Plasminogen activators of psoriatic scale extracts

Summary Psoriatic scale extracts were fractioned by using polyacrylamide gel isoelectric focusing (PAGIF) and preparative electrofocusing in granulated gel (PEGG). The largest protein fraction was found with Ip at pH 4.8–5.0, and the main protein bands within pH values 4.0–7.5.PEGG separated three main fractions with plasminogen activator or trypsin-like esterase activity with isoelectric points at pH 6.5–6.6, 5.4–6.2 and 4.9. The enzyme with Ip at pH 6.5–6.6 hydrolyzed trypsin substrates but lacked plasminogen activator capacity. The enzyme with Ip at pH 5.4–6.2 showed both activities but the third enzyme with plasminogen activator capacity with Ip at pH 4.9 was without detectable esterolytic activity towards substituted basic amino acid esters. The thrid enzyme was prominent in KCl-extract and the second in KSCN-extract. The first was equal in both extracts.The enzyme with Ip at pH 4.9 is possibly of bacterial origin while the plasminogen activator with Ip at pH 5.4–6.2 extracted in KSCN probably represents tissue activator of psoriatic scales.     

人精子甘露糖受体的部分特性研究

目的分离纯化人精子甘露糖受体并测定其分子量和等电点。方法用亲和层析法分离纯化人精子甘露糖受体,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳法测定其分子量,等电聚焦电泳法测定其等电点。结果人精子甘露糖受体的分子量为66.3 ku,等电点为pH=5.1。结论用亲和层析法分离得到人精子甘露糖受体,并测得其分子量和等电点,可为后续开展人精子甘露糖受体有关理化特性和医用价值的研究积累有价值的资料。     

NullCanada: A novel α1-antitrypsin allele with in cis variants Glu366Lys and Ile100Asn

Backgroundα₁-Antitrypsin (A1AT) deficiency predisposes patients to pulmonary disease due to inadequate protection against human neutrophil elastase released during inflammatory responses. A1AT deficiency is caused by homozygosity or compound heterozygosity for A1AT variants; individuals with A1AT deficiency most commonly have at least one Z variant allele (c.1096G > A (Glu366Lys)). Null variants that result in complete absence of A1AT in the plasma are much rarer. With one recent exception, all reported A1AT variants are characterized by a single pathogenic variant.CaseAn 8 years old patient from Edmonton, Alberta, Canada, was investigated for A1AT deficiency. His A1AT phenotype was determined to be M (wild type)/Null by isoelectric focusing (IEF) but M/Z by targeted genotyping. Gene sequencing revealed two heterozygous variants: Z and Ile100Asn (c.299 T > A). The Ile100Asn substitution is predicted to disrupt the secondary structure of an α-helix in which it resides and the neighbouring tertiary structure, resulting in intracellular degradation of A1AT prior to hepatocyte secretion.MethodsFamily testing was conducted to verify potential inheritance of an A1AT allele carrying the two mutations in cis, as this arrangement of the mutations would explain “Z” detection by genotyping but not by IEF. Molecular modeling was used to assess the effect of the variants on A1AT structure and stability.DiscussionCarrier status for a novel variant Null_Canada with in cis mutations (c.[299 T > A;1096G > A], p.[(Ileu100Asn;Glu366Lys)]) was confirmed. A sibling was identified as having A1AT deficiency on the basis of compound heterozygosity for two alleles: Null_Canada and the common Z allele. A separate pedigree from the Maritimes was subsequently recognized as carrying Null_Canada.ConclusionIn cis mutations such as Null_Canada may be more common than previously described due to failure to detect such mutations using historical testing methods. Combined approaches that include gene sequencing and segregation studies allow recognition of rare A1AT variants, including in cis mutations.     

人参叶皂甙对大鼠海马盐皮质激素受体的影响

本文应用聚丙烯酰胺凝胶板等电聚焦电泳(PAGIF)方法,检测给人参叶皂甙后,大鼠海马盐皮质激素受体(MR)的变化,实验结果表明,每日按50mg/kg给人参叶皂甙一周,大鼠海马MR平衡状态下饱和曲线的饱和点为30nmol/L,5nmol/L。海马MR结合量有下降趋势,对照组为226.21±54.8fmol/mg pro,给人参叶皂甙组为196.79±57.2fmol/mg pro.,统计学上并无显著性差异。由此我们认为大鼠海马MR对人参叶皂甙的反应不明显,可能人参叶皂甙对中枢水盐平衡的调节不是其主要功能。     

