Small bowel malabsorption: clinical and radiologic perspectives. How we see it.
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Digestion↗
Biomedical subjects
R A Rubin
Publications and source records attributed to R A Rubin.
Effect of glycemic control on the overnight dexamethasone suppression test in patients with diabetes mellitus.
Because many of the clinical features associated with Cushing's syndrome are frequently found in patients with diabetes mellitus, diabetic patients are often evaluated for Cushing's syndrome. The initial test for Cushing's syndrome is the 1 mg overnight dexamethasone suppression test (DST), but its value as a screening test in diabetic subjects, especially those with poor glycemic control, has been questioned. To address this issue, an overnight DST was administered to 100 subjects with diabetes. Only 7 patients failed to suppress their plasma cortisol to less than 140 nmol/L (5.0 micrograms/dL), achieving a specificity of 93%. There was no relation between acute glycemic control (as measured by the mean of 4 serum glucose values obtained before receiving dexamethasone) or chronic glycemic control (as measured by glycohemoglobin) and false positive responses to the 1 mg overnight DST. The mean of the measures of acute glycemic control of the 7 subjects who had false positive results, 14.4 +/- 2.8 mmol/L, was not significantly different than that of the 93 subjects with normal responses, 13.2 +/- 3.3 mmol/L. Similarly, the mean glycohemoglobin of the subjects with false positive results, 12.8 +/- 2.4%, was not significantly different than that of the subjects with normal responses, 12.9 +/- 2.5%. There was no correlation between plasma cortisol after dexamethasone and glycohemoglobin (r = 0.05), and only a weak correlation with the mean serum glucose (r = 0.21). We conclude that the 1 mg overnight DST is a valid screening test for Cushing's syndrome in patients with diabetes, regardless of glycemic control.
Adult↗
Acute esophageal obstruction: a unique presentation of a giant intramural esophageal leiomyoma.
We report the case of a 33-yr-old man who presented with an acute incomplete esophageal obstruction, mimicking food impaction, who was found to have a giant intramural esophageal leiomyoma. The leiomyoma was successfully removed through a longitudinal esophagotomy. This is the first report of a nonpedunculated intramural esophageal leiomyoma presenting as an acute esophageal obstruction.
Adult↗
Susceptibility to subacute thyroiditis is genetically influenced: familial occurrence in identical twins.
Subacute thyroiditis is thought to be virally induced in genetically predisposed individuals because a strong association has been suggested recently between HLA-B35 and patients in whom subacute thyroiditis has developed. Two identical twin brothers were seen at our clinic with the same symptoms and date of onset of hyperthyroidism and enlargement and tenderness of the thyroid, which gave us a unique opportunity to study the genetic predisposition and treatment of this thyroid disease. Diagnostic criteria for subacute thyroiditis were met in both twins, including hyperthyroxinemia, suppression of thyroidal 123I uptake, increased erythrocyte sedimentation rate, transient painful goiter, and absence of antimicrosomal antibodies. Twin B was treated with corticosteroids, and a nonsteroidal anti-inflammatory agent was prescribed for Twin A. The mode of treatment used did not make a difference in affecting the course of the disease. The erythrocyte sedimentation rate was normal after 2 months from onset of symptoms. Results of viral studies were inconclusive. The same HLA typing was found in each twin: A3, B18, B35, Cw4, DR2, DRw10, DQw1. Thus, each was heterozygous for HLA-B35. We reviewed the literature and found a strong association between HLA-B35 and subacute thyroiditis in various ethnic groups tested. Our experience with these identical twins provides additional evidence to suggest that HLA-35 and perhaps Cw4 confer genetic susceptibility in acquiring subacute painful thyroiditis in a possible dominant mode of inheritance.
Adult↗
Tet protein domains interact productively to mediate tetracycline resistance when present on separate polypeptides.
