Search PubMed⌕ Search

Biomedical subjects

M Smith

Publications and source records attributed to M Smith.

At least 559 records · Page 31Linked to original sources
PubMed1995

Trans effects on cysteine ligation in the proximal His93Cys variant of horse heart myoglobin.

Three variants of horse heart myoglobin (Mb) in which the proximal His93 residue has been replaced with a Cys residue have been constructed and studied by NMR, EPR, and MCD spectroscopy to evaluate the contributions of proximal and distal residues to the coordination environment of the heme iron in these proteins. Although no experimental conditions were identified that allowed quantitative ligation of the cysteine residue to the heme iron in the His93Cys variant, all of the spectroscopic evidence collected for the His93Cys/His64Ile and His93Cys/His64Val double variants supports the assignment of thiolate as the ligand to iron in the oxidized forms of these variants. The double metMb variants exhibit Soret maxima that are considerably blue-shifted, 1H NMR spectra with decreased mean methyl resonances, and EPR spectra with highly rhombic g values. These spectroscopic data for the Fe(III) variants resemble the corresponding properties reported for ferricytochrome P-450. The decrease in the reduction potential of the double variants by 280 mV relative to wild-type protein is also consistent with the low midpoint potential of cytochrome P-450. MCD spectroscopy of these variants confirms that the proximal cysteine residue is not bound in the reduced forms of these proteins and, in the case of the His93Cys variant, that the distal histidine is coordinated to the iron. Similar coordination environments were created in the ferrimyoglobin variants by cyanogen bromide modification, which resulted in cyanation of the sulfur atom and prevented the ligation of Cys93 to the heme iron.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Type I bipolar disorder associated with a fragile site on chromosome 1.

The objective of this paper is to study the association between chromosomal fragile sites and type I bipolar disorder. This case-control study compares bipolar patients with normal controls. Ten cases of type I bipolar disorder diagnosed according to DSM-III-R criteria and the Composite International Diagnostic Interview (CIDI) were selected from the Escola Paulista affective disorders outpatient clinic and 10 healthy controls (CIDI negative for psychiatric diagnoses) matched for sex and age were drawn from the otorhinolaryngologic outpatient clinic of the same hospital. The cytogenetic analysis was carried out with blood lymphocytes, which were cultured in a folic acid-free medium. A total of 100 mitoses per subject were blindly analyzed to the psychiatric diagnostic assignment, and fragile sites were identified according to a minimum expected frequency of events per band in conformity with a Poisson distribution. A higher frequency of chromosomal lesions for cases than controls was found for the following bands: 1q32, 5q31, and 11q23, the 1q32 being considered a fragile site. Although no evident neuropsychiatric etiological component has been mapped to the 1q32 region so far, this finding may lead to further investigation of a possible linkage between genetic markers of this region and bipolar disorder.

Bipolar Disorder↗
PubMed1995

Radiation therapy for primary orbital lymphoma.

PURPOSE: The influence of tumor size, grade, thoroughness of staging workup, and radiation dose on disease control, radiation-related complications, and incidence of systemic progression of primary orbital lymphoma is analyzed. METHODS AND MATERIALS: Twenty patients with Stage I primary orbital lymphoma were treated from August 1976 through August 1991 at Mallinckrodt Institute of Radiology. Staging workups included physical examination, chest x-ray, complete blood count (CBC), liver function test, and computerized tomography (CT) scan of the orbit, abdomen, and pelvis. Nineteen patients had bone marrow biopsy. The histological types based on the National Cancer Institute working formulation were 9 low-grade and 11 intermediate-grade, including five lymphocytic lymphomas of intermediate differentiation. The extension of disease and the volume of tumor were evaluated by CT scan of the orbit. The most commonly used radiation therapy technique was single anterior direct field with 4 MV or 6 MV photons. Lens was shielded or not treated in eight patients. Dose ranged from 20 to 43.2 Gy. Thirteen of 20 patients received 30 Gy. Minimum follow-up was 24 months (median, 4 years). RESULTS: Local control was achieved in all 20 patients. One patient with lymphocytic lymphoma with intermediate differentiation developed disseminated disease. Actuarial disease-free survival (DFS) was 100% and 90% at 2 and 5 years, respectively. No retinopathy was observed. Cataracts were noted in seven patients at 1 to 10 years following irradiation (median, 2 years). Three patients developed lacrimal function disorder; however, no corneal ulceration occurred. CONCLUSIONS: Thirty Gy in 15 fractions appears to be a sufficient dose for local control with acceptable morbidity, especially for low-grade, as well as certain types of intermediate-grade lymphomas, such as diffuse small cleaved cell and lymphocytic lymphoma of intermediate differentiation. Systemic dissemination is minimal, provided local control is achieved and initial staging workups are adequate.

