Structural polymorphism within the amino-terminal region of MM, NN, and MN glycoproteins (glycophorins) of the human erythrocyte membrane

MM, NN, and MN glycoproteins of human erythrocytes from single donors were cleaved by cyanogen bromide into three fragments-A, B, and C-which, upon gel electrophoresis, appeared to be common to the three antigens. Phenol/aqueous urea partitioning and gel filtration were used to separate the peptides quantitatively. Peptide C lacked carbohydrate and homoserine and represented the carboxyl-terminal portion of the glycoproteins. Peptides A and B contained one homoserine each and accounted for all the carbohydrate of the glycoproteins. The peptide portion of glycopeptide A from MM, NN, or MN antigens consisted of eight amino acid residues, of which six were homologous and two varied according to blood type. The variants were serine and glycine in glycopeptide A(MM), leucine and glutamic acid in A(NN), and half-residues of serine, glycine, leucine, and glutamic acid in A(MN). Serine was the amino-terminal residue in A(MM), leucine in A(NN), and one half residue of serine and leucine in A(MN). Each glycopeptide carried two tetrasaccharides (2 NANA, 1 Gal, 1 GalNAc) and one trisaccharide (NANA, Gal, GalNAc) linked O-glycosidically to one serine and two threonines as determined by beta-elimination and sulfite addition. The carbohydrate units were attached to serine and threonine located in the invariant region, because the amino-terminal serine residue could be oxidized by periodate. The M-N antigens are believed to be products of allelic genes which are expressed exclusively in homozygotes and equimolarly in heterozygotes.     

Structural basis of the multispecificity demonstrated by 17beta-hydroxysteroid dehydrogenase types 1 and 5

17Beta-hydroxysteroid dehydrogenases/ketosteroid reductases (17beta-HSDs/KSRs) catalyze the last step of sex steroid synthesis or the first step of their degradation, and are thus critical for many physiological processes. The multispecificity demonstrated by 17beta-HSDs is important for steroid metabolism in gonadal and peripheral tissues, and is a consequence of the architecture of their binding and catalytic sites. Structurally, most of the family members are short chain dehydrogenase-reductases (SDRs) except the type 5 enzyme, which is an aldo-keto reductase (AKR). 17Beta-HSD type 1, a representative of the SDR family, has been studied extensively since the 1950s. However, its structure was not determined until the 1990s. It has always been considered as estrogen specific, in accord with the narrow binding tunnel that has been structurally determined and has been found to be complementary to estrogens. A recent study revealed that, in spite of the enzyme's narrow binding tunnel, the pseudo-symmetry of C19 steroids leads to its alternative binding, resulting in the multispecificity of the enzyme. Expressed in ovary, breast and placenta, the enzyme catalyzes the formation of another estrogen A-diol from DHEA in addition to the biosynthesis of estradiol; it also inactivates the most active androgen DHT by both 17beta-hydroxysteroid oxidation and 3-ketosteroid reduction. Type 5 17beta-HSD (AKR1C3) differs significantly from the type 1 enzyme by possessing a spacious and flexible steroid-binding site. This is estimated to be about 960 or 470 A3 in ternary complex with testosterone or 4-dione, respectively, whereas the binding site volume of 17beta-HSD1 is only about 340 A3. This characteristic of the 17beta-HSD5 binding site permits the docking of various steroids in different orientations, which encompasses a wider range of activities from 20alpha-, 17beta- and 3alpha-HSD/KSR to prostaglandin 11-ketoreductase. The in vitro activities of the enzyme are significantly lower than the type 1 enzyme. In the ternary complex with testosterone, the steroid C3-C17 position is quasi-reversed as compared to the complex with 4-dione. The multi-specificity contributes significantly to steroid metabolism in peripheral tissues, due to the high levels of 17beta-HSD5 mRNA in both breast and prostate tissues.     

High degree of Plasmodium vivax diversity in the Peruvian Amazon demonstrated by tandem repeat polymorphism analysis

Molecular tools to distinguish strains of Plasmodium vivax are important for studying the epidemiology of malaria transmission. Two sets of markers-tandem repeat (TR) polymorphisms and MSP3α-were used to study Plasmodium vivax in patients in the Peruvian Amazon region of Iquitos. Of 110 patients, 90 distinct haplotypes were distinguished using 9 TR markers. An MSP3α polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using HhaI and AluI revealed 8 and 9 profiles, respectively, and 36 profiles when analyzed in combination. Combining TR and PCR-RFLP markers, 101 distinct molecular profiles were distinguished among these 110 patients. Nine TR markers arrayed along a 100 kB stretch of a P. vivax chromosome containing the gene for circumsporozoite protein showed non-linear linkage disequilibrium (I(SA) = 0.03, P = 0.001). These findings demonstrate the potential use of TR markers for molecular epidemiology studies.     