Both domains, alpha and beta, of the cytoplasmic membrane-localized Tet proteins encoded by the tet gene family (classes A through E) are required for resistance to tetracycline (Tcr) in gram-negative bacteria. Two inactive proteins, each containing a mutation in the opposite domain, are capable of complementation to produce Tcr. Similarly, inactive hybrid proteins expressed by interdomain gene hybrids constructed between tet(B) and tet(C) [tet(B) alpha/(C) beta and tet(C) alpha/(B) beta] together produce significant Tcr via trans complementation (R.A. Rubin and S. B. Levy, J. Bacteriol. 172:2303-2312, 1990). A derivative of tet(B) was constructed to express the two domains of Tet(B) as separate polypeptides, neither containing intact the central, hydrophilic interdomain region. Cells harboring this tet(B) mutant expressed Tcr at about 20% the level conferred by intact tet(B). As expected, no detectable amount of a full-length Tet protein was expressed. A polypeptide corresponding to the alpha domain was observed. Interdomain hybrids between tet(B) and tet(C) containing a frameshift at the fusion junction, designed to result in expression of each of the four domains on separate polypeptides, showed trans complementation without production of detectable full-length proteins. Levels of Tcr were greater than or equal to those previously observed in complementations using full-length hybrid proteins. These results strongly suggest that polypeptides harboring individual alpha and beta domains, lacking an intact interdomain region, can interact productively in the cell to confer Tcr.
Amino Acid Sequence↗
Gene duplication in the evolution of the two complementing domains of gram-negative bacterial tetracycline efflux proteins.
The resistance of Gram- bacteria to the broad-spectrum antibiotic tetracycline (Tc) results from energy-dependent drug efflux mediated by the tet gene product, the cytoplasmic membrane Tet protein. Amino acid (aa) sequences deduced from total tet nucleotide sequences of three different resistance determinants (classes A, B and C) indicate that the protein products [Tet(A), Tet(B), and Tet(C)] share a common ancestor. Hydropathic analysis of Tet sequences predicts twelve transmembrane segments in each protein, with six occurring in each half of the molecule. More importantly, the linear distributions of these segments in the N- and C-terminal halves are nearly identical, suggesting that the two halves of each Tet protein are related by a process of tandem gene duplication and divergence. Indeed, a variable but significant conservation of sequence was detected among the N- and C-terminal halves for all possible comparisons of the three proteins. Such conservation was not observed within other prokaryotic integral membrane proteins or when other prokaryotic proteins were compared to Tet halves. Similarity, both in sequence and in predicted transmembrane structural organization, strongly suggests that a common ancestor of Tet(A), Tet(B), and Tet(C) arose by duplication of a gene reading frame specifying a transmembrane protein of approximately 200 aa residues. The two halves of Tet proteins correspond to the two domains, alpha and beta, which have distinct, complementary roles in Tc efflux. Nevertheless, selective constraints to function in the cytoplasmic membrane have apparently led to maintenance of similar patterns of secondary structural organization in these complementary domains.
Amino Acid Sequence↗
Decreased activity of Reiter's syndrome after urethrectomy.
Reiter's syndrome is a chronic rheumatic disease that develops after infective urethritis or gastroenteritis and has a strong association with the HLA-B27 antigen. How these factors interact remains unclear. We present a patient with Reiter's syndrome who exhibited 2 novel features: unusually severe urethritis that produced strictures requiring surgery, and dramatic regression of his rheumatic manifestations following a urethrectomy.
Adult↗
Interdomain hybrid Tet proteins confer tetracycline resistance only when they are derived from closely related members of the tet gene family.
Inner membrane Tet proteins encoded by tet genes in gram-negative bacteria mediate resistance to tetracycline (Tcr) by directing its export. Total sequences for class A, B, and C tet genes demonstrate that their products have a common ancestor, with Tet(A) and Tet(C) being more closely related (78% identical) than either is to Tet(B) (45% identical). The N- and C-terminal halves of Tet(B) and Tet(C) appear to comprise separate domains, and trans-complementation observed between tetracycline sensitive mutants in either domain of Tet(B) suggests separate but interactive functions for these domains. In this present study, interdomain hybrid genes were constructed to express hybrid tet products whose N- and C-terminal halves were derived from different family members [Tet(A/C), Tet(B/C), and Tet(C/B)]. Tet(A/C) specified a level of Tcr comparable to wild-type Tet(C) and 60% that of Tet(A), indicating that domains from these closely related tet products can function in cis. Although neither Tet(B/C) nor Tet(C/B) hybrids conferred significant Tcr, cells producing both of these types of hybrid proteins expressed substantial Tcr, indicating that productive interactions can occur in trans between Tet(B/C) and Tet(C/B). Taken together, these results suggest that highly specific interactions between the N- and C-terminal domains are necessary for Tcr and do not occur in individual hybrids derived from the more distant relatives, Tet(B) and Tet(C). This requirement for specific interactions suggests that N- and C-terminal domains have coevolved in each member of the Tet family.
