Tribute to those many individuals who have collectively contributed to the successful development of this new international venture.
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Biomedical subjects
Publications and source records attributed to J Miller.
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A partial cDNA clone for the B-26 region of apolipoprotein B was isolated from an adult human liver DNA library by screening with an oligonucleotide probe derived from amino-terminal protein sequence obtained from purified B-26 peptide. Antisera against a synthetic 17-residue peptide whose amino acid sequence was encoded by the clone cross-reacts with apolipoproteins B-26, B-100, and B-48, but not with B-74. The nucleotide sequence immediately upstream from the amino terminus of B-26 codes for an apparent signal sequence, implying that the B-26 moiety is in an amino-terminal locus in the B-100 protein. That this sequence represents a 5' end region is further supported by primer extension analysis using a fragment of the cDNA clone and by S1 nuclease protection experiments using the corresponding region in a genomic clone.
A patient, who proved to have a chylous cyst of the colon, presented with bright red rectal bleeding and an abnormality on barium enema suggesting a colonic polyp. We review such an unusual presentation and other reported cases.
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Sequencing studies have shown that in somatic cells alternating runs of purines and pyrimidines are frequently associated with recombination crossover points. To test whether such sequences actually promote recombination, we have examined the effects of poly[d(pGpT).d(pApC)] and poly[d(pCpG).d(pCpG)] repeats on a homologous recombination event. The parental molecule used in this study, pSVLD, is capable of generating wild-type simian virus 40 DNA via recombination across two 751-base-pair regions of homology and has been described previously (Miller et al., Proc. Natl. Acad. Sci. USA 81:7534-7538, 1984). Single inserts of either a poly[d(pGpT).d(pApC)] repeat or a poly[d(pCpG).d(pCpG)] repeat were positioned adjacent to one region of homology in such a way that the recombination product, wild-type simian virus 40 DNA, could be formed only by recombination within the homologies and not by recombination across the alternating purine-pyrimidine repeats. We have found that upon transfection of test DNAs into simian cells, a poly[d(pCpG).d(pCpG)] repeat enhanced homologous recombination 10- to 15-fold, whereas a poly[d(pGpT).d(pApC)] repeat had less effect. These results are discussed in terms of the features of these repeats that might be responsible for promoting homologous recombination.
The nude mouse is an athymic mutant whose immunological deficiency has been exploited for transplantation of normal and diseased xenogeneic tissue. Histologically, its skin has no unusual features apart from the absence of hair. We report here a biochemical study of its epidermis, with comparison to the hairless mouse (which is devoid of hair but otherwise functionally normal). The epidermal glycoproteins were probed with the lectin, concanavalin A (Con A). Fluorescein isothiocyanate (FITC)-Con A overlays of cryostat skin sections gave a similar fluorescent pattern for both mouse strains: all the viable epidermal cell layers were labeled but not the stratum corneum. In contrast, when different populations of keratinocytes that were separated on Percoll gradients were analyzed by gel electrophoresis, and the gels then overlaid with iodinated Con A, all the epidermal layers, including the stratum corneum, were labeled. For all the epidermal cell layers there are substantial differences between the two mouse strains. We observe changes in the glycoprotein distribution with the stage of differentiation. Comparison with our earlier data for human epidermis indicates that the discrepancies between the nude mouse and the hairless mouse are much greater than those between the latter and man. The most striking difference is the absence in the stratum corneum of the nude mouse of a 40 K glycoprotein which is the dominant feature for the hairless mouse and for man. The gel patterns point to functional discrepancies in the epidermis of the nude mouse, particularly in the stratum corneum, not evident histologically or with FITC-Con A.