The low density lipoprotein receptor on human peripheral blood monocytes and lymphocytes: visualization by ligand blotting and immunoblotting techniques

Western blotting and immunoprecipitation techniques were used to study low density lipoprotein (LDL) receptors from cultured human monocytes, lymphocytes, and fibroblasts. After incubation in lipoprotein-deficient media to allow induction, receptors were solubilized, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose paper, and detected by incubation with apolipoprotein B-containing lipoproteins followed by radiolabeled antiapolipoprotein B antibody. LDL receptors of identical apparent mol wt were demonstrated in monocyte and lymphocyte cell extracts; the receptors bound LDL as well as human post-prandial lipoprotein (density, less than 1.0063) on Western blots, but did not bind acetyl-LDL. LDL receptors in mononuclear cells could also be detected by immunoblotting using immunoglobulin G-C7, a monoclonal antibody raised against the bovine LDL receptor. When cell extracts were blotted, the mononuclear cell receptors had a lower mol wt than the fibroblast receptor in unreduced sodium dodecyl sulfate-polyacrylamide gels, with an apparent mol wt difference of 5,000 [134,000 +/- 1,400 (+/- SE) for mononuclear cells vs. 139,000 +/- 1600 for fibroblasts]. Immunoprecipitation and electrophoresis of L-[35S]methionine-labeled cell extracts revealed more rapid conversion of the receptor precursor to the mature receptor in monocytes than in fibroblasts. Western blotting of mononuclear cells from a patient with an abnormally high mol wt receptor characterized in fibroblasts demonstrated that the same mutation was expressed in monocytes and lymphocytes. This report represents the first visualization of human mononuclear cell LDL receptors by Western blotting and immunoprecipitation. These techniques may find application in population screening for LDL receptor variability.     

Detection of antibodies by immunoblotting using Chlamydia trachomatis serovars

We compared reactivity between Chlamydia serovar antigens and sera from 18 patients using immunoblotting (IB) and enzyme-linked immunosorbent assay (ELISA). The antigens used were Chlamydia trachomatis serovar L2, D, E, and C organisms for IB and synthetic peptides derived from C, E, G, and L2-VDIV genes for ELISA. Eleven of 12 sera collected from Chlamydia antigen-positive women with cervicitis strongly reacted with C. trachomatis serovar E, as did one serum with serovar C in immunoblotting profiles. ELISA coated individually with peptides E and C strongly reacted with the sera of 6 different patients. The IB result between serovar L2, D, E, and C and sera from the 6 other women patients showed reactivity at E > or = D > or = L2 > or = C. ELISA using a synthetic peptide mixture including C, E, G and L2 peptides gave positive results for all 18 sera. These results indicate that IB sensitivity differes with the C. trachomatis serovar antigen used and that certain cases may produce inconsistent results between IB and ELISA. Results of ELISA and IB are thus not always consistent, indicating that different synthetic peptides should be used in ELISA for detecting of low-level C. trachomatis antibodies.     

Intrathoracic lipomas demonstrated by computed tomography

Nine cases of intrathoracic lipoma are reported. Computed tomography (CT) proved to be helpful in the diagnosis and management of these cases. The attenuations of the masses ranged from -70 to -140 Hounsfield units (HU). In 4 cases, needle biopsies were taken, confirming lipoma with mature fat cells. One patient also had a coelomic cyst with an attenuation of 20 HU, which was confirmed at thoracotomy. Another had an atypical lipoma which infiltrated the thoracic wall but was benign. In conclusion, we recommended investigation with CT scan for the diagnosis of lipoma.     

Mesenteric varices demonstrated by transhepatic portography

A case of mesenteric varices draining into the left renal vein is reported. The varices were well demonstrated by percutaneous transhepatic portography and were obliterated. This disease entity should be considered in the differential diagnosis of patients with lower gastrointestinal tract hemorrhage and portal hypertension.     

