Monoclonal antibodies to canine intra-acrosomal sperm proteins recognizing acrosomal status during capacitation and acrosome reaction

Summary.  Monoclonal antibodies Ds-1 and Ds-2 specifically labelling dog sperm acrosome were prepared by immunization of mice with acetic acid extracts of dog spermatozoa. Electron microscopy and indirect immunofluorescence localized the site of Ds-1 and Ds-2 proteins inside the acrosomal vesicle. Ds-1 antibody detected 55, 76, 115, 120 and 190kDa proteins under non-reducing conditions, and 73 kDa and 54 kDa proteins after reduction (p73/Ds-1 and p54/Ds-1). 92 kDa and 40 kDa proteins recognized by Ds-2 (p92/Ds-2 and p40/Ds-2) migrated at > 200 kDa in the absence of reducing agent. In vivo , p73/Ds-1 and p54/Ds-1 are therefore likely to be present both in free and complexed form, while all of p92/Ds-2 and p40/Ds-2 form disulfide-bonded complexes. Decrease in the rate of acrosomes stained with Ds-1 and Ds-2 was correlated with the progress of capacitation resulting in the increased rate of spontaneous acrosome reactions, as suggested by a dramatic effect of A23187. Monoclonal antibody to boar acrosin (ACR-2) recognized dog sperm acrosin homologue. A higher rate of ACR-2-negative spermatozoa was observed after capacitation and A23187 treatment compared to Ds-1 and Ds-2, indicating that proteins recognized by Ds-1 and Ds-2 are localized in a specific compartment of acrosome, distinct from acrosin and possibly representing fraction of acrosomal matrix.     

Calcium incorporation in cultured chondroblasts perturbed by an electromagnetic field

We tested the hypothesis that electric perturbation influences 45Ca incorporation in extracellular matrix (ECM) of cartilage in vitro. Hypertrophic chondroblasts of tibial epiphyses (HC), sternum (SC), and skin fibroblasts (F) were cultured from chick embryos. HC, SC, and F cells were micromass seeded three times per week and maintained at 37.5 degrees C with 5% CO2 for two weeks. Cultures were randomly designated control (C) or exposed (E) to a pulsed electromagnetic field (PEMF). A time course experiment of calcium incorporation for all cultured groups showed that 24 h of exposure produced the largest biological response in chondroblasts. Calcium incorporation required supplemental phosphate. Autoradiography data indicated that the calcium incorporation into macromolecules largely occurred in the ECM. 45Ca steady-state perturbation was enhanced by Streptomyces hyaluronidase (SH) but not by testicular hyaluronidase (TH). 45Ca incorporation experiments tested the effects of phosphate, SH, TH, and PEMF alone and in various combinations on these cultures. Only PEMF or SH plus PEMF with phosphate enhanced 45Ca incorporation. Other experiments examined the effect of rotenone or freeze-thawing on cells exposed to PEMF. PEMF plus freeze-thaw enhanced calcium incorporation in HC only. PEMF appeared to cause disruption of the ECM, enhancing the probability of matrix calcification.     

Zinc ions and alkaline PH alter the phosphorylated state of proteins 3 and 4.2 in human erythrocyte membranes

The phosphorylation patterns of isolated red blood cell (RBC) membranes labeled with [ γ-³²p]ATP are altered by Zn⁺⁺ ions. Zn⁺⁺ ions caused an increased phosphate incorporation into a 72 KDa protein and several proteins in the 40–60 KDa region and a decrease in the labeling of a 53 KDa protein. The 72 KDa and 53 KDa proteins have been identified as protein 4.2 and a protease-cleaved fragment of protein 3, respectively. Evidence suggests that the changes in phosphorylation pattern may be due to the stimulation of endogenous membrane alkaline phosphatase(s). Our results suggest that Zn⁺⁺, at physiological concentrations in the intact erythrocyte, could modulate the phosphorylation of selected proteins which may regulate their association in the cytoskeletal network.     

