Thermal gradients in microtitration plates. Effects on enzyme-linked immunoassay

Temperature studies of microtitration plates demonstrate that the use of a common bacteriology incubator for heating the plates can cause a phase lag of over 30 min for the fluid in the wells to reach 37°C from ambient temperature, and that a temperature gradient of as much as 1.6°C can exist between the peripheral and center wells. This gradient is a cause of the ‘rim’ or edge effect noted in enzyme immunoassay using microtitration plates. The problem is corrected by the use of a specially designed forced air microtitration plate incubator.     

Differential effects of interleukin 12 and interleukin 10 on superantigen-induced expression of cutaneous lymphocyte-associated antigen (CLA) and alphaEbeta7 integrin (CD103) by CD8+ T cells

At both cutaneous and mucosal sites, interleukin (IL)-10, IL-12 and transforming growth factor (TGF)-beta are important regulators of chronic inflammatory disease, where cutaneous lymphocyte-associated antigen (CLA) and alphaE integrin (CD103) may be expressed. Stimulation with streptococcal pyrogenic exotoxin C (SpeC) increased the expression of CD103 by CD8+ but not CD4+ T cells. While adding IL-12 augmented the expression of CLA, superantigen-induced expression of CD103 was markedly suppressed by IL-12, which could be reversed by TGF-beta. Antibodies against TGF-beta inhibited, and a combination of anti-TGF-beta and IL-12 completely abrogated the induced CD103 expression. IL-10 strongly decreased the frequency of CLA+ and although not increasing the frequency of CD103+CD8+ T cells, the amount of CD103 expressed per cell was markedly increased. Thus, the expression of CLA and CD103 may be antagonistically regulated by IL-10 and IL-12 and the balance between these cytokines could influence the T cell migration of inflammatory cells into epithelial tissues.     

Role of ubiquitin-mediated proteolysis in the pathogenesis of neurodegenerative disorders

Intraneuronal inclusions containing ubiquitylated filamentous protein aggregates are a common feature of many of the major human neurodegenerative disorders, including Alzheimer's and Parkinson's disease. Loss of function mutations in enzymes of the ubiquitin conjugation/deconjugation pathway are sufficient to cause familial forms of neurodegenerative diseases, suggesting that failure of ubiquitin-mediated proteolysis could also be central to inclusion formation in the more common sporadic cases. Examination of ubiquitin-positive inclusions at the protein level provides evidence of attempted proteasomal proteolysis, however close inspection of the temporal aspects of inclusion formation indicates that ubiquitylation is probably a late event. In this regard, the presence of ubiquitin within inclusions of idiopathic neurodegenerative disorders may indicate not a primary dysfunction of ubiquitin-mediated proteolysis, but rather a secondary, presumably protective cellular response. Within this model, other factors are likely to be initiating in inclusion biogenesis. Consistent with these proposals, non-ubiquitylated forms of the principal ubiquitylated components of Alzheimer's disease neurofibrillary tangles and Parkinson's disease Lewy bodies, tau and alpha-synuclein proteins, respectively, can be degraded by proteasomes in a pathway which does not have an absolute requirement for ubiquitylation. Inhibition of proteasome function in the pathological state, as has been reported in both Alzheimer's and Parkinson's disease, could therefore contribute both to accumulation of non-ubiquitylated forms of aggregation-prone neuronal proteins, as well as impaired clearance of ubiquitylated aggregates.     

^99mTc-DTPA肾动态显像及β2-m水平在评价肾移植受者肾功能中的价值

目的：探讨^99mTc-DTPA肾动态显像半定量参数和血、尿β2-m水平测定对肾移植术后早期并发症诊断与鉴别诊断的价值。方法：28例肾移植病人术后均进行放射性核素^99mTc-DTPA肾动态显像,同时测定移植肾的肾小球滤过率（GFR）、膀胱放射性计数与移植肾放射性计数比值（B/K值）和移植肾放射性1min计数与腹主动脉放射性1min计数比值（K1min/A1min比值）。在进行放射性核素肾动态显像前所有病人均收集其血液和尿液标本,采用放射免疫分析测定血、尿β2-m水平。结果：12例肾功能正常者肾动态显像示肾血流灌注及功能良好,GFR值为（49.1±6.1）ml/min,B/K值均〉3,K1min/A1min比值为8.18±1.41;4例急性排斥反应者肾血流灌注受损程度重于功能相,GFR值为（33.2±5.3）ml/min,B/K值均〈1,K1min/A1min比值为2.59±0.86,β2-m水平以血β2-m升高明显;8例慢性排斥反应者肾血流灌注和功能相均同时受损,GFR值为（19.8±7.5）ml/min,B/K值均〈1,K1min/A1min比值为2.19±0.84,β2-m水平也以血中升高明显;2例肾小管坏死者及2例环孢素A肾中毒者肾血流灌注受损均轻于功能相,GFR值分别为（38.5±4.1）ml/min和（39.4±5.81）ml/min,B/K值均〈1,K1min/A1min比值分别为5.83±0.84和6.01±0.66,β2-m水平以尿中升高显著。结论：放射性核素肾动态显像半定量参数K1min/A1min比值和B/K值,结合肾移植病人术后血、尿β2-m水平联合分析可早期初步鉴别排斥反应的类别,可作为判断移植肾受损程度、原因及预后估测的敏感指标。     

Localization of class i histocompatibility molecule assembly by subfractionation of the early secretory pathway

