Both acquired and familial amyloid neuropathies carry a poor prognosis. In addition, amyloid is sometimes difficult to visualise in nerve biopsy specimens, and the pathogenesis of nerve lesions is still a matter of controversy. In order to learn more on the subject, we studied nerve specimens from seven patients with proven amyloid neuropathy by ultrastructural immunocytochemistry in order to better understand their pathogeny and to evaluate the reliability of the method for detection of amyloid antigens in the endoneurium. An indirect immunolabelling technique using protein A-gold complex (pA-g) was applied.
Polyclonal antisera against human IgG, IgM, lambda and kappa light chains and prealbumin were assayed.
Amyloid fibrils were labelled in six of seven cases: in four cases with anti-transthyretin (TTR) antibodies and in two with anti-lambda light chain antibodies.
The type of immunolabelling correlated with the biochemical type of the amyloidosis as defined by TTR gene analysis and serum immunoelectrophoresis.
The amyloid fibrils and gold labelling were always located in the endoneurial space. No intracellular deposit or labelling was found.
The immunolabelling was highly specific, gold particles being detected only near to amyloid fibrils with no background gold labelling. Ultrastructural immunolabelling with pA-g could be used for detection of amyloid in progressive axonal neuropathy of unknown origin, with important therapeutic implications.
Micropurification of two human cerebrospinal fluid proteins by high performance electrophoresis chromatography
Using C8 reversed-phase HPLC in conjunction with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we have fractionated proteins contained in human CSFs obtained from patients with schizophrenic disorders.
When these proteins were electrophoretically blotted onto polyvinylidene difluoride membrane for direct N-terminal amino acid sequencing, several CSF proteins were identified; these included albumin, transferrin, apolipoprotein A-I, beta 2-microglobulin, and prealbumin.
We have also identified two structurally related human CSF proteins designated cerebrin 28 (M(r) 28,000) and cerebrin 30 (M(r) 30,000) that have an N-terminal amino acid sequence of NH2-APPAQVSVQPNF and NH2-APEAQVSVQPLFXQ, respectively. Comparison of these sequences with existing database at Protein Identification Resource (R 32.0), GenBank (R 72.0), SWISS-PROT (R 22.0), and EMBL (R 31.0) indicated that they are unique proteins.
These proteins were subsequently purified by high performance electrophoresis chromatography (HPEC) using an Applied Biosystems 230A HPEC system. A specific polyclonal antibody was prepared and an ELISA was established for cerebrin 30.
It was noted that HPEC is a powerful tool to purify microgram quantities of proteins from human, rabbit, and rat CSFs.
Using such a system, we have been able to micropurify as many as 10 proteins simultaneously in a single experiment because the elution of proteins occurred strictly according to their molecular weights.
More importantly, we routinely obtained a recovery of>> 90%. The potential use of this technology for micropurification of proteins was discussed.
Iron carrier proteins facilitate engraftment of allogeneic bone marrow and enduring hemopoietic chimerism in the lethally irradiated host
Cell-free supernatants of rabbit bone marrow were fractionated, separated, and purified by Ultrogel and Superose chromatography.
A single fraction promoted engraftment of allogeneic bone marrow and enduring hemopoietic chimerism across the H-2 barrier in lethally irradiated mice.
This “bio-active” fraction, analyzed by reducing SDS-PAGE electrophoresis, and transblotted on PVDF membrane, and purified by reverse-phase HPLC and SDS-PAGE electrophoresis yielded a main prealbumin band that when examined for primary structure by Edman degradation, proved to be rabbit transferrin.
This was also attested by highly specific precipitation of the prealbumin band with polyclonal antibodies to rabbit transferrin.
Iron-saturated human transferrin, lactotransferrin, and egg transferrin (conalbumin) were assayed in irradiated C57BL/6 mice infused with bone marrow from histoincompatible BALB/c donors.
Mice treated with iron-loaded transferrins survive and develop enduring allogeneic chimerism with no discernible signs of graft-versus-host disease. Iron carrier proteins thus provide an unique means of achieving successful engraftment of allogeneic bone marrow in immunologically hostile murine H-2 combinations.
Amyloid in familial amyloidosis, Finnish type, is antigenically and structurally related to gelsolin.
Immunohistochemical studies of six patients with familial amyloidosis.
Finnish type, showed that their amyloid deposits did not react with polyclonal antibodies against the amyloid proteins of other, established forms of systemic or cerebral amyloidosis.
However, strong immunoreactivity was observed with rabbit antiserum raised against a low molecular weight purified amyloid subunit isolated from one of the patients. This immunoreactivity was abolished by absorption with the low molecular weight amyloid fraction.
The amino terminal sequence of the amyloid protein subunit was homologous to gelsolin, an actin-modulating protein, and the amyloid deposits in tissues reacted with a monoclonal antibody against gelsolin.
These studies show that the amyloid protein in familial amyloidosis, Finnish type, is not related to previously identified forms of amyloid, including prealbumin (transthyretin) variants, but represents a novel amyloidogenic protein related to gelsolin, a plasma and cytoplasmic protein.
