Immunochemical Studies Research Articles

Fatty acid omega and (omega-1)-oxidation within intrasplenically transplanted fetal hepatocytes.

The expression and enzymatic activity of the cytochrome P450 LAomega within intrasplenically transplanted hepatocytes was investigated. Fetal hepatocytes were harvested from spontaneously hypertensive rats and transplanted into recipient adult spontaneously hypertensive rat spleens. Microscopic examination revealed masses of hepatocytes in the red pulp. Immunochemical studies detected cytochrome P450 LAomega in transplanted hepatocytes by 6 and 10 weeks after transplantation. Cytochrome P450 LAomega mRNA accumulates at 6 weeks after transplantation. Cytochrome P450-arachidonic acid omega/omega-1 hydroxylase activity (formation of 20/19-hydroxyeicosatetraenoic acid) was detected at 10 weeks after transplantation. These results demonstrated that fetal hepatocytes grow in the spleen and function similarly to adult hepatocytes.

Immunological and chemical studies of Salmonella haarlem somatic antigen epitopes. II. Serological investigations.

Lipopolysaccharide of Salmonella haarlem was hydrolyzed and the products separated. Native O-polysaccharide antigen was oxidised with sodium periodate followed by reduction with sodium borohydride. Native and chemically modified antigens were the subject of immunochemical studies. Monoclonal antibodies against S. haarlem and polyclonal rabbit antisera against S. haarlem, S. typhi and S. anatum bacteria were produced. The serological relationship between the lipopolysaccharide of Salmonella bacteria belonging to the two different groups D2 and E1 was demonstrated using haemagglutination reactions, inhibition of haemagglutination and immunoblotting. Cross-reactions were observed in haemagglutination reactions and in immunoblotting between antisera to S. haarlem, S. typhi and S. anatum. Factors 3,9,46 were found in the S. haarlem strain and the sugar composition for the epitopes of each factor was determined.

Immunological and chemical studies of Salmonella haarlem somatic antigen epitopes. II. Serological investigations

Lipopolysaccharide of Salmonella haarlem was hydrolyzed and the products separated. Native O-polysaccharide antigen was oxidised with sodium periodate followed by reduction with sodium borohydride. Native and chemically modified antigens were the subject of immunochemical studies. Monoclonal antibodies against S. haarlem and polyclonal rabbit antisera against S. haarlem, S. typhi and S. anatum bacteria were produced. The serological relationship between the lipopolysaccharide of Salmonella bacteria belonging to the two different groups D 2 and E 1 was demonstrated using haemagglutination reactions, inhibition of haemagglutination and immunoblotting. Cross-reactions were observed in haemagglutination reactions and in immunoblotting between antisera to S. haarlem, S. typhi and S. anatum. Factors 3,9,46 were found in the S. haarlem strain and the sugar composition for the epitopes of each factor was determined.

Adrenocortical and gonadal expression of the mammalian Ftz-F1 gene encoding Ad4BP/SF-1 is independent of pituitary control.

Ad4BP/SF-1 is a transcription factor essential for the development of the adrenal gland and the gonads as well as for the maintenance of their functions through regulating tissue-specific gene transcription. In the whole body, hypothalamo-pituitary-gonadal and -adrenal axes are known to play prominent roles in mediating the function of the gonads and adrenal. In this study, the effects of the tropic peptide hormones secreted by the pituitary on the regulation of the rat Ftz-F1 (rFtz-F1) gene encoding Ad4BP/SF-1 were investigated. Immunochemical studies revealed that Ad4BP/SF-1 was expressed even in the adrenal cortex of hypophysectomized rats. Such persistent expression of Ad4BP/SF-1 was also observed in the testes and ovaries of the hypophysectomized animals. In contrast to Ad4BP/SF-1, the expressions of steroidogenic P450s were reduced significantly. The transcriptional activities of the endogenous and transfected rFtz-F1 genes were examined with Y-1 and I-10 cells derived from mouse adrenocortical and testicular Leydig cell tumors, respectively. Neither gene appeared to be activated significantly by cAMP, whereas both endogenous and exogenous CYP11A genes encoding P450(SCC) were activated. Taken together, these observations indicate that the expression of the rFtz-F1 gene is mainly regulated by a mechanism independent of the neuroendocrine axes.

