Standard Immunoblotting Research Articles

T cell receptor-mediated signal transduction controlled by the beta chain transmembrane domain: apoptosis-deficient cells display unbalanced mitogen-activated protein kinases activities upon T cell receptor engagement.

The bases that support the versatility of the T cell receptor (TCR) to generate distinct T cell responses remain unclear. We have previously shown that mutant cells in the transmembrane domain of TCRbeta chain are impaired in TCR-induced apoptosis but are not affected in other functions. Here we describe the biochemical mechanisms by which this mutant receptor supports some T cell responses but fails to induce apoptosis. Extracellular signal-regulated protein kinase (ERK) is activated at higher and more sustained levels in TCRbeta-mutated than in wild type cells. Conversely, activation of both c-Jun N-terminal kinase and p38 mitogen-activated protein kinase is severely reduced in mutant cells. By attempting to link this unbalanced induction to altered upstream events, we found that ZAP-70 is normally activated. However, although SLP-76 phosphorylation is normally induced, TCR engagement of mutant cells results in lower tyrosine phosphorylation of LAT but in higher tyrosine phosphorylation of Vav than in wild type cells. The results suggest that an altered signaling cascade leading to an imbalance in mitogen-activated protein kinase activities is involved in the selective impairment of apoptosis in these mutant cells. Furthermore, they also provide new insights in the contribution of TCR to decipher the signals that mediate apoptosis distinctly from proliferation.

Stoichiometric Phosphorylation of Human p53 at Ser315Stimulates p53-dependent Transcription

p53 protein activity as a transcription factor can be activated in vivo by antibodies that target its C-terminal negative regulatory domain suggesting that cellular enzymes that target this domain may play a role in stimulating p53-dependent gene expression. A phospho-specific monoclonal antibody to the C-terminal Ser(315) phospho-epitope was used to determine whether phosphorylation of endogenous p53 at Ser(315) can be detected in vivo, whether steady-state Ser(315) phosphorylation increases or decreases in an irradiated cell, and whether this phosphorylation event activates or inhibits p53 in vivo. A native phospho-specific IgG binding assay was developed for quantitating the extent of p53 phosphorylation at Ser(315) where one, two, three, or four phosphates/tetramer could be defined after in vitro phosphorylation by cyclin-dependent protein kinases. Using this assay, near-stoichiometric Ser(315) phosphorylation of endogenous p53 protein was detected in vivo after UV irradiation of MCF7 and A375 cells, coinciding with elevated p53-dependent transcription. Transfection of the p53 gene with an alanine mutation at the Ser(315) site into Saos-2 cells gave rise to a form of p53 protein with a substantially reduced specific activity as a transcription factor. The treatment of cells with the cyclin-dependent protein kinase inhibitor Roscovitine promoted a reduction in the specific activity of endogenous p53 or ectopically expressed p53. These results indicate that the majority of p53 protein has been phosphorylated at Ser(315) after irradiation damage and identify a cyclin-dependent kinase pathway that plays a role in stimulating p53 function.

Equine Protozoal Myeloencephalitis

Recent advances in the understanding of the parasite life cycle, epidemiology, clinical signs, diagnosis, treatment, and prevention of EPM are reviewed. The NAHMS Equine '98 study and a controlled retrospective study from The Ohio State University College of Veterinary Medicine identified a number of risk factors associated with development of the disease. The national annual incidence of EPM was 1% or less depending on the primary use of the animals. Increased disease risk was associated with age (1-5 and > 13 years of age), season (lowest in winter months and increasing with ambient temperature), previous stressful events, the presence of opossums, the use of nonsurface water drinking systems, and failure to restrict wildlife access to feed. Horses that received treatment were 10 times more likely to improve, and those that improved were 50 times more likely to survive. A number of recent studies confirmed that horses can be experimentally infected with S. neurona; however, large numbers of sporocysts are apparently necessary to achieve infection, and clinical signs and abnormal CNS histology are only seen inconsistently. Results suggest that CNS infection and positive CSF immunoblot findings may be transient phenomena among naturally infected horses. Although immunosuppression may be involved in the development of EPM, some element of the immune response seems to be necessary for the development of clinical signs. Use of the standard immunoblot test for the detection of anti-S. neurona antibodies in CSF continues to provide the most useful adjunct to a detailed neurologic examination for the diagnosis of EPM. Test sensitivity and specificity were 89% in 295 horses euthanatized because of neurologic disease, of which 123 were confirmed cases of EPM. The PPV was 85%, and the NVP was 92%. A number of promising new EPM treatments are under investigation. In addition to standard SDZ/PYR therapy, toltrazuril, ponazuril, diclazuril, and NTZ have shown promise as possible alternatives.