Third-party-mediated graft rejection and graft-versus-host disease after T-cell-depleted bone marrow transplantation, as demonstrated by hypervariable DNA probes and HLA-DR polymorphism

Graft rejection after marrow transplantation is generally thought to be mediated by alloreactive immune effector cells of host origin. Transfused blood products also contain immune cells capable of alloreactivity against both donor graft and host. To reduce the risk of transfusion-associated graft-versus-host disease (GVHD) and graft rejection, standard procedure is to irradiate all blood products with at least 1,500 rad before transfusion. We report a patient with chronic myelogenous leukemia who developed graft rejection and GVHD after receiving a T-cell-depleted transplant from a serologically HLA-A, B, DR/DQ matched and mixed lymphocyte culture (MLC) nonreactive unrelated donor. Cytogenetic analysis of marrow cells collected at the time of graft rejection revealed a PH1-negative female karyotype that was not consistent with donor cells. Use of specific minisatellite DNA probes (YNH 24, H-RAS, and 3' HVR) revealed the exclusive presence of third-party (neither donor nor recipient) restriction-fragment-length polymorphisms (RFLP) in both peripheral blood and marrow. Repeat RFLP analysis 3 days later showed persistence of this unique third-party banding pattern. DNA-based HLA-typing, using polymerase chain reaction (PCR) and oligonucleotide probe hybridization, also showed these cells to be derived from an individual whose HLA-DR type was distinct from donor and recipient. Together, these findings suggested the presence of a proliferating population of transfused cells possessing alloreactivity against both donor graft and host, despite prior irradiation of all blood products with 2,000 rad. Limiting dilution analysis to assess the frequency of irradiated lymphocytes able to respond to mitogen revealed an approximate 5- to 6-log reduction at 1,500 to 2,000 rad as compared with unirradiated controls. These data indicate that a small percentage of lymphocytes can survive irradiation at these doses and suggest that existing blood-product irradiation guidelines may require reassessment, especially in T-cell-depleted transplant recipients.     

免疫印迹法检测老年2型糖尿病患者血清胰岛自身抗体的临床价值

目的 评价免疫印迹法检测老年2型糖尿病患者胰岛自身抗体(IAA)的临床价值. 方法 采用免疫印迹(IB)法和酶联免疫吸附(ELISA)法分别检测350例老年2型糖尿病患者和120例健康对照组血清谷氨酸脱羧酶抗体(GADA)、胰岛细胞抗体(ICA)和IAA,比较不同方法检测胰岛相关抗体的阳性率. 结果 在老年2型糖尿病患者中,IB法检测GADA阳性患者为56例,ELISA法检测阳性患者为38例,IB法检测的阳性率要明显高于ELISA法,而2种方法检测ICA、IAA的阳性率比较无明显差异.2种方法检测病程≥5年患者GADA、ICA的阳性率均低于病程＜5年患者,但IB法检测GADA的阳性率要高于ELISA法. 结论 IB法在检测老年2型糖尿病患者IAA尤其是GADA的敏感性要优于ELISA法.     

