Backscatter electron imaging of double immunogold labeled erythrocytes using two primary monoclonal IgM antibodies

The majority of mouse monoclonal antibodies reacting with blood group epitopes on erythrocytes are of the IgM class, have equal light chain type, and are available as culture supernatants only. To study the interrelationship of the blood group antigens, a method is presented which allows double labeling applying two unconjugated monoclonal antibodies of the same class and species. The method comprises two indirect, sequential labelings using mouse IgM anti-A and anti-H as primary antibodies and two goat anti-mouse IgM conjugated to 30 and 20 nm colloidal gold particles as secondary antibodies. After labeling for the first antigen, free binding sites on the primary antibody are blocked by incubation with an unconjugated goat anti-mouse antibody. The free anti-species on the secondary antibody, conjugated to 30 nm gold particles, are inactivated by silver enhancement. The silver enhancement also enlarges the gold particle for optimal discrimination between the two particle sizes, which are chosen accordingly. Semiquantitations of double labeled cells from subgroup A2 and A3 were found to be in good agreement with the counts of the corresponding single labelings as well as between experiments, irrespective of which of the two antibodies was applied in the first labeling sequence. The results were in accordance with a reciprocal but nonlinear relationship between the A and H antigens and suggest different affinities of the two antibodies for the epitopes in the subgroups investigated, indicating different biochemistry of the antigen determinants.     

Mapping the prion protein using recombinant antibodies

The fundamental event in prion disease is thought to be the posttranslational conversion of the cellular prion protein (PrPC) into a pathogenic isoform (PrPSc). The occurrence of PrPC on the cell surface and PrPSc in amyloid plaques in situ or in aggregates following purification complicates the study of the molecular events that underlie the disease process. Monoclonal antibodies are highly sensitive probes of protein conformation which can be used under these conditions. Here, we report the rescue of a diverse panel of 19 PrP-specific recombinant monoclonal antibodies from phage display libraries prepared from PrP deficient (Prnp0/0) mice immunized with infectious prions either in the form of rods or PrP 27-30 dispersed into liposomes. The antibodies recognize a number of distinct linear and discontinuous epitopes that are presented to a varying degree on different PrP preparations. The epitope reactivity of the recombinant PrP(90-231) molecule was almost indistinguishable from that of PrPC on the cell surface, validating the importance of detailed structural studies on the recombinant molecule. Only one epitope region at the C terminus of PrP was well presented on both PrPC and PrPSc, while epitopes associated with most of the antibodies in the panel were present on PrPC but absent from PrPSc.     

Long non-stop reading frames on the antisense strand of heat shock protein 70 genes and prion protein (PrP) genes are conserved between species

Several mammalian genes, including heat shock protein (Hsp70) and prion protein (PrP) genes, have been reported to have long open reading frames (ORFs) or non-stop reading frames (NRFs) in the antisense direction. A simple explanation would be that these long antisense reading frames, which are usually in the same triplet frame as the coding strand, are the fortuitous byproduct of a high overall [G+C] content with concomitant preference for G/C over A/T in the third codon position, a preference for RNY type codons (purine/any nucleotide/pyrimidine), and/or a bias against serine and leucine, the only amino acids with codons that can be read as stop codons in the antisense direction. The PrP genes and most heat shock genes with long antisense NRFs (aNRFs) are indeed relatively [G+C] rich but do not show a bias against serine and leucine. In several vertebrates investigated, at least one of the Hsp70 genes has a long antisense reading frame, and we found that some, though not all, putative stop codons in long Hsp70 antisense reading frames were due to sequencing errors. The PrP gene contains an extended antisense open reading frame in all 45 eutherian mammals tested, but not in a marsupial and in a bird. In the PrP gene, the long, protein-coding exon also harbors the antisense nonstop reading frame. In both Hsp70 and PrP genes, the putative antisense protein sequence is well conserved. Even though there is no clear evidence in Hsp70 or PrP genes for the existence of the respective antisense proteins, we speculate that such antisense proteins serve to regulate the genuine Hsp and PrP proteins under special circumstances. Alternatively, regulation might occur at the RNA level, and the antisense RNA would merely lack stop codons to prevent its rapid degradation by an mRNA quality control mechanism that is triggered by premature stop codons. We note that both Hsp and PrP are involved in physiological or pathological protein aggregation phenomena, that scrapie prions have been reported to modify the expression or localization of heat shock proteins, and that in yeast, propagation of a prion-like state (PSI+) depends on a heat shock (Hsp104) protein.     