Mitogen-induced phosphorylation of cytosolic proteins in rabbit T- and B-lymphocytes

The addition of anti-immunoglobulin (anti-Ig) to purified rabbit B-cells or concanavalin A (Con A) to purified rabbit T-cells within minutes resulted in the phosphorylation of a number of cytosolic proteins. Two-dimensional (2-D) electrophoresis and autoradiography of 32P-labeled cell sonicates was used to identify proteins whose phosphorylation was enhanced by these mitogens. Two proteins, pp58 and pp90, were phosphorylated 1.5 min after addition of anti-Ig to B-cells. Four other proteins, pp60, pp65, pp67 and pp95, were phosphorylated at later times. Three of these proteins were also phosphorylated after addition of Con A to purified T-cells. These phosphoproteins do not correspond to any previously described cytoplasmic proteins. Although all of these phosphoproteins were present in the cytosolic fraction, pp58 may be associated with the cytoskeleton. Protein pp58 is also distinguished from the rest by its absence from 2-D gels run under non-reducing conditions. Treatment of the B-cells with F(ab')2 fragments of anti-Ig stimulated phosphorylation but Fab' fragments did not--indicating that receptor cross-linking is required to induce phosphorylation. Both pp58 and pp90 contained phosphoserine, but neither phosphothreonine nor phosphotyrosine. Quantitatively the 32P-labeling of pp58 was 2.7-fold over background at 10 min after anti-Ig addition. The identification of these phosphoproteins, which may play a role in activational cascades or in cytoskeletal rearrangements, hopefully will help to clarify the interrelationships between cyclic nucleotide dependent and independent kinases in lymphocyte activation.     

The effect of myelin basic protein on the protease inhibitors alpha1 antitrypsin and alpha2macroglobulin

The effect of myelin basic protein (MBP) of central nervous tissue on the protease inhibitors, alpha 1 antitrypsin (a1AT) and alpha 2 macroglobulin (a2M) was studied in vitro. For this purpose, 2 characteristics of the protease inhibitors were used, viz. their pattern after isoelectric focusing and their trypsin-binding capacity. Both features of purified a2AT disappeared in the presence of MBP. The tests with a2M revealed that the formation and stability of the complex between a2M and protease were reduced. The results of this study suggest that MBP causes an increase in proteolytic activity by inactivating protease inhibitors. The potential relevance of these results for demyelinating processes in man and animal is discussed.     

Ultimate intrinsic signal-to-noise ratio in MRI

A method to calculate the ultimate intrinsic signal-to-noise ratio (SNR) in a magnetic resonance experiment for a point inside an arbitrarily shaped object is presented. The ultimate intrinsic SNR is determined by body noise. A solution is obtained by optimizing the electromagnetic field to minimize total power deposition while maintaining a constant right-hand circularly polarized component of the magnetic field at the point of interest. A numerical approximation for the optimal field is found by assuming a superposition of a large number of plane waves. This simulation allowed estimation of the ultimate intrinsic SNR attainable in a human torso model. The performance of six coil configurations was evaluated by comparing the SNR of images obtained by the coils with the ultimate values. In addition, the behavior of ultimate intrinsic SNR was investigated as a function of main field strength. It was found that the ultimate intrinsic SNR increases better than linearly with the main magnetic field up to 10 T for our model. It was observed that for field strengths of 4 T or higher, focusing is required to reach the ultimate intrinsic SNR.     

人血清apoE 表型等电聚焦免疫印迹分析法

为了简便、快速、准确地检测载脂蛋白Ｅ（ａｐｏＥ）表型，我们建立了人血清ａｐｏＥ等电聚焦免疫印迹分析法，并初步分析了５６例健康成人不同样品的ａｐｏＥ表型。结果显示，ａｐｏＥ表型显色图谱清晰，谱带典型，结果容易判别，可长期保存。该法样品用量少且无需特殊处理，短期内可完成大量样品分析，优于目前所用其它方法，尤其适用于大规模流行病学调查及临床实验室。     

Preferential induction of fetal versus embryonic globin chains in human leukemic cell lines

By use of a newly developed technique combining affinity chromatography of hemoglobin on haptoglobin-Sepharose and IEF of globin chains, we analyzed the globin synthetic pattern of human K562 cells in both the basal state and after addition of several potential inducers. Hemin only was found effective: its addition at 50 microM results in a quantitative increase of globin chain synthesis (from 0.3 to 1% up to 5%) and a qualitative "switch" with a striking increase of alpha and a decrease of epsilon and zeta chains (relative to the prevailing gamma chains). This system, in which hemin induces changes that mimic to some extent the normal embryonic-fetal switch, might therefore provide a cellular model for investigating molecular mechanisms of globin gene regulation. In addition similar results were obtained with a different human myeloid leukemia cell line, the KG1, thus raising the possibility that the expression of embryonic globin genes in malignant cells might not be simply the consequence of abnormal gene expression but rather reflect a possibly physiological differentiation phenomenon.     