An improved recombinant IgG immunoblot for serodiagnosis of Lyme borreliosis.

We have previously described the use of the following recombinant antigens for serodiagnostic immunoblots: p83/100, p39, OspC and p41 (flagellin) internal fragment [Wilske et al. (1993) Med Microbiol Immunol 182:255-270; Rossler et al. (1997) J Clin Microbiol 35:2752-2758]. In our currently used immunoblot p83/100 is derived from strain PKo (Borrelia afzelii), p39 (BmpA) and OspC from strains PKa2 (B. hurgdorferi sensu stricto), PKo and PBi (B. garinii), respectively; the p41 (flagellin) internal fragments were cloned from strains PKo and PBi. In this study we describe the use of two additional recombinantly expressed highly immunogenic proteins Osp 7 (derived from PKo) and p58 (derived from PBi). A clinically well-defined panel of sera from 147 Lyme borreliosis patients and 139 controls previously tested by a standardized whole cell lysate immunoblot [Hauser et al. (1997) J Clin Microbiol 35:1433-1444] was investigated in the recombinant immunoblot without (old recombinant immunoblot) and with Ospl7 and p58 (new recombinant immunoblot) for IgG antibodies. The sensitivity of the recombinant IgG immunoblot for diagnosis of stage II and stage III could be significantly improved by addition of Osp17 and p58 without loss of specificity. With the exception of sera from patients with erythema migrans the diagnostic sensitivity is comparable to the whole cell lysate IgG immunoblot. The main advantage of the recombinant immunoblot is the easy identification of diagnostic bands, whereas the identification of bands in the whole cell lysate immunoblot is difficult. The recombinant immunoblot is especially suitable where large series of sera need to be investigated.

Distribution of the alpha1 to alpha6 chains of type IV collagen in bovine follicles.

During follicular development the proliferative and differentiated state of the epithelioid granulosa cells changes, and the movement of fluid across the follicular basal lamina enables the formation of an antrum. Type IV collagen is an important component of many basal laminae. Each molecule is composed of three alpha chains; however, six different type IV collagen chains have been identified. It is not known which of these chains are present in the follicular basal lamina and whether the type IV collagen composition of the basal lamina changes during follicular development. Therefore, we immunolocalized each of the six chains in bovine ovaries using antibodies directed to the nonconserved non-collagenous (NC) domains. Additionally, dissected follicles were digested with collagenase to release the NC domains, and the NC1 domains were then detected by standard Western immunoblot methods. The follicular basal lamina of almost all primordial and preantral follicles was positive for all type IV collagen alpha chains. Colocalization of type IV collagen and factor VIII-related antigen allowed for discrimination between the follicular and endothelial basal laminae. Type IV collagen alpha1, alpha2, alpha3, alpha4, and alpha5 chains were present within the follicular basal lamina of only a proportion of antral follicles (17 of 22, 20 of 21, 15 of 18, 14 of 28, and 12 of 23, respectively), and staining was less intense than in the preantral follicles. Staining for the alpha1 and alpha2 chains was diffusely distributed throughout the theca in regions not associated with recognized basal laminae. The specificity of this immunostaining for alpha1 and alpha2 chains of type IV collagen was confirmed by Western immunoblots. As well as being detected in the basal lamina of approximately half of the antral follicles examined, type IV collagen alpha4 also colocalized with 3beta-hydroxysteroid dehydrogenase-immunopositive cells in the theca interna. Type IV collagen alpha6 was detected in the basal lamina of only one of the 16 antral follicles examined. Thus, the follicular basal lamina changes in composition during follicular development, with immunostaining levels being reduced for all type IV collagen chains and immunoreactivity for type IV collagen alpha6 being lost as follicle size increases. Additionally, immunoreactivity for alpha1 and alpha2 appears in the extracellular matrix of the theca as it develops.