Amino Acid Sequence↗
Genetic analysis of the gentamicin resistance region of pPH1JI and incorporation into a wide host range cloning vehicle.
A region of the IncP plasmid pPH1JI encoding resistance to gentamicin, spectinomycin, and streptomycin was characterized by subcloning, deletion, and insertion mutagenesis. Approximate locations of these resistance determinants were established. A 1.6-kb HindIII-SphI segment of this region expresses gentamicin resistance (Gmr) in Escherichia coli when inserted into various plasmid vectors; this DNA segment encodes a polypeptide of 17.5 kDa. Incorporation of this fragment into an IncP cloning vehicle produced a Gmr wide host range vector, pRAR209, which confers levels of Gmr comparable to those expressed by pPH1JI in E. coli, Agrobacterium tumefaciens, and Rhizobium meliloti.
Cloning, Molecular↗
Epidermal growth factor (EGF) stimulates EGF receptor synthesis.
Epidermal growth factor (EGF) binds to the extracellular domain of a specific 170,000-dalton transmembrane glycoprotein; this results in rapid removal of both ligand and receptor from the cell surface. In WB cells, a rat hepatic epithelial cell line, ligand-directed receptor internalization leads to receptor degradation. We tested whether the EGF receptor was replenished at a constitutive or enhanced rate following EGF binding by immunoprecipitating biosynthetically labeled EGF receptor from cells cultured with [35S]methionine. EGF stimulated receptor synthesis within 2 h in a dose-dependent manner; this was particularly evident when examining the nascent form of the receptor. To determine the site of EGF action, total WB cell RNA was transferred to nitrocellulose paper after electrophoresis and was hybridized to cDNA probes from both the external and cytoplasmic coding regions of the human EGF receptor. EGF increased receptor mRNA by 3-5-fold. Therefore, at least in some cells, the surface action of EGF that leads to EGF receptor degradation is counterbalanced by a positive effect on receptor synthesis.
Animals↗
Identification and genetic analysis of an Agrobacterium tumefaciens chromosomal virulence region.
A genetic analysis of Agrobacterium tumefaciens chromosomal functions required for virulence was undertaken. Large Tn5-containing cosmid clones were isolated from DNA of avirulent A. tumefaciens mutants having chromosomal Tn5 insertions and exhibiting defective attachment to plant cells. The clones from several different mutants each contained overlapping segments of a 30-kilobase A. tumefaciens chromosomal region, which were physically mapped. All chromosomal Tn5 insertions leading to the avirulent, attachment-defective phenotype were localized within an 11-kilobase portion of this chromosomal virulence region. Transposon Tn3::HoHo1 (Tn3 containing lacZ) was used to simultaneously mutagenize and create lac fusions within the virulence region. This analysis demonstrated the presence of two distinct chromosomal virulence loci, which were 1.5 and 5 kilobases long; transposon insertions into these loci led to avirulence and defective attachment. The beta-galactosidase activity associated with various Tn3::HoHo1-created lac fusions indicated that the loci are transcribed in opposite directions, and complementation studies suggested that each locus consists of a single transcriptional unit. A cosmid clone of the chromosomal virulence region containing a lac fusion in the extreme 3' portion of the 5-kilobase locus was used to demonstrate that expression of this region is dependent on the presence of sequences in the 5' portion of the locus, confirming its operon-like nature.
Chromosomes, Bacterial↗
Phosphorylation of the hepatic EGF receptor with cAMP-dependent protein kinase.