Circumventing tolerance to generate autologous monoclonal antibodies to the prion protein

Prion diseases are disorders of protein conformation and do not provoke an immune response. Raising antibodies to the prion protein (PrP) has been difficult due to conservation of the PrP sequence and to inhibitory activity of alpha-PrP antibodies toward lymphocytes. To circumvent these problems, we immunized mice in which the PrP gene was ablated (Prnp 0/0) and retrieved specific monoclonal antibodies (mAbs) through phage display libraries. This approach yielded alpha-PrP mAbs that recognize mouse PrP. Studies with these mAbs suggest that cellular PrP adopts an unusually open structure consistent with the conformational plasticity of this protein.     

Different genotypes causing indiscernible patterns of A expression on A(el) red blood cells as visualized by scanning immunogold electron microscopy

OBJECTIVE: Cerebrospinal fluid (CSF) diagnoses encompass a wide spectrum of conditions. The authors review one institution's CSF cytology results over an 11-year period. DESIGN AND SETTING: A retrospective study of 5951 CSF specimens generated between 1985 and 1995. Specimens from pediatric patients (<19 years of age) from the same time period were separately identified. RESULTS: A total of 5561 adult and 390 pediatric CSF specimens were interpreted. A diagnosis of "negative for malignant cells" was assigned in 5171 (93%) of the adult cases and in 351 (90%) of the pediatric cases. Specific infectious organisms were identified in 26 adult specimens and one pediatric specimen. Cryptococcus was the most common infectious agent observed (n = 23 adults), and Toxoplasma was the sole pediatric infectious agent. Two hundred seventy-six (5%) adult cases and 31 (8%) pediatric cases were positive for malignant cells. Diagnoses included metastatic tumors (adult, 140 [51%]; pediatric, 0); lymphoma/leukemia (adult, 112 [41%]; pediatric, 4 [13%]); malignant unclassified neoplasms (adult, 9 [3%]; pediatric, 0); and primary central nervous system neoplasms (adult, 12 [4%]; pediatric, 27 [87%]). Medulloblastoma was the most common pediatric neoplasm (n = 21). There were 105 (2%) adult cases and 8 (2%) pediatric cases with atypical cells present. Atypical lymphoid cells were the most common type in adult cases (53%). CONCLUSIONS: In our experience, infectious agents were rarely identified in pediatric CSF specimens. In adult specimens, the most commonly identified organisms was Cryptococcus. Primary central nervous system neoplasms accounted for a higher percentage of CSF specimens in the pediatric population than in the adult population. The most commonly identified malignancy in adults was metastatic neoplasms, and in children, medulloblastoma.     

Changes of extracellular matrix in a baboon (Papio hamadryas) model of insulin dependent diabetes: studies using electron microscopy and X-ray diffraction techniques

Extracellular matrix plays an important role in many physiological functions and its abnormalities are thought to play a key role in the pathogenesis of diabetic complications. In this paper we used the techniques of electron microscopy, immunostaining and X-ray diffraction to document some of the early events in the changes of extracellular matrix in a model of insulin dependent diabetes in baboons. Our results show that thickening of basement membrane and enlargement of mesangium are demonstrable in the glomeruli of prepubertal diabetic baboons within 2 years from the onset of diabetes. Concomitant with this was the accumulation of type IV collagen and laminin in the mesangium. By contrast, even the very sensitive technique of X-ray diffraction failed to demonstrate changes in the equatorial direction of collagen molecules of the skin and tendon. We conclude that changes of glomerular extracellular matrix are demonstrable early in insulin dependent diabetes even in prepubertal baboons. These can be used as endpoints in evaluating the efficacy of pharmacological agents such as aminoguanidine in preventing diabetic complications.     

Localization of the apical foramen using the newest electronic instruments: stereomicroscopy and SEM (scanning electron microscopy)

Introduction of double impedence as new parameter in root canal length electronic measurement allowed first and second generation electronic apical localizers main problems overcoming: precision failure in presence of conducting fluids. Our study's purpose was an in vitro evaluation of two third generation instruments (Apit-Osada and Root ZX-Morita Corp.) ability in apical foramen localization using sodium hypoclorite as irrigating solution. 40 human monorooted teeth with immature apex were studied. 20 samples were measured by Apit and 20 by Root ZX; measurements were recorded when apical foramen was reached. Samples were fixed for stereomicroscope observation before and after apical 3 mm worn and prepared for SEM observation. Evaluations about each system's precision were made by calculating difference between foramen position determined by electronic localizer and its real anatomical position determined by a computed image analizing system linked to SEM. All measurements were included between a minimum value of -0.45 mm and a maximum value of 0.26 mm. Mann Whithney U test was performed to compare average values of the two sample groups but his was not meaningful (p = 0.18) showing that there is no valuable difference in accuracy between Apit and Root ZX. According to most researchers, who consider a +/- 0.5 mm error range clinically acceptable, and considering that in vitro measurements never exceded this limit value we conclude confirming both instruments' safety.     