Structural polymorphism in the N-terminal oligomerization domain of NPM1

Nucleophosmin (NPM1) is a multifunctional phospho-protein with critical roles in ribosome biogenesis, tumor suppression, and nucleolar stress response. Here we show that the N-terminal oligomerization domain of NPM1 (Npm-N) exhibits structural polymorphism by populating conformational states ranging from a highly ordered, folded pentamer to a highly disordered monomer. The monomer–pentamer equilibrium is modulated by posttranslational modification and protein binding. Phosphorylation drives the equilibrium in favor of monomeric forms, and this effect can be reversed by Npm-N binding to its interaction partners. We have identified a short, arginine-rich linear motif in NPM1 binding partners that mediates Npm-N oligomerization. We propose that the diverse functional repertoire associated with NPM1 is controlled through a regulated unfolding mechanism signaled through posttranslational modifications and intermolecular interactions.Nucleophosmin (NPM1) is a highly abundant nucleolar phosphoprotein with functions associated with ribosome biogenesis (1, 2), maintenance of genome stability (1), nucleolar stress response (3), modulation of the p53 tumor suppressor pathway (4), and regulation of apoptosis (5). Importantly, genetic alterations that affect the NPM1 protein sequence or expression level are associated with oncogenesis. For example, NPM1 overexpression was observed in a variety of solid tumors, and mutations within the protein and genetic translocations involving NPM1 are associated with hematological malignancies (reviewed in ref. 6).NPM1 primarily resides in the nucleolus which is a membrane-less compartment and the site of rRNA synthesis, processing, and assembly with ribosomal proteins (7). In the nucleolus, NPM1 is involved in processing preribosomal RNA (4), chaperoning the nucleolar entry of ribosomal (1, 8) and viral (9) proteins, and stabilizing the alternate reading frame (ARF) tumor suppressor protein (4, 5, 10, 11), while also playing a role in the shuttling of preribosomal particles assembled in the nucleolus to the cytoplasm (12–14).NPM1 is a member of the nucleoplasmin protein family, which includes the histone chaperones NPM2 and NPM3. These proteins share a conserved N-terminal oligomerization domain that mediates homopentamerization (15). Disruption of NPM1 oligomerization by a small molecule (16) or an RNA aptamer (17) causes exclusive nucleoplasmic localization, loss of colocalization with ARF, and induction of p53-dependent apoptosis (16, 17). These observations suggest that changes in the oligomeric state of NPM1 may influence its biological functions. However, although it is hypothesized (1) that NPM1 function is modulated through control of its oligomeric state, experimental data are currently lacking. Intriguingly, NPM1 exhibits 40 putative phosphorylation sites, the majority of which are evolutionarily conserved (18, 19). Modification of these sites that is influenced by subcellular localization and cell cycle phase (20, 21) modulates the biological function of NPM1 (1, 6). Approximately one third of these phosphorylation sites are located within the N-terminal oligomerization domain, indicating their possible involvement in regulation of the oligomerization state of NPM1.What is the molecular mechanism that underlies the various functions of NPM1? Here we show by using in vitro biophysical and structural methods that the N-terminal oligomerization domain of NPM1 (Npm-N) exhibits structural polymorphism by populating a range of conformations with various degrees of structural disorder that span two extreme structural states: a folded pentameric state and a disordered monomeric state. Several conserved phosphorylation sites in pentameric Npm-N are positioned within the hydrophobic interior of the pentameric structure (18) and therefore are inaccessible to kinases. Interestingly, other conserved sites are solvent accessible, and we show that these posttranslational modification (PTM) sites serve as molecular switches for modulating the oligomerization and the folding state of Npm-N. We propose that, when exposed through initial destabilizing phosphorylation events, the otherwise inaccessible sites act as molecular locks that, when phosphorylated (22–24), destabilize the pentameric form and lock Npm-N in the monomeric state. We demonstrate that the monomer–pentamer equilibrium is modulated by protein binding partners and have identified a short, arginine-rich (R-rich) linear motif that mediates this interaction. Our results suggest that the diverse cellular functions and subcellular localization of NPM1 are influenced through a regulated unfolding mechanism, signaled through PTMs and intermolecular interactions.     

Multiple biatrial myxomas demonstrated by two-dimensional echocardiography

The two-dimensional and M-mode echocardiographic detection of multiple atrial myxomas is described in an asymptomatic patient with a family history of myxomas. Surgery was subsequently performed without resort to cardiac catheterization studies. Echocardiographic techniques correctly delineated the number and size of the tumors, as well as their site of attachment, mobility and degree of interference with cardiac function.     

Primary adenocarcinoma of the duodenum demonstrated by ultrasonography

A 57 year-old man presented with abdominal discomfort and melena. Abdominal ultrasonography clearly revealed a duodenal tumor as a hypoechoic mass in the transverse segment of the duodenum. The lesion was a 4 × 4-cm oval mass with partial concavity, an irregular surface, high-level central echoes, and a hypoechoic periphery. After confirmation of the diagnosis of duodenal carcinoma, a pylorus-preserving pancreatoduodenectomy was performed curatively. Pathological examination revealed moderately differentiated tubular adenocarcinoma partially invading the pancreatic parenchyma. Ultrasonography can be used to detect lesions of the transverse segment of the duodenum as the first imaging procedure. Received: January 6, 2000 / Accepted: May 26, 2000     

Case of tumor neovascularization demonstrated by cardiac catheterization

A 46-year-old man presented to his physician with nonspecific abdominal complaints. CT of the abdomen was obtained to investigate his symptoms, which showed significant pericardial effusion. One week after drainage of the pericardial fluid, his symptoms recurred and a transesophageal echo showed a right atrial tumor. Cardiac catheterization demonstrated right coronary artery neovascularization. A nonresectable primary angiosarcoma was found following median sternotomy and biopsy. Cathet. Cardiovasc. Diagn. 43:451–453, 1998. © 1998 Wiley-Liss, Inc.     

Endobronchial foreign body demonstrated by xerotomography.

A case of pneumonia secondary to aspirated foreign body is presented. Xerotomography was useful in detecting abnormalities of the tracheobronchial tree and in demonstrating endobronchial foreign bodies.