Epitopes close to the apolipoprotein B low density lipoprotein receptor-binding site are modified by advanced glycation end products.

Advanced glycation end products (AGEs) are thought to contribute to the abnormal lipoprotein profiles and increased risk of cardiovascular disease of patients with diabetes and renal failure, in part by preventing apolipoprotein B (apoB)-mediated cellular uptake of low density lipoproteins (LDL) by LDL receptors (LDLr). It has been proposed that AGE modification at one site in apoB, almost 1,800 residues from the putative apoB LDLr-binding domain, may be sufficient to induce an apoB conformational change that prevents binding to the LDLr. To further explore this hypothesis, we used 29 anti-human apoB mAbs to identify other potential sites on apoB that may be modified by in vitro advanced glycation of LDL. Glycation of LDL caused a time-dependent decrease in its ability to bind to the LDLr and in the immunoreactivity of six distinct apoB epitopes, including two that flank the apoB LDLr-binding domain. ApoB appears to be modified at multiple sites by these criteria, as the loss of glycation-sensitive epitopes was detected on both native glycated LDL and denatured, delipidated glycated apoB. Moreover, residues directly within the putative apoB LDLr-binding site are not apparently modified in glycated LDL. We propose that the inability of LDL modified by AGEs to bind to the LDLr is caused by modification of residues adjacent to the putative LDLr-binding site that were undetected by previous immunochemical studies. AGE modification either eliminates the direct participation of the residues in LDLr binding or indirectly alters the conformation of the apoB LDLr-binding site.

Immunohistochemical indicators of early brain injury: an experimental study using the fluid-percussion model in cats.

To detect early changes in neurons and astrocytes by immunohistochemical methods using antibodies against the neuron-specific enolase (NSE), neurofilament, glial fibrillary acid protein (GFAP), and S-100 protein, a fluid-percussion injury model in cats was chosen, in which a severe grade of injury (3.5-5.5 atm) was produced. Neuropathologic changes were produced through brain deformation by pressure gradients at the time of injury. The neuronal NSE immunoreactivity in the parietal cortex and the brain stem began to decrease at 1 to 2 hours after injury and were reduced markedly or even lost 4 hours after injury. Axons in the cerebral white matter and corpus callosum and in the hemorrhage regions at the brain stem were waved and enlarged <4 hours after injury. From 4 hours after injury, retraction balls were found after staining by antibody for the neurofilament. The GFAP-positive astrocytes appeared in the impact site in the parietal cortex and in the brain stem from 4 hours after injury, whereas S-100-positive astrocytes were not markedly changed, indicating that early after the injury, astrocytes manifested reactive hypertrophy without proliferation. These results suggest that immunochemical studies on NSE, neurofilament, GFAP, and S-100 are useful in pathologic and forensic practice in a patient who survives for a short time after a fatal head injury but without obvious focal damage.

Involvement of calcineurin in Ca2+ paradox-like injury of cultured rat astrocytes.

The Ca2+/calmodulin-dependent phosphatase calcineurin may have physiological and pathological roles in neurons, but little is known about the roles of the enzyme in glial cells. We have previously reported that reperfusion of cultured astrocytes in Ca2+-containing medium after exposure to Ca2+-free medium caused Ca2+ influx followed by delayed cell death. In this study, we examined if calcineurin is involved in this Ca2+-mediated astrocytic injury. FK506, an inhibitor of calcineurin, protected cultured rat astrocytes against paradoxical Ca2+ challenge-induced injury in a dose-dependent manner (10(-10)-10(-8) M). Cyclosporin A at 1 microM mimicked the effect of FK506. Rapamycin (1 microM) did not affect astrocyte injury, but it blocked the protective effect of FK506. Deltamethrin (20 nM), another calcineurin inhibitor, had a similar protective effect, whereas okadaic acid did not. FK506 affected neither paradoxical Ca2+ challenge-induced increase in cytosolic Ca2+ level nor Na+-Ca2+ exchange activity in the cells, suggesting that the calcineurin is involved in processes downstream of increased cytosolic Ca2+ level. Immunochemical studies showed that both calcineurin A (probably the A beta2 isoform) and B subunits were expressed in the cells. It is concluded that calcineurin is present in cultured astrocytes and it has a pathological role in the cells.