The 170 000 dalton hepatic epidermal growth factor (EGF) receptor is phosphorylated on serine and tyrosine residues. The evidence indicates that distinct protein kinases are involved. Since EGF and agents that elevate cAMP are believed to participate in the regulation of liver regeneration, we tested whether or not the catalytic subunit of cAMP-dependent protein kinase (catalytic subunit), a known serine kinase, would utilize the EGF receptor as a substrate. The catalytic subunit increased phosphorylation of the EGF receptor in purified rat liver plasma membranes. The serine specificity of the catalytic subunit was established by phosphoamino acid analysis of electrophoretically purified EGF receptor. The result was confirmed by catalytic subunit phosphorylation of affinity purified preparations of the EGF receptor. The rates of dephosphorylation of the membrane-associated EGF receptor phosphorylated on different residues were compared. Dephosphorylation of serine residues (after catalytic subunit phosphorylation) was considerably slower (t1/2 greater than 120 sec) than the removal of phosphotyrosine after stimulation with EGF (t1/2 less than 30 sec).
Animals↗
Vanadate stimulates tyrosine phosphorylation of two proteins in Raji human lymphoblastoid cell membranes.
A membrane fraction from Raji human lymphoblastoid cells exhibited tyrosine-specific kinase activity. Vanadate increased tyrosine phosphorylation up to 5-fold; serine and threonine phosphorylation were unchanged. The stimulation was detectable within 15 s at 0 degrees C and at concentrations of vanadate (0.3 and 1.0 microM) present in normal tissues and blood. The tyrosine phosphorylation of two substrates, M1 61 000 and 55 000, was dependent upon vanadate and incorporation into these substrates represented the majority of the vanadate-sensitive tyrosine phosphorylation.
Burkitt Lymphoma↗
Thermodynamic parameters governing interaction of EcoRI endonuclease with specific and nonspecific DNA sequences.
Equilibrium binding of EcoRI endonuclease to DNA has been analyzed by nitrocellulose filter and preferential DNA cleavage methods. Association constants for pBR322 and a 34-base pair molecule containing the EcoRI site of this plasmid in a central position were determined to be 1.9 X 10(11) M-1 and 1.0 X 10(11) M-1 at 37 degrees C, respectively, with the stoichiometry of binding being 0.8 +/- 0.1 mol of endonuclease dimer per mol of DNA. In contrast, the affinity of the enzyme for a pBR322 derivative from which the EcoRI site has been deleted is 3.2 X 10(9) M-1 as judged by competitive binding experiments. If it is assumed that each base pair can define the beginning of a nonspecific binding site, this value corresponds to an affinity for nonspecific sites of 7.4 X 10(5) M-1. Furthermore, the affinity of the endonuclease for the EcoRI-methylated sequence is at least three orders of magnitude less than that for the unmodified recognition site. The dependence on temperature and ionic strength of the equilibrium constant governing specific interactions has also been examined. The temperature dependence of the reaction indicates that entropy increase accounts for 70% of the free energy of specific binding at 37 degrees C. Affinity of the endonuclease for the EcoRI site is highly dependent on NaCl concentration. Analysis of this dependence according to the theory of Record and colleagues (Record, T. M., Jr., Lohman, T. M., and deHaseth, P. (1976) J. Mol. Biol. 107, 145-158) has implicated 8 ion pairs in the stability of specific complexes, a value identical with the number of phosphate contacts determined by ethylation interference analysis (Lu, A. L., Jack, W. E., and Modrich, P. (1981) J. Biol. Chem. 256, 13200-13206). Extrapolation to 1 M NaCl suggests that nonelectrostatic interactions account for 40% of the free energy change associated with specific complex formation.
Base Sequence↗
DMSO increases tyrosine residue phosphorylation in membranes from murine erythroleukemia cells.
Phosphorylation of membranes from murine erythroleukemia cells was performed in the presence and absence of the polar solvent dimethyl sulfoxide. Quantitation of the phosphoamino acid content revealed that DMSO stimulated phosphotyrosine accumulation by three-fold; serine and threonine phosphorylation decreased significantly. We had previously shown that DMSO stimulated tyrosine residue phosphorylation of the hepatic epidermal growth factor receptor. EGF had little effect in MEL membranes; therefore, DMSO results in accumulation of phosphotyrosine in cell membranes that do not exhibit significant EGF-dependent phosphorylation.