Clinical as well as laboratory studies have been employed to assess the complement activating potential of polycarbonate membrane hemodialyzers. Blood samples from a group of patients undergoing sequential maintenance hemodialysis with cuprophane, polyacrylonitrile and polycarbonate devices were evaluated to define plasma levels of C3a antigen and leukocyte counts during the initial phases of hemodialysis. While polyacrylonitrile dialyzers did not activate complement to a significant extent, we did observe transient elevations in the plasma concentration of C3a and corresponding diminutions in the granulocyte counts of patients dialyzed with both cuprophane and polycarbonate dialyzers. However, polycarbonate devices appeared to activate complement to a lesser degree than cellulosic dialyzers. Laboratory evaluation of these three different types of dialyzers also provided evidence that polycarbonate membranes did not appear to activate human complement as readily as cuprophane. These observations suggest that polycarbonate membranes display complement-related biocompatibility properties that are intermediate between those of cuprophane and polyacrylonitrile.
We have serially followed the function of intrahepatic canine islet autografts in 15 beagle dogs for up to 24 mo. Of these, only 20% sustained normal levels of fasting blood glucose for greater than 15 mo posttransplant. Failure of autograft function was accompanied by a preferential loss of well-granulated beta cells in the engrafted islets. The chronic stimulation of an initially marginal intrahepatic beta-cell mass ultimately resulted in metabolic deterioration and loss of beta cells below the minimal threshold required to maintain normal fasting blood glucose levels. It is possible that transplantation of a larger mass of islets would result in indefinite graft function in dogs. However, it remains to be demonstrated in larger mammals, including humans, whether an islet cell mass that is initially adequate in a heterotropic site such as the liver can remain functionally competent over a prolonged period.
The radiation dose to a series of patients referred for chest radiography has been monitored using thermoluminescent dosimetry. The postero-anterior projection was employed throughout the study. Measurements were made in two rooms of the same general hospital. Each room was equipped with automatic exposure control of the air ionisation chamber type. The effect of this equipment on patient exposure was investigated and compared with manual exposure control. In all cases the tube potential (kVp) was selected by the radiographer but tube current (mA) was determined by the generator. Anterior and posterior radiation doses were measured using sachets of lithium fluoride. For each group of patients the anterior exit dose, and therefore all of the radiation dose, was lower when automatic control was used. The standard deviation on the anterior patient dose was lower under automatic control, which resulted in fewer films needing to be repeated due to incorrect film density. Film densities were also investigated using a densitometer, to provide a further comparison between the two types of exposure control.
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Five patients with hydrocephalus were studied with carbon-11-2-deoxyglucose or 2-deoxy-2-(18F) fluoro-D-glucose and positron emission tomography both prior to and following ventricular shunting. Four subjects had communicating hydrocephalus; the fifth had aqueductal stenosis, two patients had hydrocephalus for three months or less. The three chronic patients were felt to have hydrocephalus for three years or more. After shunting ventricular size decreased in all patients, and all patients showed clinical improvement. The glucose cerebral metabolic rates increased after shunt in the two subjects with recent onset hydrocephalus but paradoxially decreased in the three chronic patients despite clinical improvement. These findings suggest that the cerebrum was metabolically hyperactive prior to shunt due to an unknown mechanism and presumably in response to the presence of hydrocephalus. A dissociation may also exist in the post-shunt period between cerebral metabolism and cerebral blood flow.
Leukotriene B4 (LTB4) induced rapid breakdown of prelabeled inositol phospholipids in rat peritoneal polymorphonuclear leukocytes (PMNs). Formation of [3H]inositol triphosphate ([3H]IP3) was rapid, with a peak of 250-300% of the control level, after 5-15 sec of exposure to LTB4. Accumulation of [3H]inositol bisphosphate was rapid, peaking after 30 sec of treatment. Accumulation of [3H]inositol monophosphate was also rapid in the presence of LiCl. The kinetics of [3H]IP3, [3H]inositol bisphosphate, and [3H]inositol monophosphate accumulation suggest that LTB4 may interact with receptors in PMNs and activate phospholipase C which in turn induces hydrolysis of inositol-phospholipids. The agonist activities of several LTB4 analogs were employed to investigate the structure-activity relationships of LTB4 receptor-mediated activation of phosphatidylinositol hydrolysis. Increases in [3H]IP3 formation were dependent upon the concentration of LTB4 and the agonist analogs. The rank order potency of these analogs was equivalent to that of the pharmacological activity of LTB4 agonists in the PMN chemotaxis assay. Furthermore, the islet activation protein isolated from Bordetella pertussis inhibited LTB4-induced [3H]IP3 formation. The tumor-promoting phorbol myristate acetate also inhibited LTB4-induced [3H]IP3 formation. The LTB4 receptors on a partially purified PMN membrane were characterized. LTB4 binding to the receptors was stereoselective and specific. The binding affinity (Kd) of [3H] LTB4 to the receptors was 1.3 +/- 0.2 nM. The maximum density of binding was 5.5 +/- 1.8 pmol/mg of protein. The rank order potency of binding affinities of several LTB4 analogs was equivalent to that of the induction of IP3 response induced by LTB4 and analogs. These results suggest that LTB4 may interact with receptors in rat PMNs, activate G protein-regulated phospholipase C, and induce [3H]IP3 formation.