Detection of CYP1A1 protein in human liver and induction by TCDD in precision-cut liver slices incubated in dynamic organ culture.

Cytochrome P4501A1 (CYP1A1) has been implicated in the conversion of numerous polycyclic aromatic hydrocarbons into electrophilic species capable of binding covalently to DNA and has therefore been postulated to be involved in the initiation of carcinogenesis. The expression of CYP1A1 protein appears not to be constitutive, but is readily inducible by aryl hydrocarbon (Ah) receptor ligands in a majority of tissues of experimental animals, especially the liver. To date, there is conflicting evidence for the expression or inducibility of CYP1A1 protein in human liver. In this present study, we report the detection of CYP1A1 in all 20 human liver microsomal samples tested by standard western immunoblotting with chemiluminescent detection using a specific monoclonal antibody (mAb 1-12-3) directed against a marine fish (scup) cytochrome P450E. mAb 1-12-3 has been shown previously to specifically recognize CYP1A1 in mammals. This system consistently demonstrated a detection sensitivity as low as 0.01-0.025 pmol CYP1A1 per lane. In the samples where CYP1A1 protein levels were quantitated, CYP1A1 ranged from approximately 0.4 to 5 pmol CYP1A1/mg microsomal protein. Additionally, the inducibility of CYP1A1 protein was demonstrated by incubating precision-cut human liver slices in dynamic organ culture for up to 96 h in the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The specificity of mAb 1-12-3 was tested using several purified human and rat cytochrome P450s to ensure that the protein being detected was CYP1A1. mAb 1-12-3 did not cross-react with human CYP1A2 or CYP3A4 or rat CYP1B1, but did strongly recognize CYP1A1. However, there was a very weak cross-reactivity of mAb 1-12-3 with human CYP2E1, approximately 75-fold less compared with CYP1A1. In order to confirm CYP1A1 as the immunoreactive protein detected in human liver, microsomal samples were subjected to two-dimensional electrophoresis involving isoelectric focusing followed by SDS-PAGE and immunoblotting. Utilizing mAb 1-12-3, the human liver microsomal samples displayed an immunoblotting profile matching that obtained from a microsomal preparation from a AHH-1 TK+/- cell line expressing solely human CYP1A1 and differing from the profile obtained using a polyclonal antibody directed against CYP2E1 and cells expressing CYP2E1. Furthermore, mAb 1-12-3 recognized only one protein of identical mobility on the two-dimensional blots from human liver microsomes and AHH-1 TK+/- cells expressing CYP1A1, while displaying no reaction to cells expressing only CYP2E1. In conclusion, CYP1A1 appears to be expressed in human liver at low levels and is inducible upon exposure to TCDD.