Adult↗
PubMed1995

Analysis of nuclear pore protein p62 glycosylation.

Glycoprotein components of the nuclear pore are essential for nuclear transport and are modified by both glycosylation and phosphorylation. The function and control of these post-translational modifications are poorly understood. Glycosylation of the major rat nuclear pore glycoprotein, p62, was examined in vitro using recombinant p62 as a substrate. Rat p62 was expressed in Escherichia coli and purified to near homogeneity. Kinetic analysis using a partially purified mammalian transferase suggests that the recombinant protein is an excellent substrate (Km = 0.30 microM) for the transfer of GlcNAc from UDP-GlcNAc (Km = 1.8 microM). Localization of the sites of O-linked GlcNAc glycosylation of rat p62 was performed by a combination of deletion analysis of in vitro translation products and by immunoprecipitation of [14C]GlcNAc-labeled proteolytic fragments. The amino terminus of rat p62 is poorly glycosylated with no O-linked GlcNAc sites between Lys22 and Lys97; the carboxyl terminus has one known glycosylation site at Ser471. The majority of the glycosylation sites in rat p62 are likely to occur on the six clustered Ser residues in the central Ser/Thr-rich region from Ser270 to Thr294. A synthetic peptide derived from this region is a good substrate for O-GlcNAc addition (Km = 30 microM) and a potent competitive inhibitor of p62 glycosylation (Ki = 15 microM). It is proposed that this Ser/Thr-rich domain functions as a linker region between the amino-terminal beta-pleated sheet and the carboxyl terminal alpha-helical domains. O-Glycosylation and phosphorylation of this linker region could provide a dynamic means of altering the conformation of p62 during nuclear pore assembly and disassembly.

Animals↗
PubMed1995

Structural studies of the roles of residues 82 and 85 at the interactive face of cytochrome c.

A combination of structural, functional, and mutagenic experiments has been used to study the roles of the invariant Phe82 and highly conserved Leu85 residues in cytochrome c, especially with respect to the complexation interface with electron transfer partners and maintenance of the hydrophobic heme pocket. Structural analyses show that the F82Y, L85A, and F82Y/L85A mutant proteins all retain the characteristic cytochrome c fold, but that conformational alterations are introduced in the direct vicinity of the mutation sites. In particular, the additional hydroxyl group of Tyr82 is in direct spatial conflict with the side chain of Leu85 in the F82Y mutant protein, leading to rotation of the side chain of Tyr82 out toward the protein surface. This strain is relieved in the F82Y/L85A mutant protein where the phenyl ring of Tyr82 is accommodated in a conformation comparable to that of the phenylalanine normally present at this location. In addition, the available space vacated by the replacement of Leu85 with an alanine allows for the inclusion of two new internal water molecules, one of which is bound to Tyr82 and the other to Arg13. In contrast, in the L85A mutant protein, no internal water molecules are observed in this exclusively hydrophobic pocket, which is partially filled by shifts in nearby side chains. Overall, the conformational changes observed result from the optimization of side chain packing to reflect the spatial requirements of new side chains, the minimization of both vacant internal space and the solvent exposure of hydrophobic groups, and the attainment of maximal hydrogen bonding between available polar groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

The intracellular polymerase chain reaction for small CMV genomic sequences within heavily infected cellular sections.

The indirect intracellular polymerase chain reaction (in situ PCR) combines the potential sensitivity of the polymerase chain reaction (PCR) with the high specificity and morphological preservation of in situ hybridization (ISH). This study describes a method for the amplification of small, specific cytomegalovirus (CMV) genomic sequences [100 base pairs (bp)] within large formalin-fixed, paraffin-embedded tissue sections. A heat-resistant glue surrounds the section, creating a well which contains a relatively large volume of isotonic reaction solution without a covering mineral oil layer; this optimizes morphological preservation, permits the evaluation of large sections, and allows both denaturation steps and up to 40 cycles of in situ PCR to be performed, whilst progressively concentrating the reaction solution by evaporation during thermal cycling. ISH was performed using a non-isotopic DNA probe with specificity for CMV, with or without prior in situ PCR amplification, both for samples on slides (fibroblasts and lung) and in suspension (fibroblasts). Samples on slides were evaluated by both blinded studies and image analysis, comparing the intensity of signal (P < 0.003) and the numbers of positive cells detected (P < 0.007), with or without intracellular amplification. Cells in suspension were analysed by blinded studies on cytospins and by gel electrophoresis of cell lysates. Successful intracellular amplification was achieved in this high copy model.