Class I molecules of the major histocompatibility complex bind peptides derived from cytosolic proteins and display them on the cell surface. This function alerts cytotoxic T cells to the presence of intracellular pathogens. Class I molecule assembly requires the association of the heavy chain with β₂-microglobulin, accompanied by peptide loading via specific transporters. This study localizes where these assembly steps take place, using monoclonal antibodies recognizing class I molecules in different assembly states to analyze subcellular fractions of the early secretory pathway. The distribution of peptide-loaded class I molecules was more localized than the distribution of the total pool of class I molecules in the early secretory pathway. Loaded molecules colocalized with the peptide transporter, free heavy chains, and the chaperone calnexin in high density rough endoplasmic reticulum (RER) membranes. These data suggest that subunit assembly and peptide acquisition occur at the same intracellular site. Class I molecules also localized to less dense subfractions of the early secretory pathway, which contained comparatively less peptide-loaded molecules than the high density RER fractions, at steady state. Following a 15 °C temperature block, class I molecules accumulated in these less dense membrane fractions, indicating that these fractions represent the intermediate compartment where empty class I molecules are trapped in mutant cells. In the presence of cycloheximide, a pool of class I molecules recycling to the RER was detected, suggesting empty molecules recycle to acquire peptide.     

Biochemical and morphological characterization of sympathetic neurons grown in a chemically-defined medium

Previous studies have demonstrated that individual neurons from neonatal rat superior cervical ganglion express a mixed adrenergic-cholinergic phenotype when grown under certain tissue culture conditions.^{9,14,15,29,30} The expression of this phenotype is critically influenced by a number of undefined components present in the culture medium.^18,23,33 In the present study, we have examined whether superior cervical ganglion neurons grown on a chemically defined serum-free medium similarly develop dual transmitter expression, or if under these conditions, neurons express only those properties characteristic of their adrenergic heritage. To address this issue, we established that superior cervical ganglion neurons could be maintained in culture for extended periods on the defined medium described by Bottenstein & Sato⁴ in the absence of supporting cells. We then studied the biochemical, immunocytochemical and ultrastructural characteristics of these neurons. We found that in defined medium, superior cervical ganglion neurons continued to express, in a modified form, certain of their expected adrenergic properties, including the development of tyrosine hydroxylase and dopamine-β-hydroxylase activities, stores of endogenous norepinephrine, synaptic vesicles with dense cores and tyrosine hydroxy lase-immunoreactive staining properties. Superior cervical ganglion neurons grown on a defined medium did not, however, acquire cholinergic traits in culture. In this paper we show that choline acetyltransferase activity did not reach detectable levels; the companion paper¹³ documents that cholinergic synapses were not formed.We conclude that superior cervical ganglion neurons, grown under serum-free culture conditions, develop certain properties characteristic of adrenergic neurons and do not express a mixed adrenergic cholinergic phenotype. A companion paper¹³ describes the electrophysiological properties of these neurons and demonstrates the frequent occurrence of electrotonic synapses in these cultures.     

The effect of surface sugars on liposomes in immunopotentiation

The effect of surface sugars of liposomes on the immunological responses to entrapped antigen has been investigated. alpha-Mannose and beta-galactose were grafted on the surface of liposomes containing lysozyme by covalent coupling of p-aminophenyl-D-glycosides to phosphatidyl ethanolamine liposomes using glutaraldehyde. Subcutaneous administration of antigen entrapped in beta-galactose liposomes stimulated an antibody response comparable to that elicited by sugar-free neutral liposomes. However, alpha-mannose bearing liposomes with entrapped lysozyme elicited an immune response similar to that induced by lysozyme in saline. Based on these observations it is suggested that alpha-mannose liposomes, that are specifically recognized by macrophages, are taken up rapidly by receptor mediated endocytosis and that the entrapped antigen is then rapidly degraded, resulting in low antibody production.     

β-Glucuronidase activation of latent aggression-promoting cues in mouse bladder urine

The present study was designed to investigate the chemical properties of the aggression-promoting cues present in bladder urine of male mice. The results of the first experiment confirmed earlier work by demonstrating the presence of an aggression-promoting chemosignal in bladder urine. In Experiment 2, behavioral assays were separately performed on the organic and aqueous layers of bladder urine obtained by repeated dichloromethane extractions. Only the combined organic layers of the initial three extractions demonstrated behavioral activity. A fourth extraction showed no behavioral activity for both organic and aqueous layers. However, the findings of Experiment 3 showed that incubation of the aqueous layer from the third CH₂Cl₂ extraction in β-glucuronidase can free additional aggression-promoting cues into a subsequent CH₂Cl₂ extraction. It is concluded that two forms of the aggression-promoting chemosignal are present in bladder urine. One is lipophilic and behaviorally active, whereas the other is conjugated, possessing latent chemosignal properties.     

NMDA受体在β-淀粉样蛋白引起海马神经元突触蛋白改变中的作用

为了研究NMDA受体活性对Aβ引发的海马神经元突触蛋白表达变化的影响,本文运用免疫细胞化学方法检测不同浓度NMDA受体激动剂以及拮抗剂对Aβ诱导的海马神经元突触蛋白变化的影响。结果显示:NMDA可浓度依赖性地缓解Aβ25-35引起的突触蛋白synaptophysin与PSD-95的减少。抑制突触内NMDA受体,NMDA缓解Aβ减少突触蛋白的作用减弱;抑制突触外NMDA受体,对抗Aβ的作用无显著变化。本研究结果提示NMDA受体活性改变影响Aβ诱导的突触蛋白减少,突触内NMDA受体激活可对抗Aβ的毒性作用。突触内NMDA受体活性减弱可能在谷氨酸兴奋毒性中发挥作用。