Leptin is Present in Human Milk ♦ 1544

Leptin, the recently cloned product of the obese (ob) gene, is a 16 kD protein secreted by adipocytes. Leptin regulates body weight through suppression of appetite and increases in energy expenditure via interaction with its receptor in the hypothalamus. A multitude of leptin receptor isoforms with unknown functions have also been identified in other tissues including the gastrointestinal tract. Serum leptin has also been identified in significant levels in the fetus and newborn raising speculation regarding its importance as a growth factor. Given studies demonstrating that exclusively breast fed infants are leaner due to decreased energy intakes than formula fed infants, we speculated that leptin absorbed from human milk could participate in mediating the earlier satiety of infants fed human milk. We undertook these studies to both identify the presence and relative levels of leptin in human milk as well as attempt to determine its bioavailability. Random term milk samples were studied from eight mothers during the first two weeks of lactation. Using a standard Western immunoblot approach, 100 micrograms of total protein from skim milk samples were separated and immunoblotted with an anti-leptin antibody and labeled proteins visualized by chemiluminescence. Significant levels of leptin were identified in all samples with no apparent increase noted over time. Utilizing a radioimmunoassay specific for human leptin, daily early morning samples from one mother were studied. The mean concentration of leptin was 2.22 ng/ml with a range of 0.8 - 8.7 ng/ml. To determine if human milk-derived leptin is absorbed from the gastrointestinal tract, human milk samples with varying concentrations of leptin (3-7 ng/ml) and exogenously added human leptin (36 mcg/ml) were fed to neonatal piglets (n= 3) and serum samples collected at Time 0 and at 15 minute intervals for two hours after feeding. However, no significant amounts of human leptin were identified in pig serum using the leptin RIA specific for human leptin. We conclude that while significant amounts appear to be present in human milk by Western analysis and RIA, we thus far have been unable to demonstrate absorption of intact leptin utilizing currently available assays and an animal model with gastrointestinal physiology similar to the human neonate. However, given the presence of leptin receptors in the gastrointestinal tract, a local effect cannot be excluded. Efforts to determine these local effects are in progress.

Biomolecular Basis for Cold-Induced Contraction of Coronary Arteries in the Newborn Lamb: Implication in Myocardial Dysfunction after Hypothermic Cardiopulmonary Bypass ♦ 98

Introduction: Despite improved overall outcome after open heart surgery, early myocardial dysfunction remains a problem in the infant and the neonate in the post hypothermic cardiopulmonary bypass recovery period. We hypothesize that a vasoconstriction of the conductance coronary arteries (CA) occurs when exposed to cold, and that this cold induced contraction involves protein tyrosine kinase (PTK)-/protein tyrosine phosphatase (PTP)-dependent signal transduction pathways. Methods: Newborn lamb 3-mm CA ring segments (1mm diameter) were studied in tissue bath (Krebs buffer, 21%O2, 5%CO2) for isometric contraction during a 2-hour exposure to hypothermia (7° & 17°C). In parallel, 8-mm CA ring segments were evaluated for changes in protein tyrosine phosphorylation using standard electrophoretic and immunoblotting techniques (monoclonal anti-phosphotyrosine antibody & chemiluminescence detection). Na-orthovanadate (SOV), a potent PTP inhibitor, and genestein (Gen), a non selective PTK inhibitor, were used separately and in combination to evaluate their effect on contractile behavior and tyrosine phosphorylation of CA during cooling (7° or 17°C) vs. 37°C. Results: Tissue bath cooling to 17°C resulted in sustained contraction (25% KCl, n=4), reversible upon rewarming. CA segments subjected to 7°C cooling demonstrated a transitory contraction at≈17°C, more prominent during rewarming (5-15% KCl). Cold-induced contraction was abolished in the presence of Gen and significantly enhanced(2-fold) in the presence of SOV, the latter being partially inhibited in the presence of Gen. There was increased tyrosine phosphorylation of 25, 30-33 and 100-110 kDa proteins detected at 17°C compared to 37°C (n=4), similarly enhanced by SOV and abolished by Gen.

B-cell epitopes and quantification of the ESAT-6 protein of Mycobacterium tuberculosis.

