03), in particular, on collagens (108.4 +/- 20.2 versus 28.0 +/- 6.1 micrometers/16 h; P= 0.001). Specific monoclonal antibodies to alphaV and beta1 integrin monomers completely inhibited the migration of astrocytoma cells on most substrates, suggesting that alphaV and beta1 integrins play a crucial role in brain tumor infiltration. These studies also suggest that although a large number of extracellular matrix molecules may promote tumor cell migration, disrupting the function of only a few tumor cell receptors may be critical for tumor infiltration in the brain.

Antibodies↗

PubMed1996

Tenascin-C expression by angiogenic vessels in human astrocytomas and by human brain endothelial cells in vitro.

The expression of the extracellular matrix glycoprotein tenascin-C (TN) is enhanced in human astrocytomas and correlates with angiogenesis. To determine whether vascular cells are able to synthesize TN, we investigated the expression of TN protein and mRNA in nine astrocytomas. Immunogold electron microscopy in two glioblastomas multiforme detected the presence of TN in an extracellular perivascular location and to a lesser extent among tumor cells, confirming light microscopy immunohistochemical findings. In situ hybridization of astrocytomas using a digoxigenin-labeled antisense riboprobe detected strong staining for TN mRNA in vascular cells, especially in hyperplastic vessels, including those at the invasive edge of the tumors but not in vessels of normal brains. We observed weaker staining in tumor cells indicating a higher level of TN mRNA in vascular than in tumor cells. No staining was detected with the sense probe. Moreover, we investigated the ability of human brain microvessel endothelial cells (HBMECs) in primary culture to synthesize TN in vitro. Western blot analysis of the culture supernatants from HBMECs detected large amounts of TN. Immunogold silver staining demonstrated the presence of TN on the surface of HBMECs and in the subendothelial matrix. The distribution of TN mRNA in vascular cells of astrocytomas and the ability of HBMECs to synthesize TN in vitro demonstrate that vascular cells, including endothelial cells, are a major source of TN associated with angiogenesis. Furthermore, our results suggest that TN expression may be associated with endothelial cell activation and may play an important role in angiogenesis.

Astrocytoma↗

PubMed1996

Age at onset of puberty following high-dose central nervous system radiation therapy.

OBJECTIVE: To determine if a relationship exists between age at irradiation, sex of the patient, and age at onset of puberty and pubarche in children treated with high-dose radiation to the central nervous system. DESIGN: Case series. SETTING: Tertiary care institutional practices and clinics. PATIENTS: Thirty-six children treated with high-dose irradiation (hypothalamic pituitary dose, 30-72 Gy) by conventional (n = 29) or hyperfractionated (n = 7) schedules. Girls were treated before age 8 years and boys before age 9 years. Twenty-six of the 36 children also received chemotherapy. All tumors were distant from the hypothalamic-pituitary region. MAIN OUTCOME MEASURE: Age at onset of puberty and pubarche. RESULTS: In girls, the median age at onset of puberty was 9.3 years vs 10.9 years for controls (P < .01); pubarche occurred at 9.4 years vs 11.2 years for controls (P < .01). In boys, the median age at onset of puberty--genital II--was 11.0 years vs 11.5 years for controls (P = .30); pubarche occurred at a median age of 10.5 years vs 12 years for controls (P = .25). A censored-data normal linear regression model was used to account for children (n = 6) who had not reached puberty. Age at diagnosis (P < .01) and sex (P = .01) were significant predictors of age at onset of puberty. Body mass index SD score (z score) was inversely related to age at onset of puberty (r = -0.77) and was greater at onset of puberty in girls than in boys. CONCLUSION: In children who have received high-dose cranial radiation therapy, a significant positive correlation exists between age at diagnosis and age at onset of puberty in boys and girls.

Adolescent↗

PubMed1996

Deferring adjuvant therapy for totally resected intracranial ependymoma.

Radical surgery is the most important treatment modality for ependymoma. The benefit of adjuvant radiotherapy and/or chemotherapy following a gross total resection of a low grade intracranial ependymoma is uncertain. Since 1990 we elected to defer adjuvant therapy in 7 pediatric patients with a median age of 7 years (range 3-16 years) who had a radical resection of an intracranial ependymoma and no evidence of central nervous system metastases. The primary tumor site was the cerebral hemisphere (6) and the cerebellum (1). A gross total resection was radiologically confirmed in 5 of the 7 patients. Two of the patients had a blood clot in the resection site on the postoperative magnetic resonance imaging scan. All patients are alive after a median follow-up of 44 months and the median progression-free survival is 38+ months. Five of the patients remain in continuous remission. The 2 patients with postoperative blood clots developed subclinical local recurrences, 10 and 11 months, respectively, after diagnosis. They remain in remission for 13+ and 27+ months after subsequent radical surgical procedures. Involved field radiotherapy was administered to 1 patient. After a limited period of follow-up, radical surgery alone appears to be sufficient for the majority of children with low grade ependymomas diagnosed at > 3 years of age when postoperative imaging confirms a gross total resection. This is more likely to occur in supratentorial ependymomas arising in older children.