Analysis of the bovine respiratory syncytial virus fusion protein (F) using monoclonal antibodies

Seven monoclonal antibodies (MAbs) directed against bovine respiratory syncytial virus (BRSV) fusion (F) protein were produced and characterized by radioimmunoprecipitation and immunofluorescence assays. These seven MAbs together with the previously described MAbs (Beeler and Van Wyke Coelingh, 1989) to the F protein of human respiratory syncytial virus (HRSV) were used to study the antigenic variation of 12 strains of ungulate RSV. All except one MAbs specific for the HRSV-F protein reacted with ungulate RSV strains less efficiently, indicating that some epitopes are conserved, and others are not conserved on the F proteins of HRSV and BRSV strains. Three MAbs specific to the BRSV-F protein neutralized virus infectivity and reacted with all the ungulate RSV strains, suggesting that these epitopes are well conserved. Based on the reactivity of three other MAbs specific to the BRSV-F protein, ungulate RSVs could be grouped into two subgroups. The results indicated that there are antigenic variations in the F protein among ungulate RSV strains.     

In vivo observations and electron microscopy in the treatment of experimental HSV keratitis with monoclonal antibodies

In vivo observations and electron microscopy showed that topical use of anti-HSV monoclonal glycoprotein antibodies produced marked antiviral effects in inhibiting the development of experimental herpetic keratitis in rabbits and in protecting the susceptible corneal cells. As a new biological product, the anti-HSV monoclonal antibodies may provide a new approach to the treatment of HSV keratitis.     

Characterisation of the major intrinsic protein (MIP) from bovine lens fibre membranes by electron microscopy and hydrodynamics

The major intrinsic protein (MIP) from bovine lens fibre membranes has been purified from unstripped membranes using a single ion-exchange chromatography step (MonoS) in the non-ionic detergent octyl-beta-D-glucopyranoside (OG). SDS-PAGE has confirmed the purity of the preparation and thin-layer chromatographic analysis has shown that the protein is virtually lipid-free. To establish a stable and monodisperse protein sample, we exchanged OG with decyl-beta-D-maltopyranoside (DeM), another non-ionic detergent, by gel-filtration column chromatography. We conclude that the resulting protein/detergent complex is composed of four copies of MIP (a tetramer) and a detergent micelle. This conclusion is based on: (1) measurement of the weight-average molecular mass (Mw,app) of the protein moiety in the protein/detergent complex by sedimentation equilibrium; (2) measurement of the apparent molecular mass of the complexes formed by MIP in OG, in DeM, in dodecyl-beta-D-maltopyranoside (DoM) and in sodium dodecylsulphate (SDS) by gel filtration; (3) measurement of the apparent molecular mass of pure detergent micelles; (4) measurement of the predicted change in the molecular mass of the MIP/DeM complex after partial enzymatic proteolysis; and (5) measurement of the size and shape of the MIP/detergent complex by electron microscopy and single-particle analysis. Therefore, the tetragonal arrangement of MIP observed in both plasma membranes and junctional membranes in lens fibre cells is maintained in solution with non-ionic detergents.     

Ultrastructural localization of cellular prion protein (PrPc) at the neuromuscular junction

We examined the localization of the normal cellular isoform of prion protein (PrPc) in mammalian skeletal muscle. Using two anti-PrP antibodies, the neuromuscular junction (NMJ) was preferentially stained after immunohistofluorescence. The mouse, hamster, and human NMJ displayed a fluorescent signal specific for PrPc. Postembedding immunoelectron microscopy analysis performed in the mouse muscle showed that the PrPc-specific colloidal gold immunolabelling was concentrated over the sarcoplasmic cytoplasm. The membrane of the postsynaptic domain was devoid of gold particles, while a weak signal was occasionally observed close to the presynaptic vesicles of the terminal axons. These results indicate that the PrP gene is expressed in mammalian muscle at the NMJ. The subsynaptic sarcoplasm of the NMJ appears to be the privileged site where PrPc presumably associated with endosome membrane may play a role in either physiological activity or maintenance of the morphological integrity of the synapse.     