ESAT-6 is an important T-cell antigen recognized by protective T cells in animal models of infection with Mycobacterium tuberculosis. In an enzyme-linked immunosorbent assay (ELISA) with overlapping peptides spanning the sequence of ESAT-6, monoclonal antibody HYB76-8 reacted with two peptides in the N-terminal region of the molecule. Assays with synthetic truncated peptides allowed a precise mapping of the epitope to the residues EQQWNFAGIEAAA at positions 3 to 15. Hydrophilicity plots revealed one hydrophilic area at the N terminus and two additional areas further along the polypeptide chain. Antipeptide antibodies were generated by immunization with synthetic 8-mer peptides corresponding to these two regions coupled to keyhole limpet hemocyanin. Prolonged immunization with a 23-mer peptide (positions 40 to 62) resulted in the formation of antibodies reacting with the peptide as well as native ESAT-6. A double-antibody ELISA was then developed with monoclonal antibody HYB76-8 as a capture antibody, antigen for testing in the second layer, and antipeptide antibody in the third layer. The assay was suitable for quantification of ESAT-6 in M. tuberculosis antigen preparations, showing no reactivity with M. bovis BCG Tokyo culture fluid, used as a negative control, or with MPT64 or antigen 85B, previously shown to cross-react with HYB76-8. This capture ELISA permitted the identification of ESAT-6 expression from vaccinia virus constructs containing the esat-6 gene; this expression could not be identified by standard immunoblotting.

Evidence for the existence of a pristanoyl-CoA oxidase gene in man

In the rat, 2-methyl branched fatty acids and the bile acid intermediates di- and tri-hydroxycoprostanic acids are desaturated by pristanoyl-CoA oxidase and trihydroxycoprostanoyl-CoA oxidase respectively. In the human, these compounds are oxidized by a single enzyme, branched-chain acyl-CoA oxidase, which according to its amino acid sequence is the human homologue of rat trihydroxycoprostanoyl-CoA oxidase. Pristanoyl-CoA oxidase is apparently absent from human tissues as indicated by immunoblot analysis [Van Veldhoven, Van Rompuy, Fransen, de Béthune and Mannaerts (1994) Eur. J. Biochem. 222, 795-801] and Northern-blot analysis [Vanhooren, Fransen, de Béthune, Baumgart, Baes, Torrekens, Van Leuven, Mannaerts and Van Veldhoven (1996) Eur. J. Biochem. 239, 302-309] of human tissues. In this paper we present evidence, however, that at least the gene for pristanoyl-CoA oxidase is present in the human. A human liver cDNA encoding a protein of 700 amino acids, showing 75% amino acid identity with rat pristanoyl-CoA oxidase and harbouring a peroxisomal C-terminal-targeting signal (SKL), was isolated. Bacterial expression of the cDNA resulted in a fusion protein that was cross-reactive with antibodies directed against rat pristanoyl-CoA oxidase and the C-terminal SKL sequence. Screening of a genomic library with the isolated cDNA as a probe resulted in a genomic clone in which four introns were localized. By means of fluorescence in situ hybridization the gene for human pristanoyl-CoA oxidase was mapped at chromosome position 4p15.3. We conclude that a gene for pristanoyl-CoA oxidase is present in the human genome. The gene appears to be expressed to such a low extent in liver that its mRNA cannot be detected by routine Northern-blot analysis and that its product remains undetected by standard immunoblotting or by enzyme activity measurements. We speculate that the gene may be expressed under special (e.g. certain developmental stages) conditions or in certain specialized tissues not examined thus far.

Clinical Comparison of Borreliacidal-Antibody Test With Indirect Immunofluorescence and Enzyme-Linked Linked Immunosorbent Assays for Diagnosis of Lyme Disease

To compare the clinical results with the borreliacidal-antibody test (BAT) and two standard screening serologic tests for Lyme disease (LD)-the indirect immunofluorescence assay (IFA) and the enzyme-linked immunosorbent assay (ELISA). The medical records of patients from an endemic LD area, who had been serologically tested during the summer of 1992, were retrospectively categorized by clinical diagnoses without results of serologic tests. Serologic testing, which included control serum samples from patients from a nonendemic LD area, was performed in a blinded fashion, and the results were compared with the clinical categories. Medical records of 307 patients who had been serologically tested for LD were reviewed. We found untreated, active LD in 43 patients (early-localized LD, 21; early-disseminated LD, 14; and late-disseminated LD, 8) and treated LD in 33. Non-LD cases were categorized into acute or chronic conditions of unknown or known cause. Overall, the BAT had a sensitivity of 11% in active LD and did not correlate with results of other conventional surface antibody assays. The IFA and ELISA were more sensitive (67 to 93%), but false-positive results frequently were noted (20 to 40%) in acute and chronic non-LD inflammatory conditions. The specificity of the BAT, IFA, and ELISA in the control group was 96%, 93%, and 97%, respectively. Until the sensitivity, as measured by prospective clinical studies, is improved without loss of specificity, the BAT should not be used clinically for the diagnosis of LD. Suspected cases of LD with atypical clinical manifestations should have positive ELISA and IFA results confirmed with a standardized immunoblot assay.