Immunohistochemical localization of intracellular plasma proteins in the human central nervous system
The regional distribution of plasma protein immunoreactivity was studied in the postmortem central nervous system (CNS) of normal subjects 18 to 78 years old. Samples taken from various areas of brain and spinal cord were processed for peroxidase-antiperoxidase immunocytochemistry using polyclonal antibodies against plasma albumin, prealbumin, alpha 1-acid glycoprotein, alpha 2-macroglobulin, IgG, transferrin, haptoglobin, hemopexin, fibrinogen, as well against the glial fibrillary acidic and S-100 proteins.
Many neurons of the spinal cord, cranial nerve nuclei, pontine nuclei, cerebellar dentate nucleus, red nucleus, thalamus and hypothalamus showed strong immunostaining for albumin and moderate to strong staining for alpha 1-acid glycoprotein, IgG, transferrin, haptoglobin, as well as relatively weak immunoreactivity against other plasma proteins. Less intense staining was seen in the nucleus basalis, putamen and Purkinje cells.
In contrast, most cerebral cortical neurons were negative except for a few positively stained pyramidal neurons in the hippocampus and in layers III and V of the association neocortex, although more positive pyramidal neurons were observed in the motor and sensory neocortices.
Reaction products were also seen in axons of motor and sensory long tracts.
These findings suggest that plasma proteins may be transported to spinal cord and brain stem neurons by peripherally projecting nerves and that a series of anterograde and retrograde transneuronal transfers are responsible for the accumulation of plasma proteins in relay nuclei and in other CNS neurons.
Purification and partial characterization of a novel thyroxine-binding protein (27K protein) from human plasma.
T4-binding globulin (TBG) prepared from human plasma by the standard three-step procedure (T4-agarose affinity chromatography, anion exchange chromatography, and gel filtration) often shows in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in addition to the expected 54K band, another with a mol wt of 27,000 (27K protein).
The two proteins can be separated after the three-step procedure by chromatofocusing (because of different isoelectric points, 4.2-4.8 for TBG and 5.0-5.2 for 27K protein) or by T4-aragose chromatography eluting with a linear gradient of T4 (TBG is eluted between 10(-10) and 10(-9) M T4, 27K protein between 10(-8) and 10(-7) M T4).
The 27K protein does not appear to be a fragment of TBG since
1) it does not displace [125I]TBG bound to anti-TBG monoclonal antibodies; and
2) absorption of polyclonal antibody reacting with both TBG and 27K protein with sera from TBG-deficient patients completely prevents [125I]27K protein binding, while only slightly affecting [125I]TBG binding. On the other hand, 27K protein is not simply a contaminant devoid of biological activity, but is a T4-binding protein, as supported by the following findings:
1) it covalently binds [125I]T4 by photoaffinity labeling, and this binding can be almost completely prevented by excess T4;
2) equilibrium dialysis shows two equivalent T4-binding sites per 66K, with an association constant of 0.85 X 10(7) M-1, intermediate between albumin and prealbumin; and
3) tryptophanyl fluorescence analysis shows quenching of 37% of the fluorescence when the protein is titrated with T4.
The 27K protein appears as a single 27K band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, pH 8.8, but under nondenaturing nonreducing conditions mostly remains at the origin of the gel; a fraction enters the gel and migrates slightly ahead of albumin.
Prealbumin Protein Polyclonal Antibody, HRP Conjugated |
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| A57605 | EpiGentek |
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Guinea pig anti-Human Prealbumin protein polyclonal Antibody, HRP conjugated |
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Guinea pig anti-Human Prealbumin protein polyclonal Antibody, HRP conjugated |
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| MBS715990-01mg | MyBiosource | 0.1mg | 270 EUR |
Guinea pig anti-Human Prealbumin protein polyclonal Antibody, HRP conjugated |
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| MBS715990-5x01mg | MyBiosource | 5x0.1mg | 1205 EUR |
Prealbumin Protein Polyclonal Antibody, FITC Conjugated |
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| A57606 | EpiGentek |
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Prealbumin Protein Polyclonal Antibody, Biotin Conjugated |
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| A57607 | EpiGentek |
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Guinea pig anti-Human Prealbumin protein polyclonal Antibody, Biotin conjugated |
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| MBS715602-005mg | MyBiosource | 0.05mg | 190 EUR |
Guinea pig anti-Human Prealbumin protein polyclonal Antibody, Biotin conjugated |
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| MBS715602-01mg | MyBiosource | 0.1mg | 270 EUR |
Guinea pig anti-Human Prealbumin protein polyclonal Antibody, Biotin conjugated |
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| MBS715602-5x01mg | MyBiosource | 5x0.1mg | 1205 EUR |
Prealbumin Conjugated Antibody |
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| C49356 | SAB | 100ul | 476.4 EUR |
Prealbumin Conjugated Antibody |
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| MBS9456984-01mLAF350 | MyBiosource | 0.1mL(AF350) | 480 EUR |
Prealbumin Conjugated Antibody |
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This electrophoretic pattern is distinct from those of albumin, prealbumin, and TBG.
In immunoelectrophoresis in agar at pH 8.6, 27K protein moves slightly faster than TBG.
The results of equilibrium sedimentation indicate a mol wt of 66,000, suggesting that the 27K protein might exist as a dimer.
These data indicate that the 27K protein is a previously unrecognized T4-binding protein with a low affinity for the hormone.
Further studies are required to clarify its physiological role in the transport of circulating thyroid hormones.