Pyruvate dehydrogenase complex deficiency and altered respiratory chain function in a patient with Kearns–Sayre/MELAS overlap syndrome and A3243G mtDNA mutation

Combined alteration of the pyruvate dehydrogenase complex and respiratory chain function is described in a 21 year-old male patient with overlapping MELAS (mitochondrial encephalomyopathy, lactic acidosis, and `stroke-like' episodes) and Kearns–Sayre syndrome. Progressive external ophthalmoplegia, pigmentary retinopathy and right bundle branch block were present when he experienced the first `stroke-like' episode at 18 years old. The A>G tRNA Leu(UUR) point mutation at nucleotide 3243 of the mitochondrial DNA was predominant in muscle tissue (79%) and present, but at lower levels in fibroblasts (49%) and blood cells (37%). Biochemical analysis revealed diminished activities of pyruvate dehydrogenase (23%) and respiratory chain complexes I and IV (57%, respectively) in muscle, but normal activities in the fibroblasts. Immunochemical studies of the muscular pyruvate dehydrogenase components showed normal content of E1α, E1β and E2 protein. Molecular screening of the E1α gene did not indicate a nuclear mutation. These observations suggest that mitochondrial DNA defects may be associated with altered nuclear encoded enzymes which are actively imported into mitochondria and constitute components of the mitochondrial matrix. Biochemical workup of mitochondrial disorders should not be restricted to the respiratory chain even if mitochondrial DNA mutations are present.

Maturation of Qa-1b Class I Molecules Requires β2-Microglobulin But Is TAP Independent

Abstract Two conformationally distinct and stable forms of Qa-1b, one strongly associated with β2-microglobulin (β2m) and the other associated with a novel molecule, gp44, were observed during immunochemical studies on the expression of Qa-1b molecules in mouse spleen cells. Both forms are efficiently processed and expressed at the cell surface. However, a large proportion of Qa-1b was found to be disulfide linked to gp44 without any detectable β2m. In TAP1-deficient mice, both forms undergo carbohydrate processing and are expressed on the cell surface, suggesting that they may traffic using a pathway not requiring a TAP association step. Consistent with this, size exclusion chromatography of newly synthesized class I molecules shows that high molecular mass complexes containing H-2Kk do not contain Qa-1b. Although Qa-1b can be stably expressed without β2m, there was no maturation of either form in cells from β2m-deficient mice where heavy chains were rapidly degraded. These results suggest that Qa-1b, like most other class I molecules, requires β2m for an initial folding step. However, β2m is not essential for subsequent processing of Qa-1b molecules.

Baculovirus expression of an insect gene that encodes multiple neuropeptides

Sex pheromone production in the corn earworm, Helicoverpa zea, is regulated by a 33-amino-acid neuropeptide named Hez-PBAN (pheromone biosynthesis activating neuropeptide). Hez-PBAN is encoded in a preprohormone that also contains four other structurally related peptides. Two recombinant baculoviruses that contain two different sequences of Hez-PBAN cDNA under the control of a strong polyhedrin promotor were constructed. The first virus, AcWT-PBAN, contains the entire prepro-Hez-PBAN coding sequence. The second virus, AcBX-PBAN, contains a synthetic chimera gene encoding a bombyxin signal peptide sequence fused to a pro-Hez-PBAN sequence. Cell extracts, culture medium of BTI-TN-5B1-4 cells, and hemolymph from 4th instar Trichoplusia ni larvae, all infected with AcBX-PBAN, showed a high level of pheromonotropic activity. Pheromonotropic activity was not detected in the cells infected with AcWT-PBAN. Results of chromatographic and immunochemical studies showed that some of the potential processing sites in the expressed pro-Hez-PBAN sequence were not used during posttranslational processing in the AcBX-PBAN-4-infected BTI-TN-5B1-4 cells and 4th instar T. ni larvae. However, the processing pattern of the recombinant pro-Hez-PBAN in AcBX-PBAN-infected 4th instar T. ni larvae was similar to that exhibited in the central nervous system of H. zea adult females, since a PBAN-like immunoreactive-peptide-band was found in the hemolymph of Ac-BX-PBAN-4-infected 4th instar T. ni larvae. In a droplet feeding assay, neonate and 3rd instar T. ni larvae infected with AcBX-PBAN-4 showed a significant reduction in survival time (26% and 19%, respectively) when compared to control larvae that were infected with a polyhedrin-deficient virus, Ac-E10.