Sensitive detection of metallothioneins-1, -2 and -3 in tissue homogenates by immunoblotting: a method for enhanced membrane transfer and retention

Standard immunoblotting procedures were unable to detect metallothioneins-1 and 2 (MT-1, MT-2) and metallothionein-3 (MT-3)/growth inhibitory factor (GIF) in unfractionated brain homogenates. We have developed a novel process which involves the inclusion of 2 mM CaCl 2 in electrophoretic transfer buffers and glutaraldehyde fixation following transfer to either nitrocellulose or polyvinylidene difluoride (PVDF) membranes. Using commercial MT antibodies and a specific MT-3 polyclonal antibody raised in our laboratory, we have been able to detect all three MTs on both membrane types with a detection limit of approx. 10 ng for MT-1 and MT-2. Nitrocellulose membrane pore size had no noticeable effect on detection sensitivity. These modifications enable more sensitive MT detection than previously described blotting methods. In addition, this technique eliminates the need for indirect monitoring approaches and simplifies quantification since sample fractionation or enrichment are not required.

Selection of a gene for apolipoprotein a1 using autoantibodies from a patient with systemic lupus erythematosus

To investigate immunoreactivity of systemic lupus erythematosus (SLE) sera with apolipoprotein A1, (Apo A1), the major lipid-binding protein of high-density lipoprotein (HDL). Since early attempts to identify Apo A1 autoantibodies using standard enzyme-linked immunosorbent assay (ELISA) and immunoblotting techniques had been unsuccessful, a mouse complementary DNA lambda phage expression library was screened. A selected clone (MA1) was found to have 82% DNA sequence homology to a segment of human Apo A1. Since there were nonconservative substitutions in the MA1 protein and lack of a complete sequence, it was possible that the SLE patient's antibodies were binding MA1 epitopes that were shared by the complete human protein but had not been conformationally accessible using the earlier techniques. Thus, gamma-irradiated ELISA plates were used as an alternative antigen-binding surface for intact human Apo A1, and high-titer anti-human Apo A1 autoantibodies were then identified in the sera of 5 more SLE patients. These findings show that Apo A1 is immunogenic. Apo A1 antibodies may play a role in the decreased HDL levels and Apo A1:Apo B ratios previously reported to occur in subgroups of SLE patients.

Downward Blotting of Proteins in a Model Based on Apolipoprotein(a) Phenotyping

Standard immunoblotting ("Western blot") involves electrotransfer of proteins from a separation gel (usually acrylamide) onto a membrane. Recently, a downward capillary method with increased hybridization efficiency was developed for DNA and RNA. The present work assessed the applicability of this method to proteins in a model based on human apolipoprotein(a) [apo(a)] isoforms which consist of a single, >200-kDa polypeptide chain varying in size with a repeat sequence. After reduction treatment and sodium dodecyl sulfate-agarose gel electrophoresis, serum proteins were transferred from the gel by upward or downward (Turboblotter) capillary action onto nitrocellulose membranes in Tris-buffered saline, pH 7.5, at room temperature. Increased detectability of apo(a) isoforms was achieved by substituting comparatively high molar concentrations of protein A for true second antibody. With downward capillary transfer and short 37°C incubations, the apo(a) phenotyping could be completed in about 26 h and required less than 8 h effective processing time. The downward transfer was about twice as fast (complete within 1 h) as the upward version and with this speed it offers a good alternative to electroblotting as well.