Base Sequence↗
PubMed1995

Replacements in a conserved leucine cluster in the hydrophobic heme pocket of cytochrome c.

A cluster of highly conserved leucine side chains from residues 9, 68, 85, 94, and 98 is located in the hydrophobic heme pocket of cytochrome c. The contributions of two of these, Leu 85 and Leu 94, have been studied using a protein structure-function-mutagenesis approach to probe their roles in the maintenance of overall structural integrity and electron transfer activity. Structural studies of the L85C, L85F, L85M, and L94S mutant proteins show that, in each case, the overall fold of cytochrome c is retained, but that localized conformational shifts are required to accommodate the introduced side chains. In particular, the side chains of Cys 85 and Phe 85 form energetically favorable interactions with Phe 82, whereas Met 85 takes on a more remote conformation to prevent an unfavorable interaction with the phenyl ring of Phe 82. In the case of the L94S mutant protein, the new polar group introduced is found to form hydrogen bonds to nearby carbonyl groups. In all proteins with substitutions at Leu 85, the hydrophobic nature of the heme pocket is preserved and no significant decrease in heme reduction potential is observed. Despite earlier predictions that Leu 85 is an important determinant in cytochrome c electron transfer partner complexation, our studies show this is unlikely to be the case because the considerable surface contour perturbations made by substitutions at this residue do not correspondingly translate into significant changes in electron transfer rates. For the L94S mutant protein, the substitution of a polar hydroxyl group directly into the hydrophobic heme pocket has a larger effect on heme reduction potential, but this is mitigated by two factors. First, the side chain of Ser 94 is rotated away from the heme group and, second, the side chain of Leu 98 shifts into a portion of the new space available, partially shielding the heme group. The Leu 94 Ser substitution does not perturb the highly conserved interface formed by the nearly perpendicular packing of the N- and C-terminal helices of cytochrome c, ruling this out as the cause of this mutant protein becoming thermally labile and having a lower functional activity. Our results show these effects are most likely attributable to disruption of the heme pocket region. Much of the ability of cytochrome c to absorb the introduction of mutations at Leu 85 and Leu 94 appears to be a consequence of the conformational flexibility afforded by the leucine cluster in this region as well as the presence of a nearby internal cavity.(ABSTRACT TRUNCATED AT 400 WORDS)

Crystallography, X-Ray↗
PubMed1995

Baseline and follow-up MRI of the internal auditory canal after suboccipital resection of acoustic schwannoma: appearances and clinical correlations.

Prospective baseline MRI was obtained on 31 patients who had "total" removal of acoustic schwannoma 6 months to 9 years previously. Follow-up MRI was performed after 1-2 years on patients with questionable abnormalities. We found four patterns (1) internal auditory canals (IAC) with nonenhancing soft-tissue strands, possibly scars or distorted residual nerves (8); (2) IAC with marginal enhancement-reactive dura mater (16); (3) IAC with contrast-enhancing globular tissues suggesting residual or recurrent tumour (5); (4) high-signal intensity in the IAC before contrast medium administration, probably related to graft with fat/fascia/muscle (2). Prospective 1-to 2-year follow-up studies were available in 8, 5, and 1 patients in groups 2, 3, and 4 respectively. In group 2, dural enhancement remained unchanged in 5 patients and decreased in 3. In group 3 follow-up showed 1 tumour recurrence (surgically confirmed) and 4 stable abnormalities. In group 4, follow-up in 1 of the 2 patients was stable. In groups 1 and 2, the MRI features correlate well with complete tumor removal. Whether follow-up in these groups is indicated needs to be determined. In group 3 residual or recurrent tumor cannot be excluded. In group 4, grafts may prevent adequate visualization of the IAC.

Adult↗
PubMed1995

A phase I trial of 5-day continuous infusion cisplatin and interferon alpha.

