Diurnal beta-endorphin changes in human cerebrospinal fluid

Plasma and cerebrospinal fluid (CSF) beta-endorphin levels were determined by a RIA method in seven hydrocephalic male patients. The samples were simultaneously collected every two hours from 8 AM to 12 midnight and every hour from 1 AM to 7 AM. In both plasma and CSF beta-endorphin levels showed significant time-related variations during the 24 hour period. These results suggest the existence of diurnal CSF beta-endorphin variations analogous to those observed in plasma.     

Isolation and immunoenzyme determination of human beta 2-microglobulin

Two procedures for isolation of homogeneous beta 2-microglobulin from urine of patients with systemic lupus erythematosus were developed: a procedure for isolation of beta 2-microglobulin with two-stage gel chromatography on Sephacryl S-200 and a procedure for isolation of homogeneous beta 2-microglobulin with affinity chromatography on rabbit polyclonal antibodies. The microglobulins isolated with the two procedures showed identical physicochemical properties and were used in development of a competitive immunoenzymatic assay method for determination of beta 2-microglobulin in the blood. The method was approved for determination of beta 2-microglobulin in blood serum of patients.     

Partial amino acid sequence of mouse beta2-microglobulin.

A highly purified preparation of mouse β₂-microglobulin has been obtained from sodium thiocyanate extracts of liver cell membranes of $\text{A}\text{J}$ strain mice and the amino acid sequence has been partially determined. The first 40 residues have been assigned except for position 34. In the sequence, the mouse protein differs only at 9 positions from human β₂-microglobulin at 8 positions from the dog homologue and at 11 positions from the rabbit homologue. The sequence has also a homology to the constant regions of mouse γG_2a, most closely to the C_H3 region. These data support the conclusion that the mouse protein is indeed the mouse homologue of human β₂-microglobulin.     

Age-related change in 7B2 (a novel pituitary polypeptide) concentrations in human cerebrospinal fluid

Concentrations of 7B2 (a novel pituitary polypeptide) immunoreactivity (7B2-IR) were measured using a specific 7B2 radioimmunoassay (RIA) in cerebrospinal fluids (CSFs) from 87 humans. The mean (+/- S.E.M.) concentration of 7B2-IR in CSF was 2022 +/- 68 pM and a statistically significant decrease with aging was observed in those concentrations (R = -0.28, t = 2.73, P less than 0.01), although it was not a strong relation based on the R-value. In the gel permeation chromatography of CSF on Sephadex G-100, a major peak with an apparent mol. wt. of 43 kDa (43K) and a minor peak with that of 11 kDa (11K) were observed.     

Purification and complete amino acid sequence of novel beta 2-microglobulin

We have previously reported that novel beta 2-microglobulin (beta 2m) is a metabolite derived from beta 2m in ultrafiltrate of patients on long-term hemodialysis (LT-HD). Chromatofocusing showed the presence of at least two major novel beta 2m's with isoelectric points of 5.38 and 5.22. In the present study we purified one of major novel beta 2m's and determined the complete amino acid sequence. We demonstrate herein that the novel beta 2m has the same sequence as native beta 2m except for the 17th residue from the N-terminus which was identified as Asp instead of Asn in native beta 2m, suggesting a possible deamidation during LT-HD.     

Properties of some variants of human beta2-microglobulin and amyloidogenesis

Three variants of human beta(2)-microglobulin (beta(2)-m) were compared with wild-type protein. For two variants, namely the mutant R3Abeta(2)-m and the form devoid of the N-terminal tripeptide (DeltaN3beta(2)-m), a reduced unfolding free energy was measured compared with wild-type beta(2)-m, whereas an increased stability was observed for the mutant H31Ybeta(2)-m. The solution structure could be determined by (1)H NMR spectroscopy and restrained modeling only for R3Abeta(2)-m that showed the same conformation as the parent species, except for deviations at the interstrand loops. Analogous conclusions were reached for H31Ybeta(2)-m and DeltaN3beta(2)-m. Precipitation and unfolding were observed over time periods shorter than 4-6 weeks with all the variants and, sometimes, with wild-type protein. The rate of structured protein loss from solution as a result of precipitation and unfolding always showed pseudo-zeroth order kinetics. This and the failure to observe an unfolded species without precipitation suggest that a nucleated conformational conversion scheme should apply for beta(2)-m fibrillogenesis. The mechanism is consistent with the previous and present results on beta(2)-m amyloid transition, provided a nucleated oligomeric species be considered the stable intermediate of fibrillogenesis, the monomeric intermediate being the necessary transition step along the pathway from the native protein to the nucleated oligomer.     

beta2-microglobulin locus on human chromosome 15.