A deficiency in light-dependent opsin phosphorylation and a slight reduction in opsin synthesis were observed during photoreceptor cell development (22-26 days) preceding photoreceptor cell loss in Irish setters with rod-cone dysplasia. In addition to opsin, two other phosphoprotein bands were found associated with the photoreceptor cell layer; synthesis and phosphorylation of one of these (band 3; 44-48 Kd) appeared reduced, while synthesis and phosphorylation of the other (band 1; 29-31 Kd) was within the normal range in 25-day-old affected setters. The deficiency in light-dependent opsin phosphorylation in affected setters was not due to a deficiency in opsin kinase, since soluble proteins from affected or normal outer segments catalyzed equally well opsin phosphorylation in partially kinase-depleted outer segment membranes from normal, while both kinase preparations failed to promote light-dependent opsin phosphorylation in those from affected setters. A deficiency in light-dependent opsin phosphorylation was also observed in rd/rd mice at all ages studied. In contrast, in Royal College of Surgeons (RCS) rats, light-dependent opsin phosphorylation was within the normal range prior to photoreceptor loss, and became nondetectable only after 50% or more of the photoreceptors had degenerated.
A 24 hour computerized four-channel esophagopharyngeal pH system is described. Using a 1.5 mm diameter esophageal probe containing four separate antimony-tipped electrodes and a small patient-worn digital recording computer, inpatient and outpatient studies are performed in the physiologic environment of the patient's workplace or home. Stored pH data in the computer are teletransmitted from satellite esophageal pH laboratories to a central esophageal pH laboratory for analysis, scoring, printout, and storage. Satellite laboratories located in hospitals, clinics, and physicians' offices use a minimum of equipment and obtain a quality computer-based printout. This preserves patient-physician relationships in the home environment and is cost-effective. Four case reports are presented identifying the advantages derived from the four-channel system localizing and quantifying the extent of cephalad transport of refluxed upper gastrointestinal content. The system has unique clinical and research potential in all age groups in such disparate problems as sleep apnea, laryngitis, bradycardia and cardiac irregularities, and aspiration pneumonia and pulmonary abscess.
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An enzyme-linked immunosorbent technique was established for the assay of serum antibodies to the group C and G streptococcal group-specific carbohydrates. The antigens consisted of formamide-extracted purified polysaccharides conjugated to poly-L-lysine. By use of hyperimmune rabbit antisera to the streptococcal group-specific polysaccharides A, C, and G, a high degree of specificity was encountered for each of the antigens tested. Antibody titers to these antigens were then measured in sera of 100 normal individuals varying in age from newborn to 20 years. The mean titer of these antibodies increased significantly between the ages of 5 and 15 years and leveled off thereafter. Assay of antibodies to the group A, C, and G carbohydrates on sera of patients with antecedent group A streptococcal infections or rheumatic fever and their matched normal controls revealed significantly elevated titers for the antibody to streptococcal group A carbohydrate only in the sera of these patients. These results support the specificity of these tests and suggest their potential usefulness for providing evidence for infection by the various streptococcal serogroups in humans.