Combination therapy of cisplatin with interferon alpha (IFN) has been shown in several in vitro as well as in vivo models to be synergistic. In order to decrease toxicity seen with cisplatin, 5-day continuous infusions, in place of bolus administration, have been introduced. This led us to investigate the combination of 5-day continuous infusion cisplatin with repeated IFN dosing in a phase I cisplatin dose escalation study. A group of 17 patients were enrolled in this trial. The maximum tolerated dose (MTD) of cisplatin was 20 mg/m2 per day when combined with 3 x 10(6) units IFN given three times a week. The dose-limiting toxicities seen included thrombocytopenia, leukopenia, and nausea and vomiting. Pharmacokinetic analyses of free (unbound or ultrafilterable) platinum revealed that the decay curve fitted a monoexponential model. Pharmacokinetic parameters of cisplatin were found to correlate with toxicity. Both increases in the maximum concentration of cisplatin achieved (Cpmax) as well as the area-under-the-curve (AUC) for free platinum, correlated with the incidence of nausea and vomiting (both acute and delayed) and hematological toxicities (leukopenia and thrombocytopenia). None of the patients exhibited significant changes in renal function while on this study. The free platinum levels were higher than found in similar studies evaluating comparable cisplatin infusions alone. The enhanced toxicities seen in this trial may be explained by the results of an in vitro study using human plasma spiked with cisplatin and IFN that revealed decreased protein binding of cisplatin by 2.5-3.0-fold. Of the 17 patients treated, two non-small cell lung cancer patients obtained a partial response and one malignant melanoma patient obtained complete resolution of a malignant pleural effusion. Considering the acceptable toxicity seen in this trial, we recommend phase II trials be conducted with continuous infusion cisplatin with IFN in the treatment of non-small cell lung cancer.

Adult↗
PubMed1995

Conservative surgical management of vaginal and vulvar pediatric rhabdomyosarcoma: a report from the Intergroup Rhabdomyosarcoma Study III.

The current management of pediatric vaginal and vulvar rhabdomyosarcoma (RMS) uses cytoreductive chemotherapy before surgical intervention. During the Intergroup Rhabdomyosarcoma Study (IRS)-III (1984 to 1988), 27 evaluable patients were entered on a preoperative chemotherapy protocol. Among 24 patients with vaginal primaries, 20 had an initial biopsy with gross residual (group III), 3 had resection with positive margins (group IIA) and 1 had metastatic disease (group IV). At subsequent surgery, 7 patients underwent partial or complete vaginectomy and 6 of them had no viable tumor identified in the specimen. Only 1 of these 7 patients underwent a cystectomy, whereas 5 underwent hysterectomy. Seventeen patients in this group have no evidence of disease 66 to 108 months after diagnosis; 2 died of chemotoxicity and 1 of unknown causes after achieving a complete response. Ten of these 17 patients were treated with biopsy and chemotherapy only. Four of these 10 had radiotherapy as well. All group IIA patients have no evidence of disease. The 1 group IV patient had rapidly progressive disease with early death. This primary chemotherapy protocol resulted in less need for operative intervention or irradiation compared with previous experience. There was no local recurrence, and 20 of 24 patients remain continuously relapse-free with no evidence of disease. All 3 patients with vulvar primaries were treated by wide local excision and chemotherapy and have no evidence of disease. Conservative surgical intervention for vaginal RMS with primary chemotherapy and adjunctive radiation when necessary appears to result in excellent disease-free survival.

Adolescent↗
PubMed1995

Neonatal soft tissue sarcomas: the influence of pathology on treatment and survival. Children's Cancer Group Surgical Committee.

INTRODUCTION: A multi-institutional study was conducted by the Children's Cancer Group (CCG) to evaluate all soft tissue sarcomas diagnosed within the first month of life. METHODS: A retrospective study by 11 CCG institutions of patient records from 1971 to 1991 were reviewed for demographic data, pathology, therapy, and outcome. RESULTS: 32 neonates with soft tissue sarcomas were identified. There were 21 boys and 11 girls. Pathology was equally divided into three groups: Congenital fibrosarcoma (CFS) (12), rhabdomyosarcoma (RMS) (11), and non-RMS soft tissue sarcomas (NRSTS) (9). Anatomic sites consisted of head/neck (11), extremity (9), trunk (8), pelvis (3), and unknown (2). Overall survival rate was 59% (19/32). CONCLUSION: Soft tissue sarcomas in the neonate comprise three general groups with survival rates dependent on pathology and extent of disease.

Combined Modality Therapy↗
PubMed1995

Strange-male-induced pregnancy disruption in mice: potentiation by administration of 17 beta-estradiol to castrated males.