We have developed an autoradiographic/electrophoretic assay capable of distinguishing mouse and human beta2-microglobulin (beta2m) in spent culture media. The method is applicable to mouse and human lines and to hybrid cell lines made from them. With this technique, mouse/human hybrid cell lines were tested for the presence of human beta2m. Isozyme and karyotype analyses were also carried out with the hybrids. The combined results of these studies show that the structural gene for human beta2m is on the long arm of chromosome 15.     

Measurement of acid monoamine metabolites in human and animal cerebrospinal fluid

Techniques for measuring 5-hydroxyindolacetic acid (5-HIAA) and homovanillic acid (HVA) have been modified to permit the use of smaller samples for measuring these acid monoamine metabolites in human and animal CSF. Levels of both HVA and 5-HIAA were approximately three times as high in human ventricular CSF as in human lumbar CSF. Lumbar CSF levels of 5-HIAA in neurologic patients were significantly higher than those in psychiatric patients. Values were obtained for HVA in dog cisternal CSF and for 5-HIAA in dog, rabbit, and cat cisternal CSF.     

The origin of indoleacetic acid and indolepropionic acid in rat and human cerebrospinal fluid

Abstract: Using a new high performance liquid chromatographic method we have measured tryptophan, 5-hydroxyindoleacetic acid (5HIAA), indoleacetic acid (IAA), and indolepropionic acid (IPA) in rat and human CSF. Experiments on rats indicate that IPA in CSF is not derived from the CNS but from bacterial metabolism in the intestine. However, IAA in CSF is derived from CNS tryptamine metabolism. Some tryptamine that is formed peripherally diffuses across the blood-brain barrier and augments the tryptamine formed within the CNS. We have concluded from our data that (i) measurements on CSF are a useful way of studying trace amine metabolism in human CNS, but it is essential to establish the anatomical and metabolic origin of any metabolite found in the CSF; and (ii) tryptamine metabolism is more important in man than in the rat.     

Identity of the HL-A common portion fragment and human beta2-microglobulin

Glycogen synthetase D from the 17,000 × g supernatant of a homogenate of human polymorphonuclear leukocytes has been purified to a specific activity of 7,4 units/mg protein in a single step, chromatography on Concanavalin A bound to agarose (Con A-Sepharose). The overall recovery of the enzyme was 66% and the entire procedure requires only 3–4 hours. After an in vitro D to I conversion, glycogen synthetase I was purified to a specific activity of 11,5 units/mg protein in a similar procedure.     

Overexpression of native human beta 2-microglobulin in Escherichia coli and its purification

beta 2-Microglobulin (beta 2M), the small subunit of human leukocyte antigen (HLA) class-I proteins, has been synthesized in Escherichia coli and purified in mg amounts. A beta 2m cDNA clone was fused in-frame behind DNA encoding the signal sequence for the outer membrane protein, OmpA. Three different constructions were made, whose products differed by the insertion of either an extra Ala residue, the hexapeptide AEFLEA [single-letter amino acid (aa) code], or no aa between the OmpA signal sequence and beta 2M-coding sequence. All three protein products were correctly processed by bacterial signal peptidase, as determined by N-terminal sequencing, and all three were secreted as soluble proteins into the periplasmic space. However, the signal sequence of the preprotein with the inserted hexapeptide, AEFLEA, was cleaved to a much greater degree than the other two preproteins. When there was no insertion, the mature protein was identical to human beta 2M, as analyzed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, circular dichroism, and native isoelectric focusing. This 'bacterial beta 2M', radiolabeled with Bolton-Hunter reagent, was able to exchange into papain-solubilized HLA-B7, as determined by Sephadex G-75 chromatography and immune precipitation, indicating that bacterial beta 2M could complex with the heavy chain of HLA-B7.     

Cross reactivity of human beta2-microglobulin with human granulocyte colony-stimulating activity.

Effects of antisera to human beta2-microglobulin (beta2 m) on factors able to stimulate colony formation in culture by human granulopoietic progenitor cells were investigated. The colony-stimulating activity (CSA) present in media conditioned by cultures of human peripheral leukocytes was suppressed by treatment with anti-beta2m. This inhibition was not due to a direct effect on the granulopietic progenitor cells; controls to test for cytotoxicity and for noncytotoxic inhibition of the progenitor cells by anti-beta2m yielded negative results. These experiments provide evidence for a relationship between human CSA and beta-microglobulin, and suggest a possible analogy between molceules involved in the in vitro regulation of granulopoiesis and products of the major histocompatibility gene complex.