Morphological observations on liposomes bearing covalently bound protein: studies with freeze-fracture and cryo electron microscopy and small angle X-ray scattering techniques

HT-29 cells, originating from a human colon carcinoma, can proliferate in standard culture conditions with an absolute requirement for polyamines. The major precursor provided in the culture medium for polyamine biosynthesis is L-arginine. L-Arginine conversion to L-ornithine by arginase is followed by stepwise conversion of this latter amino acid to putrescine, spermidine and spermine. The aim of the present work was to document the consequences of a total inhibition of L-arginine flux through arginase, resulting in a decreased L-ornithine availability, on HT-29 cell proliferation and polyamine metabolism. L-Valine, a known arginase inhibitor, when used at a high concentration, i.e., 100 mM, inhibits L-arginine flux through arginase almost totally. The addition in the culture medium of 100 mM L-valine or 50 mM NaCl used to mimic the L-valine induced increase in medium osmolality both reduced equally cellular growth. Cell viability, protein synthesis or oxidative metabolism measured in isolated cells were unaffected by the L-valine treatment, suggesting that decreased proliferation was not associated with an acute toxic effect of this aminoacid, but was rather due to the increase in the medium osmolality. L-Valine treated cells displayed an altered polyamine metabolism when compared with control cells grown in the absence of the amino acid. After 4 days of treatment with 100 mM L-valine, L-ornithine flux through ornithine decarboxylase was significantly higher as well as putrescine and spermidine cellular uptakes in treated cells. However, the changes in polyamine metabolism led to similar polyamine cell contents in untreated and L-valine treated cells. In conclusion, we propose that the observed alterations of polyamine metabolism may reflect an adaptative response of HT-29 cells to the presence of L-valine which contribute together with the low amount of L-ornithine present in the culture medium to polyamine homeostasis.     

Estimation of tissue protein synthesis in rats fed diets labeled with (U-14C)tyrosine

The rate of liver and muscle protein synthesis has been measured in 27 rats after feeding L-[U-14C]tyrosine in L-amino acid diets prepared as agar gels. Constant specific activity of the free tyrosine pool, as indicated by constant excretion of 14CO2, was reached within 2 h of feeding and was maintained for the remaining 6 h of the 8-h experiment. Muscle protein synthesis was decreased (P less than 0.05) in rats fed a 0.3% methionine diet compared with rats fed this diet supplemented with 0.51% cystine (fractional rate of synthesis, ks: 0.098 vs. 0.121). No effect (P greater than 0.05) of these diets on liver protein synthesis was observed (ks: 0.603 vs. 0.532). Protein synthetic rate was also determined by the constant-intravenous infusion technique in 17 rats fed unlabeled diets. The two techniques gave similar estimates. Restraint of the rats or the infusion of saline had no measurable effect on the rate of protein synthesis in rats fed labeled diets. This feeding technique is essentially equivalent to the constant-infusion technique and offers an easier, more physiological approach to achieving a steady state.     

Elevated levels of serum lipoprotein(a) and apolipoprotein(a) phenotype are not related to pre-eclampsia

BACKGROUND: Pre-eclampsia might result from a less effective invasion of trophoblast cells in the myometrium, caused by attenuated immunosuppression in the spiral arteries, resulting from inhibition of plasmin-mediated activation of transforming growth factor-beta-like substances. In vitro evidence indicates that lipoprotein(a) is capable of inhibiting plasmin-mediated activation of transforming growth factor-beta. Thus, high plasma levels of lipoprotein(a) might result in increased incidence of preeclampsia. METHODS: The patient group consisted of 39 patients with a history of pre-eclampsia in a previous pregnancy: Forty-seven women without pre-eclampsia in their history and matched for age were the control group. All participants gave their informed consent. In both the patient and control group blood pressure, CRP, urinalysis, cholesterol, HDL-cholesterol, triglycerides, lipoprotein(a) level and apolipoprotein(a) phenotype were determined. RESULTS: None of the participants had elevated CRP levels, excluding acute phase related elevations of lipoprotein(a). Proteinuria was present in 33% of patients and in 11% of controls (p=0.01). However, no relation was observed between proteinuria and Lp(a) level. Median Lipoprotein(a) levels in both groups were equal (300 mg/l vs. 275 mg/l; p=0.48), as well as the apo(a) phenotype distribution in both groups. CONCLUSIONS: Lipoprotein(a) and apolipoprotein(a) phenotype do not contribute significantly to the pathogenesis of pre-eclampsia.     