Animals↗
Solubilization of EGF receptor with Triton X-100 alters stimulation of tyrosine residue phosphorylation by EGF and dimethyl sulfoxide.
In isolated hepatic membranes, epidermal growth factor (EGF) and the polar solvent dimethyl sulfoxide (Me2SO) selectively stimulated the phosphorylation of the 170,000-dalton EGF receptor (p170) by 13.6 +/- 2.0- and 10.9 +/- 1.1-fold, respectively. The stimulation by maximally effective concentrations of the two substances was similar in rapidity of onset (less than 30 s at 0 degree C), time course of phosphorylation, and tyrosine residue specificity. These maximal effects were not additive when the substances were combined, indicating that the same kinase/substrate combination is activated by each. The lectin concanavalin A, which inhibits EGF receptor binding, blocked the effect of EGF but not Me2SO. In membranes solubilized with Triton X-100, EGF stimulated p170 phosphorylation by 40- to 55-fold. Me2SO also stimulated phosphorylation, indicating that it acts directly on the protein. However, the effect of the solvent was reduced by half. Additionally, Me2SO blocks the effect of EGF in the solubilized preparation. A room temperature preincubation after addition of either substance was necessary for maximal stimulation of p170 phosphorylation in solubilized membranes. With EGF, 30-40 min was necessary; with Me2SO, only 10 min was required. Thus, a secondary process appears to be involved in EGF receptor/kinase activation.
Animals↗
Dimethyl sulfoxide stimulates tyrosine residue phosphorylation of rat liver epidermal growth factor receptor.
Epidermal growth factor, a potent mitogen, stimulates phosphorylation of its 170,000-dalton plasma membrane receptor. Dimethyl sulfoxide selectively increased phosphorylation of the epidermal growth factor receptor in rat liver microsomal fraction. Maximal stimulation occurred at 15 to 25 percent dimethyl sulfoxide and resembled the effect of epidermal growth factor in magnitude and rapidity. Like epidermal growth factor, dimethyl sulfoxide selectively stimulated tyrosine residue phosphorylation of this protein.
Animals↗
Alteration of epidermal growth factor-dependent phosphorylation during rat liver regeneration.
Epidermal growth factor (EGF) stimulates membrane protein phosphorylation in a human cell line, A-431. The known hepatic mitogenic action of EGF and the reduction in EGF receptor number that occurs during liver regeneration led us to study whether EGF-dependent protein kinase activity was present in rat liver and whether its activity was altered after partial hepatectomy. Liver membranes, preincubated with or without EGF, were phosphorylated (0 degrees C, 15 sec) and subjected to NaDodSO4/polyacrylamide gel electrophoresis and autoradiography. In microsomal fractions, EGF at 5-2000 ng/ml produced a dose-related stimulation of 32P incorporation into a single 170,000-dalton protein (p170). In plasma membranes, a similar EGF-dependent phosphorylation was present and was substantially enriched relative to the microsomal fraction. Acid hydrolysis of labeled microsomal fraction followed by phosphoamino acid determination revealed that EGF stimulated 32P incorporation into phosphotyrosine residues. The EGF-dependent phosphorylation of p170 was compared in microsomal fractions isolated from rats 36 hr after partial hepatectomy or sham operation. In the absence of EGF, in vitro labeling of p170 was similar. EGF stimulated the labeling of p170 in both groups, but the response was clearly diminished after partial hepatectomy. In the presence of EGF, the labeling of p170 in microsomal fraction from regenerating livers was only 47 +/- 6% of that observed in membranes from sham-operated rats (p less than 0.005). Reduction of EGF-dependent phosphorylation during liver regeneration paralleled the loss of binding of 125I-labeled EGF. An increase in the EGF-independent phosphorylation of a 130,000-dalton protein was also observed after partial hepatectomy. The increase in the amount of this phosphoprotein was roughly equal to the loss of EGF-stimulated p170 phosphorylation. Several additional proteins showed increased phosphorylation in membranes from partially hepatectomized rats. These findings indicate that alterations in membrane tyrosine residue phosphorylation occur during regulated growth in vivo.
Animals↗