淡色库蚊不同发育阶段双向电泳的蛋白质多肽点图谱比较

用双向电泳分析淡色库蚊卵、二令幼虫、四令幼虫、蛹和成虫蛋白质组成.电泳完毕后,用考马斯亮兰G-250染色,结果以成蚊蛋白质多肽点为最多(116点),其次依次为蛹(101点)、卵(70点)、四令幼虫(53点)、二令幼虫(18点)。它们在不同分子量和不同PH 范围内的分布也不同,但总的来看,在幼体阶段(卵、二、四令幼虫及蛹)多偏低分子量和低PI 值的点,发育至成虫阶段,分布走向全面。     

Presence of antibodies reacting specifically with structural proteins of mouse mammary tumor virus (MMTV) among antibodies composing circulating immune complexes in breast cancer patients

All-Union Oncologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. N. Trapeznikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 105, No. 4, pp. 475–477, April, 1988.     

Relationship between maternal- and fetal-specific IgE

AIM: A positive correlation between maternal and cord-blood IgE levels is well documented for total IgEs, but not for specific IgEs. The difficulty in detecting specific cord-blood IgEs is due to their low concentrations, which hinder their dosage by low-sensitivity methods. The study aimed to correlate maternal and foetal specific IgEs against individual cow's milk proteins, detected by highly sensitive and specific techniques. METHODS: Cow's milk specific IgE detection was performed by chemiluminescence on 52 specimens of maternal and cord blood after cow's milk protein separation by 1D and 2D gel electrophoresis. Cow's milk protein (CMP) antigens were identified by mass spectrometry techniques. RESULTS: Specific IgEs for CMPs were found in 25/52 (48.1%) of maternal sera and in 19/52 (37%) of cord-blood sera. In order of decreasing frequency, the proteins found were BSA, IgG heavy chain, caseins and, in a single case, b-lactoglobulin. Positive cord-blood sera in all cases corresponded to a positive maternal result, and maternal and foetal immunoreactivity patterns were closely correlated. Moreover, in no case was there a positive cord-blood response with a negative maternal response. CONCLUSION: The study demonstrates a close relationship between maternal and cord-blood specific IgE patterns. The phenomenon observed could provide a model to elucidate the general production method of foetal IgEs, which might only be produced in the presence of both the corresponding maternal IgE and the related allergen.     

Perturbation of copper (Cu) homeostasis and expression of Cu-binding proteins in cadmium-resistant lung fibroblasts.

To probe mechanisms of cadmium (Cd) damage to the lung extracellular matrix (ECM), we developed Cd-resistant (CdR) rat lung fibroblasts (RFL6) by incubation with graded concentrations of Cd. CdR cells downregulated lysyl oxidase (LO), a copper (Cu)-dependent enzyme essential for crosslinking of collagen and elastin in the ECM, in conjunction with upregulation of other Cu-binding proteins including Cu,Zn-superoxide dismutase (SOD1), copper chaperone for SOD1 (CCS1), metallothionein (MT), and Menkes P-type ATPase (ATP7A), a Cu transporter in the membrane of the Golgi apparatus, as well as gamma-glutamylcysteine synthetase (gamma-GCS), an enzyme for glutathione biosynthesis. Reduction and loss of cytoplasmic distribution of LO in CdR cells were accompanied by its dislocation with the Menkes P-type ATPase and the endoplasmic reticulum marker. CdR cells displayed a defect in LO catalytic activity but an enhancement in Cu,Zn-SOD catalytic activity consistent with the protein expression levels of these enzymes. Although long-term Cd exposure of cells enhanced the Menkes P-type ATPase protein expression, actually, it reduced Cu-dependent catalytic activity of this enzyme in parallel with the deficiency of LO. The low level of 64Cu bound to the LO fraction and the high level of 64Cu bound to the MT fraction provide direct evidence for limitation of Cu bioavailability for LO existing in the CdR cells. These results suggest that downregulation of LO is linked with upregulation of other Cu-binding proteins and with alteration in Cu homeostasis in the CdR phenotype.     

基质金属蛋白酶及微血管密度和子宫内膜癌的关系

基质金属蛋白酶及其抑制剂在子宫内膜癌的侵袭和转移中发挥着重要的作用。肿瘤的新生血管对肿瘤的发生、发展、转移及预后有着重要作用。微血管密度是衡量血管生成的定量指标。基质金属蛋白酶抑制剂可抑制血管的生成和MMPs对细胞外基质的降解。现将他们与子宫内膜癌的关系综述如下。