A Salmonella enteritidis 11RX pilin induces strong T-lymphocyte responses

Our previous work, using proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to define antigens of Salmonella enteritidis 11RX able to stimulate T cells from S. enteritidis 11RX-primed (BALB/c x C57BL/6)F1 mice, had indicated the presence of a major antigenic determinant of 14 to 18 kDa (H.-M. Vordermeier and I. Kotlarski, Immunol. Cell. Biol. 68:299-305, 1990). The 14-kDa size is similar to that of the monomeric units of one of the fimbrial structures, SEF14, produced by a human enteropathogen, S. enteritidis 27655 (J. Feutrier, W. W. Kay, and T. J. Trust, J. Bacteriol. 168:221-227, 1986). Here we present data which indicate that S. enteritidis 11RX also produces this protein and that it is able to elicit delayed-type hypersensitivity reactions in S. enteritidis 11RX-primed animals and to stimulate in vitro proliferation of, and cytokine release from, T cells obtained from these animals, implying that this fimbrial protein is likely to be an important immunogen of S. enteritidis. The protein was purified to homogeneity and is free from contamination with lipopolysaccharide. Standard immunoblot analysis with unabsorbed S. enteritidis 11RX antiserum and antiserum absorbed with Salmonella typhimurium C5 and various strains of Escherichia coli, as well as a panel of anti-14-kDa-protein monoclonal antibodies, suggests that this fimbrial protein is not the common antigen expressed by a number of organisms belonging to the family Enterobacteriaceae. Immunogold electron microscopy with one of these monoclonal antibodies confirms that the 14-kDa protein and SEF14 are identical.

Identification of a Vaccinia Virus Penetration Protein

A vaccinia virus structural protein responsible for infection was identified by monoclonal antibodies (mAb). Two mAbs (2D5 and 8C2) neutralized the virus at a dilution of about 105. The 2D5 mAb reacted with VP29K under standard immunoblotting conditions and with a 23-kDa protein when virus was dissociated under nonreducing conditions. The 8C2 mAb reacted with the 23-kDa protein, but not with VP29K. Two-dimensional electrophoresis demonstrated that the 23-kDa protein was the nonreduced form of VP29K. Since they possess the same N-terminal amino acid sequence, the protein was renamed VP23-29K. The gene that encoded it was HindIII A17L ORF. The VP23-29K-dependent process of infection did not occur during the adsorption phase at 4°, and trypsin-treated virus could complete the process within 10 min at 37°. One half of the trypsin-treated intracellular mature virus (IMV) achieved the process within 20 min, but for normal IMV this time period was 2 hr. VP23-29K had function for the early step of penetration, and the functional site in the nonreduced 23-kDa form was masked to some extent in normal virus. The late cell fusion by the fusion positive (F+) D1 mutant proceeded in neutral pH. Cells infected with F- IHD-J strain virus did not fuse, but a short treatment with pH 5 medium developed cell fusion. Both of the cell fusions were inhibited by the 2D5 and 8C2 mAbs. Virion VP23-29K was suggested to be the fusion protein for the early penetration and the late cell fusion phases of vaccinia infection cycle.

Seroprevalence of hepatitis and HIV infection among rural pregnant women in Cameroon.

Since some hepatitis viruses and the human immunodeficiency viruses share common modes of transmission, such as the sexual route, we undertook to investigate the prevalence of antibodies to these and other pathogens among 384 rural pregnant women. Our study was intended to form the basis of infection management policies in pregnancy. Antibodies and other markers of the hepatitis A, B, C, and D viruses (HAV, HBV, HCV, HDV), the human immunodeficiency virus type 1 (HIV-1) and Treponema pallidum were sought. We tested for antibodies to the viruses using the appropriate enzyme-linked immunosorbent assays. HCV and HIV-1 infection were confirmed using standard immunoblotting techniques. Regarding HBV, we tested for the surface antigen (HBsAg), antibody to the surface antigen (anti-HBs) and antibody to the core antigen (anti-HBc). A non-specific test, the rapid plasma reagin test (RPR), was used for estimating Treponema pallidum (syphilis) infection. We found an overall prevalence of antibodies to HAV of 91.4%, to HCV of 6.8%, to HDV of 0%, and to HIV-1 of 3.5%. We found no IgM antibodies to HAV. The incidence of HBV markers was as follows: 5.4% for HBsAg, 61.3% for anti-HBs, and 84.6% for anti-HBc. RPR reactivity was found in 15.8% of the women. These results will be used to establish appropriate management and preventative policies for women attending the antenatal clinic. Prevention and appropriate early treatment of infections in these women will be considered.