High-voltage isoelectric focusing in ultrathin gels and enzyme-amplified immunoassay: A new method for analysis of cerebrospinal fluid proteins

Summary A procedure using high-voltage isoelectric focusing (IF) in ultrathin (0.2 mm) gels and enzyme-amplified immuno-sandwich assay was elaborated to get optimal IF separation conditions, to avoid CSF concentration, e.g. by ultrafiltration preceding IF with the risk of unequal protein losses, to minimize the amounts of CSF and expensive reagents needed, especially antibodies and to shorten the analysis time, including the selective detection of proteins. The high voltage (2000–3000V/10 cm) and efficient cooling during IF were obtained using ECPS 3000/150 and FBE 3000 (Pharmacia, Sweden). Ampholytes (Pharmalytes) of different pI intervals were used. The CSF and (diluted) serum samples were microdialysed in polyacrylamide gel before IF to minimize band curvature and to obtain optimal resolution. The IF separation was performed in about 1 h. Owing to the rapid fixation of ultrathin gels after IF, full use could be made of the high-voltage resolving capacity. The thin gels also made histochemical techniques applicable. Different immunological identification assays have been tested. An enzyme-amplified (alkaline phosphatase) immuno-sandwich method was found to be very sensitive and selective, and has so far given the best results. Many proteins in the same sample, applied as a line on the gel before IF, could be detected by overlaying antibody-soaked membrane strips. Furthermore, one specific protein could be examined in many samples simultaneously by overlaying or immersion of diluted antibody solutions. A few microlitres of unconcentrated CSF and diluted serum were used for the analysis performed within 1 day. The findings for albumin, transferrin and IgG in CSF and sera from patients with different neurological diseases, especially including cases with normal CSF, barrier damage, degenerative and demyelinating disorders, have been compared with the corresponding protein-stained (Coomassie R-250) patterns where the CSF had been concentrated by a special vacuum evaporation technique before IF.

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Zusammenfassung Ein Verfahren wurde ausgearbeitet, wo Isoelektrische Fokussierung (IF) mittels Hochspannung in ultradünnen (0.2 mm) Gelen und enzymverstärkter Zweischicht-Immunoentdeckung verwendet werden. Die CSF und (verdünntes) Serum wurden vor der IF in Polyacrylamidgel mikrodialysiert, um Bandkrümmung zu minimieren und optimale Trennung zu erzeugen. Die zweckmäßigsten IF-Bedingungen wurden ausgearbeitet. Proteinkonzentration der CSF vor der IF mittels Ultrafiltration wurden wegen unregelmäßiger Proteinverluste vermieden. Die verwendeten Mengen von CSF und teueren Reagenzien, insbesondere Antikörper, sind stark reduziert. Die Hochspannung (2000–3000 V/10 cm) und die effektive Kühlung in der IF wurden mittels der Geräte ECPS 3000/150 und FBE 3000 aus Pharmacia, Schweden, erzeugt. Ampholyten (Pharmalytes) verschiedener pI-Bereiche wurden verwendet. Die IF wurde in 1h durchgeführt. Dank der schnellen Fixierung der ultradünnen Gelen kann das Trennungsvermögen der IF voll ausgenützt werden. Die dünnen Gelen ermöglichen auch histochemische Untersuchungsmethoden. Verschiedene immunologische Identifikationsbestimmungen sind geprüft worden. Eine enzymverstärkte (Alkaline-Phosphatase) Zweischicht-Immunomethode schien sehr empfindlich und selektiv zu sein und hat bis jetzt die besten Resultate ergeben. Einige l unkonzentrierte CSF und verdünntes Serum wurden in einer eintägigen Untersuchung verwendet. Die Ergebnisse von Albumin, Transferrin und IgG in CSF und Serum aus Patienten mit verschiedenen neurologischen Krankheiten wurden mit den entsprechenden proteingefärbten (Coomassie R-250) Mustern verglichen.