Cerebral Ischemia Affects Glucose Transporter Kinetics Across Rat Brain Microvascular Endothelium: Quantitative Analysis by an In Situ Brain Perfusion Method

Background It has been reported that cerebral ischemia induces a dissociation between cerebral blood flow and blood–brain barrier glucose transport, but mechanisms of the dissociation are not yet clearly understood. Recent immunohistochemical studies reveal discrepancies of the results between physiologic and immunochemical studies. The purpose of this study was to quantify changes of the blood–brain barrier glucose transporter kinetics following cerebral ischemia by an in situ brain perfusion technique. Methods Fifty-six adult male Sprague-Dawley rats were divided into control and ischemia groups, and four-vessel occlusion was done as an ischemic insult. To obtain regional capillary permeability surface area products of glucose and regional perfusion fluid flow rates, the perfusion fluid (HCO 3-buffered saline) was dually labeled with [ 14C]-2-Deoxyglucose and [ 3H]-Diazepam, and the brain was perfused at a constant rate via the external carotid artery. After sampling tissues from three regions (frontal, frontoparietal lobe, and caudoputamen), dual scintillation counting was performed. From the results, we determined kinetic parameters, including V max, K m, and K d as described in the Michaelis–Menten equation, by weighted nonlinear least squares method. Results In the ischemia group, the affinity (1/ K m) and the maximum glucose transport rate ( V max) decreased significantly. Conclusions The results suggest that severe cerebral ischemia downregulates the blood–brain barrier glucose transporter kinetics, and the discrepancies between physiologic and immunohistochemical studies may be derived from redistribution of transporters, some deformation of transporters, production of some inhibitors, recruitment of capillaries with different types of transporters, and/or the effect of surrounding glial reaction.

Regional quantitation of preprodynorphin mRNA in guinea pig gastrointestinal tract.

The endogenous opioid peptide dynorphin has been shown by immunochemical studies to be widely distributed in the gastrointestinal tract. The aim of this study was to determine basal levels of preprodynorphin (ppDyn) mRNA in different regions of the gastrointestinal tract of the guinea pig. A modified sensitive and specific solution hybridization RNase protection assay was used to quantitate ppDyn mRNA, with confirmation by gel analysis of the RNase protected hybrids and PCR amplified cDNA. This method combines high sensitivity and sufficient throughput to analyze large number of samples in a single assay. Low but measurable amounts of ppDyn mRNA were detected in fundus, duodenum, jejunum, ileum, cecum, and rectum. The rectum contained significantly more ppDyn mRNA than the stomach, small bowel, and cecum. The muscularis/myenteric plexus layer of both ileum and rectum contained a higher concentration of ppDyn mRNA per microg total RNA compared to the mucosa/submucosa/submucosal plexus. However, a greater absolute amount of ppDyn mRNA (80-85%) localized to the mucosal layer. The greater absolute amount of ppDyn mRNA in the mucosal layer may indicate the presence of dynorphin in the endocrine cells of the mucosa.

Differentiation of human pluripotent teratocarcinoma stem cells induced by bone morphogenetic protein-2.

Pluripotent human teratocarcinoma stem cells cultured in vitro provide a resource for the study of early embryonic development in man, as well as a means for discovery of novel factors controlling cell differentiation and commitment. We previously reported that the human teratocarcinoma stem cell line GCT 27X-1 could be induced to differentiate into an endodermal progenitor cell by treatment with high doses of retinoic acid. A search for polypeptide inducers of differentiation in this system has identified bone morphogenetic protein-2 (BMP-2) as a potent inducer of differentiation. In cell line GCT 27X-1, treatment with BMP-2 reduces proliferation, induces morphological changes similar to obtained following treatment with retinoic acid, and causes a decrease in the expression of transcripts for the stem cell markers CD30 and Oct-4. Preliminary immunochemical studies indicate that the differentiated cells produced by BMP-2 are endodermal precursors with a pattern of marker expression similar to that found in retinoic acid treated cells. Models of endoderm differentiation in humans will be useful for identifying the molecules which mediate cell interactions in development, and in achieving directed differentiation of cells for use in transplantation.