Previous evidence suggests that androgen activity is necessary for strange males to disrupt early pregnancy in mice. Inseminated females were housed below castrated males, separated by a wire-mesh grid. Castrated males did not disrupt pregnancy, whereas those given daily injections of 27 or 81 micrograms of 17 beta-estradiol did so. In conjunction with previous evidence, these data suggest a similarity between the hormones involved in the capacity of males to disrupt pregnancy and the hormones directly implicated in the females' vulnerability to pregnancy disruption.

Animals↗
PubMed1995

Changing p53 mutations with the evolution of chronic myeloid leukaemia from the chronic phase to blast crisis.

The frequent involvement of chromosome 17p abnormalities in the progression of chronic myeloid leukaemia (CML) led us to investigate the involvement of the p53 tumour suppressor gene located on chromosome 17p. We analysed 31 samples from four patients sequentially, and 16 patients in blast crisis only, using single stranded conformational polymorphism (SSCP) analysis of exons 5-8, followed by cloning and sequencing. The sequential samples ranged from diagnosis through to late disease. We found that 15% of our blast crisis samples had p53 abnormalities. In our sequential studies we found two of the four patients analysed in more detail had p53 mutations in the late chronic phase of disease (11 and 5 months prior to blast crisis becoming apparent). These chronic phase mutations differed from the p53 abnormalities found in the blast crisis samples from these patients. One patient also had the same chronic phase mutation at post bone marrow transplant relapse. Our results suggest that, in some cases, sequential investigations through CML disease progression of p53 mutations and other oncogenes/proto-oncogenes may provide early indications of the routes of disease progression to blast crisis.

Base Sequence↗
PubMed1995

Sexually abused and nonabused mothers' discussions about sex and their children's sexual knowledge.

This study investigated the impact of a mother's experience of childhood sexual abuse on her discussion of sex with her child. Two groups of sexually abused and nonabused mothers, drawn from a larger community sample and matched for social class and the sex and age of their index children, were asked about discussion of sexual information within their family. Children were interviewed about their sexual knowledge. Significantly more mothers who reported an incident of sexual abuse during their childhood said that they had detailed discussions with their child about sexual development and contraception than mothers who reported no childhood incidence of abuse. Although there was no difference between the two groups in independent interviewers' ratings of the children's overall sexual knowledge, more children from the abused mothers' group mentioned their parents as a definite source of sexual information, in particular, regarding contraception. There were also differences between the two groups in the children's responses to a set of ambiguous pictures. More children in the abused mothers' group than the nonabused group gave stories related to child abductions and the possibility of sexual abuse in response to two of the pictures. A mothers' willingness to acknowledge childhood sexual abuse is related to more open discussion of sexual information within the family.

Adolescent↗
PubMed1995

In situ hybridization detection of short viral amplicon sequences within cultured cells and body fluids after the in situ polymerase chain reaction.

Using single primer pairs, intracellular gene sequences of cytomegalovirus (CMV-Towne's strain) and alpha-tubulin were amplified (in situ PCR) from cells in human body fluids and in suspensions. Visualization of CMV amplificants was carried out by in situ hybridization (ISH), using both a biotinylated double-stranded DNA probe and a radiolabelled oligonucleotide probe. Visualization of alpha-tubulin amplificants was achieved using both radiolabelled single-stranded cRNA and oligonucleotide probes. Liberated amplificants were also identified by bands of expected size by gel electrophoresis. The specificity of the PCR products was confirmed by Southern blot analysis. Intracellular amplification was identified both in unfixed cells and, optimally, after brief alcohol fixation, whilst maintaining relative isotonicity in all working solutions. For CMV, enhanced signal was observed in cells (cultured fibroblasts or urine sediment) undergoing in situ PCR using either biotinylated or radiolabelled probes compared with controls undergoing ISH alone. For alpha-tubulin, radiolabelled riboprobes and oligoprobes only produced signals within cells (human peripheral lymphocytes, ascitic fluid and bladder washings from routine cytological specimens) after in situ PCR, but not after ISH alone. Morphological evaluation was superior with biotinylated probes, and minimal back-diffusion effect was found compared with radiolabelled probes. Up to 80% of cells survived thermal cycling. In situ PCR detected short sequence (100 bp) foreign DNA and low copy number genomic DNA, and was superior to ISH alone. In contrast to radiolabelled probes, very small CMV amplificants could be detected without a significant 'back-diffusion' effect when using the large biotinylated probe in this model system.

Animals↗