Adolescent↗

PubMed1996

Evaluation of autosensing as an automatic means of maintaining a 2:1 sensing safety margin in an implanted pacemaker. Autosensing Investigation Team.

As intracardiac signal amplitudes fluctuate due to patient activity, drug intake, and other factors, it is imperative that an adequate sensing safety margin in an implanted pacemaker be used to compensate. We studied an investigational autosensing feature that automatically adjusts the device's sensitivity. Data were collected from 55 patients, with Intermedics model 292-03 or 294-03 pacemakers, upon inclusion of the study (Visit 1); 1 month postinclusion (Visit 2); 1 month, 1 day postinclusion (Visit 3); then 1 month, 2 days postinclusion (Visit 4). Atrial (N = 45) and/or ventricular (N = 54) thresholds were assessed at each visit; during Visit 2, myopotential tests were performed at two sensitivity settings. Autosensing was activated following Visit 1, then programmed on randomly at Visit 2 or Visit 3. From Visit 2-Visit 4, patients were monitored during daily activities (D), exercise (E), and sleep (S) with 24-hour Holter. With Autosensing on, atrial undersensing episodes were D = 33 (P > 0.2), and S = 28 (p < 0.05); ventricular undersensing episodes were D = 6 (p > 0.5). Ventricular oversensing episodes were S = 2; atrial oversensing episodes were S = 34 (p > 0.5), D = 2, and E = 3. Comparing Autosensing adjusted sensitivity with the recommended 2:1 safety margin, 4 patients (p = 0.15) experienced atrial myopotential oversensing, and 2 patients (p = 0.15) ventricular. No unanticipated clinical events occurred. Compared with the recommended 2:1 sensing safety margin, the Autosensing feature performed equal to manual programming in preventing episodes of under/oversensing, and was better for atrial undersensing during sleep. Autosensing obviates the need for periodic reprogramming of a fixed sensitivity value.

Action Potentials↗

PubMed1996

Exclusion of candidate loci and cholesterol biosynthetic abnormalities in familial Pallister-Hall syndrome.

Pallister-Hall syndrome (PHS) was originally described in 1980 in six sporadic cases of children with structural anomalies including hypothalamic hamartoma, polydactyly, imperforate anus, and renal and pulmonary anomalies. In 1993, the first familial cases were reported, including affected sibs and vertical transmission. Three of these families are sufficiently large to allow initial evaluation by linkage studies to candidate genes or loci. We have evaluated candidate loci for PHS based on three clinical observations. The first is a patient with PHS-like malformations, including a hypothalamic hamartoma, and an unbalanced translocation involving 7q and 3p. The second is a family with familial PHS where the founder's father had an autosomal dominant hand malformation previously mapped to 17q. The third is the phenotypic overlap of PHS and Smith-Lemli-Opitz syndrome. In this report, we exclude these loci as candidates for linkage to the PHS phenotype on the basis of lod scores of less than-2.0. We conclude that hypothalamic hamartoma is not specific to PHS and that the dominant hand malformation in one of the families was a coincidence. To evaluate the relationship of PHS to Smith-Lemli-Opitz syndrome, we analysed levels of cholesterol and intermediate metabolites of the later stages of cholesterol biosynthesis. There is no evidence of a generalised disorder of cholesterol biosynthesis in patients with familial PHS. On genetic and biochemical grounds, we conclude that PHS and Smith-Lemli-Opitz syndrome are not allelic variants of a single locus.

Cholesterol↗

PubMed1996

Amino-terminal processing of the catalytic subunit from Na(+)-K(+)-ATPase.

The first five amino acids of the catalytic alpha 1-subunit predicted from its cDNA are not found in purified mammalian Na(+)-K(+)-ATPase, suggesting co- or posttranslational cleavage. To facilitate evaluation of amino-terminal structure and the cleavage process, we developed a site-directed antibody (anti-VGR) specific for the first nine residues of nascent alpha 1 from rat. In immunoblots of polypeptides generated by in vitro translation, anti-VGR detected a prominent band with a mobility appropriate for the alpha 1-subunit (100 kDa). Immunoblots of total protein from various rat organs, however, revealed no significant binding, implying that virtually all the alpha 1-subunit expressed in vivo was modified. We also assessed amino-terminal structure in various heterologous expression systems. Binding of anti-VGR was observed in Escherichia coli transformed with a vector containing an alpha 1/troponin fusion protein and in insect cells infected with baculovirus containing full-length alpha 1 or alpha 1T. This suggests that modification of the introduced alpha 1 in these expression systems was absent or different from that in mammals. In contrast, green monkey kidney cells (COS-1) transfected with alpha 1 did not reveal significant binding of the antibody, indicating that the introduced isoform was processed appropriately. These results demonstrate that the structure of the alpha 1-subunit's amino terminus differs among various expression systems. The results further imply that efficient co- or posttranslational processing of nascent alpha 1 is conserved among various organs within the rat, yet the required modification enzymes are not present in distant phyla.

Amino Acid Sequence↗