Cerebellar degeneration associated with human immunodeficiency virus infection

Cerebellar disorders associated with HIV infection are typically the result of discrete cerebellar lesions resulting from opportunistic infections such as toxoplasmosis and progressive multifocal leukoencephalopathy or primary CNS lymphoma. Clinical symptoms and pathologic abnormalities related to the cerebellum may also be observed with HIV dementia. A primary cerebellar degeneration with HIV has not previously been reported. Ten patients were identified over an 8-year period at five medical centers. All patients had clinical, laboratory, and radiologic evaluations, and three had neuropathologic examinations. Patients presented with progressively unsteady gait, slurred speech, and limb clumsiness. Examination revealed gait ataxia, impaired limb coordination, dysarthria, and abnormal eye movements. Cognition, strength, and sensory function remained normal. CD4 lymphocyte counts varied between 10 and 437 cells/mm3. Neuroimaging studies showed prominent cerebellar atrophy. Neuropathology showed focal degeneration of the cerebellar granular cell layer and unusual focal axonal swellings in the brainstem and spinal cord. Cultures, histopathology, and immunochemical studies showed no conclusive evidence of infection. We report a syndrome of unexplained degeneration of the cerebellum occurring in association with HIV infection.

Structural and immunochemical studies of two cross-reactive Proteus mirabilis O-antigens, O6 and O23, containing beta1-->3-linked 2-acetamido-2-deoxy-D-glucopyranose residues.

A marked serological cross-reactivity was observed by ELISA and a precipitation test between anti-Proteus mirabilis O23 serum and the lipopolysaccharide as well as the O-specific polysaccharide from the Proteus mirabilis strain belonging to serogroup O6. The structures of the O-specific polysaccharides were elucidated using chemical and NMR spectroscopic analyses, and the only common component, 2-acetamido-2-deoxy-beta-D-glucopyranose (beta-D-GlcNAc), was revealed, which was suggested to be responsible for the cross-reactivity observed. Both anti-O23 and anti-O6 sera were shown to react with 1, 3-Linked beta-D-GlcNAc-containing O-antigen from Salmonella enterica ssp. arizonae O59 also. The lack of reactivity of Smith-degraded P. mirabilis O6 O-specific polysaccharide with homologous antiserum indicated the crucial role of alpha-D-glucuronic acid in specific antibody binding.

Contact-dependent disruption of the host cell membrane skeleton induced by Trichomonas vaginalis.

This report presents evidence showing that the pathogenetic process of the protozoan parasite Trichomonas vaginalis involves degradation of the target cell membrane skeleton; spectrin, the most representative protein within this structure, has been identified as the main molecular target. Degradation of the target cell spectrin is accomplished only upon contact with the parasite, and immunochemical and immunofluorescence studies performed with the erythrocyte as a model demonstrate that degradation of the protein takes place before target cell lysis. A preliminary characterization of the effectors involved has led to the identification of a nonsecreted 30-kDa proteinase which is characterized by a high specificity for spectrin. This molecule is suggested as the main effector responsible for cytoskeletal disruption.

The Renal Handling of IgG in the Aging Rat and in Experimental Kidney Disease

The urinary excretion of total protein, low-MW proteins, albumin, high-MW proteins, and intact IgG was measured in male Wistar rats between the ages of 5-52 weeks, and in rats with experimentally induced glomerular or tubular proteinuria. About 25% of aging rats spontaneously developed focal glomerulosclerosis and a mild glomerular proteinuria. By age 52 weeks, total protein excretion in rats with glomerulosclerosis exceeded that of unaffected rats by a factor of seven (39.5 vs 5.4 mg/24 hr x 100 g body wt), and albumin excretion was seven times higher than IgG excretion in affected rats (21.2 vs 2.9 mg/24 hr x 100 g body wt). Rats with chromate toxicity exhibited a reversible tubular proteinuria, with low-molecular weight protein excretion reaching 16.8 mg/24 hr x 100 g body wt (75% of total protein excretion) at the time of peak toxicity. IgG excretion remained less than 0.6 mg/24 hr x 100 g body wt. Aminonucleoside induced a massive but reversible glomerular proteinuria (204 mg/24 hr x 100 g body wt), with IgG excretion reaching 11.4 mg/24 hr x 100 g body wt (6% of total protein excretion) at the time of peak toxicity. Biochemical and immunochemical studies showed that, while some intact IgG is present in normal rat urine, most IgG immunoreactivity is derived from low-molecular weight catabolic fragments of IgG which interfere with the immunoassay of intact urinary IgG. One of these fragments, probably Fc fragment, may be involved in the pathogenesis of focal segmental glomerulosclerosis.