Plants transformed with a mutant alfalfa mosaic virus coat protein gene are resistant to the mutant but not to wild-type virus

Transgenic tobacco plants expressing the wild-type (wt) coat protein (CP) gene of alfalfa mosaic virus (AIMV) have been shown to be resistant to infection with viral particles and RNAs or to infection with viral particles only. The difference in resistance of these plants to RNA inocula was found to correlate with a difference in the expression level of the transgene. Plants expressing a mutant AIMV CP with the N-terminal serine residue changed to glycine have been shown to be susceptible to infection with wt viral particles or RNAs. By site-directed mutagenesis of AIMV cDNA a viable mutant virus encoding CP with the same N-terminal mutation was obtained. Plants expressing wt or mutant CP were resistant to the mutant virus, demonstrating that a single amino acid substitution in CP did not permit the virus to overcome CP-mediated resistance. Although the mutant CP did not confer resistance to wt virus when expressed in transgenic plants, it was still effective in classical cross-protection: plants infected with the mutant virus were resistant to severe strain of AIMV. A model to explain the data is discussed.     

Double-stranded (ds) RNA binding and not dimerization correlates with the activation of the dsRNA-dependent protein kinase (PKR)

Upon binding to double-stranded (ds) RNA, the dsRNA-dependent protein kinase (PKR) sequentially undergoes autophosphorylation and activation. Activated PKR may exist as a dimer and phosphorylates the eukaryotic translation initiation factor 2 alpha subunit (cIF-2 alpha) to inhibit polypeptide chain initiation. Transfection of COS-1 cells with a plasmid cDNA expression vector encoding a marker gene, activates endogenous PKR, and selectively inhibits translation of the marker mRNA, dihydrofolate reductase (DHFR). This system was used to study the dsRNA binding and dimerization requirements for over-expressed PKR mutants and subdomains to affect DHFR translation. DHFR translation was rescued by expression of either an ATP hydrolysis defective mutant PKR K296P, the amino-terminal 1-243 fragment containing two dsRNA binding motifs, or the isolated first RNA binding motif (amino acids 1-123). Mutation of K64E within the dsRNA binding motif 1 destroyed dsRNA binding and the ability to rescue DHFR translation. Immunoprecipitation of T7 epitope-tagged PKR derivatives from cell lysates detected interaction between intact PKR and the amino-terminal 1-243 fragment as well as a 1-243 fragment harboring the K64E mutation. Expression of adenovirus VAI RNA, a potent inhibitor of PKR activity, did not disrupt this interaction. In contrast, intact PKR did not interact with fragments containing the first dsRNA binding motif (1-123), the second dsRNA binding motif (98-243), or the isolated PKR kinase catalytic domain (228-551). These results demonstrate that the translational stimulation mediated by the dominant negative PKR mutant does not require dimerization, but requires the ability to bind dsRNA and indicate these mutants act by competition for binding to activators.     

Genetic markers at the leptin (OB) locus are not significantly linked to hypertension in African Americans

Increased body mass index (BMI) has been correlated with increased blood pressure in human populations. To examine the role of the leptin gene (OB) in essential hypertension in African Americans, we performed affected sib pair analysis on a set of 103 hypertensive African American sibships using four highly polymorphic markers at the human leptin locus. No evidence of linkage was detected between these markers and the phenotype of essential hypertension either in these sibships or in a severely obese subset of 46 sibships in which each sibling had a BMI > or = 85th percentile for the US population. Using BMI rather than hypertension as a quantitative trait, we found significant linkage for the marker D7S504 (P=0.029) but not for the other markers. Significance strengthened in the overweight subset of sibships for this marker (P=0.001), and there was a trend of lower P values for the other three markers. However, multipoint analysis with the use of all four markers simultaneously to estimate linkage between BMI and the leptin locus did not demonstrate a statistically significant relationship. Analysis of the coding region of the leptin gene (exons 2 and 3) by single-strand conformational polymorphism revealed a rare Ile-Val polymorphism at amino acid 45 but revealed no other alterations. These results suggest that the OB gene is not a major contributor to the phenotype of essential hypertension in African Americans, although a minor contribution to the phenotype of extreme obesity in this group cannot be ruled out.