Skin Explant Culture: A Reliable Method for Detecting Pemphigoid Antibodies in Pemphigoid Sera that Are Negative by Standard Immunofluorescence and Immunoblotting

We investigated the presence of bullous pemphigoid antibodies in bullous pemphigoid sera that are negative by standard indirect immunofluorescence. We incubated each of four indirect immunofluorescence-positive bullous pemphigoid sera, seven indirect immunofluorescence-negative bullous pemphigoid sera, one indirect immunofluorescence-negative herpes gestationis serum, three indirect immunofluorescence-positive epidermolysis bullosa acquisita sera, five indirect immunofluorescence-negative epidermolysis bullosa acquisita sera, and two normal human sera with fresh human skin explants in medium 199 at 4 degrees C for 48 h. All bullous pemphigoid sera, herpes gestations serum, and the three indirect immunofluorescence-positive epidermolysis bullosa acquisita sera had IgG that bound the basement membrane zone of skin explants with moderate to marked intensity as demonstrated by immunofluorescence. Normal sera and indirect immunofluorescence-negative epidermolysis bullosa acquisita sera failed to bind the explant basement membrane zone. Immunoblotting of bullous pemphigoid sera showed five of seven indirect immunofluorescence-negative bullous pemphigoid sera to bind high-molecular weight and/or low-molecular weight bullous pemphigoid antigens from epidermal extracts. We conclude that the skin explant culture system is a very sensitive method for the detection of bullous pemphigoid antibodies in all bullous pemphigoid sera.

Cultured Human Melanocytes Express the Intermediate Filament Vimentin

Human melanocytes are neural crest-derived cells that synthesize the pigment melanin. These cells migrate from a central location to the dermal-epidermal junction early in gestation and situate themselves between keratinocytes of the basal layer of the epidermis, extending thin dendritic processes upwards into the epidermis. In vitro, neonatal melanocytes can assume a variety of morphologies, depending on the culture conditions. Using standard immunofluorescent, immunoblotting, and Northern blotting techniques, we investigated the expression of intermediate filament proteins and demonstrate here that cultured human melanocytes express vimentin gene and protein under a variety of culture conditions. Vimentin is a 57-kD intermediate filament protein synthesized primarily by cells of mesenchymal origin. It is transcribed as a single-messenger RNA species of 2.0 kb and the human gene is located on chromosome 10. As a member of the intermediate filament family of proteins, we suggest that vimentin is an important component of the cytoskeleton of neonatal, human melanocytes.

Direct immunodetection of surface adsorbed proteins

A modification of standard immunoblotting techniques gives rise to a novel method of imaging surface associated proteins without removing them from their original arrangement on the surface. The original proteins and subsequent antibody probe reactions are performed directly on the surface. The substrate is adsorbed into the nitrocellulose support which, in semi-dry contact, reacts only in the regions in which the target protein is antigenically present. The sensitivity of the method is comparable to other immunological techniques and diffusion of the resulting images is negligible resulting in reproducible reblots from the same surface with high lateral resolution. The method can be applied to a wide variety of reasonably flat surfaces. It demonstrates two dimensional protein patterns as a function of varying surface characteristics as well as differing protein detectabilities in their purified form as opposed to the same molecule as a plasma protein component adsorbed onto a surface. The method promises to be a useful tool in circumstances where surface bound proteins are not completely removable from surfaces or are lost in commonly used extraction or analytical techniques.