Emery-Dreifuss syndrome.

Emery-Dreifuss syndrome is a heterogeneous entity characterized by the following clinical triad: early contracture of the elbows. Achilles tendons and postcervical muscles; slowly progressive muscle wasting and weakness with a humeroperoneal distribution early in the course of disease; and a cardiomyopathy usually presenting as an atrioventricular block ranging from sinus bradycardia to complete heart block. As the heart block is the major problem, insertion of a cardiac pacemaker can be life saving. Recent advances through genetic and immunochemical studies have provided valuable clues to the understanding and the early diagnosis of this disease.

Brain-derived neurotrophic factor-like immunoreactivity in the adult rat central nervous system predominantly distributed in neurons with substantial amounts of brain-derived neurotrophic factor messenger RNA or responsiveness to brain-derived neurotrophic factor

Distribution of brain-derived neurotrophic factor-like immunoreactivity was investigated in the adult rat brain using two types of antibodies against peptides, V2 and V4, unique to the brain-derived neurotrophic factor. Western blot analysis showed that both antibodies specifically bound brain-derived neurotrophic factor, but not other neurotrophins, and that they recognized identical molecules of 18 000 mol. wt, but not the 14 500 mol. wt mass of mature form, in extracts from the rat hippocampus. Both antibodies recognized an identical precursor form (30 000 mol. wt) in lysates of COS7 cells transfected with brain-derived neurotrophic factor gene. These indicated that both antibodies predominantly recognized identical precursor protein(s) or its derivative(s) probably because of their much higher amounts than the amount of mature protein. Immunochemical studies showed that anti-V2 predominantly stained the cytoplasm of cells; whereas the anti-V4 bound to the nucleus, suggesting that the tertiary structure of immunoreactive molecules changed depending on their location. Cell populations with the immunoreactivity were similar in most brain sections stained with either anti-V2 or anti-V4 antibodies. These results suggest that brain-derived neurotrophic factor-like immunoreactivity distributes, in most cases, in neurons responding to brain-derived neurotrophic factor and in neurons expressing abundant brain-derived neurotrophic factor messenger RNA. These, taken together with other results concerning distributions of messenger RNAs of brain-derived neurotrophic factor and TrkB, provide additional information to elucidate the function of brain-derived neurotrophic factor in the rat central nervous system.

Noncomplement Fixing, IgG4 Autoantibodies Predominate in Patients With Anti-Epiligrin Cicatricial Pemphigoid

This study characterized the specific reactivity, IgG subclass, and complement fixing ability of anti-laminin-5 IgG from 12 patients with anti-epiligrin cicatricial pemphigoid. Circulating IgG from all patients bound the dermal side of 1 M NaCl split skin, immunoprecipitated laminin-5 produced by biosynthetically radiolabeled human keratinocytes, and (in 10 of 12 cases) immunoblotted the laminin-alpha3 subunit. Analysis of the distribution of IgG subclasses in these patients' circulating anti-laminin-5 autoantibodies by semiquantitative indirect immunofluorescence microscopy using the HP series of subclass-specific monoclonal antibodies revealed: (i) IgG4 predominant autoantibodies in seven of 11 sera; (ii) IgG1 and IgG2 at substantially lower levels in a smaller number of sera; and (iii) no specific IgG3 anti-laminin-5 autoantibodies in any patients. The same IgG4-dominant profile of anti-laminin-5 autoantibodies was found in enzyme-linked immunosorbent assay studies of purified human laminin 5. Direct immunofluorescence microscopy of six skin biopsies from three patients found that IgG4 was also the predominant subclass of IgG in epidermal basement membranes in situ. Consistent with these findings, sera from 11 of 11 patients with anti-laminin-5 IgG autoantibodies did not fix C3 to epidermal basement membranes in vitro. These immunochemical studies suggest that complement activation does not play a major role in the pathophysiology of this disease and that subepidermal blisters in these patients may develop via a direct effect of anti-laminin-5 IgG itself.